key: cord- -rhld dy authors: zhong, dongwei; liu, mingming; cao, yang; zhu, yelin; bian, shihui; zhou, jiayi; wu, fengjie; ryu, kum-chol; zhou, lu; ye, deyong title: discovery of metal ions chelator quercetin derivatives with potent anti-hcv activities date: - - journal: molecules doi: . /molecules sha: doc_id: cord_uid: rhld dy analogues or isosteres of α,γ-diketoacid (dka) a show potent inhibition of hepatitis c virus (hcv) ns b polymerase through chelation of the two magnesium ions at the active site. the anti-hcv activity of the flavonoid quercetin ( ) could partly be attributed to it being a structural mimic of dkas. in order to delineate the structural features required for the inhibitory effect and improve the anti-hcv potency, two novel types of quercetin analogues, -o-arylmethylquercetins and quercetin- -o-benzoic acid esters, were designed, synthesized and evaluated for their anti-hcv properties in cell-based assays. among the newly synthesized compounds, -o-substituted derivative i and -o-substituted derivative f were found to be the most active in the corresponding series (ec( ) = . μm and . μΜ, respectively). docking studies suggested that the quercetin analogues are capable of establishing key coordination with the two magnesium ions as well as interactions with residues at the active site of hcv ns b. it was estimated that about million people worldwide were chronically infected by hepatitis c virus (hcv) and were consequently at risk of developing liver cirrhosis and/or hepatocellular carcinoma [ ] . a protective vaccine is not yet available and the previous standard of care (soc) , which is pegylated-interferon, combined with ribavirin, is often difficult to tolerate and results in a sustained viral response (svr) in only % of patients infected with the predominant genotype [ , ] . recent advances in the development of direct acting antivirals (daas) have significantly improved svr in patients, providing a new hope for cure in infected patients. three ns / a protease inhibitors were approved by fda for the treatment of genotype hepatitis c, and in late , sofosbuvir, a first-in-class ns b polymerase inhibitor, was launched and became a cornerstone of recommended hcv therapy against almost all hcv genotypes [ ] [ ] [ ] [ ] . however, due to the potential of drug resistant strains [ ] and the high price of the new hcv drugs [ ] , the development of novel anti-hcv agents is still an urgent necessity. the rna-dependent rna polymerase (rdrp) of hcv, also known as protein ns b, is a key enzyme in the synthesis of a complementary minus-strand rna and the subsequent synthesis of a genomic plus-strand rna from this minus-strand rna template [ ] . in the enzymatic reactions, two divalent cations in the active site, such as magnesium (mg + ) or manganese (mn + ) play a critical role in the ligation of the ribonucleotide triphosphate (rntp) substrates and the promotion of a favorable geometry of the active site. the pivotal role of the divalent cations in the active site of the hcv polymerase supports the rational basis of the inhibition of this enzyme by metal chelating motifs. the α,γ-diketoacid (dka) a ( figure ) has been identified as a selective and reversible inhibitor against ns b through high-throughput screening approaches. subsequent structure-activity relationship (sar) optimization led to b (figure ), a potent hcv polymerase inhibitor [ ] . the mode of action of dkas involves chelation of the metal ions present in the active site of the hcv rdrp [ ] . however, due to their unfavorable physicochemical and pharmacokinetic properties [ ] , many dka analogues or isosteres with ns b inhibitory activities, including meconic acid [ ] ( c, figure ) and dihydroxypyrimidine carboxylic acid [ ] ( d, figure ), have been designed and prepared, by keeping the metal-binding moieties to achieve effective chelation of the two mg + ions. quercetin ( , figure ) is a naturally occurring flavonoid with various biological activities such as antioxidant, antiviral, anticancer, antimicrobial and anti-inflammatory properties; hence, quercetin and its derivatives have potential as drug development leads [ ] . as a part of our studies on anti-hcv agents, quercetin was identified as an hcv inhibitor with moderate antiviral activity (ec = . μm) [ ] . furthermore, it had been reported that quercetin could inhibit hcv rdrp at low micromolar concentration ( . % inhibition at the concentration of μm in an enzyme assay) [ ] . we reasoned that the adjacent carbonyl and hydroxyl groups on a and c rings of quercetin make it a perfect dkas mimic to chelate the two metal ions. in consideration of the structures of a and b (figure ), introduction of a -cyano benzyl ether group at the aromatic meta-position relative to the chelating moieties was shown to significantly improve the antiviral activity. thus, we anticipated that substituents of quercetin at the -o position ( -o-arylmethyl quercetins , figure ) would mimic the meta-substituted dka b and enhance the anti-hcv activities. herein, we report the design and synthesis of novel -o-arylmethyl quercetin derivatives with various aromatic substituents and the evaluation of their anti-hcv activities. moreover, it can be hypothesized that another introduced carbonyl group at -position of quercetin, together with the adjacent carbonyl and hydroxyl group, would be capable of chelating the two metal ions. hence, this paper also described a series of novel quercetin- -o-benzoic acid esters (compounds , figure ) in an attempt to investigate whether the introduction of another carbonyl group at -position would result in an improved anti-hcv activity. in addition, several quercetin derivatives with different aliphatic groups substituted at the -position were also synthesized for further sar investigations. synthesis of -o-substituted quercetin derivatives is well documented [ ] . as depicted in scheme , peracetylation of quercetin ( ) followed by regioselective deacetylation of -oac group with thiophenol and imidazole in n-methyl- -pyrrolidone (nmp) at °c gave the -o-monodeprotected flavonol . alkylation of with variously substituted benzyl bromides, followed by deacetylation by treatment with methanolic ammonia provided the desired -o-arylmethylquercetins a- s. [ , ] , tribenzylation of rutin ( ) with potassium carbonate and benzyl bromide in dmf followed by removal of the c -rutinose with hydrochloric acid and ethanol gave ', ', -tri-o-benzylquercetin ( ) . compound was condensed with variously substituted benzoic acids, -ethyl- -( -dimethylaminopropyl) carbodiimide (edci) and a catalytic amount of -dimethylamiopryidine (dmap) to afford the relevant quercetin- -o-ester derivatives a- o. alternatively, the etherification of compound was performed in the presence of potassium carbonate with diverse bromides or iodides to afford relevant quercetin- -o-ether derivatives a- d. finally, the desired final products quercetin derivatives a- o and a- d were obtained by hydrogenolysis and further purification by silica gel chromatography. the inhibition of hcv by all synthesized quercetin derivatives was evaluated following a previously reported procedure [ ] . all newly synthesized compounds were tested in authentic hcv infection/replication system in the human hepatoma cell lines huh . . using mycophenolic acid [ ] (mpa) as control compound. the cytotoxicity effect of the test compounds was also evaluated in the same cell line by mtt method. antiviral effect and cytotoxicity effect were summarized as ec and cc respectively, as well as the selective index (si) which is the ratio of cc to ec . the stability of quercetin- -o-ester compounds were also tested in the same buffer as the antiviral activity assay at °c and the hydrolysate was measured by hplc. only trace quercetin as hydrolyzate was monitored over h which demonstrated that quercetin- -o-ester derivatives were stable under the biological evaluation conditions. as clearly shown in table , all -o-substituted quercetin derivatives a- s exhibited potent anti-hcv activities with ec values ranging from . μm to . μm. among these compounds, ''-cl substituted derivatives possessed the highest activities (for i, ec = . μm) and a decent selectivity ratio (for i, si = . ). these results tend to indicate the importance of introducing an aromatic group at -o position. substituents of quercetin at the -o position may mimic the meta-substituents of dka b and thus enhance the anti-hcv activities. it should be noted that all the -o-substituted analogues evaluated in this study were found in a narrow range of inhibition activity. this high level of tolerance will be discussed in the following section. to examine the effects of the additional carbonyl group at the -position, -o-substituted quercetin derivatives were designed by adding substituted aroyl groups. all the substituted benzoic acid esters a- o exhibited similar or better anti-hcv activities in cell-based assays as compared to the lead compound quercetin (table ) . among these compounds, ''-f substituted derivative ( f) showed more than a two-fold increase in potency over quercetin (ec of . μm for f versus . μm for ). in order to study the significance of the coordination between the additional carbonyl group and the magnesium ions, we synthesized several compounds a- d in which the carbonyl group at -o position was replaced by aliphatic groups of various lengths. it is not surprising that a decrease in potency was observed for these analogues, mainly because the aliphatic substituents at the -position, especially when they are bulky, could not coordinate to the magnesium ions. the maintenance of anti-hcv activities for quercetin- -o-benzoic acid-ester derivatives instead of the -o-aliphatic substituents revealed that the introduction of an electronegative carbonyl group at -o position could enhance the chelation ability between the inhibitor and metal ions, which contribute to the binding affinity in a similar way as the -hydroxyl group. to rationalize the observed activities of different substituents of quercetin and investigate the binding modes with ns b, docking calculations were performed on the basis of the reported x-ray crystallographic structure of hcv genotype b rdrp complexed with uridine triphosphate (utp) and divalent metal ions (pdb code: gx [ ] ). initially, the lead compound quercetin was docked into ns b active site after the removal of the original utp. as depicted in figure a , -, -, and -o derivatives of quercetin chelate to the two magnesium ions ( . Å- . Å) together with four conserved key residues (asp , thr , asp , asp ) and two water molecules in the active site to form a double-octahedral center. the three acidic residues (asp , asp , asp ) were found to be indispensable to the polymerase-catalyzed nucleotidyl transfer reaction [ ] . the '-hydroxyl in b ring of quercetin forms a hydrogen bond ( . Å) with the side chain of asp , the same residue that is hydrogen bonded to the '-hydroxyl group of utp. this binding mode is in accordance with our previous results [ ] that kaempferol (lacking a '-hydroxyl as compared to quercetin, . % inhibition at μm) and isorhamnetin (with '-hydroxy methylated, . % inhibition at μm) are less potent against hcv than quercetin (ec = . μm). were also docked into the active site of ns b. in addition to the similar chelation and hydrogen bond found in quercetin-ns b system, the -o phenyl group of i forms a hydrophobic interaction with phe ( figure b ). these findings could potentially account for the much better activities of -o-substituent compared to quercetin (table ). however, due to the relatively large size of the hydrophobic pocket around phe , modification of -o phenyl group did not give a noticeable gain of activity. as to the -o-substituted analogue f, the carbonyl group at -position interacts with one magnesium ion with a distance of . Å, the same distance as that in quercetin-ns b system (figure a,c) . interestingly, an extra hydrogen bond ( . Å) was found between the carbonyl group at -position and the backbone nh of phe , which may contribute to the better activities of quercetin- -o-substituted derivatives compared to quercetin (table ). all solvents and reagents were purchased from commercial sources and used directly without purification. progress of all the reactions were monitored by thin-layer chromatography (tlc) with precoated silica gel plates gf , - µm (huanghai, yantai, china). melting points (m.p.) were determined using an x- microscope melting point apparatus and are uncorrected. nuclear magnetic resonance (nmr) spectroscopic analysis was performed on a bruker drx- spectrometer at mhz for h-nmr and mhz for c-nmr. chemical shifts are reported in δ ppm (parts per million) relative to tms ( . ) as internal standard. splitting parterns were assigned as s (singlet), d (doublet), t (triplet), q (quartet), m (multiplet), or br s (broad singlet). mass spectra (esi) were performed on an agilent s mass spectrometer, and the values are reported in m/z. ( ) quercetin ( , . g, . mmol), acetic anhydride ( . g, . mmol), and pyridine ( ml) were heated to reflux under argon atmosphere for h. after cooling down to room temperature, the mixture was poured into ml of ice water and filtered to give the product in % yield. ( ) to a solution of ( . g, . mmol) in anhydrous nmp ( ml) at °c was added imidazole ( mg, . mmol) followed by thiophenol ( . ml, . mmol). after stirring at °c until the disappearance of the starting material as monitored by tlc, the mixture was diluted with ch cl , and then washed succesively with n aq. hcl and saturated brine. the organic phase was dried over anhydrous na so and concentrated. the residue was purified by silica gel column chromatography (ch cl /meoh, / , v/v) to give compound as white powder in % yield. k co ( mg, . mmol) and appropriate benzyl bromide ( . mmol) were added to solution of ( mg, . mmol) in acetone ( ml). the reaction mixture was stirred for h at room temperature and then filtered. the filtrate was concentrated under reduced pressure to offer the crude product a- s for the next step directly without further purification. the solution of crude compound a- s in nh /meoh ( ml) was stirred for h at °c and concentrated under reduced pressure. this residue was purified by silica gel column chromatography (ch cl /meoh, / , v/v) to give the desired compound a- s. a mixture of rutin ( , . g, . mmol) and potassium carbonate ( . g, . mmol) in dry dmf ( ml) was stirred under argon atmosphere for . h. benzyl bromide ( . ml, . mmol) was then added dropwise. after stirring for h at °c, the mixture was acidified to ph with % aqueous acetic acid solution, and the precipitate was collected by centrifugation. ethanol ( ml) was added to the precipitate and then conc. aqueous hydrochloric acid ( ml) of was add in portions. this reaction mixture was stirred at °c for h. after cooling to room temperature, the precipitate was filtered and washed with water. the crude product was recrystallized from ch cl /etoh to afford as yellow powder in % yield, m.p. - °c. a mixture of compound ( mg, . mmol), edci ( mg, . mmol), dmap ( mg, . mmol) and appropriate benzoic acid ( . mmol) in dmf ( ml) was stirred at °c overnight. the mixture was poured into ml of water and filtered to give the crude products a- o for the next step directly without further purification. to a solution of the appropriate benzyl-protected quercetin- -o-ester derivatives a- o in ethanol and , -dioxane ( ml, : ) was added % palladium on carbon ( mg). the mixture was stirred under hydrogen atmosphere at room temperature for h. the resulting mixture was filtered, washed with etoh and purified by silica gel column chromatography (ch cl /meoh, / , v/v) to give the desired compounds a- o. to a mixture of compound ( mg, . mmol) and potassium carbonate ( mg, . mmol) in dry dmf ( ml), the appropriate halide ( . mmol) in dry dmf ( ml) was add, and the reaction mixture was stirred at °c for h. the mixture was then poured into water ( ml) and filtered to give the crude product a- o used or the next step directly without further purification. % palladium on carbon ( mg) was added to the crude product a- o in a solution of ethanol and , -dioxane ( ml, : ), then the mixture was stirred under hydrogen atmosphere at room temperature for h. the resulting mixture was filtered, washed with etoh and purified by silica gel column chromatography (ch cl /meoh, / , v/v) to give the desired compound a- d. the biological assays were performed following a previously reported procedure [ ] . the huh . . cell line and the hcv infectious virus j em were kindly provided by xin-wen chen, wuhan institute of virology, chinese academy of sciences. huh . . cells were cultured in dulbecco's modified eagle's medium (dmem) containing % (v/v) fetal bovine serum, . mg/ml g , iu/ml penicillin, and mg/ml streptomycin. j em is an infectious hcv virus derived from the jfh virus (genotype a) by insertion of enhanced green fluorescent protein (egfp) into the hcv ns a region. the establishment of the infectious j em hcv virus has been described previously [ ] . j em was propagated in huh . . cells. briefly, huh . . cells were infected with j em at moi = . and cultured for days. culture supernatant was collected and filtered through a . μm membrane and stored at − °c as virus stock. virus titer was tested in huh . . cells. stocks were serially diluted, and the foci forming units (ffu) were determined as the number of fluorescent colonies formed in infected huh . . . all the compounds were first dissolved in dmso at mm, and then diluted to various concentrations ( μm, μm, . μm, . μm and . μm). approximately × huh . . cells per well were plated in an opaque -well tissue culture plate (costar ). the next day, the medium was replaced by j em virus at an moi of . . four hours later, the virus inoculum was removed and medium containing different concentrations of compounds was added. after an additional h of incubation, the culture medium was removed and the egfp autofluorescence was measured by using a luminometer with excitation at nm and emission at nm. compounds and controls were performed in duplicate and each experiment repeated independently at least twice. after the egfp signal was read, cell viability was measured in the same plate by the mtt [ -( , -dimethylthiazol- -yl)- , -diphenyltetrazolium bromide; sigma-aldrich, st. louis, mo, usa] method. briefly, mtt ( mg/ml) was dissolved in pbs. cell culture supernatants were removed and replaced with mtt and further cultured for h; cells were then lysed with % sodium dodecyl sulfate (sds), % n,n-dimethylformamide, ph . . od values were read. the percentage of cell death was calculated against the control well without compounds. docking studies were carried out on quercetin and relative substituents using schrodinger software package [ ] . the high-resolution ( . Å) x-ray co-crystal structure of hcv ns b in complex with mn + and utp (pdb code: gx [ ] ) was downloaded from the protein data bank. water molecules were deleted except for those involved in coordination with the two divalent cations in the active site. the crystallographic mn + ions were replaced with mg + ions. all missing hydrogen atoms were added by standard protein preparation protocol within maestro followed by energy minimization using opls force field. restrained minimization was later performed on heavy atoms of the protein with root-mean-square deviation (rmsd) converged to . Å. the structures of lead compound quercetin and the most active compound of corresponding series ( i and f) were built and optimized by maestro with default settings. docking of the three compounds were carried out using glide with standard precision protocol with the mg + ions defined as required constraints. the binding conformation with the lowest score was selected to represent the predicted binding mode with hcv ns b polymerase. dka analogues or isosteres show potent inhibition of hcv ns b through chelation of the two magnesium ions at the active site. following our recent report which evidenced the anti-hcv potential of the dkas mimic quercetin ( ), efforts were continued in order to delineate the structural features required for the inhibitory effect and improve the anti-hcv potency. accordingly, two novel quercetin analogues, -o-arylmethylquercetin derivatives and quercetin- -o-benzoic acid ester derivatives, were synthesized and evaluated for anti-hcv activities. experimental and modeling results highlighted the importance of introducing an aromatic group at -o position, which is exemplified by compound i, where a -chlorobenzyl substitution at quercetin -o position confers low micromolar inhibitory activity (ec = . μm) and a decent selectivity ratio (si = . ). moreover, the electronegative carbonyl groups at quercetin -o position may maintain the coordination with the magnesium ions and more importantly, introduce other interactions with the surrounding residues and thus enhance the anti-hcv activities, which is manifested by compound f (ec = . μm and si > . ). docking studies suggested that the quercetin structures are capable of establishing key coordination with the two magnesium ions as well as interactions with residues at the active site of hcv rdrp. these results demonstrated the feasibility and potential of modifications on quercetin scaffold and provided a promising fundamental to optimize the structures of flavonoids to obtain potent anti-hcv agents. supplementary materials can be accessed at: http://www.mdpi.com/ - / / / /s . l.z. and d.y. conceived and designed the research; d.z. and m.l. performed the experimental work and wrote the manuscript; y.c. performed the docking study; y.z., s.b., j.z., f.w. and k.c.r. participated in the experimental work, analyzed the data and discussed the results. all the authors read and approved the final manuscript. global epidemiology of hepatitis c virus infection: new estimates of age-specific antibody to hcv seroprevalence peginterferon alfa- a plus ribavirin for chronic hepatitis c virus infection mechanism of action of interferon and ribavirin in treatment of hepatitis c discovery and development of telaprevir: an ns - a protease inhibitor for treating genotype chronic hepatitis c virus boceprevir, an ns protease inhibitor of hcv discovery and development of simeprevir (tmc ), a hcv ns / a protease inhibitor sofosbuvir for the treatment of hepatitis c virus rapid emergence of telaprevir resistant hepatitis c virus strain from wildtype clone in vivo the high price of the new hepatitis c virus drugs crystal structure of the rna-dependent rna polymerase of hepatitis c virus discovery of α, γ-diketo acids as potent selective and reversible inhibitors of hepatitis c virus ns b rna-dependent rna polymerase hcv ns b rna-dependent rna polymerase inhibitors: from α, γ-diketoacids to , -dihydroxypyrimidine-or -methyl- -hydroxypyrimidinonecarboxylic acids. design and synthesis -dihydroxypyrimidine carboxamides and n-alkyl- -hydroxypyrimidinone carboxamides are potent, selective hiv integrase inhibitors with good pharmacokinetic profiles in preclinical species the monoethyl ester of meconic acid is an active site inhibitor of hcv ns b rna-dependent rna polymerase pharmacological applications of quercetin and its derivatives: a short review discovery of flavonoid derivatives as anti-hcv agents via pharmacophore search combining molecular docking strategy inhibition of rna binding to hepatitis c virus rna-dependent rna polymerase: a new mechanism for antiviral intervention ntpase/helicase by dihydroxychromone derivatives discovering novel quercetin- -o-amino acid-esters as a new class of src tyrosine kinase inhibitors metabolism-based synthesis, biologic evaluation and sars analysis of o-methylated analogs of quercetin as thrombin inhibitors mycophenolic acid inhibits hepatitis c virus replication and acts in synergy with cyclosporin a and interferon-α structural analysis of the hepatitis c virus rna polymerase in complex with ribonucleotides discovering novel anti-hcv compounds with inhibitory activities toward hcv ns / a protease compensatory mutations in ns and ns a proteins enhance the virus production capability of hepatitis c reporter virus schrodinger software suite the authors acknowledge xin-wen chen from wuhan institute of virology, chinese academy of the authors declare no conflict of interest. key: cord- - oca mrm authors: shen, wen-jun; cui, wenjuan; chen, danze; zhang, jieming; xu, jianzhen title: rpirls: quantitative predictions of rna interacting with any protein of known sequence date: - - journal: molecules doi: . /molecules sha: doc_id: cord_uid: oca mrm rna-protein interactions (rpis) have critical roles in numerous fundamental biological processes, such as post-transcriptional gene regulation, viral assembly, cellular defence and protein synthesis. as the number of available rna-protein binding experimental data has increased rapidly due to high-throughput sequencing methods, it is now possible to measure and understand rna-protein interactions by computational methods. in this study, we integrate a sequence-based derived kernel with regularized least squares to perform prediction. the derived kernel exploits the contextual information around an amino acid or a nucleic acid as well as the repetitive conserved motif information. we propose a novel machine learning method, called rpirls to predict the interaction between any rna and protein of known sequences. for the rpirls classifier, each protein sequence comprises up to diverse amino acids but for the rpirls- g classifier, each protein sequence is represented by using -letter reduced alphabets based on their physiochemical properties. we evaluated both methods on a number of benchmark data sets and compared their performances with two newly developed and state-of-the-art methods, rpi-pred and ipminer. on the non-redundant benchmark test sets extracted from the pridb, the rpirls method outperformed rpi-pred and ipminer in terms of accuracy, specificity and sensitivity. further, rpirls achieved an accuracy of % on the prediction of lncrna-protein interactions. the proposed method can also be extended to construct rna-protein interaction networks. the rpirls web server is freely available at http://bmc.med.stu.edu.cn/rpirls. the interactions of proteins with other proteins, peptides, dnas and rnas govern most the essential molecular function. rna-protein interactions (rpis) have a critical influence on post-transcriptional gene regulation [ ] [ ] [ ] , viral assembly [ ] [ ] [ ] , cellular defence [ ] , protein synthesis [ , ] and various other fundamental biological processes [ , ] . a significant portion of transcripts is long non-coding rnas (lncrnas) which are not translated into proteins and are longer than nucleotides [ ] . lncrnas normally function with their interacting proteins [ ] . for instance, the lncrna hotair regulated the hoxd locus in trans by interacting with pcg proteins [ ] ; several lncrnas were shown to be able to interact with auf , a protein linked to aging and cancer [ ] ; lncrnas binding to jarid protein were essential for the recruitment of prc to the chromatin [ ] ; lncrna gas inhibited hepatitis c virus replication by decoying hcv ns protein [ ] . hence, the study of rpis is essential for understanding their functions. compared to those of protein-protein interactions and dna-protein interactions, current knowledge regarding rna-protein interactions, especially lncrna-protein interactions is still limited. in this study, we propose a novel machine learning method, which we call rna-protein interaction prediction based on regularized least squares (rpirls), to quantitatively predict the potential rna-protein interactions. the experimental determination of rpis remains expensive and time-consuming [ ] [ ] [ ] , but fortunately, the accumulated rpi experimental data facilitate the development of computational models for rpi prediction [ ] [ ] [ ] . in , pancaldi and b .. ahler [ ] introduced a computational approach for rbp (rna binding protein)-mrna interaction prediction. they employed support vector machines (svms) and random forests (rfs) based on more than physical and functional features of rpis, including gene ontology, chromosomal position, gene and protein physical properties, protein localization, experimental translation, mrna properties, predicted protein structure, utr properties and genetic interactions. bellucci et al. [ ] proposed a method called catrapid for the prediction of protein lncrna interaction. they evaluated the interaction propensities of protein-rna based on their physicochemical properties, including secondary structure, hydrogen bonding and van der waals. muppirala et al. [ ] developed a method called rpiseq, which predicted rpis solely based on primary sequences. the rpiseq method still employed svms and rfs but exploited different features. they represented each sequence of proteins and rnas as the normalized frequencies of the corresponding -mer and -mer, respectively. in , based on the same feature vectors presented in muppirala et al., wang et al. [ ] first reduced the dimensionality of feature vectors, and then performed the rpis prediction by using naive bayes classifier which assumed the independence of attributes and by using extended naive bayes classifier which considered the correlation between attributes. lu et al. [ ] integrated the information on the secondary structure, hydrogen bonding propensities and van der waals of lncrnas and proteins with fisher's linear discriminant model. in , suresh et al. [ ] developed a method called rpi-pred to predict rpis by considering the high-order d protein and rna structure information. in , pan et al. [ ] proposed a new method named ipminer that integrated deep learning with stacked ensembling to improve the prediction performance of ncrna-protein interactions. in this paper, we classified rna-protein pairs as interacting or non-interacting by integrating derived kernel with regularized least squares (rls) [ ] . the motivation is to relate the sequence information of proteins and rnas to their biological functions, i.e., interactions. our method attempted to extract discriminant subsequence features from amino acid sequences and nucleotide sequences. the derived kernel measures the similarity between two biological sequences by capturing nucleic acid or amino acid compositions and repetitive sequence patterns. we used regularized least squares in learning as the computations performed by rls algorithms can be expressed using just inner products, hence allowing efficient implementation of kernel-based learning, in addition the rls algorithms often perform comparable to the best batch classifiers [ ] . since the dimensionality of feature space increases exponentially with the template size, for computational sake, we set upper limit for template size. on the other hand, we categorized amino acids into several groups based on their physiochemical properties [ ] [ ] [ ] , the reduced alphabet representation of the protein sequence allows larger template size and also decreases the dimensionality of feature space. we considered the derived kernel with two-layer architecture, hence there were two template sets, denoted as t p and t r needed to be constructed for protein and rna, respectively. here we considered all possible substrings of the same length making up a template set. the template set t p for amino acid sequences was composed of substrings with k continuous amino acids, while the template set t r for nucleic acid sequences was composed of substrings with l continuous nucleic acids. in order to extract discriminant subsequence features from amino acid sequences and nucleotide sequences, we explored the effect on rpi prediction over a range of choices for the template sizes of protein and rna. the training set rpi was used to determine these parameters. in our case, we used different template sizes of protein and rna chosen from set { , , . . . , } ∪ { , , . . . , } for rpirls and { , , . . . , } ∪ { , , . . . , } for rpirls- g. with different combination of protein template size and rna template size, the combined kernelk dk was integrated with rls to predict rna-protein interactions. the ten-fold stratified cross-validation has been verified to be the best algorithm for model selection on a large scale experiments [ ] , therefore on the data set rpi , we tuned the parameter λ by ten-fold stratified cross-validation with the optional parameter set {λ = e n , n = − , · · · , }. the data set rpi was divided into ten mutually exclusive folds and the mean response of each fold was approximately equal. in each test we merged parts of the samples as the training set and left the other part as the test set. the parameter λ was chosen by leave-one-out cross-validation on the training set. for rpirls, in all the ten sets, λ = e − uniformly achieved the best performance in the training data. tables and showed the performance of the proposed method in terms of auc and accuracy with different combination of parameters k and l, respectively. the experiment results showed that when the protein template size k = and the rna template size l = , the model performs best with auc score of . and accuracy of . . the other measurements of specificity (sp) and sensitivity (se) were . and . , respectively. while for rpirls- g, λ = e − uniformly performed best in all the ten sets. table showed that the method achieved the best prediction accuracy of . when the protein template size k = and the rna template size l = . the other measurements (auc, sp and se) were observed as . , . and . , respectively. the computational results showed that the rpirls classifier outperformed the rpirls- g classifier in terms of various performance measurements, indicating that the diversity of amino acids at a sequence is important for the prediction of rpis. the performance of predicting rpis was evaluated by using -fold stratified cross-validation on the rpi data set. different combinations of parameters k and l were evaluated. remark on the symbols of template sizes: k stands for template size of amino acid sequences; l stands for template size of nucleic acid sequences. the best auc in the table is marked in bold. remark on the symbols of template sizes: k stands for template size of amino acid sequences; l stands for template size of nucleic acid sequences. the best accuracy in the table is marked in bold. in order to evaluate the reliability and robustness of rpirls and rpirls- g, we compared them with other two state-of-the-art methods rpi-pred and ipminer. the rpi and rpi data sets after removing overlapping rpis with the training data were evaluated. both the rpirls and rpirls- g classifiers were trained on the rpi data set, and tested on the rpi and rpi data sets, respectively. as shown in tables and , rpirls outperformed the rpirls- g, rpi-pred and ipminer methods on both data sets. for the rpi data set as shown in table , the performance of the rpirls method was . , . , . and . for predictive accuracy, auc, specificity and sensitivity, respectively. while the predictive accuracy of the rpi-pred and ipminer methods were just . and . , respectively which were much lower than rpirls's. the remaining measurements (specificity and sensitivity) were observed as . and . , respectively for rpirls, and . and . , respectively for ipminer. the rpirls method outperformed rpi-pred and ipminer in terms of accuracy, specificity and sensitivity on the rpi data set. table . predictive performance of, rpirls- g in terms of the accuracy on the, rpi training data set over varying template sizes. the performance of predicting rpis was evaluated by using -fold stratified cross-validation on the rpi data set. different combinations of parameters k and l were evaluated. remark on the symbols of template sizes: k stands for template size of amino acid sequences; l stands for template size of nucleic acid sequences. the best accuracy in the table is marked in bold. similar results were observed on the rpi data set in table . the specificity of the rpi-pred and ipminer methods was just . and . , respectively, indicating there was a positive bias in their predictions of performance. a low specificity increases the labor, cost, and time needed to perform the required experimental tests, but our rpirls method achieved both reasonable specificity and sensitivity. furthermore, we evaluated the rpirls classifier on large-scale rna-protein pairs in the currently available rpintdb data base. the rpirls method correctly predicted out of rpis, reaching the predictive accuracy of %. to explore the effectiveness of the proposed method on predicting ncrna-protein interactions, a large-scale ncrna-protein interaction data set (we called nrpi ) was retrieved from the npinter data base [ ] . we trained rpirls and rpirls- g on the rpi data set, and tested it on the nrpi . table showed the prediction results compared with the rpi-pred classifier on the nrpi data set. the ipminer method showed a significantly positive bias on predicting ncrna-protein interactions, thus here we didn't include it into the comparison. the predictive accuracy for different organisms were separately computed. for the six organisms, our method rpirls performed best for the homo sapiens and saccharomyces cerevisiae, rpi-pred performed best for drosophila melanogaster, escherichia coli and mus musculus, and both methods obtained the same predictive accuracy for the caenorhabditis elegans. rpirls outperformed rpirls- g over all six organisms. for ncrna-protein pairs, the rpirls method achieved an accuracy of % compared to % for rpirls- g and % for the rpi-pred method. we further tested rpirls and rpirls- g on the lnrpi data set which was a subset of the nrpi data set and consisted of only lncrna-protein interactions (lncrpis). our model achieved an overall accuracy of % compared to % for rpirls- g and % for the rpi-pred classifier as shown in table . the predictive performance of rpirls was improved in out organisms compared with its performance on the nrpi data set. the results indicated the effectiveness of our method to predict lncrna-protein interactions only by using primary sequences of proteins and rnas. predicting lncrna-protein interaction networks is useful to explore the molecular mechanisms that are regulated by lncrnas [ , ] . in this experiment, we evaluated the performance of rpirls on building lncrpi networks and further compared its performance with rpi-pred. on the basis of the data in npinter, we analyzed the results of four organisms, i.e., caenorhabditis elegans, drosophila melanogaster, escherichia coli and saccharomyces cerevisiae, consisting of , , and lncrpis, respectively. for caenorhabditis elegans, the rpirls method correctly identified all lncrpis (blue edges) while the rpi-pred method correctly identified out of . as shown in figure , rpi-pred made incorrect prediction for the pair of n -g ebf (red edges). in figure , rpi-pred correctly predicted all lncrpis, whereas rpirls missed out of lncrpis for drosophila melanogaster. these out of incorrect predictions which were observed between two proteins p and q vss (yellow rectangle) and three signal recognition particle (srp) rnas n , n and n (green ellipse), formed the srp rna-protein interactions. for escherichia coli, the rpirls classifier made much more errors than the rpi-pred method, with predictive accuracies of % vs. %. the performance of rpirls for escherichia coli was much poorer than that for the other five organisms. in order to analyze why rpirls had relative poor performance on escherichia coli, we estimated the amino acid composition of escherichia coli compared with that of the other five organisms. as shown in figure , we found that escherichia coli had relative higher observing frequencies of alanine and valine as well as much lower content of serine compared with that of the other five organisms. the amino acid composition bias in escherichia coli probably leaded to poor results. as shown in figure , among incorrect predicted pairs, rpis corresponded to protein hubs, e.g., p a h , p afz , p ag , p ce , p ce , p and p , each of which appearing as a yellow rectangle node was shown to interact with six transfer-messenger rnas (tmrnas), e.g., n , n , n , n , n and n (green ellipse). for saccharomyces cerevisiae, as showed in figure , among incorrect predicted pairs, rpis were involved in protein hubs (p , q and q ), in which each protein interacted with small nuclear rnas (snrnas: n , n , n and n ), and other rpis corresponded to protein hubs (p , p , p , q , q , q and q ), each of which interacted with three small nucleolar rnas (snornas: n , n and n ). the rpirls classifier correctly identified out of rpis, achieving a high accuracy of %, compared of % (correctly predicted out of pairs) for rpi-pred. in this work, we illustrated the effectiveness and reliability of rpirls in predicting rpis for eukaryotic organisms in networks which comprised a variety protein hubs and rna hubs. mammalian cells contain more than different proteins interacting with rna [ ] . normally, any individual rna can interact with multiple proteins [ , ] . conversely, most proteins are capable of interacting with multiple rnas [ ] . given the number of rnas and rna-binding proteins, the number of possible rpis is enormous. high-throughput sequencing methods have accumulated huge amount of rna-protein binding experimental data and opened new possibilities to measure and understand rna-protein interactions by computational methods. most of the previous computational works on rpis focus on the prediction of rna-binding proteins or rna-binding residues in a protein sequence [ , [ ] [ ] [ ] . to our knowledge, very limited works have been developed to predict the specific associations between rnas and proteins, which play a critical role in post-transcriptional gene regulatory networks. complex networks of rpis mediate post-transcriptional gene regulation and therefore prediction of rpis helps us to gain insight into regulatory networks [ , ] . the work presented here provided a computational method, called rpirls, to classify rna-protein pairs as interacting or non-interacting by integrating a sequence-based derived kernel with regularized least squares. the derived kernel exploited the contextual information around an amino acid or a nucleic acid as well as the repetitive conserved motif information. our results demonstrated that only the sequence structures of rnas and proteins provide sufficient information to accurately predict rna-protein interactions, especially long non-coding rna-protein interactions. specifically, the rpirls classifier considered each protein sequence comprising up to diverse amino acids, while the rpirls- g classifier encoded each protein sequence by using the -letter reduced alphabets according to amino acid physiochemical properties. the computational results showed that the rpirls classifier was superior to the rpirls- g classifier in reliability and effectiveness, indicating that the diversity of amino acids at a sequence has critical impact on the function of rna-binding proteins. on two non-redundant benchmark data sets extracted from the pridb, the rpirls method outperformed rpi-pred and ipminer in terms of accuracy, specificity and sensitivity. compared with rpi-pred and ipminer, the rpirls method obtained a reasonable sensitivity at a lower false positive rate. further, rpirls achieved an accuracy of % compared to % for rpi-pred on the prediction of lncrna-protein interactions. the rpirls method can be extended to construct rna-protein interaction networks and therefore helps us to gain insights into post-transcriptional gene regulation. the reason for the good performance of the proposed method may be due to several factors. firstly, the use of similarity scores is a significant conceptual change in protein/rna evaluation, quantifying the overall similarity between proteins, rnas and their interactions. combining kernels by tensor product for the set of rna-protein pairs allowing to share information across the rna-binding proteins considerably improved the prediction, especially in the case of rnas with few known binding proteins. secondly, we have found that contiguous k-mer frequencies alone captured rich statistical information on the repetitive conserved motif of rna-protein pairs and the diversity of amino acids at a sequence has also contributed to an observed improvement contrast to rpi-pred which just applied -letter frequency for both protein and rna. finally, a kernel works as a measure of similarity and supports the application of powerful machine learning algorithms such as regularized least squares which we used in this paper. the rls mehod enables us to efficiently search for an optimized parameter λ at essentially no additional cost [ ] . further, our model was trained on a large data set which contained rna-protein pairs, and yielded more robust results. in contrast, ipminer had much more model parameters to fit as combining deep learning with stacked ensembling, however, was trained on a small data set of just rna-protein pairs, and thus showed a significantly positive bias on predicting ncrna-protein interactions. the main disadvantage of the proposed method is that the method is purely data-driven, in the sense that it relies solely on information derived from amino acid sequences and nucleic acid sequences, and thus does not consider higher structural information of protein and rna. while this may be seen as an advantage, since it can predict any rna-protein pair of known sequences. the increase of the number of protein-rna complexes in protein data bank [ ] has opened possibilities for researchers to develop secondary data bases and to gain valuable insight into the structure and function of these complexes. the protein-rna interface data base (pridb) v . [ ] identifies interfacial residues in rna-protein complexes and also calculates atomic distances between interfacial residues. the rb and rb are two precalculated data sets in the pridb, which respectively consist of and rna-binding protein chains. after combining the rb and rb data sets, we obtained a total of experimentally validated non-redundant rna-protein pairs, which had at least two atoms respectively coming from rna and protein with distance no more than Å. next, we removed redundant rna-protein pairs, which are the same protein chains interacting with the same rna chains. further, we removed those rna-protein pairs with amino acid sequence length < or nucleic acid sequence length < . finally, we obtained a positive sample set which consisted of experimentally validated rna-protein pairs. so far there are no definite negative samples of rna-protein interactions that are available. to construct a balanced negative sample set ("rna-protein non-interacting pairs"), we made it by randomly permute the proteins in the positive sample set but kept the rna fixed. we repeated the permutation process until no negative pairs existed in the positive sample set. as a result, the training set, called rpi , was composed of rna-protein interacting pairs and rna-protein non-interacting pairs. several data sets were employed to evaluate the performance of the proposed methods. our rpirls method was first evaluated using two popular non-redundant data sets of rpis studied in [ ] . the rpi data set consisted of experimentally validated rna-protein pairs extracted from the pridb data base. while the rpi data set eliminated all rpi pairs with ribosomal proteins or ribosomal rnas from the rpi data set. to avoid overlapping between training and testing data sets, those rpis overlapping the training data were removed, leaving the rpi data set of rpis and the rpi data set of rpis. their corresponding negative pairs were generated by following the same steps as developing the training negative pairs. next, we tested the performance of the rpirls method on a large scale data set extracted from the rna-protein interaction data base (rpintdb) (http://pridb.gdcb.iastate.edu/rpiseq/download.php). this data set consisted of , experimentally validated rpis from several sources, including the rpidb, npinter data base and high-throughput experiments published in literature. the fourth data set were extracted from the npinter data base which we called nrpi . the nrpi data set consisted of , experimentally validated ncrna-protein pairs from six model organisms, i.e., caenorhabditis elegans, drosophila melanogaster, escherichia coli, homo sapiens, mus musculus and saccharomyces cerevisiae. we constructed the fifth data set called lnrpi by extracting only lncrna-protein pairs from the npinter data base. this data set contains , experimentally validated lncrna-protein pairs. in this paper, we proposed two classifiers for predicting rpis based on different representations of protein sequences. for the rpirls classifier, each amino acid sequence comprised up to different amino acids. while for the rpirls- g classifier, we adopted the same amino acid classification approach as [ , ] . the derived kernel was proposed by smale et al. [ ] on images inspired by neuroscience of visual cortex. in what follows, we briefly described the construction of derived kernel on sequences. suppose a is a finite set called the alphabet. in the work here a is the set of amino acids (for rpirls), alphabets (for, rpirls- g) or nucleic acids. let a = a and define a k+ = a k × a recursively for any k ∈ n. we say s is a string if s ∈ ∪ ∞ k= a k , and s = (s , . . . , s k ) is a k-mer (e.g., a sequence of length k) if s ∈ a k for some k ∈ n with s i ∈ a . the process of computing the derived kernel mainly includes three steps as below: step . set an initial kernelk at the first layer. here the initial kernel is defined as: where x, y ∈ a k . x = {x , . . . , x k } and y = {y , . . . , y k } are substrings of the same length k. x = y if and only if x i = y i for i = , . . . , k. step . let f = ( f , . . . , f n ), denote by | f | the length of f , so here | f | = n. then define the second layer neural response of f at t : where t is the template set at the first layer, here we consider all possible substrings of length k making up the template set t , so here t = a k . h is the transformation set at the first layer. step . compute the second layer derived kernel by normalizing the inner product of two neural responses: where ·, · l (t ) denotes the l inner product with respect to the uniform measure |t | ∑ t∈t δ t , where |t | is the cardinality of the template set t and δ t is the dirac measure; with correlation normalization: . this process can continue if appropriate higher level templates are defined. at each layer (local) derived kernels are built by recursively pooling over previously defined local kernels. here, for the -layer derived kernel, pooling is accomplished by taking an average over a set of transformations which calculating the frequency of a template that occurs in a sequence. in this paper we deal with inner product kernels k which satisfies the mercer condition, are known to be an instance of reproducing kernels. next with correlation normalization,k is also a reproducing kernel andk(x, x) = for all x ∈ x. the kernel function is symmetric (i.e., k( f , g) = k(g, f )), and non-negative (i.e., k( f , f ) ≥ ), therefore it can be interpreted as a measure of similarity. we first apply the kernel to the set r which contains nucleic acid sequences, and denote it byk r , and then apply the kernel to the set of amino acid sequences p, denote it byk p , and lastly combine two kernels in a natural way by tensor product for the set of rna-protein pairs . the reproducing kernel for two rna-protein pairs (r, p), (r , p ) ∈ r × p is defined by: k dk ((r, p), (r , p )) =k r (r, r )k p (p, p ). since bothk r (r, r ) andk p (p, p ) are positive definite kernels,k dk ((r, p), (r , p )) is obviously a positive definite kernel too [ ] . after combining the kernel with other kernel-based supervised learning algorithm, we can predict rpis to any rna-protein pairs with known primary sequences. the rls algorithm is one of the most widely used models for regression. let k be a kernel on a finite set x. write h k to denote the inner product space of functions on x defined by k. supposez = {(x i , y i )} m i= is a sample set (called the training set) with x i ∈ x and y i ∈ r for each i. the rls can be written as follows: we integrated rls with the combined kernel k =k dk , hence the main construction is to computē herein, we aim to develop a novel method to distinguish rna-protein interaction pairs from non-rna-protein interaction pairs. therefore, for the binary classification case with y i ∈ {− , } for each i, iff ≤ , the predicted class is − ( denotes non-interaction), otherwise it is (denotes interaction). one important step of rls is to find a "good" value of the regularization parameter λ > in equation ( ). they were selected from an optional set Λ by leave-one-out cross-validation [ ] on the training data. we never used testing data for parameter selection which is under the risk of over-fitting. the sensitivity (se) and specificity (sp) are used to measure the ability of identifying positive and negative instances, respectively. they are defined by the accuracy which is used to measure the prediction quality, is defined by accuracy = tp + tn tp + tn + fp + fn . the auc (area under the receiver operating characteristic curve) is further employed to measure the predictive performance, which is for perfect prediction and . for random prediction. rna regulons: coordination of post-transcriptional events rpicool: a tool for in silico rna-protein interaction detection using random forest a census of human rna-binding proteins sequence-specific interaction of r coat protein with its ribonucleic acid binding site rna-rna and rna-rotein interactions in coronavirus replication and transcription diverse roles of host rna binding proteins in rna virus replication rna-protein interactions in human health and disease the three-dimensional structure of the ribosome and its components ribosomal protein structures: insights into the architecture, machinery and evolution of the ribosome emerging roles of rna and rna-binding protein network in cancer cells rna processing and its regulation: global insights into biological networks long noncoding rnas in interaction with rna binding proteins in hepatocellular carcinoma long noncoding rnas: functional surprises from the rna world functional demarcation of active and silent chromatin domains in human, hox loci by noncoding rnas par-clip analysis uncovers, auf impact on target rna fate and genome integrity jarid is implicated in the initial xist-induced targeting of, prc to the inactive x chromosome long non-coding rna gas inhibited hepatitis c virus replication by binding viral ns protein rip-chip: the isolation and identification of mrnas, micrornas and protein components of ribonucleoprotein complexes from cell extracts hits-clip yields genome-wide insights into brain alternative rna processing transcriptome-wide identification of rna-binding protein and microrna target sites by par-clip protein-rna interactions: structural analysis and functional classes advances in rip-chip analysis: rna-binding protein immunoprecipitation-microarray profiling quantitative analysis of rna-protein interactions on a massively parallel array reveals biophysical and evolutionary landscapes in silico characterization and prediction of global protein-mrna interactions in yeast predicting protein associations with long noncoding rnas predicting rna-protein interactions using only sequence information de novo prediction of rna-protein interactions from sequence information computational prediction of associations between long non-coding rnas and proteins predicting ncrna-protein interaction using sequence and structural information ipminer: hidden ncrna-protein interaction sequential pattern mining with stacked autoencoder for accurate computational prediction applications of regularized least squares to pattern classification simulations of the dynamics at an rna-protein interface prediction of rna-binding proteins from primary sequence by a support vector machine approach prediction of rna binding sites in proteins from amino acid sequence a study of cross-validation and bootstrap for accuracy estimation and model selection the noncoding rnas and protein related biomacromolecules interaction database molecular mechanisms of long noncoding rnas function of lncrnas and approaches to lncrna-protein interactions principles and properties of eukaryotic mrnps transcriptome-wide analysis of protein-rna interactions using high-throughput sequencing a neural network method for identification of rna-interacting residues in protein a server for the computational prediction of rna-binding residues in protein sequences prediction of protein-rna binding sites by a random forest method with combined features dissecting the expression dynamics of rna-binding proteins in posttranscriptional regulatory networks deciphering the role of rna-binding proteins in the post-transcriptional control of gene expression the protein data bank pridb: a protein-rna interface database introduction to the peptide binding problem of computational immunology: new results. found generalized cross-validation as a method for choosing a good ridge parameter this article is an open access article distributed under the terms and conditions of the creative commons attribution (cc by) license author contributions: w.s. and j.x. conceived and designed the experiments; w.s., w.c. and j.z. performed the experiments; w.s. and w.c. analyzed the data; w.s. and d.c. contributed web tools; w.s. and j.x. wrote the paper. the authors declare no conflict of interest. key: cord- -wmmbkmrg authors: wang, de-guo; brewster, jeffrey d.; paul, moushumi; tomasula, peggy m. title: two methods for increased specificity and sensitivity in loop-mediated isothermal amplification date: - - journal: molecules doi: . /molecules sha: doc_id: cord_uid: wmmbkmrg the technique of loop-mediated isothermal amplification (lamp) utilizes four (or six) primers targeting six (or eight) regions within a fairly small segment of a genome for amplification, with concentration higher than that used in traditional pcr methods. the high concentrations of primers used leads to an increased likelihood of non-specific amplification induced by primer dimers. in this study, a set of lamp primers were designed targeting the prfa gene sequence of listeria monocytogenes, and dimethyl sulfoxide (dmso) as well as touchdown lamp were employed to increase the sensitivity and specificity of the lamp reactions. the results indicate that the detection limit of this novel lamp assay with the newly designed primers and additives was fg per reaction, which is ten-fold more sensitive than a commercial isothermal amplification kit and hundred-fold more sensitive than previously reported lamp assays. this highly sensitive lamp assay has been shown to detect strains of listeria monocytogenes, and does not detect other listeria species (including listeria innocua and listeria invanovii), providing some advantages in specificity over commercial isothermal amplification kits and previously reported lamp assay. loop-mediated isothermal amplification, developed and reported by notomi et al., in [ ] , can specifically, sensitively and rapidly amplify nucleic acids with two pairs of primers recognizing independent sequences of a target gene under isothermal conditions. moreover, nagamine et al., has advanced the method by putting forward loop primers that accelerate the lamp reaction [ ] . therefore, the lamp assay theoretically has the advantage of specificity, selectivity, and rapidity over polymerase chain reaction (pcr) [ , ] , nucleic acid sequence based amplification (nasba) [ , ] , strand displacement amplification (sda) [ ] , rolling circle amplification (rca) [ ] , helicase dependent amplification (hda) [ ] , and cross-priming amplification assay (cpa) [ , ] . for practical application of lamp as well as reduction of the rate of false positive results in lamp reactions, most researches currently focus on development of closed-tube detection to reduce aerosol pollution and cross pollution, which include the use of turbidity [ ] , sybr green i [ ] , picogreen [ ] , gelred tm [ , ] , lateral flow dipstick [ ] , hydroxynaphthol blue dye [ ] , malachite green [ ] , microfluidic chips and gmr sensors as well as calcein used by eiken chemical co., ltd [ , ] . however, there is still no report studying non-specific amplification and cause of false positive results in lamp reactions at present. the objective of this paper is to study the cause and limit the rate of false positive results in lamp reactions targeting l. monocytogenes as well as to increase the specificity and sensitivity of these lamp reactions using dmso and touchdown lamp. although there were only two primers, non-specific amplification occurred in the isothermal amplification of four primer combinations out of seven combinations, and it was more obvious in the reaction of three primer combinations (hlya-fip + hlya-lf, hlya-fip + hlya-lb, hlya-fip + hlya-b ), as table shown. analysis on the three combinations indicated that they had the common situation that - bases at ' end of both primers had two complementary sequences on a same primer, and such situation had been avoided when lamp primers for prfa of l. monocytogenes were designed and screened in this study. moreover, it was proved by the experiment that non-specific amplification caused by primer dimers was one reason for false positive results of lamp. the lamp reaction mixtures containing varying concentrations of dmso were heated at °c for min ( s per cycle), as indicated in figure . when % dmso was added, the detection time for pg l. monocytogenes genomic dna was less than min; however, one of the two negative controls amplified as well. the tm value and melt curve were obviously different from those of the positive controls, and were therefore as attributed to non-specific amplification, which may be caused by partial complementation among primers of lamp. the detection time with % dmso was slightly longer than with . %. overall, the results showed that the lower concentration of dmso does not inhibit non-specific amplification while the higher concentration of dmso may inhibit the activity of bst . warmstart dna polymerase, and therefore, . % dmso was determined to be the optimal among these three concentrations. . % dmso was added to lamp reaction mixtures and the reactions were carried out at varying temperatures for min, as shown in table . with a reaction temperature of °c, only one of two positive controls ( pg l. monocytogenes genomic dna) was detected. the threshold time obtained using a reaction temperature of °c was shorter than that obtained using °c reaction temperature and slightly shorter than that obtained using the other temperatures. therefore, °c was chosen as the most suitable reaction temperature. the optimized lamp reaction conditions were used with the conventional lamp methodology with a serial dilution of l. monocytogenes dna template was and these mixtures were heated at °c for min. as shown in table , the detection limit of the optimized reaction mixture using the conventional lamp technique was found to be fg l. monocytogenes dna template. the optimized lamp mixture was used with the touchdown methodology to detect a serial dilution of l. monocytogenes dna template. after the mixtures were preheated at °c for min and bst . warmstart dna polymerase (new england biolabs, beverly, ma, usa) were added, they were heated at °c for min, at °c for min, at °c for min and then at °c for min, and, as indicated in table , the sensitivity of touchdown lamp was found to be fg of l. monocytogenes dna. therefore comparing these identical reaction mixtures in the conventional lamp assay and the touchdown lamp assay shows that the touchdown lamp method increases the overall sensitivity of lamp assay. the detection limit of the original reported lamp method by tang, et al (tang method) tested using fg l. monocytogenes dna template, as well. only one of positives controls amplified using these conditions. moreover, one of four negative controls showed non-specific amplification, as reported in table . sensitivity of both the isothermal master mix using our own designed lamp primers as well as the loopamp ® listeria monocytogenes detection kit to detect l. monocytogenes was tested. the results indicate that the detection limit of both commercial lamp kits is fg l. monocytogenes dna template per reaction, as shown in table . therefore, the sensitivity of the newly developed lamp assay presented here was ten-fold higher than that obtained using the commercial isothermal amplification kits and hundred-fold higher than the originally reported tang lamp assay. this newly developed lamp assay was tested with listeria monocytogenes strains ( stereotypes) and as shown in table , all were successfully detected. the assay was also tested with five other listeria species. in the initial experiment, listeria invanovii atcc was falsely detected and one of three reactions amplified, while the other species had negative results. the experiment with l. invanovii atcc was repeated four times and all four repeated reactions were negative. therefore, the initial false positive result may have been caused by slight aerosol pollution of dna templates. [ ] ; however, the optimized lamp assay required an extended amplification time of min, and even with the lengthy reaction time, two strains (listeria monocytogenes j - (stereotype: / b) and listeria monocytogenes atcc (stereotype: b) were not detected, as shown in table . extending the amplification time further to h, led to non-specific amplification of negative controls. the two commercial lamp kits were able to distinguish listeria monocytogenes from other listeria species. there were, however, two negative controls that exhibited non-specific amplification, and, sometimes, l. monocytogenes j - (stereotype: a) was not detected, as shown in table . therefore, the newly developed lamp assay presented here can detect stereotypes of listeria monocytogenes selectively while not detecting other listeria species (including listeria innocua and listeria invanovii), and had some advantages over commercial isothermal amplification kit and the original tang lamp assay in specificity. the lamp primers targeting specific gene hlya of listeria monocytogenes reported by tang, et al., are used for studying non-specific amplification of lamp [ ] , as shown in table . targeting the specific gene prfa (genbank locus: ay . ) of l. monocytogenes, a set of lamp primers were designed and selected with primerexplorer and oligo according to the reported methodology [ ] , and are listed in table . the isothermal amplification was performed in a total μl reaction mixture containing . [ , ] . %, . % and % dmso were added into different reaction tubes. lamp was carried out at °c for min and a melt curve was obtained using a stepone tm system. lamp was performed as above in a μl reaction mixture containing pg l. monocytogenes dna template as well as the optimized concentration dmso at , , , and °c for min, and a melt curve was obtained using a stepone tm system. the optimized lamp mixture was combined with serial dilutions of dna template of listeria monocytogenes ranging from to fg, and the reaction mixtures were heated at selected temperature °c for min in stepone tm system, and the detection limit of conventional lamp was determined. in the case of touchdown lamp, the reaction mixture was preheated at °c for min. after min, bst . warmstart dna polymerase (large fragment) was added and the reaction mixture was heated at temperatures °c higher than selected temperature for min, at temperature of °c higher than selected temperature for min, at temperature of °c higher than selected temperature for min and then at selected temperature for min, and the sensitivity of touchdown lamp was determined and compared with that of conventional lamp. for comparison, the detection limit of the reported method by tang, et al., (tang lamp assay) was determined by carrying out lamp reactions according to the conditions specified in their publication [ ] . isothermal master mix and loopamp ® listeria monocytogenes detection kits were purchased from optigene limited (west sussex, uk) and eiken chemical co., ltd (tochigi, japan), respectively, and lamp was carried out according to the manufacturers' instructions using a set of serially diluted dna template of l. monocytogenes ranging from to fg. eleven strains of l. monocytogenes (different stereotype) and other listeria species (including l. innocua and l. invanovii) were used for the specificity study (table ) . listeria strains were cultured overnight at °c in difco tm buffered listeria enrichment broth base (becton, dickinson and company, san jose, ca, usa) and the others in luria-bertani (lb) broth. dna from these pure cultures was extracted according to the manufacturer's handbook of dneasy ® blood and tissue kit (qiagen ltd, north manchester, uk), and these dna templates was used for determining the specificity of the optimized touchdown lamp assay, the tang lamp assay and the lamp assay utilizing the commercial isothermal amplification kit. the amount of dna template used is pg per reaction. it is difficult to avoid primer dimers and non-specific amplification when couple numerous sets of primers are used in lamp assays. this is especially true when the concentrations of primers, mg + , dntps and dna polymerase in reaction mixtures are as high as those used in real-time pcr. the concentrations of these factors must be strictly controlled to avoid non-specific amplification in real-time pcr [ ] as well as lamp reactions. there are instances in which standard pcr amplification reaction conditions do not produce acceptable results. addition of dmso and use of touchdown temperature conditions have been used improve pcr results. we investigate these approaches for the first time for optimization of lamp reactions. unfortunately, with the information presently available it is not possible to predict which enhancing agent is best for any particular target. but dmso has been frequently used in this capacity [ ] . the results presented here using dmso in lamp reaction mixtures indicate that, dmso can increase amplification with lamp at low concentration and can inhibit activity of bst . warmstart dna polymerase. we had tried to enhance the reaction of lamp with betaine, tetramethylammonium chloride, tetramethylene sulfoxide, and formamide, but their effect was not as good as that of dmso, because of the limitation of length, no more tautology here. while dmso may not necessarily be the best enhancing agent [ , ] , i.e., the manufacturer of the commercial isothermal amplification kit used in this experiment may have found some favorable enhancing agent, but their reagents are proprietary, and dmso served to decrease non-specific amplification in the specific experiments presented here. touchdown pcr offered a simple and rapid means to optimize pcrs, increasing specificity, sensitivity and yield, without the need for lengthy reaction times and/or the redesigning of primers [ , ] . touchdown lamp, compared to conventional lamp methods, results in increased sensitivity and yield of lamp. this improvement may be due to the high temperature inhibiting the formation of primer dimers and promoting the correct combination of primers and template. the biggest advantage of lamp is that the reaction can be performed isothermally, and people argue all the time that all we need is a simple water bath for the rapid detection, so the advantage may be compromised by the developed touchdown lamp assay, we made such efforts here just to reveal or verify the main cause for false positive results of lamp and inspire people to modify and improve lamp technology, my colleagues and i have also been looking for a more suitable method, which can not only keep the advantage but also improve the sensitivity and specificity of lamp. in summary, non-specific amplification was a limiting factor in the applicability of the lamp methodology. a few different options to eliminate this issue have been reported here to successfully selectively and sensitively detect l. monocytogenes. designing ideal primers, additives such as dmso, and method modifications such as touchdown lamp may be favorable alternatives for increased specificity and sensitivity in lamp in other applications as well. loop-mediated isothermal amplification of dna accelerated reaction by loop-mediated isothermal amplification using loop primers enzymatic amplification of β-globin genomic sequences and restriction site analysis for diagnosis of sickle cell anemia primer-directed enzymatic amplification of dna with a thermostable dna polymerase nucleic acid sequence-based amplification self-sustained sequence replication ( sr): an isothermal transcription-based amplification system alternative to pcr strand displacement amplification-an isothermal, in vitro dna amplification technique mutation detection and single-molecule counting using isothermal rolling-circle amplification helicase-dependent isothermal dna amplification cross-priming amplification for rapid detection of mycobacterium tuberculosis in sputum specimens the development and evaluation of cross-priming amplification for the detection of avian reovirus optimisation of reverse transcriptase loop-mediated isothermal amplification assay for rapid detection of macrobrachium rosenbergii noda virus and extra small virus in macrobrachium rosenbergii rapid identification of virus-carrying mosquitoes using reverse transcription-loop-mediated isothermal amplification development of reverse transcription loop-mediated isothermal amplification for rapid detection of porcine epidemic diarrhea virus loop-mediated isothermal amplification for the detection of goose circovirus a loop-mediated isothermal amplification (lamp) method for the identification of species within the echinococcus granulosus complex the development of loop-mediated isothermal amplification combined with lateral flow dipstick for detection of vibrio parahaemolyticus visual detection of turkey coronavirus rna in tissues and feces by reverse-transcription loop-mediated isothermal amplification (rt-lamp) with hydroxynaphthol blue dye development of a loop-mediated isothermal amplification method for rapid mass-screening of sand flies for leishmania infection a novel hbv genotypes detecting system combined with microfluidic chip, loop-mediated isothermal amplification and gmr sensors a new surveillance and response tool: risk map of infected oncomelania hupensis detected by loop-mediated isothermal amplification (lamp) from pooled samples rapid and sensitive detection of listeria monocytogenes by loop-mediated isothermal amplification simple and rapid method for detecting foodborne shigella by a loop-mediated isothermal amplification kinetic pcr analysis: real-time monitoring of dna amplification reactions betaine and dmso: enhancing agents for pcr a specificity enhancer for polymerase chain reaction. nucl. acid res improvement of pcr amplified dna sequencing with the aid of detergents touchdown pcr for increased specificity and sensitivity in pcr amplification touchdown" pcr to circumvent spurious priming during gene amplification this article is an open access article distributed under the terms and conditions of the creative commons attribution license the authors would like to acknowledge the china scholarship council. this work was supported by natural science foundation of china ( ), nsfc-henan talent the authors declare no conflict of interest. key: cord- - jyui ca authors: lai, zheng-zong; ho, yi-jung; lu, jeng-wei title: harringtonine inhibits zika virus infection through multiple mechanisms date: - - journal: molecules doi: . /molecules sha: doc_id: cord_uid: jyui ca mosquito-borne zika virus (zikv) is a flavivirus that came under intense study from to for its well-known ability to cause congenital microcephaly in fetuses and neurological guillain–barré disease in adults. substantial research on screening antiviral agents against zikv and preventing zikv infection are globally underway, but food and drug administration (fda)-approved treatments are not available yet. compounds from chinese medicinal herbs may offer an opportunity for potential therapies for anti-zikv infection. in this study, we evaluated the antiviral efficacy of harringtonine against zikv. harringtonine possessed anti-zikv properties against the binding, entry, replication, and release stage through the virus life cycle. in addition, harringtonine have strong virucidal effects in zikv and exhibited prophylaxis antiviral ability prior zikv infection. the antiviral activity also observed in the treatment against japanese encephalitis reporter virus (rp -gfp strain). overall, this study demonstrated that harringtonine would be a favorable potential candidate for the development of anti-zikv infection therapies. zika virus (zikv) is a member of flavivirus genus of the flaviviridae family and is a mosquito-borne virus with positive single-stranded rna. other flaviviruses include yellow fever virus (yfv), dengue virus (denv), west nile virus (wnv), tick-borne encephalitis viruses (tbev), and japanese encephalitis virus (jev). like these, the genome size of zikv is approximately , nucleotides in length and encodes approximately amino acids, which translate a single polyprotein. the polyprotein produces three structural proteins (capsid, pre-membrane, and envelope) and seven nonstructural proteins (ns , ns a, ns b, ns , ns a, ns b, and ns ) via viral and cellular proteolysis [ , ] . among these viral proteins, the envelope protein is responsible for viral entry and influences host attachment [ ] . however, the nonstructural proteins are related to viral rna replication and virion assembly [ ] . zikv was first discovered in rhesus macaques in ugandan forests in . the first outbreak of zikv occurred in on yap island in the western pacific ocean, followed by a large epidemic in central and south american countries in - [ ] . the spread of zikv has caused a global health concern. in addition to transmission by infected mosquitoes, zikv can also be transmitted the cytotoxicity profiles of harringtonine in non-infected vero cells were evaluated. vero cells were grown in fresh medium with raising concentrations of harringtonine in -well microplates; cytotoxicity was assessed for h using a cck- assay, which evaluated cell proliferation by measuring cellular metabolic activity. the cell viability of vero cells was approximately or % at harringtonine concentrations up to or nm after h treatment, respectively ( figure a ). to avoid drug-induced cell cytotoxicity of harringtonine treatment, this was limited to nm in subsequent antiviral experiments. to investigate the anti-zikv activity of harringtonine, we assessed the inhibition of virus infection in vero cells with moi = . under different concentrations of harringtonine for h. for this, vero cells monolayer were cultured in -well microplates overnight, infected with ffu zikv per well, and incubated with raising concentrations of harringtonine from , and nm for h. intracellular viral rna levels, protein expression levels and virus progeny in supernatants were respectively determined by rt-qpcr, western blotting and fluorescent focus assay (ffa). the dose-dependent anti-zikv activities of harringtonine were observed to decrease viral rna/protein production and progeny yield ( figure b-d) , indicating that virus propagation was suppressed. results of ifa assay also showed that harringtonine treatment inhibited zikv infection in a dose-dependent manner ( figure e ,f). taken together, our data indicated that harringtonine inhibited zikv infection by suppressing the production of zikv rna, viral protein and reducing virion yield in vitro. the experiments were carried out in triplicates, and the error bars represent standard deviation (a). vero cells were infected with zikv and were treated with different concentrations of harringtonine for h. the anti-zikv ability of harringtonine was analyzed by measuring viral rna levels (b). the cell supernatants were harvested, and virus titers were assessed by fluorecent focus assay (c). the intracellular protein expression of env was assessed by western blot (d). the severity of zikv infection was determined by ifa (e) and quantification of vero cells (f). statistical significance was analyzed from t-test compared with the zikv group: * p < . ; ** p < . ; *** p < . . scale bar: μm. to further evaluate the individual stages of the virus life cycle, a time of addition assay was performed ( figure a ). harringtonine ( nm) was administered at different stages of zikv infection with moi = . , and then the levels of zikv rna in cells, as well as virus titers in supernatants, were determined after h treatment. the zikv group and full-treatment group were, respectively, negative control and positive control. both levels of zikv rna production ( figure b ) and progeny yield ( figure c ) reduced at all processes of the virus life cycle. the strongest inhibition effect of harringtonine was observed at the post-treatment stage (approximately log inhibition of viral rna level and progeny yield), which suggested that the compound may mainly inhibit at the late stage of zikv infection. in addition, both levels of zikv rna and progeny yield were also suppressed at the co-treatment stage, which suggested that the compound may also affect at the early stage of zikv infection. in order to investigate the effect after zikv entry, vero cells were seeded and inoculated with zikv at moi = . for h absorption. then, harringtonine nm was added at , , and h after the inoculum removed. the viral rna level was detected after h incubation. based on the above data, we have determined that harringtonine effectively interferes at late stage of zikv infection ( figure d ). the viability was determined with a cell counting kit assay. the experiments were carried out in triplicates, and the error bars represent standard deviation (a). vero cells were infected with zikv and were treated with different concentrations of harringtonine for h. the anti-zikv ability of harringtonine was analyzed by measuring viral rna levels (b). the cell supernatants were harvested, and virus titers were assessed by fluorecent focus assay (c). the intracellular protein expression of env was assessed by western blot (d). the severity of zikv infection was determined by ifa (e) and quantification of vero cells (f). statistical significance was analyzed from t-test compared with the zikv group: * p < . ; ** p < . ; *** p < . . scale bar: µm. to further evaluate the individual stages of the virus life cycle, a time of addition assay was performed ( figure a ). harringtonine ( nm) was administered at different stages of zikv infection with moi = . , and then the levels of zikv rna in cells, as well as virus titers in supernatants, were determined after h treatment. the zikv group and full-treatment group were, respectively, negative control and positive control. both levels of zikv rna production ( figure b ) and progeny yield ( figure c ) reduced at all processes of the virus life cycle. the strongest inhibition effect of harringtonine was observed at the post-treatment stage (approximately log inhibition of viral rna level and progeny yield), which suggested that the compound may mainly inhibit at the late stage of zikv infection. in addition, both levels of zikv rna and progeny yield were also suppressed at the co-treatment stage, which suggested that the compound may also affect at the early stage of zikv infection. in order to investigate the effect after zikv entry, vero cells were seeded and inoculated with zikv at moi = . for h absorption. then, harringtonine nm was added at , , and h after the inoculum removed. the viral rna level was detected after h incubation. based on the above data, we have determined that harringtonine effectively interferes at late stage of zikv infection ( figure d ). . zikv rna levels were determined using rt-qpcr at different infection processes (b). virus titers in supernatants were determined using fluorescent focus assay (ffa) (c). the nm of harringtonine was added at , , and hpi (hour post infection). at hpi, the viral rna level was analyzed by rt-qpcr (d). statistical significance was analyzed from t-test compared with the zikv group: * p < . ; ** p < . ; *** p < . . statistical significance was analyzed from t-test compared with the zikv group: * p < . ; ** p < . ; *** p < . . our previous studies have shown that virus could bind onto cells, but could not enter into the cells at • c. thus, °c utilized to verify the effect of harringtonine on zikv absorption and then removed the inoculum and washed which could focus on the effect of virus binding. to further clarify the inhibitory process of harringtonine at the co-treatment stage, temperature difference-based binding assay and entry assay, replication, and release assay were conducted. the nm harringtonine was added during zikv infection (moi = . ) at °c, then the treated cells were washed, and fresh medium was added at °c. after h, rna and virus titer were detected and used to verify the effect of viral binding. furthermore, the cells were infected with zikv (moi = . ) at • c for h incubation, then the inoculum was removed, washed to replace with nm harringtonine, and incubated at • c for another hour. subsequently, nm harringtonine was replaced with fresh medium, which was used to investigate the effect of viral entry. the results demonstrated that harringtonine could inhibit zikv infection with moi = . through virus binding ( figure a ,b) and virus entry processes ( figure c,d) . besides, the cells were infected with zikv (moi = . ) at • c for h, and then incubated at • c for another hour. when zikv entered into the cell, the specified concentration of harringtonine was added. the rna level was used to verify whether harringtonine affected virus rna replication, and the viral progeny yield was used to further determine whether harringtonine influenced virus release. these results indicated that harringtonine also reduced the viral rna replication and virion release ( figure e ,f) by rt-qpcr and ffu assay. moreover, we also assessed whether harringtonine affects zikv stability. the zikv supernatant was added with the specified concentration of harringtonine for h incubation to verify the virucidal ability. then, the zikv supernatant was progressed -fold serial-diluted and ffa was applied to determine the reduction of virus stability. therefore, the above experiments could verify the effects of different stages. the results of harringtonine were to inhibit zikv stability at a concentration of and nm ( figure g ). we produced a timeline of time of binding, entry, replication, and release assays ( figure h ). interestingly, the antiviral effect of harringtonine was also observed even at the pre-treatment stage, when cells were pre-treated with harringtonine for h before virus infection (figure a,b) . the result of the analysis of molecular docking was used to measure the likelihood of harringtonine binding to the zikv envelope proteins, the highest patchdock score was ( figure a,b) . overall, the above evidence indicated that harringtonine possessed antiviral activities by not only disrupting viral replication, release but also blocking viral binding, entry, as well as exhibiting prophylactic effect before zikv infection. binding, entry, replication and release assays. the red line refers to the zikv absorption period and dotted blue line refers to the harringtonine administration period (h). statistical significance was analyzed from t-test compared with the zikv group: * p < . ; ** p < . . to further assess the antiviral activity of harringtonine against other virus, the japanese encephalitis reporter virus (rp -gfp strain) with moi = . was used. briefly, vero cells were seeded in -microplates overnight. cells were then infected with ffu rp -gfp virus in the presence of harringtonine at different concentrations for h. intracellular viral rna levels were determined using rt-qpcr. the inhibitory effect on viral infection was evaluated by observing virus fluorescent protein expression under an inverted fluorescence microscope. results showed that to nm harringtonine exhibited a dose-dependent anti-rp -gfp activity ( figure a-c) . consequently, harringtonine exhibited an antiviral potential to be used in the treatment of jev infection. to further assess the antiviral activity of harringtonine against other virus, the japanese encephalitis reporter virus (rp -gfp strain) with moi = . was used. briefly, vero cells were seeded in -microplates overnight. cells were then infected with ffu rp -gfp virus in the presence of harringtonine at different concentrations for h. intracellular viral rna levels were determined using rt-qpcr. the inhibitory effect on viral infection was evaluated by observing virus fluorescent protein expression under an inverted fluorescence microscope. results showed that to nm harringtonine exhibited a dose-dependent anti-rp -gfp activity ( figure a-c) . consequently, harringtonine exhibited an antiviral potential to be used in the treatment of jev infection. zikv infection in neonates with congenital microcephaly born from zikv-infected pregnant women was identified as an emerging health issue in brazil from to . to prevent the severe consequences of zikv infections and reduce zikv-induced neurological defects, an effective anti-zikv agent is required. compounds isolated from natural plants have been widely evaluated in many antiviral studies [ , ] . previous research on the natural cephalotaxus alkaloids harringtonine, homoharringtonine, and cephalotaxine majorly showed antitumor effects, such as antileukemic activity [ , , ] . cephalotaxus alkaloid antitumor effects were suggested via their inhibitory effects on protein synthesis and partly on dna synthesis [ , ] . however, further research on cephalotaxus alkaloids demonstrated antiviral effects against hepatitis b virus (hbv), bovine viral diarrhea virus (bvdv), chikv, vzv, foot and mouth disease virus (fmd), vesicular stomatitis virus (vsv), newcastle disease virus (ndv), and sars-cov- [ , , [ ] [ ] [ ] [ ] [ ] [ ] . in this study, the drug-induced cytotoxicity of harringtonine was first assessed in vero cells, the vero cells have been previously reported to be highly permissive for zikv growth and replication [ ] . the concentration of harringtonine used in this anti-zikv study was no more than nm to avoid drug-induced cytotoxicity and kept the cell viability of vero cells remained above % ( figure a ). this concentration of harringtonine was much lower than that used in the treatment of chikv with µm harringtonine in bhk cells [ ] . this distinction may be due to the different cell lines and experimental approaches used. a time of addition experiment was conducted to determine which stage of zikv life cycle was disrupted. harringtonine treatments exhibited anti-zikv effects on all virus life stages including co-treatment, post-treatment, and even pre-treatment ( figure ) . noticeably, post-treatment of harringtonine showed that the most potent inhibitory effects on intracellular viral rna level and viral progeny yield in supernatants. harringtonine also exhibited a prophylactical antiviral activity before zikv infection in vitro, suggesting that harringtonine probably entered and was retained in the cells and then exerted inhibitory effects. a previous study demonstrated that harringtonine decreased in chikv rna synthesis and protein production and also reduced viral progeny. furthermore, harringtonine also presented the prophylactical antiviral activity in chikv infection. [ ] . denv-infected cells revealed the increase of subpolysomal mrnas which might correlate to the repression of translation at the initiation stage [ ] . harringtonine, through a block following s subunit joining, could inhibit the initiation of translation, and be used to verify if denv infection could affect the translation elongation. denv infection could disrupt the host cell translation at the starting stage, but does not change the translation elongation [ ] . other studies reported that harringtonine inhibited protein synthesis by blocking poly(u)-directed polyphenylalanine synthesis and peptide bond formation [ ] , and interfered with large ribosome subunit [ ] . therefore, the decrease of viral protein expression might also relate to down-regulating protein synthesis. previous evidences indicated that harringtonine was an inhibitor of protein synthesis, which is most likely to inhibit the large ribosomal unit of eukaryotes, thereby inhibiting the translation of non-structural or structural proteins [ , ] . the above evidence implies that the antiviral activities of harringtonine might occur on host factors. harringtonine was effective against sindbis virus (sinv) and chikv and exhibits a dose-dependent inhibitory effect, but it is not effective in inhibiting the growth of encephalomyocarditis virus (emcv). therefore, the antiviral effect of harringtonine may be limited to related viruses transmitted by mosquitoes [ , ] . to further clarify the inhibitory activities of harringtonine that occurred at the co-treatment stage, binding assay and entry assay were performed. the results indicated that harringtonine could block both viral binding and entry into host cells based on the decrease in viral rna production ( figure a ,c) and virion progeny ( figure b,d) . harringtonine also revealed the virucidal ability which could destroy the virion stability ( figure g ). through molecular docking, harringtonine was predicted the binding affinity of the envelope of zikv, which might be the reason of harringtonine blocking the early stage of zikv infection and affecting the virion stability. compared to our previous study of cephalotaxine, harringtonine obviously possessed more complex mechanisms against zikv infection than cephalotaxine, in blocking virus binding, entry, stability and possessing prophylactical antiviral ability. the results mean that harringtonine treatment could be used in more complicated medical conditions against zikv infection. both of the in vivo and clinical treatments, these results proved the safety or lower drug toxicity of harringtonine [ ] [ ] [ ] . in our previous study, we also demonstrated that cephalotaxine exhibited anti-zikv and denv activity in vitro. although both harringtonine and cephalotaxine belong to cephalosporin isolates, they still have different anti-zikv abilities. first of all, the ec and cc of harringtonine are . nm and > µm, while the ec and cc of cephalotaxine are µm and > µm. the selectivity index (si) of harringtonine is . , while the si of cephalotaxine is . . second, both harringtonine and cephalotaxine have inhibitory effects after virus entry, because they have inhibitory effects in post-infection treatment. however, harringtonine revealed more effects on zikv binding and entry, whereas cephalotaxine did not observe the same effect. third, the virucidal assay showed that µm cephalotaxine reduced the infection ability of zikv by about %, but did not show more effects at a concentration of µm (data not shown) [ ] . however, the harringtonine has a better inhibitory effect ( . % at nm and . % at nm) in the virucidal assay, and it is dose-dependent. in this study, harringtonine possessed the ability against zikv infection during virus binding, entry, and virucidal assay. furthermore, the molecular docking showed that harringtonine could bind with zikv envelope protein ( figure ). the envelope protein was the most important structural protein of zikv and responded for virus binding and entry. based on the above new findings, harringtonine is more conducive to becoming a new candidate drug against zikv infection than cephalotaxine. despite these compounds sharing similar structures, their multiple biological and pharmacological activities may vary with different side chains [ ] . in this study, the anti-jev effects of harringtonine was also investigated and demonstrated that harringtonine possessed dose-dependent antiviral activities ( figure a -c). in conclusion, harringtonine was proved to suppress zikv and jev infection, and all of those evidence indicated that cephalotaxus alkaloids might possess the broad-spectrum anti-viral effects in flaviviruses through multiple mechanisms. african green monkey kidney cells (vero; atcc, ccl- ) was used in this study, as it is more permissive to zikv (pravabc ; genbank sequence accession: ku ) replication; vero cells were cultured in dulbecco's modified eagle medium (dmem) supplemented with % fetal bovine serum (fbs) and antibiotics under a % co incubator at • c. harringtonine was purchased from chemfaces (catalog number: cfn ), dissolved in % dimethyl sulfoxide (dmso) as a stock of mm, and stored at − • c until use. green fluorescence protein-expressing japanese encephalitis reporter virus (rp -gfp strain) was kindly given to us by dr. lin ren-jye. the propagation and titration of zikv were performed using vero cells. virus titer was determined using the fluorescent focus assay (ffa). the propagation and titration of jev were conducted using c / mosquito cells (c / ; atcc, crl- ) and vero cells, respectively. cytotoxicity of harringtonine was determined using the cell counting kit (cck- , dojindo laboratories, kumamoto, japan). increasing concentrations of harringtonine with fresh medium were added to cells in -well microplates in triplicates and incubated at • c in a % co incubator. after a or h incubation period, the medium was replaced with µl of fresh medium containing µl of cck- reagent for h. the absorbance at nm was measured using an elisa reader (synergy ht, biotek, winooski, vt, usa). the cell viability values for treated cells were normalized with those of untreated cells. total rna including viral genomic rna was extracted from infected cells using total rna reagent (bioman, tri ), after that rna levels were measured using the one-step × rt-qpcr mix sybr green kit (bioman; catalog number: qrp ). rt-qpcr was performed on a roche lightcycler (roche applied science, indianapolis, in) at the following conditions: • c, min; • c, min; ( • c, s; • c s; • c s) for cycles. the primers used to detect zikv, jev and β-actin (internal control) were as follows: zikv: forward primer '-ttggtcatgatactgctgatgc- and reverse primer '-ccttccacaaagtccctattgc- ', jev: forward primer '-tccgtcaccatgccagtctt- ' and reverse primer '-gaggatgattctgtaagtatctaggtatagagccc- ', and b-actin: forward primer '-aggcaccagggcgtgat- ' and reverse primer '-gcccacataggaatccttc tgac- ' [ ] . data were analyzed using the -ct method. viral titers were performed by using ffa. briefly, the virus solution was serially diluted and added to monolayer vero cells. the medium was discarded after a h incubation time, and cells were overlaid under semisolid dmem containing . % methylcellulose for h. subsequently, assay of immunofluorescence was conducted and fluorescent viral foci were counted under an inverted fluorescence microscope (whited). results of viral titers were expressed as fluorescent focus units per ml (ffu/ml). cells cultured in -well plates were fixed with % formaldehyde at room temperature for h and were then permeabilized with an equal ratio of chilled methanol and acetone ( : ) for min. cells were then washed three times with phosphate-buffered saline (pbs) and blocked with % skimmed milk for h and were stained with anti-flavivirus envelope protein g primary antibody ( : dilution; produced in-house) for h. subsequently, cells were washed three times with pbs again, and alexa fluor -conjugated goat anti-mouse igg secondary antibody ( : dilution; jackson immunoresearch laboratories, inc.; west grove, pa, usa) was added at • c for another h incubation. stained cells were visualized using an inverted fluorescence microscope (olympus ckx , olympus, japan), as previously reported [ ] . total cell lysates were harvested by adding ripa lysis buffer supplemented with protease inhibitors. anti-flavivirus envelope protein g antibodies ( : dilution; produced in-house) and rabbit anti-β-actin polyclonal antibodies ( : dilution; finetest, wuhan, china, catalog number: fnab ) were used as primary antibodies. anti-mouse or anti-human horseradish peroxidase-conjugated antibodies were used as secondary antibodies. blots were developed by adding enhanced chemiluminescence (ecl) reagent. vero cell monolayers were cultured overnight in -well microplates. drug-containing medium ( nm of harringtonine) was added at different time points relative to the h period of cell infection with approximately ffu of zikv (moi = . ). for the pre-treatment group (pre), harringtonine was added h before the virus infection. for the co-treatment group (co), harringtonine was added at the beginning of the virus infection. for the post-treatment group (po), harringtonine was added after the h period of infection. for the full-duration treatment group (full), harringtonine was added throughout the infection. cells were washed twice with pbs in each stage. after h incubation, cells and supernatants in all groups were harvested. the levels of intracellular viral rna and virus titers from supernatants were determined by rt-qpcr and ffa, respectively, as previously described [ , , ] . for binding assay, zikv (moi = . ) were inoculated onto vero cell monolayers in culture medium with or without nm of harringtonine for h at • c. cells were then washed twice with pbs, and fresh medium was added for h incubation at • c in a % co incubator. the levels of intracellular viral rna and virus titers in supernatants were determined by rt-qpcr and ffa, respectively. for entry assay, fresh medium containing zikv (moi = . ) was added to vero cells at • c for h. cells were then washed twice with pbs, and fresh medium with or without nm of harringtonine was added for another h incubation at • c in a % co incubator. thereafter, cells were washed twice with pbs again before being added to fresh medium. after a h incubation period, the levels of intracellular viral rna and virus titers in supernatants were determined by rt-qpcr and ffa, respectively [ , ] . the cells were infected with zikv (moi = . ) at • c for h, and then incubated at • c for another hour. when zikv entered into the cell and added the specified concentration of harringtonine. after day incubation, the cells lysate were collected for viral rna replication assay by rt-qpcr and the supernatant was assessed by the ffu assay to determine the viral release [ , ] . zikv ( × ffu) was respectively mixed with harringtonine at , , , and nm at • c for h; virus titers were then determined by ffa [ ] . the zikv envelope proteins ( ire) crystal structure was obtained from the protein data bank (pdb). three-dimensional ligand structures of (chemspider id: ) was obtained from the chemspider database and converted into the pdb format using pymol software, as previously reported [ ] . patchdock was used in this project to conduct molecular docking analyses. finally, the conformations were then ranked according to docking scores [ ] . the data obtained from this study were statistically analyzed in triplicate and using graphpad prism . software (graphpad software inc., san diego, ca, usa), and values were expressed as mean ± standard deviation. the statistical analyses of data were calculated using a two-tailed student's t-test, where a p-value < . was considered significant. full-length sequencing and genomic characterization of bagaza, kedougou, and zika viruses zika virus: history, emergence, biology, and prospects for control structures of the zika virus envelope protein and its complex with a flavivirus broadly protective antibody pathogenesis of flavivirus infections: using and abusing the host cell zika virus outbreak on yap island, federated states of micronesia potential sexual transmission of zika virus risk of zika virus transmission by blood donations in brazil zika virus infection and the eye microcephaly and zika virus infection study links zika virus to guillain-barre syndrome structures of harringtonine, isoharringtonine, and homoharringtonine anti-varicella-zoster virus activity of cephalotaxine esters in vitro inhibition of chikungunya virus replication by harringtonine, a novel antiviral that suppresses viral protein expression identification of inhibitory compounds against singapore grouper iridovirus infection by cell viability-based screening assay and droplet digital pcr comparative in vitro antitumor activity of homoharringtonine and harringtonine against clonogenic human tumor cells cephalotaxine inhibits zika infection by impeding viral replication and stability herb-target interaction network analysis helps to disclose molecular mechanism of traditional chinese medicine effect of feeding chinese herb medicine ageratum-liquid on intestinal bacterial translocations induced by h n aiv in mice new natural products in cancer chemotherapy molecular modes of action of cephalotaxine and homoharringtonine from the coniferous tree cephalotaxus hainanensis in human tumor cell lines inhibition of translation in eukaryotic systems by harringtonine harringtonine, an inhibitor of initiation of protein biosynthesis in vitro" models of hepatitis b virus (hbv) and bovine viral diarrhoea virus (bvdv) replication inhibitory effects of homoharringtonine on foot and mouth disease virus in vitro the natural compound homoharringtonine presents broad antiviral activity in vitro and in vivo shedding light on the effect of natural anti-herpesvirus alkaloids on sars-cov- : a treatment option for covid- remdesivir, lopinavir, emetine, and homoharringtonine inhibit sars-cov- replication in vitro comparative analysis of different cell systems for zika virus (zikv) propagation and evaluation of anti-zikv compounds in vitro flavivirus infection uncouples translation suppression from cellular stress responses u determines the species specificity of the a-site cleft antibiotics: the structures of tiamulin, homoharringtonine, and bruceantin bound to the ribosome specificity of protein synthesis inhibitors in the inhibition of encephalomyocarditis virus replication uptake, initial effects, and chemotherapeutic efficacy of harringtonine in murine leukemic cells sensitive and resistant to vincristine and other chemotherapeutic agents antitumor activities of harringtonine and homoharringtonine, cephalotaxus alkaloids which are active principles from plant by intraperitoneal and oral administration long survival in an elderly patient with acute myeloid leukaemia after treatment with harringtonine palmatine inhibits zika virus infection by disrupting virus binding, entry, and stability antiviral activities of niclosamide and nitazoxanide against chikungunya virus entry and transmission micafungin is a novel anti-viral agent of chikungunya virus through multiple mechanisms suramin inhibits chikungunya virus entry and transmission patchdock and symmdock: servers for rigid and symmetric docking we wish to thank yen-mei lee and hsin-hsuen shen for their experimental assistance. the authors declare no conflict of interest.molecules , , key: cord- -zhauwxye authors: wu, huaxing; li, beili; wang, xue; jin, mingyuan; wang, guonian title: inhibitory effect and possible mechanism of action of patchouli alcohol against influenza a (h n ) virus date: - - journal: molecules doi: . /molecules sha: doc_id: cord_uid: zhauwxye in the present study, the anti-influenza a (h n ) virus activity of patchouli alcohol was studied in vitro, in vivo and in silico. the cc( ) of patchouli alcohol was above µm. patchouli alcohol could inhibit influenza virus with an ic( ) of . ± . µm. mtt assay showed that the inhibition by patchouli alcohol appears strongly after penetration of the virus into the cell. in the influenza mouse model, patchouli alcohol showed obvious protection against the viral infection at a dose of mg/kg/day. flexible docking and molecular dynamic simulations indicated that patchouli alcohol was bound to the neuraminidase protein of influenza virus, with an interaction energy of – . kcal mol(– ). the invariant key active-site residues asp , arg , glu , glu and tyr played important roles during the binding process. based on spatial and energetic criteria, patchouli alcohol interfered with the na functions. results presented here suggest that patchouli alcohol possesses anti-influenza a (h n ) virus properties, and therefore is a potential source of anti-influenza agents for the pharmaceutical industry. the influenza virus, which is one of the main causes of acute respiratory infections in humans, can lead to annual epidemics and infrequent pandemics. the two influenza pandemics of the th century, "asian influenza ( /h n )" and "hong kong influenza ( /h n )" resulted in the deaths of an estimated - million people globally [ , ] . today, their descendants continue to cause the majority of influenza infections in humans [ ] . so far as it is learned that the most effective antiviral drug is the neuraminidase (na) inhibitor, which target the na glycoproteins of influenza a and b virus [ , ] . the release of new virions from the infected cell is a key step in the influenza life cycle and need neuraminidase (na) to cleave the α-ketosidic linkage between terminal sialic acid and an adjacent sugar residue [ ] . the na inhibitors were designed to prevent the key step by blocking the active site of enzyme and thus allow sufficient time for the host immune systems to remove infected viruses [ ] . consistent efforts have been devoted to the development of na inhibitors, using the crystal structure of the n sub-type na protein [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] . indeed, oseltamivir (tamiflu) is the representative na inhibitor that has proven to be uniquely applicable oral drug in clinical practice for the treatment of influenza infection [ , , ] . however, with an increase in medical use, the oseltamivir-resistant strains have been found and probably lead to a large scale outbreak of novel pandemic flu [ , ] . patchouli alcohol ( figure ) has been well known for over a century. it is a major constituent of the pungent oil from the east indian shrub pogostemon cablin (blanco) benth, and widely used in fragrances. patchouli oil is an important essential oil in the perfume industry, used to give a base and lasting character to a fragrance [ , ] . the essential oil is very appreciated for its characteristic pleasant and long lasting woody, earthy, and camphoraceous odor, as well as for its fixative properties, being suitable for use in soaps and cosmetic products [ , ] . the aerial part of pogostemon cablin has wildly been used for the treatment of the common cold and as an antifungal agent in china [ , ] . moreover, the plant is widely used in traditional chinese medicine as it presents various types of pharmacological activity according to the composition of the oil [ , ] . patchouli alcohol, as the major volatile constituent of patchouli oil, has been found to strongly inhibit h n replication and weakly inhibit b/ibaraki/ / replication [ ] . to the best of our knowledge, the anti-influenza virus (h n ) activities of patchouli alcohol have not been evaluated yet. therefore, the aim of the present study was to evaluate the anti-influenza a virus (h n ) activity of patchouli alcohol by mtt assay and mouse influenza model. on such basis, explicitly solvated docking and molecular dynamic (md) methods were applied to investigative the binding mode involving patchouli alcohol with influenza virus na protein. we anticipate that the insight into the understanding of inhibiting mechanism will be of value in the rational design of novel anti-influenza drugs. first the efficacy of patchouli alcohol on influenza a (h n ) virus replication and cell viability were examined. cc was used to express the cytotoxicity of patchouli alcohol on mdck. the cc of patchouli alcohol was above mm, which indicated that patchouli alcohol did not affect the growth of mdck (table ) . thus, it seems that the antiviral effects of patchouli alcohol were not due to the cytotoxicity. moreover, patchouli alcohol was found to inhibit influenza a (h n ) virus with an ic of . ± . µm. based on the ic and cc values, the selectivity index (si) was calculated as > . . it is reported that a si of or more is appropriate for an antiviral agent [ ] , suggesting that patchouli alcohol can be judged to have anti-influenza a (h n ) virus activity. until now, it has been found that patchouli alcohol showed dose-dependent anti-influenza virus (a/pr/ / , h n ) activity, with an ic value of . µm. furthermore, it showed weak activity against b/ibaraki/ / (ic = . µm) [ ] . with the addition of the above h n inhibitory activity, we have a comprehensively view of the anti-influenza activity of patchouli alcohol. cells were pretreated with patchouli alcohol prior to virus infection (pretreatment cells), viruses were pretreated prior to infection (pretreatment virus), and patchouli alcohol was added during the adsorption period (adsorption) or after penetration of the viruses into cells (replication). experiments were repeated independently three times and data presented are the average of three experiments. the symbols * indicated very significant difference p < . with respect to other mode (pretreatment virus, adsorption and pretreatment cell). as shown in figure , patchouli alcohol showed anti-influenza a (h n ) virus activity in a timedependent manner. it showed best antiviral activity when added at a concentration of µm during the replication period with inhibition of the viral replication of . % ± . % for influenza a (h n ) at h. however, no significant effect was detected when patchouli alcohol was used for pretreatment of cells or viruses or when patchouli alcohol was only added during the adsorption phase. these results suggested that the inhibition of influenza a (h n ) virus by patchouli alcohol appears to occur much more strongly after penetration of the virus into the cell. besides, biochemical studies have indicated that the bioactivity of na protein is essential determinant after the replication of influenza a (h n ) virus [ ] [ ] [ ] . hence, we conclude that the function of na protein may be suppressed by patchouli alcohol. to evaluate the toxicity of patchouli alcohol, the mean value of body weight of mice in each group was statistically analyzed. the mean weights of mice administered at the mg/kg/dose oseltamivir, mg/kg/dose patchouli alcohol and mg/kg/dose of patchouli alcohol one time daily for days were not significantly different compared with the normal control mice, showing no toxicity of patchouli alcohol and oseltamivir within the testing concentration (p > . ). physiological status was observed in virus infection mice. three days after viral infection, some mice, especially mice in the h n infected control group showed changes in behavior, such as a tendency to huddle, diminished vitality, and ruffled fur, etc. in the mouse influenza model, viral infection leads to loss of body weight and high mortality. therefore, the efficacy of patchouli alcohol and oseltamivir were evaluated on the basis of survival rate measured for days post-infection, for treated infected animals relative to untreated infected (control) animals. a comparison of efficacy of patchouli alcohol and oseltamivir in vivo mouse influenza model (oral treatment) showed that at a dose of mg/kg/day, patchouli alcohol showed obvious protection against the influenza virus, as the mean day to death was detected as . ± . (table ) . when the dose was lowered to mg/kg/day, patchouli alcohol showed weaker protection (measured by survivors/total) than that of mg/kg/day, the mean day to death was . ± . . whereas oseltamivir at this dose level ( mg/kg/day) showed % protection (measured by survivors/total) against the influenza virus. in the h n infected control group, there were no survivors. in view of both in vitro and in vivo data, we conclude that patchouli alcohol could be used in the treatment of human influenza virus infections. based on the above experiment data, patchouli alcohol is determined to be bound within na protein. as the total energies and backbone root-mean-square-deviations (rmsd) in figure indicate, the energy-minimized patchouli alcohol-na complex has been in equilibrium since about . ns, and then retains quite stable in the last . ns. it is consistent with the previous md results of other na inhibitors [ ] [ ] [ ] [ ] [ ] [ ] . accordingly, the geometric and energetic analyses were made on the average structures of . ~ . ns md trajectories, where the system has been already at equilibrium. the interaction energy (e inter ) of patchouli alcohol with na was calculated at − . kcal mol − , where the vdw rather than electrostatic interactions were found to play a dominant role, contribute to about % (− . kcal mol − ). as shown in figure , the patchouli alcohol was bound at the active site which also bound to oseltamivir and zanamivir [ ] . as figure shows, the oxygen atom of patchouli alcohol was oriented towards the sidechains of residues glu and tyr , with one h-bond formed with each residue. the values of distances in figure further reveal that the docked complex remains rather stable throughout the simulation, with the average distances of glu :oe patchouli alcohol:o and tyr :oh -patchouli alcohol:o less than . Å. the sum contributions (e sum ) of residues glu and tyr amounted to − . and − . kcal mol − , respectively (table ) . besides, patchouli alcohol was stabilized by residues arg , asp , arg , trp , ala , glu , arg , asn and gln , especially residues asp , arg and glu ( figure and table ). as a matter of fact, residues asp , arg , glu , glu and tyr of the na protein have already received enough attention from rational drug designs [ , , ] . the catalytic residues asp , arg and glu are crucial to the na functions and the residues glu and tyr are important to stabilize the na active sites [ , ] . it suggests that the na functions will be affected by the presence of patchouli alcohol, consistent with the above experiments. patchouli alcohol matches with the na active site and has an acceptable interaction energy. considering the obvious structure discrepancies against current na inhibitors, it represents an ideal lead compound for the designs of novel anti-influenza agents. patchouli alcohol and oseltamivir were obtained from sigma chemical co. (st. louis, mo, usa, purity > %) and was stored in glass vials with teflon sealed caps at − ± . °c in the absence of light. mdck (madin-darby canine kidney) was purchased from harbin veterinary research institute (harbin, heilongjiang, china). the cells were grown in monolayer culture with eagle's minimum essential medium (emem) supplemented with % fetal calf serum (fcs), u/ml penicillin and μg/ml streptomycin. the monolayers were removed from their plastic surfaces and serially passaged whenever they became confluent. cells were plated out onto -well culture plates for cytotoxicity and anti-influenza assays, and propagated at °c in an atmosphere of % co . the influenza strain a/leningrad/ / / h n ) was purchased from national control institute of veterinary bioproducts and pharmaceuticals (beijing, china). virus was routinely grown on mdck cells. the stock cultures were prepared from supernatants of infected cells and stored at − °c. the cellular toxicity of patchouli alcohol on mdck cells was assessed by the mtt method. briefly, cells were seeded on a microtiter plate in the absence or presence of various concentrations ( µm - . µm) of patchouli alcohol (eight replicates) and incubated at °c in a humidified atmosphere of % co for h. the supernatants were discarded, washed with pbs twice and mtt reagent ( mg/ml in pbs) was added to each well. after incubation at °c for h, the supernatants were removed, then μl dmso was added and incubated at °c for another min. after that the plates were read on an elisa reader (thermo molecular devices co., union city, usa) at / nm. the mean od of the cell control wells was assigned a value of %. the maximal non-toxic concentration (td ) and % cytotoxic concentration (cc ) were calculated by linear regression analysis of the dose-response curves generated from the data. inhibition of virus replication was measured by the mtt method. serial dilution of the treated virus was adsorbed to the cells for h at °c. the residual inoculum was discared and infected cells were added with emem containing % fcs. each assay was performed in eight replicates. after incubation for h at °c, the cultures were measured by mtt method as described above. the concentration of patchouli alcohol and oseltamivir which inhibited virus numbers by % (ic ) was determined from dose-response curves. cells and viruses were incubated with patchouli alcohol at different stages during the viral infection cycle in order to determine the mode of antiviral action. cells were pretreated with patchouli alcohol before viral infection, viruses were incubated with patchouli alcohol before infection and cells and viruses were incubated together with patchouli alcohol during adsorption or after penetration of the virus into the host cells. patchouli alcohol was always used at the nontoxic concentration. cell monolayers were pretreated with patchouli alcohol prior to inoculation with virus by adding patchouli alcohol to the culture medium and incubation for h at °c. the compound was aspirated and cells were washed immediately before the influenza a (h n ) inoculum was added. for pretreatment virus, influenza a (h n ) was incubated in medium containing patchouli alcohol for h at room temperature prior to infection of mdck cells. for analyzing the anti-influenza a (h n ) inhibition during the adsorption period, the same amount of influenza a (h n ) was mixed with the drug and added to the cells immediately. after h of adsorption at °c, the inoculum was removed and dmem supplemented with % fcs were added to the cells. the effect of patchouli alcohol against influenza a (h n ) was also tested during the replication period by adding it after adsorption, as typical performed in anti-influenza a (h n ) susceptibility studies. each assay was run in eight replicates. kunming mice, weighing - g ( weeks of age) were purchased from harbin veterinary research institute animal co., ltd. (harbin, heilongjiang, china) . first, the toxicity of patchouli alcohol and oseltamivir was assessed in the healthy mice by the loss of body weight compared with the control group ( % dmso in physiological saline). the mice were orally administered with mg/kg/dose patchouli alcohol, mg/kg/dose patchouli alcohol or mg/kg/dose oseltamivir (dissolved in % dmso in physiological saline) one time daily for days. the weight of mice was determined daily. we conducted procedures according to principle of laboratory animal care (nih publication no. - , revised ) and the guidelines of the peking university animal research committee. kunming mice were anesthetized with isoflurane and exposed to virus (a/leningrad/ / / ) by intranasal instillation. drugs were prepared in % dmso in physiological saline and administered h prior to virus exposure and continued daily for days. all mice were observed daily for changes in weight and for any deaths. parameters for evaluation of antiviral activity included weight loss, reduction in mortality and/or increase in mean day to death (mdd) determined through days. the n sub-type neuraminidase crystal structure (pdb code ivd) was obtained from the rcsb protein data bank [ ] . for convenience, the structure is named as na hereafter. geometry and partial atomic charges of the patchouli alcohol ( figure ) were calculated with the discover . module (insight ii ) [ ] by applying the bfgs algorithm [ ] and the consistent-valence force-field (cvff), with a convergence criterion of . kcal mol − Å − . the docking and molecular dynamics (md) simulations were performed by the general protocols in the insight ii software packages, consistent with the previous literatures [ , , , , [ ] [ ] [ ] . during the md simulations, the canonical ensemble (nvt) was employed at normal temperature ( k). the md temperature was controlled by the velocity scaling thermostat [ ] . integrations of the classical equations of motion were achieved using the verlet algorithm. the systems were solvated in a large sphere of tip p water molecules [ ] with the radius of . Å, which is enough to hold the ensembles [ ] . the md trajectories were generated using a . -fs time step for a total of . ns, saved at . -ps intervals. the interaction energies of patchouli alcohol with na and the respective residues at the na active site were calculated by the docking module [ ], over the . ~ . ns md trajectories. all results are expressed as mean values ± standard deviations (sds) (n = ). the significance of difference was calculated by one-way analysis of variance, and values p < . were considered to be significant. in conclusion, patchouli alcohol possesses anti-influenza a (h n ) virus activity via interference with the na function that cleaves the α-glycosidic bond between sialic acid and glycoconjugate. our results provide the promising information for the potential use of patchouli alcohol in the treatment of influenza a (h n ) virus infectious disease. further mechanistic studies on the anti-influenza a virus activity are needed to support this point of view. global epidemiology of influenza: past and present emerging and re-emerging infectious diseases: influenza as a prototype of the host-pathogen balancing act pandemic influenza: an inconvenient mutation influenza virus neuraminidase inhibitors neuraminidase inhibitors for influenza avian influenza a (h n ) infection: targets and strategies for chemotherapeutic intervention controlling influenza by inhibiting the virus's neuraminidase rational design of potent sialidase-based inhibitors of influenza virus replication influenza neuraminidase inhibitors possessing a novel hydrophobic interaction in the enzyme active site: design, synthesis, and structural analysis of carbocyclic sialic acid analogues with potent anti-influenza activity drug design against a shifting target: a structural basis for resistance to inhibitors in a variant of influenza virus neuraminidase hydrophobic benzoic acids as inhibitors of influenza neuraminidase influenza virus neuraminidase inhibitors pyrrolidinobenzoic acid inhibitors of influenza virus neuraminidase: modifications of essential pyrrolidinone ring substituents the war against influenza: discovery and development of sialidase inhibitors design, synthesis, inhibitory activity, and sar studies of hydrophobic p-aminosalicylic acid derivatives as neuraminidase inhibitors molecular mechanisms of influenza virus resistance to neuraminidase inhibitors resistant influenza a viruses in children treated with oseltamivir: descriptive study patchouli alcohol: in vitro direct anti-influenza virus sesquiterpene in pogostemon cablin benth antiviral activity of natural occurring flavonoids in vitro the nucleocapsid protein of coronavirus infectious bronchitis virus: crystal structure of its n-terminal domain and multimerization properties amino acid residues critical for rna-binding in the n-terminal domain of the nucleocapsid protein are essential determinants for the infectivity of coronavirus in cultured cells x-ray structures of the n-and c-terminal domains of a coronavirus nucleocapsid protein: implications for nucleocapsid formation investigation of neuraminidase-substrate recognition using molecular dynamics and free energy calculations molecular dynamics and free energy analysis of neuraminidase-ligand interactions scoring binding affinity of multiple ligands using implicit solvent and a single molecular dynamics trajectory: application to influenza neuraminidase the conformational analysis and proton transfer of the neuraminidase inhibitors: a theoretical study computer-based de novo designs of tripeptides as novel neuraminidase inhibitors synergistic effects in the designs of neuraminidase ligands: analysis from docking and molecular dynamics studies structural and functional basis of resistance to neuraminidase inhibitors of influenza b viruses influenza neuraminidase inhibitors: structure-based design of a novel inhibitor series on the lower susceptibility of oseltamivir to influenza neuraminidase subtype n than those in n and n structure of the catalytic and antigenic sites in influenza virus neuraminidase influenza virus neuraminidase: structure, antibodies, and inhibitors structures of aromatic inhibitors of influenza virus neuraminidase a broyden-fletcher-goldfarb-shanno optimization procedure for molecular geometries a computational investigation on the interaction mechanisms of neuraminidases and -( -pentyloxy)benzoic acid anti-infectious bronchitis virus (ibv) activity of , -cineole: effect on nucleocapsid (n) protein affinity user guide generalized langevin equation approach for atom/solid-surface scattering: general formulation for classical scattering off harmonic solids sample availability: samples of patchouli alcohol are available on request from the authors this article is an open access article distributed under the terms and conditions of the creative commons attribution license we are grateful for the computing support provided by harbin medical university. this research was supported by funds from the important research project of heilongjiang province of china (no. , the outstanding youth science foundation (no. jc ), and initial foundation project inside of the third affiliated hospital of harbin medical university (no. jj ). key: cord- -tazd dvm authors: yang, kun; jin, ming-ji; quan, zhe-shan; piao, hu-ri title: design and synthesis of novel anti-proliferative emodin derivatives and studies on their cell cycle arrest, apoptosis pathway and migration date: - - journal: molecules doi: . /molecules sha: doc_id: cord_uid: tazd dvm emodin is a cell arrest and apoptosis-inducing compound that is widely distributed in different plants (rhubarb, aloe), lichens and terrestrial fungi, and also isolated from marine-derived fungi and marine sponge-associated fungi. in this study, we designed and synthesized a novel series of emodin derivatives by binding emodin to an amino acid using linkers of varying lengths and composition, and evaluated their anti-proliferative activities using hepg cells (human hepatic carcinoma), mcf- cells (human breast cancer) and human normal liver l cells. most of these derivatives showed moderate to potent anti-proliferative activities. notably, compound a exhibited potent anti-proliferative activity against hepg cells with the half maximal inhibitory concentration (ic( )) value of . µm, which was enhanced . -fold compared to the parent compound emodin (ic( ) = . µm), and it also exhibited better selective anti-proliferative activity and specificity than emodin. moreover, further experiments demonstrated that compound a displayed a significant efficacy of inducing apoptosis through mitochondrial pathway via release of cytochrome c from mitochondria and subsequent activation of caspase- and caspase- , inducing cell arrest at g /g phase, as well as suppression of cell migration of tumor cells. the preliminary results suggested that compound a could be a promising lead compound for the discovery of novel anti-tumor drugs and has the potential for further investigations as an anti-cancer drug. emodin ( -methyl- , , -trihydroxyanthraquinone, figure ) is a naturally occurring anthraquinone derivative that is widely distributed in different plants (rhubarb, aloe), lichens and terristrial fungi, that has also been isolated from marine-derived fungi and marine sponge-associated fungi [ ] [ ] [ ] [ ] . it is also an active ingredient in chinese medicinal herbs used in the treatment of constipation, jaundice, gastro-intestinal hemorrhage, and ulcers. emodin has the same tricyclic planar chromophore skeleton as certain anti-tumor antibiotics such as daunorubicin, doxorubicin, epirubicin, and several synthetic analogs distributed such as mitoxantrone and pixantrone ( figure ) ; all of which are in used for clinical treatment of various cancers. many reports have demonstrated that emodin possesses a wide spectrum of pharmacological effects such as anti-tumour [ ] [ ] [ ] , anti-inflammatory [ , ] , antiviral [ ] , antibacterial [ ] , anti-allergic [ ] , anti-osteoporotic [ ] , anti-diabetic [ , ] , immunosuppressive [ ] , neuroprotective [ ] , and hepatoprotective [ , ] activities, etc. among these effects, anti-tumor anti-osteoporotic [ ] , anti-diabetic [ , ] , immunosuppressive [ ] , neuroprotective [ ] , and hepatoprotective [ , ] activities, etc. among these effects, anti-tumor activity is the most widely reported. in recent decades, pharmacological studies have shown that emodin is capable of inhibiting cellular proliferation [ ] , inducing of cell differentiation [ ] and apoptosis [ ] , and activating of caspase cascade pathway [ , ] and mitochondrial death pathway [ , ] in different cancer cells. chemical transformation of bioactive compounds of natural products is one of the most common approaches in drug discovery to improve therapeutic properties. to date, the anti-tumor activity of emodin has been improved by structural modification, mainly to the side chain including methyl, hydroxyl, and/or aryl ring groups. for example, wang et al. reported that emodin quaternary ammonium salt derivatives showed significant anti-cancer activities against hepatoma cells [ ] [ ] [ ] . xing et al. reported the effects of emodin rhamnoside derivatives against human cancer cells [ ] , and tan et al. reported dna-binding pyrazole emodin derivatives [ ] . however, emodin is not an ideal chemotherapeutic agent for cancer due to its poor bioavailability, low solubility, and high toxicity in vivo. the bioavailability of a drug is positively correlated with its solubility; thus amino acids, which possess the carboxylic and amino functionality, are ideal as a moiety for the structural modification of bioactive natural products. amino acids can interact with other biomolecules via secondary interactions such as hydrogen bonding to improve their pharmacological profiles in both potency and bioavailability [ ] . for example, conjugation of an amino acid to betulinic acid is able to improve its water-solubility as well as its anti-melanoma activity [ ] . these findings prompted us to design and synthesize a series of novel emodin derivatives linked with various amino acids as tfa salt ( figure ). among these derivatives, compound b derived from (r)- -aminopropanoic acid exhibited the most anti-proliferative activity against both hepg cells and mcf- cells. this prompted us to screen the different length of diol linker between -aminopropanoic acid and emodin. our results also suggest that compared to compound a, compound a exhibited a slightly more potent chemical transformation of bioactive compounds of natural products is one of the most common approaches in drug discovery to improve therapeutic properties. to date, the anti-tumor activity of emodin has been improved by structural modification, mainly to the side chain including methyl, hydroxyl, and/or aryl ring groups. for example, wang et al. reported that emodin quaternary ammonium salt derivatives showed significant anti-cancer activities against hepatoma cells [ ] [ ] [ ] . xing et al. reported the effects of emodin rhamnoside derivatives against human cancer cells [ ] , and tan et al. reported dna-binding pyrazole emodin derivatives [ ] . however, emodin is not an ideal chemotherapeutic agent for cancer due to its poor bioavailability, low solubility, and high toxicity in vivo. the bioavailability of a drug is positively correlated with its solubility; thus amino acids, which possess the carboxylic and amino functionality, are ideal as a moiety for the structural modification of bioactive natural products. amino acids can interact with other biomolecules via secondary interactions such as hydrogen bonding to improve their pharmacological profiles in both potency and bioavailability [ ] . for example, conjugation of an amino acid to betulinic acid is able to improve its water-solubility as well as its anti-melanoma activity [ ] . these findings prompted us to design and synthesize a series of novel emodin derivatives linked with various amino acids as tfa salt ( figure ). anti-osteoporotic [ ] , anti-diabetic [ , ] , immunosuppressive [ ] , neuroprotective [ ] , and hepatoprotective [ , ] activities, etc. among these effects, anti-tumor activity is the most widely reported. in recent decades, pharmacological studies have shown that emodin is capable of inhibiting cellular proliferation [ ] , inducing of cell differentiation [ ] and apoptosis [ ] , and activating of caspase cascade pathway [ , ] and mitochondrial death pathway [ , ] in different cancer cells. chemical transformation of bioactive compounds of natural products is one of the most common approaches in drug discovery to improve therapeutic properties. to date, the anti-tumor activity of emodin has been improved by structural modification, mainly to the side chain including methyl, hydroxyl, and/or aryl ring groups. for example, wang et al. reported that emodin quaternary ammonium salt derivatives showed significant anti-cancer activities against hepatoma cells [ ] [ ] [ ] . xing et al. reported the effects of emodin rhamnoside derivatives against human cancer cells [ ] , and tan et al. reported dna-binding pyrazole emodin derivatives [ ] . however, emodin is not an ideal chemotherapeutic agent for cancer due to its poor bioavailability, low solubility, and high toxicity in vivo. the bioavailability of a drug is positively correlated with its solubility; thus amino acids, which possess the carboxylic and amino functionality, are ideal as a moiety for the structural modification of bioactive natural products. amino acids can interact with other biomolecules via secondary interactions such as hydrogen bonding to improve their pharmacological profiles in both potency and bioavailability [ ] . for example, conjugation of an amino acid to betulinic acid is able to improve its water-solubility as well as its anti-melanoma activity [ ] . these findings prompted us to design and synthesize a series of novel emodin derivatives linked with various amino acids as tfa salt ( figure ). among these derivatives, compound b derived from (r)- -aminopropanoic acid exhibited the most anti-proliferative activity against both hepg cells and mcf- cells. this prompted us to screen the different length of diol linker between -aminopropanoic acid and emodin. our results also suggest that compared to compound a, compound a exhibited a slightly more potent among these derivatives, compound b derived from (r)- -aminopropanoic acid exhibited the most anti-proliferative activity against both hepg cells and mcf- cells. this prompted us to screen the different length of diol linker between -aminopropanoic acid and emodin. our results also suggest that compared to compound a, compound a exhibited a slightly more potent anti-proliferative activity against hepg cells and mcf- cells. following this, the anti-cancer mechanism of compound a was evaluated against hepg cells. a novel series of emodin derivatives linked with amino acids were designed and synthesized. the synthesis strategies for these emodin derivatives are outlined in schemes - . for the synthesis of compounds a- z, compound was subjected to an alkylation reaction with -iodoethanol and cs co in dmf that resulted in compound . then coupling reactions under the conventional coupling condition (dcc/dmap) between compound and various commercially available n-boc protected amino acids resulted in the formation of condensation products. after removal of the protecting group usinng % tfa in dcm, the target compounds a- z were obtained as tfa salts. molecules , , x for peer review of anti-proliferative activity against hepg cells and mcf- cells. following this, the anti-cancer mechanism of compound a was evaluated against hepg cells. a novel series of emodin derivatives linked with amino acids were designed and synthesized. the synthesis strategies for these emodin derivatives are outlined in scheme - . for the synthesis of compounds a- z, compound was subjected to an alkylation reaction with -iodoethanol and cs co in dmf that resulted in compound . then coupling reactions under the conventional coupling condition (dcc/dmap) between compound and various commercially available n-boc protected amino acids resulted in the formation of condensation products. after removal of the protecting group usinng % tfa in dcm, the target compounds a- z were obtained as tfa salts. following the dcc/dmap coupling condition and deprotection under % tfa in dcm for the synthesis of compounds a- z, the target compounds a and b were obtained as tfa salts (scheme ). scheme . synthesis of compounds a- z. reagents and conditions: (a) -iodoethanol, cs co , dmf, • c, %; (b) (i) various n-boc amino acids, dcc, dcm, • c; (ii) % tfa in dcm, r.t., %- % over two steps. following the dcc/dmap coupling condition and deprotection under % tfa in dcm for the synthesis of compounds a- z, the target compounds a and b were obtained as tfa salts (scheme ). these products were then coupled with formaldehyde ( % aqueous solution) and reduced utilizing nabh cn to provide a and b as tfa salt in % and % yields, respectively. as shown in scheme , by employing the route for the synthesis of compounds a- z, compounds a- l were obtained as tfa salts. scheme . synthesis of compounds a- l. reagents and conditions: (a) various hydroxyalkyl bromides or iodides, cs co , dmf, °c, %- %; (b) (i) r/s-n-boc-ala-oh, dcc, dcm, °c; (ii) % tfa in dcm, r.t., %- % over two steps. the in vitro anti-proliferative activities of all the novel synthesized compounds were evaluated against hepg cells and mcf- cells by celltiter-glo ® luminescent cell viability assay, using paclitaxel (a clinically used drug) as a positive control. emodin ( ) and compound were also included for comparison. as shown in table , emodin ( ) showed weak inhibitory activity against hepg cells and mcf- cells with ic values of . ± . µm and . ± . µm, respectively. compared to emodin ( ), compound showed reduced activity due to the introduction of the hydroxyethyl group at the -position of emodin. among compounds a- z containing amino acid groups, it is worth noting that most of the compounds displayed more potent activity than emodin ( ) . compound a with the gly group displayed near double the activity of emodin ( ) against hepg cells, but had slightly decreased activity against mcf- cells. compound b with the d-ala group displayed the most potent anti-proliferative activity against hepg cells and mcf- cells with ic values of . ± . µm and . ± . µm, respectively. compound c with the l-ala group displayed slightly weaker activity than compound b, but still displayed stronger activity than emodin ( ). compounds d- j containing alkyl chain amino acid groups also displayed stronger inhibitory activities than emodin ( ) . overall, with the exception of compound n, compounds k- m with cyclic alkyl amino acid groups displayed worse inhibitory activities than emodin ( ); especially compound m containing a heterocycle. interestingly, compound n which contains a cyclopentane amino acid group displayed higher anti-proliferative activity against hepg cells and mcf- cells with ic values of . ± . µm scheme . synthesis of compounds a, b, a and b. reagents and conditions: (a) (i) boc-n-me-r/ s-ala-oh, dcc, dcm, • c; (ii) % tfa in dcm, r.t., %- % over two steps; (b) formaldehyde ( % aqueous solution), nabh cn, meoh, r.t., %- %. these products were then coupled with formaldehyde ( % aqueous solution) and reduced utilizing nabh cn to provide a and b as tfa salt in % and % yields, respectively. as shown in scheme , by employing the route for the synthesis of compounds a- z, compounds a- l were obtained as tfa salts. these products were then coupled with formaldehyde ( % aqueous solution) and reduced utilizing nabh cn to provide a and b as tfa salt in % and % yields, respectively. as shown in scheme , by employing the route for the synthesis of compounds a- z, compounds a- l were obtained as tfa salts. the in vitro anti-proliferative activities of all the novel synthesized compounds were evaluated against hepg cells and mcf- cells by celltiter-glo ® luminescent cell viability assay, using paclitaxel (a clinically used drug) as a positive control. emodin ( ) and compound were also included for comparison. as shown in table , emodin ( ) showed weak inhibitory activity against hepg cells and mcf- cells with ic values of . ± . µm and . ± . µm, respectively. compared to emodin ( ), compound showed reduced activity due to the introduction of the hydroxyethyl group at the -position of emodin. among compounds a- z containing amino acid groups, it is worth noting that most of the compounds displayed more potent activity than emodin ( ) . compound a with the gly group displayed near double the activity of emodin ( ) against hepg cells, but had slightly decreased activity against mcf- cells. compound b with the d-ala group displayed the most potent anti-proliferative activity against hepg cells and mcf- cells with ic values of . ± . µm and . ± . µm, respectively. compound c with the l-ala group displayed slightly weaker activity than compound b, but still displayed stronger activity than emodin ( ). compounds d- j containing alkyl chain amino acid groups also displayed stronger inhibitory activities than emodin ( ) . overall, with the exception of compound n, compounds k- m with cyclic alkyl amino acid groups displayed worse inhibitory activities than emodin ( ); especially compound m containing a heterocycle. interestingly, compound n which contains a cyclopentane amino acid group displayed higher anti-proliferative activity against hepg cells and mcf- cells with ic values of . ± . µm the in vitro anti-proliferative activities of all the novel synthesized compounds were evaluated against hepg cells and mcf- cells by celltiter-glo ® luminescent cell viability assay, using paclitaxel (a clinically used drug) as a positive control. emodin ( ) and compound were also included for comparison. as shown in table , emodin ( ) showed weak inhibitory activity against hepg cells and mcf- cells with ic values of . ± . µm and . ± . µm, respectively. compared to emodin ( ), compound showed reduced activity due to the introduction of the hydroxyethyl group at the -position of emodin. among compounds a- z containing amino acid groups, it is worth noting that most of the compounds displayed more potent activity than emodin ( ) . compound a with the gly group displayed near double the activity of emodin ( ) against hepg cells, but had slightly decreased activity against mcf- cells. compound b with the d-ala group displayed the most potent anti-proliferative activity against hepg cells and mcf- cells with ic values of . ± . µm and . ± . µm, respectively. compound c with the l-ala group displayed slightly weaker activity than compound b, but still displayed stronger activity than emodin ( ). compounds d- j containing alkyl chain amino acid groups also displayed stronger inhibitory activities than emodin ( ) . overall, with the exception of compound n, compounds k- m with cyclic alkyl amino acid groups displayed worse inhibitory activities than emodin ( ); especially compound m containing a heterocycle. interestingly, compound n which contains a cyclopentane amino acid group displayed higher anti-proliferative activity against hepg cells and mcf- cells with ic values of . ± . µm and . ± . µm, respectively. compounds o and p with d-and l-ala groups exhibited similar inhibitory activity to compound n. compared to emodin ( ), compounds q- z with aryl (phenyl, substituted phenyl, benzyl, substituted benzyl, etc.) amino acid groups displayed enhanced activity in varying degrees when tested against two cancer cell lines, with ic values ranging from . ± . to . ± . µm. following our initial results, we performed further modification of compounds b and c to improve the anti-proliferative activity, thus mono-and dimethylation of the amino moiety in the amino acid afforded compounds a, b, a and b. unfortunately, this modification led to decreased activity to varying degrees against both the hepg and mcf- cell lines, with ic values ranging from . ± . to . ± . µm, as shown in table . notably, compound b displayed a -fold activity reduction against mcf- cell lines compared to emodin ( ). the results revealed that "nh " was the moiety that favors improving the anti-proliferative activity. the preliminary explanation was that amino group can interact with other biomolecules to improve the anti-proliferative activity via formation of hydrogen bond. mono-or dimethylation of amino group might weaken the trend of hydrogen bonding and decrease the anti-proliferative activity. next, as shown in table , the linking group of compounds b and c was further optimized by increasing the linker length in compounds b and c by one additional methylene group. the resulting compounds, a and b, displayed further enhanced potency at inhibiting cell growth against both hepg cells and mcf- cells. however, compounds c- f initially failed to increase the potency of cell growth inhibition against both tested cancer cell lines; this was successfully addressed by increasing the linker length using additional methylene bridge. on the other hand, elongation of the linker by two to four ethanediol groups resulted in compounds g- l, which showed decreased activity compared to compounds b and c. three pairs of compounds possessing stronger anti-proliferative activities were screened out and their cytotoxic activities were preformed against human normal liver cells (l ) in vitro. as shown in table , the parent compound emodin exhibited stronger cytotoxic activity against l cells with an ic value of . ± . µm than against cancer cell lines (ic = . ± . µm against hepg cells and ic = . ± . µm against mcf- cells). however, all the six compounds exhibited weaker cytotoxic activities against l cells compared with their corresponding cancer cell lines. above all, we finally chose to investigate the mechanism of action for compound a because it had the strongest anti-proliferative activity against hepg cells. lack of selective cytotoxicity is the main factor that hinders conventional chemotherapeutic agents. thus, to evaluate the selective anti-proliferative activity of the compound a, the selectivity index (si) between cancer and normal cells was calculated and the results are summarized in table . the si was calculated by dividing the ic values in normal cells by the ic values in cancer cells. emodin displayed stronger cytotoxic activity, with si values of . (hepg ) and . (mcf- ), respectively, indicating that both normal cells and cancer cells would be killed. however, compound a exhibited weaker cytotoxic activity with si value of . (hepg ) and . (mcf- ), respectively. it means that a exhibited a better selective anti-proliferative activity and specificity than emodin. in order to verify whether compound a is able to induce apoptosis in hepg cells, we utilized fitc-annexin v/pi staining and estimated the percentage of apoptotic cells by flow cytometry. we noted a concentration-dependent increase in the percentage of apoptotic cells when the cells were treated with compound a for h at concentrations . , , and µm. as shown in figure a , few ( . %) apoptotic cells were present in the control panel. in contrast, the percentage of apoptotic cells increased to . % after treatment with compound a at µm for h and further increased to . % after treatment with a at the concentration of µm. as illustrated in figure b , the quantitative analysis of apoptosis strongly suggests that treatment with compound a effectively induced apoptosis in hepg cells in a concentration-dependent manner in comparison to the control. in order to verify whether compound a is able to induce apoptosis in hepg cells, we utilized fitc-annexin v/pi staining and estimated the percentage of apoptotic cells by flow cytometry. we noted a concentration-dependent increase in the percentage of apoptotic cells when the cells were treated with compound a for h at concentrations . , , and µm. as shown in figure a , few ( . %) apoptotic cells were present in the control panel. in contrast, the percentage of apoptotic cells increased to . % after treatment with compound a at µm for h and further increased to . % after treatment with a at the concentration of µm. as illustrated in figure b , the quantitative analysis of apoptosis strongly suggests that treatment with compound a effectively induced apoptosis in hepg cells in a concentration-dependent manner in comparison to the control. statistical significance is determined by two-tailed student t-test: "***" denote p < . , "**" denote p < . , respectively (supplementary table s ). (c, e) western blot analysis effect of compound a on the levels of bax, bcl- , cytochrome c, procaspase- , caspase- and procaspase- expression in hepg cells. (d, f) an equal amount of protein was loaded on sds-page gel for western blot analysis. data are expressed as means ± sd of the percentages of apoptotic cells from three independent experiments. statistical significance is determined by two-tailed student t-test: "***" denote p < . , "**" denote p < . , "*" denote p < . , respectively (supplementary table s ). to verify the molecular mechanisms of apoptosis induction of compound a, we performed a western blot assay. it is well known that the bcl- family of pro-apoptotic and anti-apoptotic the quantitative analysis of apoptosis. data are expressed as means ± sd of the percentages of apoptotic cells from three independent experiments. statistical significance is determined by two-tailed student t-test: "***" denote p < . , "**" denote p < . , respectively (supplementary table s ). (c, e) western blot analysis effect of compound a on the levels of bax, bcl- , cytochrome c, procaspase- , caspase- and procaspase- expression in hepg cells. (d, f) an equal amount of protein was loaded on sds-page gel for western blot analysis. data are expressed as means ± sd of the percentages of apoptotic cells from three independent experiments. statistical significance is determined by two-tailed student t-test: "***" denote p < . , "**" denote p < . , "*" denote p < . , respectively (supplementary table s ). to verify the molecular mechanisms of apoptosis induction of compound a, we performed a western blot assay. it is well known that the bcl- family of pro-apoptotic and anti-apoptotic proteins regulates the mitochondrial pathway of apoptosis. these bcl- family proteins stimulate the permeabilization of the mitochondrial outer membrane, which results in the release of cytochrome c into the cytosol and in turn promotes the activation of the caspase cascade. the activation of the caspase cascade ultimately leads to the induction of apoptotic cell death. as shown in figure c and d, in comparison with the control cells, compound a induced an increase in the levels of bax and a decrease in the expression of bcl- in a concentration-dependent manner. meanwhile, the release of cytochrome c from mitochondria increased after the treatment of compound a, while procaspase and procaspase decreased after treatment with a, indicating that the caspase and caspase were activated. as shown in figure e ,f, the increased expression of cleaved caspase- after treatment with a provided a further evidence that compound a induced cell apoptosis through mitochondrial pathway in a concentration-dependent manner. the apoptosis process can be summarized as follows: the mitochondrial apoptosis-induced channel (mac) of hepg cells was formed by pro-apoptotic protein bax after the treatment of compound a. the formation of mac led to the releasing of cytochrome c from mitochondria. once cytochrome c was released, it binded with apoptotic protease activating factor- (apaf- ) and atp, which then binded to procaspase- to create a protein complex known as apoptosome. the apoptosome cleaved the pro-caspase- to its active form of initiator caspase- , which in turn activated procaspase- and then the effector caspase- and finally resulted in cell apoptosis. to further examine how compound a suppressed the growth of hepg cells, the effect of compound a on cell cycle distribution with different concentrations was investigated by flow cytometric analysis following staining the dna with propidium iodide (pi). the results of a typical experiment are shown in figure a . as determined by flow cytometry, the exposure of hepg cells to compound a for h resulted in an obvious increase in the percentage of cells in g /g phase in comparison with the control. treatment with compound a resulted in an increase of g phase cells ( . %) compared to the control ( . %). inversely, s phase cell population decreased to . % compared to the control ( . %). therefore, compound a resulted in a significant g /g phase arrest in a concentration-dependent manner with a concomitant decrease in the number of cells in the s phase of the cycle. proteins regulates the mitochondrial pathway of apoptosis. these bcl- family proteins stimulate the permeabilization of the mitochondrial outer membrane, which results in the release of cytochrome c into the cytosol and in turn promotes the activation of the caspase cascade. the activation of the caspase cascade ultimately leads to the induction of apoptotic cell death. as shown in figure c and d, in comparison with the control cells, compound a induced an increase in the levels of bax and a decrease in the expression of bcl- in a concentration-dependent manner. meanwhile, the release of cytochrome c from mitochondria increased after the treatment of compound a, while procaspase and procaspase decreased after treatment with a, indicating that the caspase and caspase were activated. as shown in figure e ,f, the increased expression of cleaved caspase- after treatment with a provided a further evidence that compound a induced cell apoptosis through mitochondrial pathway in a concentration-dependent manner. the apoptosis process can be summarized as follows: the mitochondrial apoptosis-induced channel (mac) of hepg cells was formed by pro-apoptotic protein bax after the treatment of compound a. the formation of mac led to the releasing of cytochrome c from mitochondria. once cytochrome c was released, it binded with apoptotic protease activating factor- (apaf- ) and atp, which then binded to procaspase- to create a protein complex known as apoptosome. the apoptosome cleaved the pro-caspase- to its active form of initiator caspase- , which in turn activated procaspase- and then the effector caspase- and finally resulted in cell apoptosis. to further examine how compound a suppressed the growth of hepg cells, the effect of compound a on cell cycle distribution with different concentrations was investigated by flow cytometric analysis following staining the dna with propidium iodide (pi). the results of a typical experiment are shown in figure a . as determined by flow cytometry, the exposure of hepg cells to compound a for h resulted in an obvious increase in the percentage of cells in g /g phase in comparison with the control. treatment with compound a resulted in an increase of g phase cells ( . %) compared to the control ( . %). inversely, s phase cell population decreased to . % compared to the control ( . %). therefore, compound a resulted in a significant g /g phase arrest in a concentration-dependent manner with a concomitant decrease in the number of cells in the s phase of the cycle. table s ). to evaluate the effect of compound a on cancer migration, a wound healing assay was conducted to determine whether compound a could prevent hepg cell migration. after culturing hepg cells for h in the presence and absence of compound a at , . , . , and µm, a pipette tip was streaked through the cell culture, resulting in a cell deficient space. the territory recovered by the hepg cells was used to observe the migration inhibition capability of compound a. the empty space reduced significantly in size in the absence of compound a because of cell proliferation and migration and results indicate compound a suppressed the mobility of tumor cells in a concentration-dependent manner ( figure ). cell cycle distribution after treatment of compound a. data are expressed as means ± sd of the percentages of apoptotic cells from three independent experiments. statistical significance is determined by two-tailed student t-test: "***" denote p < . , "*" denote p < . and ns means no significance, respectively (supplementary table s ). to evaluate the effect of compound a on cancer migration, a wound healing assay was conducted to determine whether compound a could prevent hepg cell migration. after culturing hepg cells for h in the presence and absence of compound a at , . , . , and µm, a pipette tip was streaked through the cell culture, resulting in a cell deficient space. the territory recovered by the hepg cells was used to observe the migration inhibition capability of compound a. the empty space reduced significantly in size in the absence of compound a because of cell proliferation and migration and results indicate compound a suppressed the mobility of tumor cells in a concentration-dependent manner ( figure ). table s ). the starting materials and reagents, purchased from commercial suppliers, were used without further purification. emodin extracted from polygonum cuspidatum was purchased from china xi'an sino-herb bio-technology co., ltd. (xi'an, china). all reactions were monitored by thin-layer chromatography (tlc) on aluminum sheets (silica gel -f , e. merck, darmstadt, germany). compounds were visualized by uv light. column chromatography was carried out using silica gel ( - mesh). all reaction solvents were dried prior to use according to standard procedures. all primary reagents were commercially available. silica gel chromatography solvents were of analytical grade. nmr spectra were recorded in dmso-d on a bruker- spectrometer (bruker biospin, fällanden, switzerlahd), at mhz for h-nmr, mhz for c-nmr and mhz for f-nmr with tms as the internal standard. chemical shifts were expressed in δ (ppm) and coupling constants (j) in hz. multiplicity was indicated as follows: s (singlet), d (doublet), t (triplet), p (quintet), dd (doublet of doublets), brs (broad singlet), etc. mass spectra were obtained on an agilent series lc/msd trap mass spectrometer (esi-ms, agilent, santa clara, ca, usa). table s ). the starting materials and reagents, purchased from commercial suppliers, were used without further purification. emodin extracted from polygonum cuspidatum was purchased from china xi'an sino-herb bio-technology co., ltd. (xi'an, china). all reactions were monitored by thin-layer chromatography (tlc) on aluminum sheets (silica gel -f , e. merck, darmstadt, germany). compounds were visualized by uv light. column chromatography was carried out using silica gel ( - mesh). all reaction solvents were dried prior to use according to standard procedures. all primary reagents were commercially available. silica gel chromatography solvents were of analytical grade. nmr spectra were recorded in dmso-d on a bruker- spectrometer (bruker biospin, fällanden, switzerlahd), at mhz for h-nmr, mhz for c-nmr and mhz for f-nmr with tms as the internal standard. chemical shifts were expressed in δ (ppm) and coupling constants (j) in hz. multiplicity was indicated as follows: s (singlet), d (doublet), t (triplet), p (quintet), dd (doublet of doublets), brs (broad singlet), etc. mass spectra were obtained on an agilent series lc/msd trap mass spectrometer (esi-ms, agilent, santa clara, ca, usa). , -dihydroxy- -( -hydroxyethoxy)- -methylanthracene- , -dione ( ) to a mixture of emodin ( . g, . mmol) in dry dmf ( ml) were added cs co ( . g, . mmol) and -iodoethanol ( . g, mmol) at room temperature. after stirring for h at • c, the resulting mixture was evaporated under reduced pressure and then mixed with water ( ml). the ph value of aqueous phase was adjusted to around with % hydrochloric acid solution. the yellow precipitate was collected and washed with water to give the crude product, which was in further purification by triturating twice with ethyl acetate ( ml) and filtered to afford compound ( . g, %) as a brown solid; general procedure a for preparation of compounds a- z, a- b, and a- l to a mixture of compound ( . mmol) in dry dichloromethane ( ml) were added various n-boc amino acids ( . mmol), dicyclohexyl carbodiimide (dcc) ( . mmol) and -(n,n-dimethlyamino) pyridine (dmap) ( . mmol) at • c. after stirring about min to h at • c, tlc analysis showed the complete consumption of compound , and then the resulting mixture was added dropwise tfa ( ml) at the same temperature and kept stirring for anther about h. the insoluble side product was filtered out and the filtrate was evaporated to give the residue, which was purified by reverse phase flash chromatography with the following conditions: column: spherical c , - µm, g; mobile phase a: water (plus mm tfa); mobile phase b: acn; flow rate: ml/min; gradient: % b gradient in min, % b- % b gradient in min; detector: nm. the fractions containing the desired product were collected at around % b and concentrated under reduced pressure to afford compounds a- z in % to % yields. -( -( , -dihydroxy- -methyl- , -dioxo- , -dihydroanthracen- -yloxy)ethoxy)- -oxoethanaminium , , -trifluoroacetate ( a). according to the general procedure a, compound was treated with n-boc-glycine and then purified by reverse phase flash chromatography to give compound a: yellow solid; yield, %; (s)- -( -( , -dihydroxy- -methyl- , -dioxo- , -dihydroanthracen- -yloxy)ethoxy)- -oxopropan- -aminium , , -trifluoroacetate ( c). according to the general procedure a, compound was treated with n-boc-l-alanine and then purified by reverse phase flash chromatography to give compound c: yellow solid; yield, %; h-nmr (s)- -( -( , -dihydroxy- -methyl- , -dioxo- , -dihydroanthracen- -yloxy)ethoxy)- -hydroxy- -oxopropan- -aminium , , -trifluoroacetate ( d). according to the general procedure a, compound was treated with n-boc-o-tert-butyl-l-serine and then purified by reverse phase flash chromatography to give compound d: yellow solid; yield, %; (r)- -( -( , -dihydroxy- -methyl- , -dioxo- , -dihydroanthracen- -yloxy)ethoxy)- -methyl- -oxobutan- -aminium , , -trifluoroacetate ( e). according to the general procedure a, compound was treated with n-boc-d-valine and then purified by reverse phase flash chromatography to give compound e: yellow solid; yield, %; . , . , . , . , . , . , . , . , . , . , . , . , . , . , . , . , . , . . , . , . , . , . , . , . , . , . , . , . , . -( -( , -dihydroxy- -methyl- , -dioxo- , -dihydroanthracen- -yloxy)ethoxy)- -methyl- -oxopropan- aminium , , -trifluoroacetate ( j). according to the general procedure a, compound was treated with n-boc-aib-oh and then purified by reverse phase flash chromatography to give compound j: yellow solid; yield, %; h-nmr δ . (br, h), . (br, h), . (s, h), . - . (m, h), -(( -( , -dihydroxy- -methyl- , -dioxo- , -dihydroanthracen- -yloxy)ethoxy)carbonyl)cyclopropan-aminium , , -trifluoroacetate ( k). according to the general procedure a, compound was treated with -(boc-amino)cyclopropanecarboxylic acid and then purified by reverse phase flash chromatography to give compound k: yellow solid; yield, %; h-nmr δ . (brs, h), . , . , . , . , . , . , . , . , . , . , . , . , . , . , . , . , . -(( -( , -dihydroxy- -methyl- , -dioxo- , -dihydroanthracen- -yloxy)ethoxy)carbonyl)cyclobutan-aminium , , -trifluoroacetate ( l). according to the general procedure a, compound was treated with -(boc-amino)cyclobutanecarboxylic acid and then purified by reverse phase flash chromatography to give compound l: yellow solid; yield, %; h-nmr δ . (brs, h), -(( -( , -dihydroxy- -methyl- , -dioxo- , -dihydroanthracen- -yloxy)ethoxy)carbonyl)oxetan- -aminium , , -trifluoroacetate ( m). according to the general procedure a, compound was treated with -boc-amino- -oxetanecarboxylic acid and then purified by reverse phase flash chromatography to give compound m: yellow solid; yield, %; h-nmr δ . (brs, h), -(( -( , -dihydroxy- -methyl- , -dioxo- , -dihydroanthracen- -yloxy)ethoxy)carbonyl)cyclopentan-aminium , , -trifluoroacetate ( n). according to the general procedure a, compound was treated with n-boc-aminocyclopentanecarboxylic acid and then purified by reverse phase flash chromatography to give compound n: yellow solid; yield, %; h-nmr δ . (brs, h), (r)- -(( -( , -dihydroxy- -methyl- , -dioxo- , -dihydroanthracen- -yloxy)ethoxy)carbonyl)-pyrrolidinium , , -trifluoroacetate ( o). according to the general procedure a, compound was treated with n-boc-d-proline and then purified by reverse phase flash chromatography to give compound o: yellow solid; yield, %; h-nmr δ . (d, j = . (s)- -(( -( , -dihydroxy- -methyl- , -dioxo- , -dihydroanthracen- -yloxy)ethoxy)carbonyl)-pyrrolidinium , , -trifluoroacetate ( p). according to the general procedure a, compound was treated with n-boc-l-proline and then purified by reverse phase flash chromatography to give compound p: (r)- -( -( , -dihydroxy- -methyl- , -dioxo- , -dihydroanthracen- -yloxy)ethoxy)- -oxo- -phenylethanaminium , , -trifluoroacetate ( q). according to the general procedure a, compound was treated with n-boc-d-phenylglycine and then purified by reverse phase flash chromatography to give compound q: yellow solid; yield, %; h-nmr δ . (brs, h), (r)- -( -( , -dihydroxy- -methyl- , -dioxo- , -dihydroanthracen- -yloxy)ethoxy)- -( -hydroxyphenyl)- -oxoethanaminium , , -trifluoroacetate ( s). according to the general procedure a, compound was treated with n-boc-d- -hydroxyphenylglycine and then purified by reverse phase flash chromatography to give compound s: yellow solid; yield, %; h-nmr δ . (brs, h), . (s)- -( -( , -dihydroxy- -methyl- , -dioxo- , -dihydroanthracen- -yloxy)ethoxy)- -( -hydroxyphenyl)- -oxoethanaminium , , -trifluoroacetate ( t). according to the general procedure a, compound was treated with n-boc-l- -hydroxyphenylglycine and then purified by reverse phase flash chromatography to give compound t: yellow solid; yield, %; h-nmr δ . (brs, h), . (r)- -( -( , -dihydroxy- -methyl- , -dioxo- , -dihydroanthracen- -yloxy)ethoxy)- -( -fluorophenyl)- -oxoethanaminium , , -trifluoroacetate ( u). according to the general procedure a, compound was treated with (r)-n-boc- -fluorophenylglycine and then purified by reverse phase flash chromatography to give compound u: yellow solid; yield, %; h-nmr δ . (brs, h), (s)- -( -( , -dihydroxy- -methyl- , -dioxo- , -dihydroanthracen- -yloxy)ethoxy)- -( -fluorophenyl)- oxoethanaminium , , -trifluoroacetate ( v). according to the general procedure a, compound was treated with (s)-n-boc- -fluorophenylglycine and then purified by reverse phase flash chromatography to give compound v: yellow solid; yield, %; h-nmr ( mhz, dmso-d ) δ . (brs, h), (r)- -( -( , -dihydroxy- -methyl- , -dioxo- , -dihydroanthracen- -yloxy)ethoxy)- -oxo- -phenyl-propan- -aminium , , -trifluoroacetate ( w). according to the general procedure a, compound was treated with n-boc-d-phenylalanine and then purified by reverse phase flash chromatography to give compound w: yellow solid; yield, %; h-nmr δ . brs, h), (s)- -( -( , -dihydroxy- -methyl- , -dioxo- , -dihydroanthracen- -yloxy)ethoxy)- -oxo- -phenyl-propan- -aminium , , -trifluoroacetate ( x). according to the general procedure a, compound was treated with n-boc-l-phenylalanine and then purified by reverse phase flash chromatography to give compound x: yellow solid; yield, %; h-nmr δ . (brs, h), (r)- -( -( , -dihydroxy- -methyl- , -dioxo- , -dihydroanthracen- -yloxy)ethoxy)- -( -hydroxyphenyl)- -oxopropan- -aminium , , -trifluoroacetate ( y). according to the general procedure a, compound was treated with n-boc-o-tert-butyl-l-tyrosine and then purified by reverse phase flash chromatography to give compound y: yellow solid; yield, %; h-nmr δ . (s, h), . (d, j = . (s)- -( -( , -dihydroxy- -methyl- , -dioxo- , -dihydroanthracen- -yloxy)ethoxy)- -( -hydroxyphenyl)- -oxopropan- -aminium , , -trifluoroacetate ( z). according to the general procedure a, compound was treated with n-boc-l-tyrosine and then purified by reverse phase flash chromatography to give compound z: yellow solid; yield, %; h-nmr δ . (s, h), . (d, j = . (r)- -( -( , -dihydroxy- -methyl- , -dioxo- , -dihydroanthracen- -yloxy)ethoxy)-n-methyl- -oxo-propan- -aminium , , -trifluoroacetate ( a). according to the general procedure a, compound was treated with boc-n-methyl-d-alanine and then purified by reverse phase flash chromatography to give compound a: yellow solid; yield, %; h-nmr δ . (brs, h), . (s)- -( -( , -dihydroxy- -methyl- , -dioxo- , -dihydroanthracen- -yloxy)ethoxy)-n-methyl- -oxo-propan- -aminium , , -trifluoroacetate ( b). according to the general procedure a, compound was treated with boc-n-methyl-l-alanine and then purified by reverse phase flash chromatography to give compound b: yellow solid; yield, %; h-nmr δ . (brs, h), . (brs, h), . general procedure b for preparation of compounds a and b to a solution of compound a or b ( mg, . mmol) and paraformaldehyde ( % aqueous solution, mg, . mmol) in meoh ( ml) was added nabh cn ( mg, . mmol) at • c. after stirring h at room temperature, the reaction was quenched by tfa ( . ml). the resulting reaction solution was used directly in purification by reverse phase flash chromatography with the following conditions: column: spherical c , - µm, g; mobile phase a: water (plus mm tfa); mobile phase b: acn; flow rate: ml/min; gradient: % b gradient in min, % b- % b gradient in min; detector: nm. the fractions containing the desired product were collected at around % b and concentrated under reduced pressure to afford compounds a or b in % or % yield, respectively. (r)- -( -( , -dihydroxy- -methyl- , -dioxo- , -dihydroanthracen- -yloxy)ethoxy)-n,n-dimethyl- -oxopropan- -aminium , , -trifluoroacetate ( a). yellow solid; yield, %; general procedure c for preparation of compounds a- f to a mixture of emodin ( mmol) in dry dmf ( ml) were added cs co ( mmol) and hydroxybromides or iodides ( mmol) at room temperature. after stirring for h at • c, the resulting mixture was evaporated under reduced pressure and then mixed with water ( ml). the ph value of aqueous phase was adjusted to around with % hydrochloric acid solution, extracted with dichloromethane ( × ml). the combined organic layer was washed with brine ( ml), dried over anhydrous sodium sulfate and evaporated to dryness. the crude product was purified by silica gel column chromatography with %- % ethyl acetate in petroleum to afford compounds a- f. total cell lysates from cultured hepg cells treated with different concentrations of compound a for h were obtained by lysing the cells in ice-cold ripa buffer ( pbs, % np- , . % sodium deoxycholate and . % sds) containing mg/ml pmsf, mg/ml aprotinin, mg/ml leupeptin, mg/ml pepstatin and mg/ml naf. after centrifugation at , rpm for min, the protein in the supernatant was quantified by the bradford method (bio-rad, hercules, ca, usa) using a multimode varioscan instrument (thermo fischer scientific, waltham, ma, usa). twenty micrograms of protein per lane was applied in % sds polyacrylamide gel. after electrophoresis, the protein was transferred to a polyvinylidine difluoride membrane (amersham biosciences, marlborough, ma, usa). the membrane was blocked at room temperature for h in tbst containing % blocking powder (santa cruz, dallas, tx, usa). the membrane was washed with tbst for min, and the primary antibody was added and incubated at • c overnight (o/n). bax ( , cst, danvers, ma, usa), bcl- ( , cst), cytochrome c ( , cst), procaspase- (ab , abcam, cambridge, ma, usa), procaspase- (ab , abcam), caspase- ( - -lg, proteintech group, rosemont, il, usa) and gapdh (ab , abcam) antibodies were employed. after three tbst washes, the membrane was incubated with the corresponding horseradish peroxidase-labeled secondary antibody ( : ) (santa cruz) at room temperature for h. membranes were washed with tbst for min five times and the protein blots were visualized with chemiluminescence reagent (thermo fischer scientific ltd.). the x-ray films were developed with a developer and fixed with fixer solution. the grey levels were analyzed using imagequant las system (ge, marlborough, ma, usa). the hepg cells were treated with indicated concentrations of compound a. after incubation for h, cells were washed twice with ice-cold pbs, fixed and permeabilized with ice cold % ethanol at − • c overnight. the cells were treated with µg/ml rnase a at • c for min, then washed with ice-cold pbs and finally stained with mg/ml pi in the dark at • c for min. the cellular dna content for the cell cycle distribution analysis was performed with the system software (cellquest; bd biosciences), plotting at least , events per sample. the percentage of cells in the g , s and g phases of the cell cycle were determined using the modfit lt version . software package (verity software, topsham, me, usa). hepg cells were grown in dmem medium containing growth factors at a cell density of × cells/ml for h. a disposable ml plastic pipette tip was used to scratch the monolayer of cells in a streaking motion. compounds were added to the streaked cell culture at the indicated concentrations. the streaked cells were then cultured in serum-free medium for an additional h and photographed. to quantify the experimental results, the % cell inhibitory rate was calculated by the equation: cell inhibitory rate (%) = ( − d drug /d control ) × %, where d drug is the mean distance of cell migration in drug group and d control is the mean distance of cell migration in control group. pictures of the initial wounded monolayers were compared with the corresponding pictures of cells at the end of the incubation, and data were presented as mean ± sd from three independent experiments. in conclusion, a novel series of emodin derivatives via the introduction of an amino acid were designed and synthesized. their in vitro anti-proliferation tests revealed that these derivatives exhibited moderate to potent anti-proliferative activity against hepg cells and mcf- cells. among these compounds, the most potent compound, a, exhibited better selective anti-proliferative activity and specificity than emodin, displayed a significant effect in inducing cell cycle arrest at g /g phase and inducing cell apoptosis in hepg cells via release of cytochrome c and subsequent activation of caspase- and caspase- , which revealed that the possible molecular mechanism of a apoptosis induction may mainly through the mitochondrial death pathway. moreover, compound a also resulted in inhibition of hepg cells migration in the wound healing assay. these preliminary molecular mechanism results suggest that compound a could be a promising lead compound for the development of novel antitumor drugs and has the potential for further investigations as an anti-cancer drug. supplementary materials: the following are available online at http://www.mdpi.com/ - / / / /s , tables s -s : biochemistry analytical data from three independent experiments; figures s -s : h-nmr, c-nmr and f-nmr spectra of these compounds. according to the general procedure c, emodin was treated with -iodopropan- -ol and then purified by silica gel column chromatography to give compound a: brown solid; yield, %; h-nmr δ . (s, h), . (s, h), . (s, h according to the general procedure c, emodin was treated with -bromobutan- -ol and then purified by silica gel column chromatography to give compound b: brown solid; yield, %; h-nmr δ . (s, h), . (s, h), . (s, h) according to the general procedure c, emodin was treated with -bromopentan- -ol and then purified by silica gel column chromatography to give compound c: brown solid; yield, %; h-nmr δ . (s, h), . (s, h) according to the general procedure c, emodin was treated with -( -bromoethoxy)ethanol and then purified by silica gel column chromatography to give compound d: brown solid; yield, %; h-nmr δ . (s, h), . (s, h), . (d, j = -bromoethoxy)ethoxy)ethanol and then purified by silica gel column chromatography to give compound e: brown solid; yield, %; h-nmr δ . (s, h) -bromoethoxy)ethoxy)ethoxy) ethanol and then purified by silica gel column chromatography to give compound f: brown solid h-nmr δ . (s, h), . (s, h), . (d, j = . hz, h), . (t, j = . hz, h), . (d, j = . hz, h) according to the general procedure a, compound a was treated with n-boc-d-alanine and then purified by reverse phase flash chromatography to give compound a: yellow solid; yield, %; h-nmr δ . (d, j = according to the general procedure a, compound a was treated with n-boc-l-alanine and then purified by reverse phase flash chromatography to give compound b: yellow solid according to the general procedure a, compound b was treated with n-boc-d-alanine and then purified by reverse phase flash chromatography to give compound c: yellow solid according to the general procedure a, compound b was treated with n-boc-l-alanine and then purified by reverse phase flash chromatography to give compound d: yellow solid hz, h), . (s, h), . - . (m, h), . (d, j = . hz, h) according to the general procedure a, compound c was treated with n-boc-d-alanine and then purified by reverse phase flash chromatography to give compound e: yellow solid; yield, %; h-nmr δ . (brs, h), . (brs, h), . (d, j = according to the general procedure a, compound c was treated with n-boc-l-alanine and then purified by reverse phase flash chromatography to give compound f: yellow solid (s, h), . - . (m, h), . - . (m, h), . - . (m, h), . (d, j = . hz, h) according to the general procedure a, compound d was treated with n-boc-d-alanine and then purified by reverse phase flash chromatography to give compound g: yellow solid; yield, %; h-nmr δ according to the general procedure a, compound d was treated with n-boc-l-alanine and then purified by reverse phase flash chromatography to give compound h: yellow solid according to the general procedure a, compound e was treated with n-boc-d-alanine and then purified by reverse phase flash chromatography to give compound i: yellow solid; yield, %; h-nmr δ according to the general procedure a, compound e was treated with n-boc-l-alanine and then purified by reverse phase flash chromatography to give compound j: yellow solid; yield, %; h-nmr δ according to the general procedure a, compound f was treated with n-boc-d-alanine and then purified by reverse phase flash chromatography to give compound k: yellow solid; yield, %; h-nmr δ . (brs, h), . (d, j = . hz, h) according to the general procedure a, compound f was treated with n-boc-l-alanine and then purified by reverse phase flash chromatography to give compound l: yellow solid; yield, %; h-nmr δ ) containing % fetal bovine serum (fbs) and % penicillin ( units/ml)-streptomycin ( µg/ml) in a humidified incubator at • c under % co and % relative humidity (rh) atmosphere. the cells were harvested for metabolomics experiments using cell scrapers cell viability assay hepg cells and mcf- cells in µl dmem supplemented with % fbs were seeded at a density of cells/well in -well cell culture plate ( , corning), respectively. after h, the medium was substituted with fresh medium containing various sample solutions in dmso usa) was added to each well containing cells and the contents were mixed for min on an orbital shaker to induce cell lysis. the plates were then incubated at room temperature for min to stabilize the luminescent signal. the luminescence was recorded on an envision cells were seeded at × /well in % fbs-dmem into -well plates and treated with compounds a for h. the cells were washed twice with cold phosphate buffered saline (pbs) and then analysis is as follows: lower left quadrant, viable cells (annexin v−/pi−); lower right quadrant, early apoptotic cells (annexin v+/pi−); upper right quadrant, late apoptotic cells (annexin v+/pi+); upper left quadrant, necrotic cells a new diphenyl ether from marine-derived fungus aspergillus sp b-f- a: a new antimicrobial anthraquinone from a sea urchin-derived fungus monodictys sp a new ergosterol analog, a new bis-anthraquinone and anti-obesity activity of anthraquinones from the marine sponge-associated fungus talaromyces stipitatus kufa . mar. drugs bis-indolyl benzenoids, hydroxypyrrolidine derivatives and other constituents from cultures of the marine sponge-associated fungus aspergillus candidus kufa emodin and aloe-emodin suppress breast cancer cell proliferation through er alpha inhibition. evid.-based complement emodin suppresses wnt signaling in human colorectal cancer cells sw and sw emodin suppresses hyperglycemia-induced proliferation and fibronectin expression in mesangial cells via inhibiting cflip emodin suppresses inflammatory responses and joint destruction in collagen-induced arthritic mice emodin blocks the sars coronavirus spike protein and angiotensin-converting enzyme interaction effect of emodin on the cariogenic properties of streptococcus mutans and the development of caries in rats emodin, a naturally occurring anthraquinone derivative, suppresses ige-mediated anaphylactic reaction and mast cell activation ct imaging biomarker for evaluation of emodin as a potential drug on lps-mediated osteoporosis mice formula optimization of the jiashitang scar removal ointment and antiinflammatory compounds screening by nf-kappa b bioactivity-guided dual-luciferase reporter assay system emodin protects against diabetic cardiomyopathy by regulating the akt/gsk- beta signaling pathway in the rat model emodin prolongs recipient survival time after orthotopic liver transplantation in rats by polarizing the th /th paradigm to th emodin induces neurite outgrowth through pi k/akt/gsk- beta-mediated signaling pathways in neuro a cells emodin ameliorates ethanol-induced fatty liver injury in mice emodin protects against concanavalin a-induced hepatitis in mice through inhibiting activation of the p mapk-nf-kappa b signaling pathway emodin suppresses cell proliferation and fibronectin expression via p mapk pathway in rat mesangial cells cultured under high glucose emodin accelerates osteoblast differentiation through phosphatidylinositol -kinase activation and bone morphogenetic protein- gene expression emodin-induced apoptosis through p -dependent pathway in human hepatoma cells emodin azide methyl anthraquinone derivative triggers mitochondrial-dependent cell apoptosis involving in caspase- -mediated bid cleavage aloe-emodin induces apoptosis of human nasopharyngeal carcinoma cells via caspase- -mediated activation of the mitochondrial death pathway emodin inhibits tnf-alpha-induced human aortic smooth-muscle cell proliferation via caspase-and mitochondrial-dependent apoptosis emodin induces apoptosis in human lung adenocarcinoma cells through a reactive oxygen species-dependent mitochondrial signaling pathway synthesis and antitumor activity of emodin quaternary ammonium salt derivatives synthesis and biological activity, evaluation of emodin quaternary ammonium salt derivatives as potential anticancer agents synthesis, sar and pharmacological characterization of novel anthraquinone cation compounds as potential anticancer agents antitumor effects and mechanism of novel emodin rhamnoside derivatives against human cancer cells in vitro synthesis, dna binding and cytotoxicity of new pyrazole emodin derivatives synthesis and cytotoxic activities of beta-carboline amino acid ester conjugates preparation of amino acid conjugates of betulinic acid with activity against human melanoma this article is an open access article distributed under the terms and conditions of the creative commons attribution (cc by) license the authors declare no conflict of interest. key: cord- -fewy y a authors: wang, ming-yang; liang, jing-wei; mohamed olounfeh, kamara; sun, qi; zhao, nan; meng, fan-hao title: a comprehensive in silico method to study the qstr of the aconitine alkaloids for designing novel drugs date: - - journal: molecules doi: . /molecules sha: doc_id: cord_uid: fewy y a a combined in silico method was developed to predict potential protein targets that are involved in cardiotoxicity induced by aconitine alkaloids and to study the quantitative structure–toxicity relationship (qstr) of these compounds. for the prediction research, a protein-protein interaction (ppi) network was built from the extraction of useful information about protein interactions connected with aconitine cardiotoxicity, based on nearly a decade of literature and the string database. the software cytoscape and the pharmmapper server were utilized to screen for essential proteins in the constructed network. the calcium-calmodulin-dependent protein kinase ii alpha (camk a) and gamma (camk g) were identified as potential targets. to obtain a deeper insight on the relationship between the toxicity and the structure of aconitine alkaloids, the present study utilized qsar models built in sybyl software that possess internal robustness and external high predictions. the molecular dynamics simulation carried out here have demonstrated that aconitine alkaloids possess binding stability for the receptor camk g. in conclusion, this comprehensive method will serve as a tool for following a structural modification of the aconitine alkaloids and lead to a better insight into the cardiotoxicity induced by the compounds that have similar structures to its derivatives. the rhizomes and roots of aconitine species, a genus of the family ranunculaceae, are commonly used in treatment for various illnesses such as collapse, syncope, rheumatic fever, joints pain, gastroenteritis, diarrhea, edema, bronchial asthma, and tumors. they are also involved in the management of endocrinal disorders such as irregular menstruation [ , ] . however, the usefulness of this aconitine species component intermingles with toxicity after it is administered to a diseased patient. so far, few articles have recorded the misuse of aconitine medicinals with strong emphasis and thus have referenced that the misuse of this medicinal can result in severe cardio-and neurotoxicity [ ] [ ] [ ] [ ] [ ] . in our past research, it was evidenced that the aconitine component is the main active ingredient in this species' root and rhizome, and is responsible for both therapeutic and toxic effects [ ] . this medicinal has been tested for cancerological and dermatological activities. its application to disease conditions proved to exhibit an activity that slowed down cancer tumor growth and to cure serious cases of dermatosis. it was also found to have an effect on postoperative analgesia [ ] [ ] [ ] [ ] . however, a previous safety study has revealed that aconitine toxicity is responsible for its restriction in clinical settings. further studies are needed to explain the cause of aconitine toxicity as well as to show whether the toxicity supersedes its usefulness. a combined network analysis and in silico study was once performed to obtain insight on the relationship between aconitine alkaloid toxicity and the aconitine structure, and it was found that the cardiotoxicity of aconitine is the primary cause of patient death. the aconitine poison is similar to the poison created by some pivotal proteins such as the ryanodine receptor (ryr and ryr ), the gap junction α- protein (gja ), and the sodium-calcium exchanger (slc a ) [ ] [ ] [ ] [ ] . however, among all existing studies about the aconitine medicinal, no one has reported detail of its specific binding target protein linked to toxicity. protein-protein interactions (ppis) participate in many metabolic processes occurring in living organisms such as the cellular communication, immunological response, and gene expression control [ , ] . a systematic description of these interactions aids in the elucidation of interrelationships among targets. the targeting of ppis with small-molecule compounds is becoming an essential step in a mechanism study [ ] . the present study was designed and undertaken to identify the critical protein that can affect the cardiotoxicity of aconitine alkaloids. a ppi network built by the string database is a physiological contact for the high specificity that has been established for several protein molecules and has stemmed from computational prediction, knowledge transfer between organisms, and interactions aggregated from other databases [ ] . the analysis of the ppi network is based on nodes and edges and is always performed via cluster analysis and centrality measurements [ , ] . in cluster analysis, highly interconnected nodes and protein target nodes are divided and used to form sub-graphs. the reliability of the ppi network is identified by the content of each sub-graph [ ] . the variability in centrality measurements is connected to the quantitative relationship between the protein targets and its weightiness in the network [ ] . hence, ppi networks with protein targets related to aconitine alkaloid cardiotoxicity must enable us to find the most relevant protein for aconitine toxicity and to understand the mechanism at the network level. in our research, the evaluation and visualization analysis of essential proteins related to cardiotoxicity in ppis were performed by the clusterone and cytonca plugins in cytoscape . , designed to find the potential protein targets via combination with conventional integrated pharmacophore matching technology built in the pharmmapper platform. structural modification of a familiar natural product, active compound, or clinical drug is an efficient method for designing a novel drug. the main purpose of the structural modification is to reduce the toxicity of the target compound while enhancing the utility of the drug [ ] . the identification of the structure-function relationship is an essential step in the drug discovery and design, the determination of the d protein structures was the key step in identifying the internal interactions in the ligand-receptor complexes. x-ray crystallography and nmr were the only accepted techniques of determining the d protein structure. although the d structure obtained by these two powerful techniques are accurate and reliable, they are time-consuming and costly [ ] [ ] [ ] [ ] [ ] . with the rapid development of structural bioinformatics and computer-aided drug design (cadd) techniques in the last decade, computational structures are becoming increasingly reliable. the application of structural bioinformatics and cadd techniques can improve the efficiency of this process [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] . the ligand-based quantitative structure-toxicity relationship (qstr) and receptor-based docking technology are regarded as effective and useful tools in analysis of structure-function relationships [ ] [ ] [ ] [ ] . the contour maps around aconitine alkaloids generated by comparative molecular field analysis (comfa) and comparative molecular similarity index analysis (comsia) were combined with the interactions between ligand substituents and amino acids obtained from docking results to gain insight on the relationship between the structure of aconitine alkaloids and their toxicity. scores from functions were used to evaluate the docking result. the value-of-fit score in moe software reflects the binding stability and affinity of the ligand-receptor complexes. when screening for the most potential target for cardiotoxicity, the experimental data was combined with the value-of-fit score by the ndcg (normalized discounted cumulative gain). the possibility of a protein being a target of cardiotoxicity corresponds with the consistency of this experimental data. since the pioneering paper entitled "the biological functions of low-frequency phonons" [ ] was published in , many investigations of biomacromolecules from a dynamic point of view have occurred. these studies have suggested that low-frequency (or terahertz frequency) collective motions do exist in proteins and dna [ ] [ ] [ ] [ ] [ ] . furthermore, many important biological functions in proteins and dna and their dynamic mechanisms, such as cooperative effects [ ] , the intercalation of drugs into dna [ ] , and the assembly of microtubules [ ] , have been revealed by studying the low-frequency internal motions, as summarized in a comprehensive review [ ] . some scientists have even applied this kind of low-frequency internal motion to medical treatments [ , ] . investigation of the internal motion in biomacromolecules and its biological functions is deemed as a "genuinely new frontier in biological physics," as announced in the mission of some biotech companies (see, e.g., vermont photonics). in order to consider the static structural information of the ligand-receptor complex, dynamical information should be also considered in the process of drug discovery [ , ] . finally, molecular dynamics was carried out to verify the binding affinity and stability of aconitine alkaloids and the most potential target. this present study may be instrumental in our future studies for the synergism and attenuation of aconitine alkaloids and for the exploitation of its clinical application potential. a flowchart of procedures in our study is shown in figure . of-fit score by the ndcg (normalized discounted cumulative gain). the possibility of a protein being a target of cardiotoxicity corresponds with the consistency of this experimental data. since the pioneering paper entitled "the biological functions of low-frequency phonons" [ ] was published in , many investigations of biomacromolecules from a dynamic point of view have occurred. these studies have suggested that low-frequency (or terahertz frequency) collective motions do exist in proteins and dna [ ] [ ] [ ] [ ] [ ] . furthermore, many important biological functions in proteins and dna and their dynamic mechanisms, such as cooperative effects [ ] , the intercalation of drugs into dna [ ] , and the assembly of microtubules [ ] , have been revealed by studying the low-frequency internal motions, as summarized in a comprehensive review [ ] . some scientists have even applied this kind of low-frequency internal motion to medical treatments [ , ] . investigation of the internal motion in biomacromolecules and its biological functions is deemed as a "genuinely new frontier in biological physics," as announced in the mission of some biotech companies (see, e.g., vermont photonics). in order to consider the static structural information of the ligand-receptor complex, dynamical information should be also considered in the process of drug discovery [ , ] . finally, molecular dynamics was carried out to verify the binding affinity and stability of aconitine alkaloids and the most potential target. this present study may be instrumental in our future studies for the synergism and attenuation of aconitine alkaloids and for the exploitation of its clinical application potential. a flowchart of procedures in our study is shown in figure . the whole framework of the comprehensive in silico method for screening potential targets and studying the quantitative structure-toxicity relationship (qstr). the compounds were aligned over, under the superimposition of the common moiety and template compound . the statistical parameters for database alignment-q , r , f, and see-were the whole framework of the comprehensive in silico method for screening potential targets and studying the quantitative structure-toxicity relationship (qstr). the compounds were aligned over, under the superimposition of the common moiety and template compound . the statistical parameters for database alignment-q , r , f, and see-were table . the comfa model with the optimal number of components presented a q of . , an r of . , an f of . , and an see of . , and contributions of the steric and electrostatic fields were . and . , respectively. the comsia model with the optimal number of components presented a q of . , an r of . , an f of . , and an see of . , and the contributions of steric, electrostatic, hydrophobic, hydrogen bond acceptor, and hydrogen bond donor fields were . , . , . , . , and . , respectively. the statistical results proved that the aconitine alkaloids qstr model of comfa and comsia under the database alignment have adequate predictability. experimental and predicted pld values of both the training set and test set are shown in figure , and the comfa ( figure a ) and comsia ( figure b ) model gave the correlation coefficient (r ) value of . and . , respectively, which demonstrated the internal robustness and external high prediction of the qstr models. experimental and predicted pld values of both the training set and test set are shown in figure residuals vs. leverage williams plots of the aconitine qstr models are shown in figure a ,b. all values of standardized residuals fall between σ and − σ, and the values of leverage are less than h*, so the two models demonstrate potent extensibility and predictability. residuals vs. leverage williams plots of the aconitine qstr models are shown in figure a ,b. all values of standardized residuals fall between σ and − σ, and the values of leverage are less than h*, so the two models demonstrate potent extensibility and predictability. under mesh (medical subject headings), a total of articles ( articles were received from web of science, and others were received from pubmed) were retrieved. after selecting cardiotoxicity-related and excluding repetitive articles, articles were used to extract the correlative proteins and pathways for building a ppi network in the string server. the correlative proteins or pathways are shown in table . all proteins were taken as input protein in the string database to find its direct and functional partners [ ] , and proteins and its partners were then imported into the cytoscape . to generate the ppi network with nodes and edges ( figure ). potassium voltage-gated channel h scn a sodium voltage-gated channel type , scn a sodium voltage-gated channel type scn a sodium voltage-gated channel type scn a sodium voltage-gated channel type scn a sodium voltage-gated channel type kcnj potassium inwardly-rectifying channel j during the case of screening of the essential proteins in ppi network, three centrality measurements (subgraph centrality, betweenness centrality, and closeness centrality) in cytonca were utilized to evaluate the weight of nodes. after removing the central node "ac," the centrality measurements of nodes were calculated by cytonca and documented in table s . the top % of three centrality measurement values of all node are painted with a different color in figure a . to screen the node with the high values of each three centrality measures, nodes with three colors were overlapped and merged into sub-networks in figure b . under mesh (medical subject headings), a total of articles ( articles were received from web of science, and others were received from pubmed) were retrieved. after selecting cardiotoxicity-related and excluding repetitive articles, articles were used to extract the correlative proteins and pathways for building a ppi network in the string server. the correlative proteins or pathways are shown in table . all proteins were taken as input protein in the string database to find its direct and functional partners [ ] , and proteins and its partners were then imported into the cytoscape . to generate the ppi network with nodes and edges ( figure ). table . proteins related to aconitine alkaloids induced cardiotoxicity extracted from articles. classification frequency ryanodine receptor ryr ryanodine receptor gja gap junction α- protein (connexin ) slc a sodium/calcium exchanger atp a calcium transporting atpase fast twitch kcnh potassium voltage-gated channel h scn a sodium voltage-gated channel type , scn a sodium voltage-gated channel type scn a sodium voltage-gated channel type scn a sodium voltage-gated channel type scn a sodium voltage-gated channel type kcnj potassium inwardly-rectifying channel j during the case of screening of the essential proteins in ppi network, three centrality measurements (subgraph centrality, betweenness centrality, and closeness centrality) in cytonca were utilized to evaluate the weight of nodes. after removing the central node "ac," the centrality measurements of nodes were calculated by cytonca and documented in table s . the top % of three centrality measurement values of all node are painted with a different color in figure a . to screen the node with the high values of each three centrality measures, nodes with three colors were overlapped and merged into sub-networks in figure b . in the sub-networks, the voltage-gated calcium and sodium channel accounted for a large proportion, which is consistent with our research in clustering the network (clusters , , and ). all proteins in the sub-networks will be utilized to predict the results of the pharmmapper server to receive the potential target of cardiotoxicity induced by aconitine alkaloids (in figure a ,b). in the meantime, v o (camk g) and vz (camk a) were identified as the potential targets with higher fit scores. in the sub-networks, the voltage-gated calcium and sodium channel accounted for a large proportion, which is consistent with our research in clustering the network (clusters , , and ). all proteins in the sub-networks will be utilized to predict the results of the pharmmapper server to receive the potential target of cardiotoxicity induced by aconitine alkaloids (in figure a ,b). in the meantime, v o (camk g) and vz (camk a) were identified as the potential targets with higher fit scores. all compounds were docked into three potential targets. the values of ndcg are shown in table . the dock study of three proteins with an ndcg of . and . , respectively (the detailed in the sub-networks, the voltage-gated calcium and sodium channel accounted for a large proportion, which is consistent with our research in clustering the network (clusters , , and ). all proteins in the sub-networks will be utilized to predict the results of the pharmmapper server to receive the potential target of cardiotoxicity induced by aconitine alkaloids (in figure a ,b). in the meantime, v o (camk g) and vz (camk a) were identified as the potential targets with higher fit scores. all compounds were docked into three potential targets. the values of ndcg are shown in table . the dock study of three proteins with an ndcg of . and . , respectively (the detailed all compounds were docked into three potential targets. the values of ndcg are shown in table . the dock study of three proteins with an ndcg of . and . , respectively (the detailed docking result is shown in table s ) proves that the result of the dock study of v o is consistent with the experimental pld , so the protein v o was utilized for the ligand interaction analysis. table . ranking results by experimental and predicted pld and fit score. experimental pld fit score ( v o) fit score ( vz ) ndcg . . the d-qstr contour maps were utilized to visualize the information on the comfa and comsia model properties in three-dimensional space. these maps used characteristics of compounds that are crucial for activity and display the regions around molecules where the variance of activities is expected based on physicochemical property changes in molecules [ ] . the analysis of favorable and unfavorable regions of steric, electrostatic, hydrophobic, hbd, and hba atom fields contribute to the realization of the relationship between the aconitine alkaloid's toxic activity and its structure. steric and electrostatic contour maps of the comfa qstr model are shown in figure a ,b, respectively. hydrophobic, hbd, and hba contour maps of the comsia qstr model are shown in figure c -e. compound has the most toxic activity, so it was chosen as the reference structure for the generation of the comfa and comsia contour maps. in the case of the comfa study, the steric contour map around compound is shown in figure a . the yellow regions near r , r , and r showed the substituents of the molecule, which proved that these positions were not ideal for sterically favorable functional groups. therefore, compounds , , and (with pld values of . , . , and . , respectively), which consist of sterically esterified moieties at positions r and r , were less toxic than compounds and (with pld values of . and . ), which were substituted by a small hydroxyl group, and compound (with a pld value of . ) has less toxic activity due to the esterified moiety in r . the green regions, sterically favorable the comfa electrostatic contour map is shown in figure b . the blue region near the r and r substitution revealed that the replacement of electropositive groups is in favor of toxicity. this can be proven by the fact that the compounds with hydroxy in these two positions had higher pld values than the compound with acetoxy or no substituents. the red region surrounding molecular scaffolds was not distinct, which revealed that there was no connection between the electronegative and the toxicity. the comsia hydrophobic contour map is shown in figure c . the r , r , and r around the white region indicated that the hydrophobic groups were unfavorable for the toxicity, so the esterification of hydrophilic hydroxyl or dehydroxylation decreased the toxicity, which is consistent with the steric and electrostatic contour map. the yellow contour map near the r manifested that the hydrophilic hydroxy was unfavorable to the toxicity, which can be validated by the fact that aconitine alkaloids with hydroxy substituents in r (compound , with a pld the ppi network of aconitine alkaloids cardiotoxicity was divided into nine clusters using clusterone. statistical parameters are shown in figure . six clusters, namely clusters , , , , , and , which possess quality scores higher than . , a density higher than . , and a p-value less than . , were selected for further analysis (in figure ) . clusters , , and consisted of proteins mainly involved in the effects of various calcium, potassium, and sodium channels. cluster mainly the comsia contour map of hbd is shown in figure d . the cyan regions at r , r , and r represented a favorable condition for the hbd atom, which clearly validated the fact that the compounds with hydroxy in this region show potent toxicity. a purple region was found near r , which proved that the hbd atom (hydroxyl) in this region has an adverse effect on toxicity. the hba contour map is shown in figure . the magenta region around r substitution proved that this substitution was favorable to the hba atom, so compounds , , , and with the hba atom in the r substitution exhibit more potent toxicity (with pld values of . , . , . , and . ) than compounds with methoxymethyl substituents (compounds , , and with pld values of . , . , and . ). the red contour map where hba atoms are unfavorable for the toxicity was positioned around r and r . these contours were well validated by the lower pld value of compounds with carbonyl in these substitutions. the ppi network of aconitine alkaloids cardiotoxicity was divided into nine clusters using clusterone. statistical parameters are shown in figure . six clusters, namely clusters , , , , , and , which possess quality scores higher than . , a density higher than . , and a p-value less than . , were selected for further analysis (in figure ) . clusters , , and consisted of proteins mainly involved in the effects of various calcium, potassium, and sodium channels. cluster mainly consisted of three channel types related to the cardiotoxicity of aconitine alkaloids, cluster contained calcium and sodium channels and some channel exchangers (such as ryr and ryr ), and cluster mainly consisted of various potassium channels. all of these findings are consistent with previous research about the arrhythmogenic properties of the toxicity of aconitine alkaloids: the aconitine binds to ion channels and affects their open state, and thus the corresponding ion influx into the cytosol [ ] [ ] [ ] . the channel exchangers play a crucial role in keeping the ion transportation and homeostasis inside and outside of the cell. cluster contained some regulatory proteins that can activate or repress the ion channels through the protein expression level. atp a , ryr , ryr , cacna c, cacna d, and cacna s mediate the release of calcium, thereby playing a key role in triggering cardiac muscle contraction and maintaining the calcium homeostasis [ , ] . aconitine may cause aberrant channel activation and lead to cardiac arrhythmia. clusters and consisted of camp-dependent protein kinase (capk), cgmp-dependent protein kinase (cgpk), and guanine nucleotide binding protein (g protein). they have not been fully studied to prove whether the cardiotoxicity induced by aconitine alkaloids is linked to the capk, cgpk, and g proteins; however, some studies have shown that cardiotoxicity-related protein kcnj (potassium inwardly-rectifying channel) is controlled by g proteins and the cardiac sodium/calcium exchanger and is said to be regulated by capk and cgpk [ , ] . the result of clusterone indicated that the constructed network is consistent with existing studies and that the network can be used to screen essential proteins in the cytonca plugin. the protein v o belonging to the camkii (calcium/calmodulin (ca + /cam)-dependent serine/threonine kinases ii) isozyme protein family plays a central role in cellular signaling by transmitting ca + signals. the camkii enzymes transmit calcium ion (ca + ) signals released inside the cell by regulating signal transduction pathways through phosphorylation. ca + first binds to the small regulatory protein cam, and this ca + /cam complex then binds to and activates the kinase, which then phosphorylates other proteins such as ryanodine receptor and sodium/calcium exchanger. thus, these proteins are related to the cardiotoxicity induced by aconitine alkaloids [ ] [ ] [ ] . the excessive activity of camkii has been observed in some structural heart disease and arrhythmias [ ] , and past findings demonstrate neuroprotection in neuronal cultures treated with inhibitors of camkii immediately prior to excitotoxic activation of the camkii [ ] . the acute cardiotoxicity of the aconitine alkaloids is possibly related to this target. based on the analysis of the ppi network above, camkii was selected as the potential target for further molecular docking and dynamic simulation. the dock result of v o is shown in figure a . compound has the highest fit scores, so it was selected as the template for conformational analysis. the mechanisms of camkii activation and inactivation are shown in figure b . compound affects the normal energy metabolism of the myocardial cell via binding in the atp-competitive site in figure c . the inactive state of the camkii was regulated by cask-mediated t /t phosphorylation, and this state can be inhibited by the binding of compound in the atp-competitive site. such binding moves camkii toward a ca + /cam-dependent activation active state and a ca + /cam-dependent activation through structural rearrangement of the inhibitory helix caused by ca + /cam binding and the subsequent autophosphorylation of t [ ] , which will induce the excessive activity of camkii and dynamic imbalance of the calcium ions in the myocardial cell, eventually leading to heart disease and arrhythmias. molecules , , x for peer review of channel) is controlled by g proteins and the cardiac sodium/calcium exchanger and is said to be regulated by capk and cgpk [ , ] . the result of clusterone indicated that the constructed network is consistent with existing studies and that the network can be used to screen essential proteins in the cytonca plugin. the protein v o belonging to the camkii (calcium/calmodulin (ca + /cam)-dependent serine/threonine kinases ii) isozyme protein family plays a central role in cellular signaling by transmitting ca + signals. the camkii enzymes transmit calcium ion (ca + ) signals released inside the cell by regulating signal transduction pathways through phosphorylation. ca + first binds to the small regulatory protein cam, and this ca + /cam complex then binds to and activates the kinase, which then phosphorylates other proteins such as ryanodine receptor and sodium/calcium the information of a binding pocket of a receptor for its ligand is very important for drug design, particularly for conducting mutagenesis studies [ ] . as has been reported in the past [ ] , the binding pocket of a protein receptor to a ligand is usually defined by those residues that have at least one heavy atom within a distance of Å from a heavy atom of the ligand. such a criterion was originally used to define the binding pocket of atp in the cdk -nck a complex [ ] , which was later proved to be very useful in identifying functional domains and stimulating the relevant truncation experiments. a similar approach has also been used to define the binding pockets of many other receptor-ligand interactions important for drug design [ , , , [ ] [ ] [ ] [ ] . the information of a binding pocket of camkii for the aconitine alkaloids will serve as a guideline for designing drugs with similar scaffolds, particularly for conducting mutagenesis studies. in figure a , four top fit scores-compounds , , , and -generated similar significant interactions with amino acid residues around the atp-competitive binding pocket. four compounds formed with many van der waals interactions within the noncompetitive inhibitor pocket through amino acid residues such as asp , lys , glu , lys , and leu . the ligand-receptor interaction showed that the hydroxy in r formed a side chain donor interaction with asp . in addition, the hydroxy in r and r also formed a side chain acceptor interaction with glu and ser , respectively (the docking result of compounds and in figure a ). these results correspond to the comfa and comsia contour maps. however, the small electropositive and hydrophilic group in r , r , and r possess a certain enhancement function to toxicity. there were aromatic interactions between the phenyl group in r and amino acid residues. the phenyl group in r formed aromatic interactions with leu , leu , and phe , while the small group hydroxyl did not form any interaction with asp , which demonstrate that bulky phenyl group is crucial to this binding pattern and toxicity. this was mainly equal to the comfa steric contour map, where r was ideal for sterically favorable groups. the methoxymethyl r generated backbone acceptor with lys , which correspond to the comsia hba contour map, where r was favorable for the hba atom. compound docked into v o, and the atp-competitive pocket was painted green; the t , t , and t phosphorylation sites were painted green, orange, and yellow, respectively; the inhibitory helix was painted red. the result of md simulation is shown in figure . the red plot represented the rmsd values of the docked protein. the values of rmsd reached . Å in . ns and then remained between and . Å throughout the simulation for up to ns. the averaged value of the rmsd was . Å. the md simulation demonstrated that the ligand was stabilized in the active site. finally, we combined the ligand-based d-qstr analysis with the structure-based molecular docking study to identify the necessary moiety related to the cardiotoxicity mechanism of the aconitine alkaloids (in figure ). finally, we combined the ligand-based d-qstr analysis with the structure-based molecular docking study to identify the necessary moiety related to the cardiotoxicity mechanism of the aconitine alkaloids (in figure ). to build the ppi network of aconitine alkaloids, literature from january to february was retrieved from pubmed (http://pubmed.cn/) and web of science (http://www.isiknowledge.com/) with the mesh word "aconitine" and "toxicity" and without language restriction. all documents about cardiotoxicity caused by aconitine alkaloids were collected. the proteins related to the aconitine alkaloids cardiotoxicity of this decade were gathered and taken as the input protein in the string (https://string-db.org/) database [ , ] , used to search for related proteins or pathways that had been reported. finally, all the proteins and its partners were recorded in excel in order to import information and build a ppi network in cytoscape software. cytoscape is a free, open-source, java application for visualizing molecular networks and integrating them with gene expression profiles [ , ] . plugins are available for network and molecular profiling analyses, new layouts, additional file format support, making connections with figure . crucial requirement of cardiotoxicity mechanism was obtained from the ligand-based d-qstr and structure-based molecular docking study. to build the ppi network of aconitine alkaloids, literature from january to february was retrieved from pubmed (http://pubmed.cn/) and web of science (http://www.isiknowledge.com/) with the mesh word "aconitine" and "toxicity" and without language restriction. all documents about cardiotoxicity caused by aconitine alkaloids were collected. the proteins related to the aconitine alkaloids cardiotoxicity of this decade were gathered and taken as the input protein in the string (https://string-db.org/) database [ , ] , used to search for related proteins or pathways that had been reported. finally, all the proteins and its partners were recorded in excel in order to import information and build a ppi network in cytoscape software. cytoscape is a free, open-source, java application for visualizing molecular networks and integrating them with gene expression profiles [ , ] . plugins are available for network and molecular profiling analyses, new layouts, additional file format support, making connections with databases, and searching within large networks [ ] . clusterone (clustering with overlapping neighborhood expansion) of cytoscape was utilized to cluster the ppi network into overlapping sub-graphs of highly interconnected nodes. clusterone is a plugin for detecting and clustering potentially overlapping protein complexes from ppi data. the quality of a group was assessed by the number of sub-graphs, p-values, and density. the cluster was discarded when the number of sub-graphs was smaller than , the density was less than . , the quality was less than . , and the p-value was under . [ ] . the clustering results of the clusterone are instrumental to understanding how the reliability of the ppi network relates to aconitine alkaloids' cardiotoxicity. cytonca is a plugin in cytoscape integrating calculation, evaluation, and visualization analysis for multiple centrality measures. there are eight centrality measurements provided by cytonca: betweenness, closeness, degree, eigenvector, local average connectivity-based, network, subgraph, and information centrality [ ] . the primary purpose of the centrality analysis was to confirm the essential proteins in the pre-built ppi network. the three centrality measurements in the cytonca plugin-subgraph centrality, betweenness centrality, and closeness centrality-were used for evaluating and screening the essential protein in the merged target network. the subgraph centrality characterizes the participation of each node in all subgraphs in a network. smaller subgraphs are given more weight than larger ones, which makes this measurement an appropriate one for characterizing network properties. the subgraph centrality of node "u" can be calculated by [ ] µ l (u) is the uth diagonal entry of the lth power of the weight adjacency matrix of the network. v , v , . . . , v n is be an orthonormal basis composed of r n composed by eigenvectors of a associated to the eigenvalues λ , λ , . . . , λ n v u v , which is the uth component of v v [ ] . the betweenness centrality finds a wide range of applications in network theory. it represents the degree to which nodes stand between each other. betweenness centrality was devised as a general measure of centrality. it is applicable to a wide range of problems in network theory, including problems related to social networks, biology, transport, and scientific cooperation. the betweenness centrality of a node u can be calculated by [ ] ρ (s, t) is the total number of shortest paths from node s to node ρ (s, u, t), which is the number of those paths that pass through u. closeness centrality of a node is a measure of centrality in a network, calculated as the sum of the length of the shortest paths between the node and all other nodes in the graph. thus, the more central a node is, the closer it is to all other nodes. the closeness centrality of a node u can be calculated by [ ] |nu| is the number of node u's neighbors, and dist (u, v) is the distance of the shortest path from node u to node v. pharmmapper serves as a valuable tool for identifying potential targets for a novel synthetic compound, a newly isolated natural product, a compound with known biological activity, or an existing drug [ ] . of all the aconitine alkaloids in this research, compounds , , and exhibited the most toxic activity and were used for the potential target prediction. the mol format of three compounds was submitted to the pharmmapper server. the parameters of generate conformers and maximum generated conformations was set as on and , respectively. other parameters used default values. finally, the result of the clusterone and pharmmapper will be combined together to select the potential targets for the following docking study [ ] . comparative molecular field analysis (comfa) and comparative molecular similarity index analysis (comsia) are efficient tools in ligand-based drug design and are in use for contour map generation and identification of favorable and unfavorable regions in a moiety [ , ] . the comfa consists of a steric and electrostatic contour map of molecules that are correlated with toxic activity, while the comsia consists of hydrophobic field, hydrogen bond donor (hbd)/hydrogen bond acceptor (hba) [ ] , and steric/electrostatic fields that are correlated with toxic activity. the comfa and comsia have been utilized to generate a d-qstr model [ ] . all molecule models and the generation of d-qstr were performed with sybyl x . . alkaloids in mice with ld values listed in table were extracted from recent literature [ ] . the ld values of all aconitine alkaloids were converted into pld with a standard tripos force field. these pld values were used as a dependent variable, while comfa and comsia descriptors were used as an independent variable. the sketch function of sybyl x . was utilized to illustrate structure and charges, and was calculated by the gasteiger-huckel method. additionally, the tripose force field was utilized for energy minimization of these aconitine alkaloid molecules [ ] . the molecules were divided into a ratio of : . the division was done in a way that showed that both datasets are balanced and consist of both active and less active molecules [ ] . the reliability of the d-qstr model depends on the database molecular alignment. the most toxic aconitine alkaloids (compound ) was selected as the template molecule, and the tetradecahydro- h- , , -(epiethane [ , , ] triyl)- , -methanonaphtho [ , -b] azocine was selected as the common moiety. pls (partial least squares) techniques are associated with field descriptors with activity values such as [ ] leave one out (loo) values, the optimal number of components, the standard error of estimation (see), cross-validated coefficients (q ), and the conventional coefficient (r ). these statistical data are pivotal in the evaluation of the d-qstr model and can be worked out in the pls method [ ] . the model is said to be good when the q value is more than . and the r value is more than . . the q and r values reflect a model's soundness. the best model has the highest q and r values, the lowest see, and an optimal number of components [ , , ] . in the case of comfa and comsia analysis, the values of the optimal number of components, see, and q can be worked out by loo validation, with use sampls turned on and components set to , while in the process of calculating r , the use sampls was turned off and the column filtration was set to . kcal mol − in order to speed up the calculation without the need to sacrifice information content [ ] [ ] [ ] [ ] . therefore, components were set to and , respectively, which were optimal numbers of components calculated by performing a sampls run. see and r were utilized to assess the non-cross validated model. the applicability domain (ad) of the topomer comfa and comsia model was confirmed by the williams plot of residuals vs. leverage. leverage of a query chemical is proportional to its mahalanobis distance measure from the centroid of the training set [ , ] . the leverages are calculated for a given dataset x by obtaining the leverage matrix (h) with the equation below: x is the model matrix, while xt is its transpose matrix. the plot of standardized residuals vs. leverage values was drawn, and compounds with standardized residuals greater than three standard deviation units (± σ) were considered as outliers [ ] . the critical leverage value is considered p/n, where p is the number of model variables plus one, and n is the number of objects used to calculate the model. h > p/n mean predicted response is not acceptable [ ] [ ] [ ] . (cadd) software program that incorporates the functions of qsar, molecular docking, molecular dynamics, adme (absorption, distribution, metabolism, and excretion), and homologous modeling. all of these functions are regarded as conducive instruments in the field of drug discovery and biochemistry. the molecular docking and dynamics technology were performed in moe software to detect the stability and affinity between the ligands and predictive targets [ , ] . the docking process involves the prediction of ligand conformation and orientation within a targeted binding site. docking analysis is an important step in the docking process. it has been widely used to study the reasonable binding mode and obtain information of interactions between amino acids in active protein sites and ligands. the molecular docking analysis was carried out to determine the toxicity-related moiety of aconitine alkaloids through the ligand-amino-acid interaction function in moe . the pdb format of v o and vz was downloaded from the pdb (protein data bank) database (https://www.rcsb.org/), and the mol format of compounds was from the sybyl software of qstr research. the structure preparation function in moe software will be carried out to minimize the energy and optimize the structure of the protein skeleton. based on the london dg score and induced fit refinement, all compounds will be docked into the active site of every potential target by taking score values as the scoring function [ ] . the dcg (discounted cumulative gain) algorithm was utilized to examine the consistency between the ranking result of pld and our research (fit scores of dock study). they rely on the formula that refers to pld . the idcg (ideal dcg) refers to the ordered pld values. the closer the normalized discounted cumulative gain (ndcg) value is to , the better the consistency [ ] . preliminary md simulations for the model protein were performed using the program namd (nanoscale molecular dynamics program, v . ), and all files were generated using visual molecular dynamics (vmd). namd is a freely available software designed for high-performance simulation of large biomolecular systems [ ] . during the md simulation, minimization and equilibration of original and docked proteins occurred in a Å size water box. a charmm force field file was applied for energy minimization and equilibration with gasteiger-huckel charges using boltzmann initial velocity [ , ] . integrator parameters also included fs/step for all rigid bonds and nonbonded frequencies were selected for Å and full electrostatic evaluations for Å were used with steps for each cycle [ ] . the particle mesh ewald method was used for electrostatic interactions of the simulation system periodic boundary conditions with grid dimensions of . Å [ ] . the pressure was maintained at . kpa using the langevin piston and the temperature was controlled at k using langevin dynamics. covalent interactions between hydrogen and heavy atoms were constrained using the shake/rattle algorithm. finally, ns md simulations for original and docked protein were carried out for comparing and verifying the binding affinity and stability of the ligand-receptor complex. the method combining network analysis and the in silico method was carried out to illustrate the qstr and toxic mechanisms of aconitine alkaloids. the d-qstr was built in sybyl with internal robustness and external high prediction, enabling identification of pivotal molecule moieties related to toxicity in aconitine alkaloids. the comfa model had q , r , optimum component, and correlation coefficient (r ) values of . , . , , and . , respectively, and the comsia model had q , r , optimum component, and correlation coefficient (r ) values of . , . , , and . . the network was built with cytoscape software and the string database, which demonstrated the reliability of cluster analysis. the v o and vz proteins were identified as potential targets with the cytonca plugin with pharmmapper server for interactions between the aconitine alkaloids and key amino acids in the dock study. the result of the dock study demonstrates the consistency of the experimental pld . the md simulation indicated that aconitine alkaloids exhibit potent binding affinity and stability to the receptor camk g. finally, we incorporate pivotal molecule moieties and ligand-receptor interactions to realize the qstr of aconitine alkaloids. this research serves as a guideline for studies of toxicity, including neuro-, reproductive, and embryo-toxicity. with a deep understanding of the relationship between toxicity and structure of aconitine alkaloids, subsequent structural modification of aconitine alkaloids can be carried out to enhance their efficacy and to reduce their toxic side effects. based on such research, aconitine alkaloids can bring us closer to medical and clinical applications. in addition, as pointed out in past research [ ] , user-friendly and publicly accessible web servers represent the future direction of reporting various important computational analyses and findings [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] . they have significantly enhanced the impacts of computational biology on medical science [ , ] . the research in this paper will serve as a foundation for constructing web servers for qstr studies and target identifications of compounds. immunomodulating agents of plant origin. i: preliminary screening chinese drugs plant origin aconitine poisoning: a global perspective ventricular tachycardia after ingestion of ayurveda herbal antidiarrheal medication containing aconitum fatal accidental aconitine poisoning following ingestion of chinese herbal medicine: a report of two cases five cases of aconite poisoning: toxicokinetics of aconitines a case of fatal aconitine poisoning by monkshood ingestion determination of aconitine and hypaconitine in gucixiaotong ye by capillary electrophoresis with field-amplified sample injection a clinical study in epidural injection with lappaconitine compound for post-operative analgesia therapeutic effects of il- combined with benzoylmesaconine, a non-toxic aconitine-hydrolysate, against herpes simplex virus type infection in mice following thermal injury aconitine: a potential novel treatment for systemic lupus erythematosus aconitine-containing agent enhances antitumor activity of dichloroacetate against ehrlich carcinoma complex discovery from weighted ppi networks prediction and analysis of the protein interactome in pseudomonas aeruginosa to enable network-based drug target selection the string database in : quality-controlled protein-protein association networks, made broadly accessible identification of functional modules in a ppi network by clique percolation clustering united complex centrality for identification of essential proteins from ppi networks the ppi network and cluster one analysis to explain the mechanism of bladder cancer the progress of novel drug delivery systems mitochondrial uncoupling protein structure determined by nmr molecular fragment searching structural basis for membrane anchoring of hiv- envelope spike unusual architecture of the p channel from hepatitis c virus architecture of the mitochondrial calcium uniporter structure and mechanism of the m proton channel of influenza a virus computer-aided drug design using sesquiterpene lactones as sources of new structures with potential activity against infectious neglected diseases successful in silico discovery of novel nonsteroidal ligands for human sex hormone binding globulin in silico discovery of novel ligands for antimicrobial lipopeptides for computer-aided drug design structural bioinformatics and its impact to biomedical science coupling interaction between thromboxane a receptor and alpha- subunit of guanine nucleotide-binding protein prediction of the tertiary structure and substrate binding site of caspase- study of drug resistance of chicken influenza a virus (h n ) from homology-modeled d structures of neuraminidases insights from investigating the interaction of oseltamivir (tamiflu)with neuraminidase of the h n swine flu virus prediction of the tertiary structure of a caspase- /inhibitor complex design novel dual agonists for treating type- diabetes by targeting peroxisome proliferator-activated receptors with core hopping approach heuristic molecular lipophilicity potential (hmlp): a d-qsar study to ladh of molecular family pyrazole and derivatives fragment-based quantitative structure & ndash; activity relationship (fb-qsar) for fragment-based drug design investigation into adamantane-based m inhibitors with fb-qsar hp-lattice qsar for dynein proteins: experimental proteomics ( d-electrophoresis, mass spectrometry) and theoretic study of a leishmania infantum sequence the biological functions of low-frequency phonons: . cooperative effects low-frequency collective motion in biomacromolecules and its biological functions quasi-continuum models of twist-like and accordion-like low-frequency motions in dna collective motion in dna and its role in drug intercalation biophysical aspects of neutron scattering from vibrational modes of proteins biological functions of soliton and extra electron motion in dna structure low-frequency resonance and cooperativity of hemoglobin solitary wave dynamics as a mechanism for explaining the internal motion during microtubule growth designed electromagnetic pulsed therapy: clinical applications steps to the clinic with elf emf molecular dynamics study of the connection between flap closing and binding of fullerene-based inhibitors of the hiv- protease molecular dynamics studies on the interactions of ptp b with inhibitors: from the first phosphate-binding site to the second one the cambridge structural database: a quarter of a million crystal structures and rising molecular similarity indices in a comparative analysis (comsia) of drug molecules to correlate and predict their biological activity single channel analysis of aconitine blockade of calcium channels in rat myocardiocytes conversion of the sodium channel activator aconitine into a potent alpha -selective nicotinic ligand aconitine blocks herg and kv . potassium channels inactivation of ca + release channels (ryanodine receptors ryr and ryr ) with rapid steps in [ca + ] and voltage targeted disruption of the atp a gene encoding the sarco(endo)plasmic reticulum ca + atpase isoform (serca ) impairs diaphragm function and is lethal in neonatal mice cyclic gmp-dependent protein kinase activity in rat pulmonary microvascular endothelial cells different g proteins mediate somatostatin-induced inward rectifier k + currents in murine brain and endocrine cells cardiac myocyte calcium transport in phospholamban knockout mouse: relaxation and endogenous camkii effects inhibition of camkii phosphorylation of ryr prevents induction of atrial fibrillation in fkbp . knock-out mice regulation of ca + and electrical alternans in cardiac myocytes: role of camkii and repolarizing currents the role of calmodulin kinase ii in myocardial physiology and disease excitotoxic neuroprotection and vulnerability with camkii inhibition structure of the camkiiδ/calmodulin complex reveals the molecular mechanism of camkii kinase activation a model of the complex between cyclin-dependent kinase and the activation domain of neuronal cdk activator binding mechanism of coronavirus main proteinase with ligands and its implication to drug design against sars an in-depth analysis of the biological functional studies based on the nmr m channel structure of influenza a virus molecular therapeutic target for type- diabetes novel inhibitor design for hemagglutinin against h n influenza virus by core hopping method the string database in : functional interaction networks of proteins, globally integrated and scored cytoscape: a software environment for integrated models of biomolecular interaction networks detecting overlapping protein complexes in protein-protein interaction networks cytonca: a cytoscape plugin for centrality analysis and evaluation of protein interaction networks subgraph centrality and clustering in complex hyper-networks ranking closeness centrality for large-scale social networks enhancing the enrichment of pharmacophore-based target prediction for the polypharmacological profiles of drugs comparative molecular field analysis (comfa). . effect of shape on binding of steroids to carrier proteins sample-distance partial least squares: pls optimized for many variables, with application to comfa a qsar analysis of toxicity of aconitum alkaloids recent advances in qsar and their applications in predicting the activities of chemical molecules, peptides and proteins for drug design unified qsar approach to antimicrobials. . multi-target qsar modeling and comparative multi-distance study of the giant components of antiviral drug-drug complex networks comfa qsar models of camptothecin analogues based on the distinctive sar features of combined abc, cd and e ring substitutions applicability domain for qsar models: where theory meets reality comparison of different approaches to define the applicability domain of qsar models molecular docking and qsar analysis of naphthyridone derivatives as atad bromodomain inhibitors: application of comfa, ls-svm, and rbf neural network concise applications of molecular modeling software-moe medicinal chemistry and the molecular operating environment (moe): application of qsar and molecular docking to drug discovery qsar models of cytochrome p enzyme a inhibitors using comfa, comsia and hqsar estimating a ranked list of human hereditary diseases for clinical phenotypes by using weighted bipartite network biomolecular simulation on thousands processors molecular dynamics and docking investigations of several zoanthamine-type marine alkaloids as matrix metaloproteinase- inhibitors salts influence cathechins and flavonoids encapsulation in liposomes: a molecular dynamics investigation review: recent advances in developing web-servers for predicting protein attributes irna-ai: identifying the adenosine to inosine editing sites in rna sequences iss-psednc: identifying splicing sites using pseudo dinucleotide composition irna-pseu: identifying rna pseudouridine sites ploc-mplant: predict subcellular localization of multi-location plant proteins by incorporating the optimal go information into general pseaac ploc-mhum: predict subcellular localization of multi-location human proteins via general pseaac to winnow out the crucial go information iatc-misf: a multi-label classifier for predicting the classes of anatomical therapeutic chemicals psuc-lys: predict lysine succinylation sites in proteins with pseaac and ensemble random forest approach irnam c-psednc: identifying rna -methylcytosine sites by incorporating physical-chemical properties into pseudo dinucleotide composition ikcr-pseens: identify lysine crotonylation sites in histone proteins with pseudo components and ensemble classifier iacp: a sequence-based tool for identifying anticancer peptides ploc-meuk: predict subcellular localization of multi-label eukaryotic proteins by extracting the key go information into general pseaac iatc-mhyb: a hybrid multi-label classifier for predicting the classification of anatomical therapeutic chemicals ihsp-pseraaac: identifying the heat shock protein families using pseudo reduced amino acid alphabet composition irna-psecoll: identifying the occurrence sites of different rna modifications by incorporating collective effects of nucleotides into pseknc impacts of bioinformatics to medicinal chemistry an unprecedented revolution in medicinal chemistry driven by the progress of biological science this article is an open access article distributed under the terms and conditions of the creative commons attribution (cc by) license key: cord- -wybmer o authors: chung, dong hoon; jonsson, colleen b.; maddox, clinton; mckellip, sara n.; moore, blake. p.; heil, marintha; white, e. lucile; ananthan, subramaniam; li, qianjun; feng, shuang; rasmussen, lynn title: hts-driven discovery of new chemotypes with west nile virus inhibitory activity date: - - journal: molecules doi: . /molecules sha: doc_id: cord_uid: wybmer o west nile virus (wnv) is a positive sense, single-stranded rna virus that can cause illness in humans when transmitted via mosquito vectors. unfortunately, no antivirals or vaccines are currently available, and therefore efficient and safe antivirals are urgently needed. we developed a high throughput screen to discover small molecule probes that inhibit virus infection of vero e cells. a primary screen of a , compound library at a μm final concentration was conducted using the -well format. z′ values ranged from . – . with a median of . . average s/b was and s/n for each plate ranged from . to . . twenty-six compounds showed a dose response in the ht screen and were further evaluated in a time of addition assay and in a titer reduction assay. seven compounds showed potential as small molecule probes directed at wnv. the hit rate from the primary screen was . % ( compounds out of , compounds) and from the secondary screens was . % ( out of , compounds) respectively. contrast to dye formation or dye-uptake methods, which have a low dynamic range, the celltiter-glo® assay shows a higher dynamic range with less background. we implemented the wnv assay in the -well format in a screen of , compounds. a diverse group of small molecules targeting various specific steps in virus replication was discovered in the screening campaign. to optimize the assay for robust and reproducible hts data, we explored the assay media, number of cells per well, multiplicity of infection (moi) and incubation time post-infection. we tested complete dmem, mem-e with or without non-essential amino acids (neaa) and mem-e with reduced nahco ( . g/l) [ ] . we did not find a significant difference among the three mem-e based media with regard to luminescence (data not shown). however, cytopathic effect (cpe) was more evident in cells grown and infected with wnv in mem-e than in dmem. the signal from the virus control with dmem showed a higher luminescence signal than that with mem-e while the luminescence from cell controls was identical ( figure a ). the mem-e media also shortened the assay incubation period by two days compared to dmem media and hence was chosen for the hts. we tested the moi from . to . in -well plates. we did not witness a significant difference in cell viability between different mois ( figure b ). the z′ value was > . at any moi tested confirming the development of a very robust assay. the efficacy of the positive control compound, mycophenolic acid (mpa), varied depending on infectious dose. at moi . , mpa showed % protection without decreasing z′ value. based on these findings we have developed optimized conditions as described in materials and methods. assay optimization for assay media, incubation period, and moi. (a) vero e cells were infected with wnv at . moi using the -well plate format and developed on successive days. virus cultured in mem-e produced a higher cpe between day and day than in dmem. (b) assay robustness with z´ factor and inhibition profiles using mpa (positive control) and different amounts of virus in a -well plate format. z´ factor was higher than . in a . ~ moi range. the inhibitory activity of mpa was greater at the lower moi. a primary screen of a , compound library at a µm final concentration was conducted using the -well format. z′ values ranged from . - . with a median of . . average s/b was and s/n for each plate ranged from . to . with a mean of . and standard deviation of . (figure a and b) . these statistical values indicate that the assay provided high quality and the required robustness and reproducibility of an hts assay. in this assay the overall average inhibitions were . % with a standard deviation of . % and the control drug mpa showed . % inhibition on average. the percent inhibition cutoff was . % that represents the viral mean + three standard deviations, a common method of statistically defining hits in a single dose screen. using this cutoff method, . %, compounds were identified as active wnv inhibitors ( figure c ). to confirm the activity and to test the cytotoxicity, the top inhibitory compounds were evaluated in a dose response assay using the identical screen. the secondary assay revealed compounds with a dose dependent response and fifteen of those compounds showed ec values less than µm. three compounds, however, were cytotoxic with low cc values ranging from . to . µm. remaining compounds gave cc values greater than µm (table ) . ten compounds showed an si (selectivity index , ratio of cc to ec ) > and these were evaluated further (table ) . the compounds selected by the primary and dose response screening were re-supplied from the suppliers as a solid form except compounds; smr , smr , smr and sri- , which were not available at that time ( table ) . time of addition assays were conducted in a dose response format with the compounds. serially diluted compounds were added to vero e cells at - , , and h post-infection. luminescent data was calculated (as above) to obtain ec values for each compound at each time point of addition. seventeen compounds showed ec values less than µm and compounds were not active. interestingly, some compounds showed no significant change in ec over the time of addition ( figure a) . a similar pattern was seen with the positive control (mpa). the ec of mpa was within a range of . ~ . µm regardless of when it was added. in contrast, other compounds showed a dramatic change in ec according to the time of addition ( figure b ). sri- showed a minor change ( . to . µm) in ec , and the remaining six compounds in this category showed an ec value of over µm when the compounds were added h post-infection. the ec values of the compounds in this class were less than µm when the compounds were delivered prior to h post-infection. we classified the compounds according to the pattern of change in the values of the ec as shown in table and figure . the results from the time of addition assay led us to examine the ns protease as a potential target of the hit compounds. ns protease is an important enzyme responsible for the maturation of viral proteins at early stage during the replication of virus in the host cells. we tested the compounds for an inhibitory activity on ns protease using a fret based, in vitro, enzymatic assay. as a result, we identified sri- with an inhibitory effect on the protease and with ic of . µm, suggesting the molecular target of these two compounds may be the ns pro of wnv (figure ). we also tested n-phenylanthranilic acid for ns pro inhibitory activity as this moiety was common for two compounds, sri- and smr , but we could not detect any significant activity in this system (data not shown). finally, we employed a titer reduction assay to confirm the antiviral activity of the seven compounds confirmed in the assays. the titer of the progeny virus produced in the presence of the compounds was measured in tcid format. infection of vero e cells using a . moi resulted in around . × tcid /ml in two days post-infection. treatment of infected cells with the compounds decreased the progeny titer up to fold ( figure ). for example, smr and sri- showed . % inhibition followed by sri- and sri- at . and . % inhibition, respectively, at µm concentration. sri- was most potent in this assay with ec of . µm and sri- was least active with ec of . µm. the positive control drug, mpa, worked extremely well as a potent inhibitor with an ec of . µm in this assay. the titer reduction assay confirmed the antiviral activity of the hit compounds selected through our pathway ( figure ). herein we report the discovery of several new chemotypes from an ht screen of a , compound library that inhibit wnv replication ( table ). the endpoint we developed and employed in the ht screen was cpe caused by viral replication, which has advantages over other target based hts. a cpe based antiviral screen is expected to elucidate a wider range of antiviral compounds than other target based assays. a cpe based assay is likely to discover cytotoxic compounds since the cytotoxic compounds will decrease the endpoint signal and are read as inactive. this has an advantage over reporter-based assays in that they can read false positives. in fact, we found only compounds with cc less than µm among compounds subjected to the dose response assay. the pathway for probe discovery is depicted in figure . the top compounds, based on their inhibitory activities were examined by a dose response and cytotoxicity assay. twenty-four compounds showing a dose response inhibition were further evaluated in a time of addition assay and in a titer reduction assay. finally, seven compounds are presented as anti-wnv probes. hit rate for the secondary assay and the final confirmation were . % ( compounds out of , compounds) and . % ( out of , compounds) respectively. we categorized the compounds into chemical groups based on their chemical structures ( table ). the first group are uridine derivatives; sri- , sri- and sri- (full chemical structure not disclosed). similar compounds have been known for wnv antiviral activity [ ] . the second group is composed of sri- (redoxal) and smr . sri- was also active against flaviviruses with inhibitory activity on dihydroorotate dehydrogense [ ] . the third group of compounds, sri- and smr , are new chemotypes with anti-flavivirus activity. smr has shown activity in other hts assays, such as anti-tnf-α-specific nf-kb induction and nod and inhibition assays (aid: , and http://pubchem.ncbi.nlm.nih.gov/). all seven compounds screened in the time of addition experiment were confirmed to have anti-wnv activity based on the titer reduction assay and ec values between . ~ . µm. this suggests that hts and following assays are an effective method for probe discovery and evaluation. none of the compounds showed anti-wnv activity as good as the positive control drug, mpa, however, the mechanism of mpa is not specific to the virus making the probes we identified of significant interest. our time of addition analysis showed interesting patterns in the value of ec . these studies suggest possible targets for each compound. we showed that there are two types of patterns in ec that change according to the time of addition; ) compounds with minimal change in ec , and ) the compounds with an increased ec mpa reflected the first pattern and nucleoside derivatives, sri- and sri- produced the second pattern. this suggests that compounds that do not have a change in their ec may target the host while those that change, target the virus. we identified one compound, sri- , that inhibited viral protease in vitro and time of addition studies. these results suggest it might target the viral protease ns b-ns pro. sri- exhibited low activity in the primary, dose response (ec of µm), and time of addition assays. all work with wnv was performed at bsl- following cdc guidelines. vero e cells (atcc crl ) were maintained in dulbecco's modified eagle's medium (dmem) supplemented with % fetal bovine serum (fbs) and mm l-glutamine. wnv strain ny- was used for the assays and was propagated in a complete minimum essential medium eagle (mem-e) media obtained from sigma-aldrich (st. louis, usa), supplemented with % fbs, units/ml penicillin, µg/ml streptomycin and mm l-glutamine. the virus stock was divided into ml aliquots and stored at - ºc. virus plaque forming units were measured using an agar overlay method described previously [ ] with minor modifications. for primary ht screening, two compound sets were used for a total of , compounds: the southern research institute (sri) proprietary compound library composed of , compounds and a selection of compounds from the national institutes of health molecular libraries screening network library of , compound that were active in a previous screen against bluetongue virus (http://pubchem.ncbi.nlm.nih.gov/assay/-assay.cgi?aid= &loc=ea_ras/). the sri proprietary library consists of the historical, unique compounds designed and synthesized at sri over many years as potential drug candidates. the members of this collection have high biological relevance, particularly to cancer and infectious disease. major classes of compounds that are represented in this collection include nucleosides, purines, pyrimidines, pteridines, imidazoles, pyridines, quinolines, triazines, disaccharides, guanidines, ureas and carbamates. the stock concentrations of both compound sets were mm in % dmso. in the single dose primary screen, the stock compounds were diluted : in a × solution using the biomek fx giving a µm working concentration in % dmso. a µl aliquot of the working concentration plus an additional µl of assay media was transferred into each well. control stocks were prepared in assay media at a fold higher than the target concentration: . % dmso for cell and viral control and µg/ml mycophenolic acid (mpa) at . % dmso for positive control. mpa was purchased from sigma-aldrich co. (st. louis, usa) and solubilized in % dmso to a mg/ml stock concentration. controls were added separately to the assay plate in µl aliquots via the biomek fx. with the addition of µl of cells and µl of virus, the final test concentration for the compounds was µm and for mpa was µg/ml. the final dmso was . %, which is well below the . % maximum allowable for this assay. after reviewing the data from the primary screen, compounds were selected for a doseresponse study. the same compound stocks ( mm in % dmso) were used to select µl of the compounds into a low-volume, -well plate. the dose response plates were prepared using a "stacked-plate" format using a : dilution series for a total of ten concentrations. by using this format, each entire plate represents one of the ten concentrations in the dilution series. a µl aliquot of each of the serial dilution plates was transferred to the assay plates along with an equal volume but separate vero e cells were suspended in complete mem-e at , cells/ml and dispensed at µl into black, clear-bottom, tissue culture treated, -well plates ( , cells/well) using a matrix wellmate® (matrix, thermofisher). the plated cells were maintained overnight at ºc with % co in an actively humidified incubator. test compounds were then transferred at µl per well for a final dmso concentration of . % (see above). each plate consisted of wells representing the cell control, wells for virus control, wells for positive control (mpa), and wells for testing individual compounds. cell plates were transferred into the bsl and infected by addition of µl of wnv suspension at . × pfu/ml in complete mem-e using a matrix wellmate®. plates were incubated for h at ºc and % co to promote virus replication. µl of celltiter-glo® (promega, madison, wi) was added to each well using a matrix wellmate and incubated at room temperature for min. luminescence was measured using a perkin elmer envision plate reader (wellesley, ma) with an integration time of . s. cytotoxicity and antiviral efficacy were performed in a dose-response format in parallel. compounds were serially diluted in complete media (mem-e) with a . % dmso final concentration and adding µl to cell plates, identical to the stacked plate method [ ] . a ten point, two-fold dilution series of compound concentrations ranging from µm to . µm were used in the assay. virus ( . moi) or media was added for antiviral effect or cytotoxicity respectively. the plates were incubated h in a % co incubator at ºc. cell viability was measured as stated above. the serially diluted compounds were also used for time of addition studies adding to the infected cells in -well plates at - , , and h post-infection with wnv ( . moi). the plates were then developed as above h after infection. the ec value was calculated for each time of addition point. the z′ value was employed to evaluate the assay's robustness and was calculated from -( ×standard deviation of cell control (σ c ) + * standard deviation of the virus control (σ v )/ [mean cell control signal (µ c ) minus mean virus control signal (µ v )]). the signal/background (s/b) was calculated from the mean cell control signal (µ c ) divided by the mean virus control signal (µ v ). the signal/noise (s/n) was calculated from mean cell control signal (µ c ) minus mean virus control signal (µ v ) divided by the (standard deviation of the cell control signal (σ c ) minus the standard deviation of the virus control signal (σ v ) ) / [ ] . the effective concentration at which the drug inhibited cell death at % in the presence of virus (ec ) and the cytotoxicity of the drug alone at % (cc ) were calculated using activitybase software (idbs, inc,guildford, uk). cpe inhibition (%) and cell viability were calculated as described in elsewhere [ ] . the standard curve analysis function in sigmaplot™ was used to calculate the inhibitory concentration (ic and ic values) for the protease and titer reduction assays. a recombinant ns protease expressed and purified from e.coli with the cofactor ns b (residues - ) and a fluorescence resonance energy transfer (fret) based assay kit were purchased from anaspec inc (ca, usa). we followed an assay protocol published on line at pubchem by university of pittsburgh molecular library screening center (http://www.ncbi.nlm.nih.gov/, aid: ). the assay was performed in a -well format with ng/well of ns b-ns pro enzyme. compounds that showed specific inhibitory effects on virus replication steps were further validated for their efficacy using a tcid reduction assay. vero e cells were seeded in a -well plate in a volume of ml and incubated overnight at ºc and % co . the next day media was aspirated from the cells and they were infected by adsorption of wnv ( . moi) for one hour in µl media. after aspirating the virus and washing the cells with phosphate buffered saline, media was replenished containing compounds at various concentrations. the plates were incubated for h. progeny virus titer was measured by tcid assay in -well plate format with wells per dilution of virus. celltiter-glo® reagent was employed to determine a cell death which is a sign of infection. a well with a luminescence signal less than the mean of the non-infected control signal, minus times the standard deviation of the control, was regarded as a positive infection. the tcid was calculated with the numbers of positive infection and negative infection by the reed-muench method [ ] . in summary, we report our approaches in the development and use of an ht screen to discover specific and effective anti-wnv probes. as a result of hts and secondary assays, we have identified compounds as specific and effective anti-wnv probes. further optimization of these compounds is needed to develop effective antiviral treatment for wnv infection. in addition, the compounds we have identified should serve as novel molecular probes to help us understand the biology of wnv. future studies will focus on how these compounds target virus replication and the mechanism of action. in conclusion, identification of previously known and novel moieties from a screening library is proof that the wnv assay is an effective ht screen. west nile virus infection identification of a kunjin/west nilelike flavivirus in brains of patients with new york encephalitis outbreak of west nile-like viral encephalitis pathogenesis of west nile virus infection: a balance between virulence, innate and adaptive immunity, and viral evasion west nile virus: epidemiology and clinical features of an emerging epidemic in the united states the continuing spread of west nile virus in the western hemisphere the molecular biology of west nile virus: a new invader of the western hemisphere evidence that the n-terminal domain of nonstructural protein ns from yellow fever virus is a serine protease responsible for site-specific cleavages in the viral polyprotein functional characterization of cis and trans activity of the flavivirus ns b-ns protease the mechanism of cell death during west nile virus infection is dependent on initial infectious dose inhibition of apoptosis prevents west nile virus induced cell death west nile virus-induced baxdependent apoptosis an infectious west nile virus that expresses a gfp reporter gene potential high-throughput assay for screening inhibitors of west nile virus replication adaptation of west nile virus replicons to cells in culture and use of replicon-bearing cells to probe antiviral action high-throughput assays using a luciferase-expressing replicon, virus-like particles, and full-length virus for west nile virus drug discovery identification of compounds with anti-west nile virus activity discovery of small molecule inhibitors of west nile virus using a high-throughput sub-genomic replicon screen a cell-based luminescence assay is effective for high-throughput screening of potential influenza antivirals high-throughput screening of a , -compound library for inhibitors of influenza a virus (h n ) identification of active antiviral compounds against a new york isolate of west nile virus compounds for the treatment of viral-mediated disease simple statistical parameter for use in evaluation and validation of high throughput screening assays development and validation of a high-throughput screen for inhibitors of sars cov and its application in screening of a , -compound library a simple method of estimating fifty percent endpoints this work was supported by southern research institute sip program and nih mlpcn grant number -u hg - (pi c.b.j.). we thank to anna manouvakhova for data handling. key: cord- -z w zeq authors: nguyen, thi thanh hanh; lee, sun; wang, hsi-kai; chen, hsin-yen; wu, ying-ta; lin, simon c.; kim, do-won; kim, doman title: in vitro evaluation of novel inhibitors against the ns b-ns protease of dengue fever virus type date: - - journal: molecules doi: . /molecules sha: doc_id: cord_uid: z w zeq the discovery of potent therapeutic compounds against dengue virus is urgently needed. the ns b-ns protease (ns b-ns (pro)) of dengue fever virus carries out all enzymatic activities needed for polyprotein processing and is considered to be amenable to antiviral inhibition by analogy. virtual screening of , compounds using autodock on the gvss platform was conducted to identify novel inhibitors against the ns b-ns (pro). thirty-six compounds were selected for in vitro assay against ns b-ns (pro) expressed in pichia pastoris. seven novel compounds were identified as inhibitors with ic( ) values of . ± . – . ± . μm. three strong ns b-ns (pro) inhibitors were further confirmed as competitive inhibitors with k(i) values of . ± . , . ± . , and . ± . μm, respectively. hydrophobic and hydrogen bond interactions between amino acid residues in the ns (pro) active site with inhibition compounds were also identified. among members of the flavivirus genus dengue virus (denv) is responsible for the highest rate of disease and mortality and consists of a group of four serologically related viruses referred to as denv types - . infection with these viruses results in a range of clinical diseases such as dengue fever, dengue hemorrhagic fever, and dengue shock syndrome [ ] [ ] [ ] . global epidemics of denv have occurred over the past few years. there were million ( % credible interval - million infections) dengue infections per year, of which million ( - million infections) apparently manifested some level of disease severity [ ] . denv are transmitted to humans by the bite of infective female mosquitoes of the genus aedes. denv has a single-stranded rna genome that is packaged by the virus capsid protein in a hostderived lipid bilayer and is surrounded by copies of two glycoproteins. the complete virion is approximately nm in diameter and contains an approximate . kb positive-sensed rna genome that has one open reading frame encoding a single polyprotein [ ] . the '-end encodes three structural proteins: the capsid (c); membrane precursor protein (prm) proteolytically cleaved by the host protease furin to form the membrane protein in mature virions; and the envelope (e), constituting the enveloped virus particle [ , ] . seven non-structural (ns) proteins essential for viral replication are encoded by the remainder of the genome. the order of proteins encoded is '-c-prm-e-ns -ns a-ns b-ns -ns a-ns b-ns - ' [ , ] . ns pro is responsible for cleaving both in the cis and trans directions to generate viral proteins that are essential for viral replication and maturation of infectious dengue virions. a number of different strategies have been employed to search for denv ns b-ns protease inhibitors, including high-throughput screening [ ] [ ] [ ] , synthesizing rationally designed substrate-based peptidomimetics [ , ] , cyclopeptide [ ] , and structure-based virtual screening (sbvs) [ ] [ ] [ ] as well as screening natural products [ , ] . however, only a few peptide or small molecule inhibitors of the denv ns b-ns protease with moderate activity have so far been reported [ , ] . there are currently no effective antiviral agents to treat dengue infection [ ] as well as no licensed vaccine against dengue infection is available, and the most advanced dengue vaccine candidate did not meet expectation in a recent large trial [ , , ] . over the past decade, high-throughput virtual screening (vs), and particularly substrate-based virtual screening (sbvs) has emerged as a reliable, cost-effective and time-saving technique for discovering lead compounds as an alternative to high-throughput screening [ ] . vs, as applied to new enzyme inhibitor discovery, involves docking, computational fitting of compound structures to the enzyme active site, and scoring and ranking of each compound [ , ] . grid application platform virtual screening service (gvss) has been developed with the autodock . . docking engine [ ] . thus, a user friendly grid service, gvss, was developed to conduct large-scale molecular docking easily [ ] . in this study, ns b-ns pro was expressed in pichia pastoris culture. this enzyme was used for in vitro enzyme inhibition assays against compounds selected from vs of ns pro using the gvss. the novel compound inhibitors were tested for their ic values and used for an enzyme kinetic study. . . expression of active ns b-ns pro in p. pastoris denv ns b-ns pro , fused to the α-factor secretion signal sequence and placed under the control of the methanol inducible alcohol oxidase promoter, was constructed to express the secreted protein in p. pastoris. the expressed ns b-ns pro was kda based on western blot using the anti-his antibody ( figure ). the ns b-ns pro proteolytic activity was monitored with fluorogenic tetrapeptide substrate containing the -amino- -methylcoumarin (amc) leaving group (benzoyl-norleucine-lysinearginine-arginine- -amino- -methylcoumarin, bachem, bubendorf, switzerland) for an increase in fluorescence at a λ ex = nm and λ em = nm at °c. no enzyme activity was detected before induction or in the p. pastoris negative control without ns b-ns pro gene (data not shown). ns b-ns pro activity increased steadily with increasing culture time (and induction time) and reached a final activity of u·l − after h of induction with methanol. ns b-ns pro was purified from p. pastoris culture supernatants. after dialysis to remove the potassium phosphate buffer, . mg of proteins remained in the culture medium supernatant. a total of . mg of ns b-ns pro was isolated by ammonium sulfate precipitation. the final preparation of ns b-ns pro was . mg following ion-exchange chromatography using a deae-sepharose column, which resulted in a . -fold purification ( table ). the recombinant protein was expressed in approximately . mg·l − of the induced culture medium and specific activity of the purified recombinant ns b-ns pro was . u·mg − . the michaelis-menten constant (k m ) derived with amc peptide substrates from the lineweaver-burk plot was . ± . μm ( figure s ). gvss was developed with the autodock . . docking engine [ ] . a java-based graphical user interface and grid application platform (gap) allows end-users to specify target and compound libraries, set up docking parameters, monitor docking jobs and computing resources, visualize and refine docking results, and download the final results. gvss was designed for conducting large-scale molecular docking more easily by providing a user-friendly grid service [ ] . a total of , compounds were used for the large-scale screening with gvss against ns pro protein (pdb accession number vbc [ ] ) by using , cpu per day, and the results were completed in days. a top scoring of % was selected from the first post-docking filtering strategy based on the free binding energy of the lowest energy conformation. thirty-six compounds were selected for in vitro assay based on their free energy binding (table s ) and h-bonds interactions with amino acid residues at the ns pro active site. each compound obtained after vs was tested in duplicate at μm for its ability to inhibit ns b-ns pro activity. the primary inhibition assay of the selected compounds is shown in table s . among them, seven compounds that showed higher inhibition activities against ns b-ns pro were selected for determining their ic values ( table ). their physicochemical properties were shown in table s . the chemical structure of each compound is depicted in table . the seven compounds displayed ns b-ns pro inhibitory activities with ic values of . ± . - . ± . μm ( table ) . compounds , , and , which displayed over % inhibition activity at μm, were subjected to kinetic characterization. inhibitory kinetic experiments were performed at different constant inhibitor concentrations and different substrate concentrations. lineweaver-burk and dixon plots were used to analyze the inhibition modes of these compounds. compounds , , and were analyzed and compared by molecular docking as potent binders to the ns pro active site pocket ( figure a) . figure b provides the details of the specific interactions between compound and ns pro ; carbon atoms of compound formed hydrophobic interactions with met , trp , val , arg , asp , pro , gly , asn , ala , and thr of ns pro . the the ns b-ns pro gene was synthesized for p. pastoris after codon optimalization (dna . , menlo park, ca, usa) based on the amino acid sequence of ns b-ns pro (ambl aaw . ) [ ] . the protein encoding the ns b-ns pro comprises ns b amino acid residues (amino acid residues - ), which are linked by a flexible ggggsgggg linker with the ns pro amino acid residues (amino acid residues - ) ( figure s ). the ns b-ns pro gene ( bp) was isolated from the vector (pj ) with the ns b-ns pro gene by cutting with ecori/noti (takara, shiga, japan) and was ligated into the ecori/noti-digested ppiczαa vector (invitrogen, carlsbad, ca, usa). this construct allowed secretion of ns pro into the culture medium because of the presence of an in frame n-terminal α-factor secretion signal peptide. the novel construct was named ppiczαa-ns b-ns pro and transformed into e. coli dh α (invitrogen) using a standard heat shock method. transformants harboring ppiczαa-ns b-ns pro were selected from low salt medium lb agar [ . % (w/v) tryptone, . % (w/v) yeast extract, . % (w/v) nacl, ph . ] containing μg·ml − zeocintm (invitrogen). the ppiczαa-ns b-ns pro plasmid was amplified in e. coli dh α, linearized by saci digestion, and transformed into p. pastoris gs using a modified lithium chloride method. the ppiczαa vector was also linearized by saci digestion and transformed into p. pastoris gs as a negative control strain. after transformation, cells were plated on ypd agar [ % (w/v) yeast extract, % (w/v) peptone, and % (w/v) glucose] containing µg zeocin·ml − and incubated for days at °c. positives were screened by polymerase chain reaction (pcr) with two sets of primers (bioneer, deajeon, korea): set one contained the ns b-ns pro primers [dv -f ( '-gaattcgctgatctta gtttag- ') and dv -r ( '-gcggccgctttctttctaaa- ')] and set two contained the α-factor and aox primers. the selected clone was inoculated for gene expression into ml bmgy [ % (w/v) yeast extract, % (w/v) peptone, mm potassium phosphate, ph . , . % (w/v) yeast nitrogen base, × − % (w/v) biotin, and % glycerol] in a ml flask and incubated at °c with shaking at rpm for h. the cells were harvested and resuspended in ml bmmy [ % (w/v) yeast extract, % (w/v) peptone, mm potassium phosphate, ph . , . % (v/v) yeast nitrogen base, × − % (w/v) biotin, and . % (v/v) methanol] medium in a l flask to an optical density at nm of . and then incubated at °c with shaking at rpm. to maintain methanol induction by the aox promoter, . % (v/v) methanol was fed every h during the fermentation period [ ] . culture aliquots ( ml each) were collected every h for analyses of ns b-ns pro expression and enzyme activity. ppiczαa-transformed p. pastoris x- proteins were expressed as a negative control. the yeast cells were then separated from the broth by centrifugation at , ×g for min at °c. the pellet was discarded, and the supernatant was exchanged with mm tris buffer (ph . ) using a kda membrane (millipore, billerica, ma, usa). the supernatant was used for ammonium sulfate fractionation (from %- %). the proteins obtained were dissolved in mm tris buffer (ph . ) and dialyzed against mm tris buffer (ph . ). concentrated protein sample was loaded onto a deae-sepharose column equilibrated with a low-salt buffer containing mm nacl and mm tris-hcl (ph . ). the recombinant protein was eluted with a salt gradient of - , mm nacl in a buffer containing mm tris-hcl (ph . ). one ml fractions were collected and analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (sds-page) and checked for enzyme activity. the fractions containing recombinant ns b-ns pro were pooled and concentrated using a , da molecular weight cut-off pierce ® concentrator. ns b-ns pro proteolytic activity was measured in a spectramax gemini xps apparatus (molecular devices, sunnyvale, ca, usa) (λex = nm, λem = nm) performed in a final volume of µl containing mm tris-hcl (ph . ) and µm of fluorogenic tetrapeptide substrate, benzoyl-norleucine-lysine-arginine-arginine- -amino- -methyl coumarin (bachem, bubendorf, switzerland), at °c using a fluorescence plate reader. the proteolytic reaction was monitored by an increase in fluorescence (relative fluorescence units min- ), which was subsequently converted to µm·min − from a standard -amino- -methyl coumarin (amc) calibration curve [ ] . one unit of ns b-ns pro activity was defined as the quantity of enzyme required to produce μm of amc per min at °c and ph . in mm tris-hcl. ns b-ns pro kinetic parameters were obtained using - μm amc peptide substrates in the fluorescent assay with a min measurement period. reaction responses were linear within this time period. the michaelis-menten constant (k m ) was calculated from a lineweaver-burk using the sigmaplot program (systat software, san diego, ca, usa). the vs process for denv has been described previously [ ] . the three-dimensional coordinates in the x-ray crystal structure of ns pro (pdb accession number vbc) [ ] obtained from the protein data bank [ , ] were used as the receptor model for the structural-based vs docking simulations. the ns pro docking library comprised , compounds from chemdiv inc. (san diego, ca, usa), a commercially available compound library, was used for vs. autodock version . . was used for the computational molecular docking simulation of flexible small molecules to rigid proteins using ligand and rigid proteins [ , ] . large-scale computations were conducted between vbc and , compounds using the gvss [ ] . important docking parameters for the lamarckian genetic algorithm were a population size of individuals, maximum of . million energy evaluations, maximum of , generations, mutation rate of . , crossover rate of . , and docking runs (each docking job produced docked conformations). the probability of performing a local search on an individual in the population was set to . and the maximum number of iterations per local search was set to . one percentage top scoring function was extracted and the potential hydrogen bond among ligands and key residues in the active site pocket of hma was identified using chimera software [ ] , and selected compounds for next step were analyzed for their hydrophobic and h-bond interactions using the ligplot program [ ] . among , compounds, were selected for testing in vitro inhibitory activity against ns b-ns pro based on hydrogen bond interactions [ , ] . each compound was tested in duplicate at a concentration of μm for its ability to inhibit ns b-ns pro activity during initial screening. each compound was dissolved in dimethyl sulfoxide (dmso) as a mm stock solution. assays were performed in a reaction mixture (final volume μl) containing . u enzymes, . μm amc peptide substrate, μm of inhibitor, and mm tris buffer, ph . . reactions were run for min at °c with continuous fluorescence monitoring using a spectramax gemini xps apparatus (molecular devices, eugene, or, usa) with excitation and fluorescence emission wavelengths of nm and nm, respectively. inhibitor kinetic studies were performed for compounds , , and against ns b-ns pro . the ns b-ns pro kinetic parameters were obtained using . , , . , . , and . μm amc peptide substrate in the fluorescent assay with a min measurement period. reaction responses were linear within this time period. the inhibitor concentration used was - μm for compound , - μm for compound , and - μm for compound . the type of inhibition was determined using lineweaver-burk plots, and k i values were determined with a dixon plot ( /v as a function of [i], inhibitor concentration) from different inhibitor concentrations [ , ] . thirty-six compounds from vs were tested for their inhibitory activities towards ns b-ns pro expressed from p. pastoris. the ic values of seven were . ± . - . ± . μm, and three of the compounds ( , , and ) were competitive inhibitors of ns b-ns pro with k i values of . ± . , . ± . , and . ± . μm, respectively. detailed docking simulation binding mode analyses showed that the inhibitors were stabilized by formation of h-bonds with catalytic residues and the establishment of hydrophobic interactions with amino acids in the ns pro active site pocket. more detailed inhibition studies are underway to elucidate the inhibitory mechanism of compounds , , and . supplementary materials can be accessed at: http://www.mdpi.com/ - / / / /s . activity of recombinant dengue virus ns protease in the presence of a truncated ns b co-factor, small peptide substrates, and inhibitors structure of dengue virus: implications for flavivirus organization, maturation, and fusion expression of dengue virus e glycoprotein domain iii in non-nicotine transgenic tobacco plants the global distribution and burden of dengue the dengue viruses biological characteristics of dengue virus and potential targets for drug design identification and biochemical characterization of small-molecule inhibitors of west nile virus serine protease by a high-throughput screen a novel, potential dengue virus ns b/ns protease inhibitor, bp , was discovered through high-throughput screening with dengue replicon cells use of parallel validation high-throughput screens to reduce false positives and identify novel dengue ns b-ns protease inhibitors peptide inhibitors of dengue virus ns protease. part : warhead peptide inhibitors of dengue virus ns protease. part : sar study of tetrapeptide aldehyde inhibitors synthesis and disulfide bond connectivity-activity studies of a kalata b -inspired cyclopeptide against dengue ns b-ns protease novel dengue virus-specific ns b/ns protease inhibitor, bp , discovered by a high-throughput screening assay structure-guided fragment-based in silico drug design of dengue protease inhibitors discovery of novel small molecule inhibitors of dengue viral ns b-ns protease using virtual screening and scaffold hopping inhibitory activity of cyclohexenyl chalcone derivatives and flavonoids of fingerroot, boesenbergia rotunda (l.), towards dengue- virus ns protease structure and anti-dengue virus activity of sulfated polysaccharide from a marine alga towards the design of antiviral inhibitors against flaviviruses: the case for the multifunctional ns protein from dengue virus as a target strategies for development of dengue virus inhibitors dengue: guidelines for diagnosis, treatment, prevention and control; world health organization protective efficacy of the recombinant, live-attenuated, cyd tetravalent dengue vaccine in thai schoolchildren: a randomised, controlled phase b trial dengue vaccine development: a % solution? recent development and application of virtual screening in drug discovery: an overview virtual screening and its integration with modern drug design technologies virtual screening identification of novel severe acute respiratory syndrome c-like protease inhibitors and in vitro confirmation gvss: a high throughput drug discovery service of avian flu and dengue fever for egee and euasiagrid crystal structure of the ns protease-helicase from dengue virus recombination in the nonstructural gene region in type dengue viruses efficient expression and purification of recombinant alcohol oxidase in pichia pastoris a fluorescence quenching assay to discriminate between specific and nonspecific inhibitors of dengue virus protease the protein data bank automated docking using a lamarckian genetic algorithm and an empirical binding free energy function inhibitory effects of epigallocatechin gallate and its glucoside on the human intestinal maltase inhibition ucsf chimera-a visualization system for exploratory research and analysis ligplot: a program to generate schematic diagrams of protein-ligand interactions discovery of novel inhibitors for human intestinal maltase: virtual screening in a wisdom environment and in vitro evaluation flavonoid-mediated inhibition of sars coronavirus c-like protease expressed in pichia pastoris sample availability: all tested compounds are commercially available from chemdiv inc this work was partially supported by the national research foundation of korea (nrf) grant funded by the korea government (mest) (no. ) . the authors declare no conflict of interest. key: cord- -j z q x authors: akaji, kenichi; konno, hiroyuki title: design and evaluation of anti-sars-coronavirus agents based on molecular interactions with the viral protease date: - - journal: molecules doi: . /molecules sha: doc_id: cord_uid: j z q x three types of new coronaviruses (covs) have been identified recently as the causative viruses for the severe pneumonia-like respiratory illnesses, severe acute respiratory syndrome (sars), middle east respiratory syndrome (mers), and corona-virus disease (covid- ). neither therapeutic agents nor vaccines have been developed to date, which is a major drawback in controlling the present global pandemic of covid- caused by sars coronavirus (sars-cov- ) and has resulted in more than , , cases and , deaths. each of the c-like ( cl) proteases of the three covs is essential for the proliferation of the covs, and an inhibitor of the cl protease ( cl(pro)) is thought to be an ideal therapeutic agent against sars, mers, or covid- . among these, sars-cov is the first corona-virus isolated and has been studied in detail since the first pandemic in . this article briefly reviews a series of studies on sars-cov, focusing on the development of inhibitors for the sars-cov cl(pro) based on molecular interactions with the cl protease. our recent approach, based on the structure-based rational design of a novel scaffold for sars-cov cl(pro) inhibitor, is also included. the achievements summarized in this short review would be useful for the design of a variety of novel inhibitors for corona-viruses, including sars-cov- . the term coronavirus (cov) is derived from the crown-like spikes on the surface of the virus. covs are enveloped positive-strand rna viruses that infect various vertebrates, including humans. in the s, two human coronaviruses, the human alpha coronavirus e (hcov- e) and the human beta coronavirus oc (hcov-oc ) were discovered as causative agents of disorders such as the common cold or respiratory illnesses of mild to moderate severity [ , ] . additionally, two human coronaviruses, the alpha coronavirus nl [ ] [ ] [ ] and the beta coronavirus hku [ , ] , were then identified in and . at the same time, a contrasting new human beta coronavirus causing life-threatening illnesses (severe acute respiratory syndrome (sars)-cov) was identified in [ ] [ ] [ ] . sars spread rapidly worldwide from its likely origin in southern china; the subsequent sars epidemic involved approximately patients, with more than fatalities. after nearly a decade, in , a new respiratory illness similar to sars, named middle east respiratory syndrome (mers), affected more than patients with a fatality rate of % [ , ] . even after these pandemics, no effective therapy has been developed for covs infections, and the present worldwide pandemic of corona-virus disease has resulted in more than , , cases and , deaths [ ] . the causative agent of covid- is a virus called sars-cov- since its single-stranded rna genome is % identical to that of sars-cov [ ] [ ] [ ] . sars-cov recognizes angiotensin-converting enzyme (ace ) [ ] on the host cell membrane as a specific receptor using the spike (s) protein of the virus. the interaction of the viral s protein with the host cell receptor is followed by membrane fusion of the virus and host cell, which transports the virus rna into the host cell. thus, an agent such as a soluble ace , or an antibody to this protein, could be a possible inhibitor of the virus-cell interactions. the viral genome injected into the host cell is then translated and processed to virus-derived structural proteins, including spike (s), envelope (e), membrane (m), and nucleocapsid (n) proteins, as well as nonstructural proteins that are used for the construction of viral particles. therefore, inhibition of the processing reaction, essential for the generation of viral proteins, is a promising approach for the suppression of viral proliferation. papain-like protease (pl pro ) and chymotrypsin-like protease (cl pro ) are the essential proteases for the processing reaction; pl pro cleaves the n-terminal region of the viral precursor protein at three sites whereas cl pro cleaves the c-terminal region of the precursor protein at sites. the features of the sars-cov cl pro , which cleaves the precursor protein at three times more sites than pl pro and has no known homologs in the host cell [ , ] , making it an ideal target for antiviral agents. in addition, the sequence of sars-cov- cl pro shares % homology with that of sars-cov cl pro , initially identified from the sars causative coronavirus [ ] . these findings indicate that the studies on the sars-cov cl pro are robust bases for designing therapeutic agents for covid- . in this short review article, the efforts to develop therapeutic agents for sars focusing on the inhibitors of sars-cov cl pro are described. instead of an exhaustive survey of the inhibitors [ ] , we provide an overview of several typical inhibitors, and our recent efforts for the rational design of new scaffolds are discussed based on the inhibitory mechanism and structural interactions with sars-cov cl pro . first, the protein chemistry of the target enzyme, sars-cov cl pro , is described in brief, as a basis for the structural analyses of protease-inhibitor interactions. the . kb positive-strand rna genome of sars-cov contains two open reading frames (orfs a and b) encoding two large replicative polyproteins, pp a ( kda) and pp ab ( kda) [ , ] . the expression of the orf b-encoded region of pp ab is involved in the orf produced by ribosomal frameshifting into the - frame just upstream of the orf a translation termination codon. the pp a and pp ab polyproteins are processed by two cysteine proteases: papain like protease (pl pro ) and c-like protease ( cl pro , also called main protease, m pro ). the name c-like is derived from picornavirus c proteases having a similar substrate specificity, which is encoded on the noncapsid region c [ ] . the sars-cov cl pro cleaves sites of the pp ab [ ] and is indispensable for viral replication, but is not found in the host cells, which makes the sars-cov cl pro an ideal target for antiviral agents [ , ] . sars-cov cl pro consists of amino acid residues and contains the catalytic dyad defined by his and cys . the n-terminal part ( - , domains i and ii) is composed of a two-β-barrel fold forming the chymotrypsin-like architecture as in picornavirus c pro . the substrate-binding site is located in a cleft between these two domains. the c-terminal part ( - , domain iii), containing five α-helices, adopts a globular fold. domain iii, connected to domain ii via a long loop, is a globular cluster of five helices, which is an essential architecture to hold the active dimer-structure of the protease (figure a ) [ ] . most cov-derived cl proteases recognize a conserved (leu/ile)-gln ↓ (ser, ala, or gly) core sequence as a canonical sequence (the cleavage site is indicated by ↓) at the active center ( figure b) [ ] . a comparison of the cleavage efficiencies of synthetic substrates containing the cleavage sites of sars cl pro confirmed that the most suitable substrate was the n-terminal site of sars cl pro itself, suggesting that sars cl pro cleaves itself most efficiently. a study using a fluorescent-dodecapeptide as an experimental substrate also revealed a similar tendency of sars-cov cl pro substrate recognition, as well as differences between sars-cov cl pro and mers-cov cl pro [ ] . moreover, recent studies on the sars-cov polyprotein processing using native mass spectrometry (ms) combined with collision-induced dissociation (cid) revealed a dynamic reaction, including substrate consumption, the rise and fall of intermediate products and complexation [ ] . sars cl pro is a cysteine protease in which cys and his form a catalytic dyad ( figure ). the initial step of the hydrolysis is the deprotonation of cys -thiol by the imidazole group of his to increase the nucleophilicity of the thiol group, which then attacks the substrate carbonyl carbon. therefore, sars cl pro shows the highest enzymatic activity at approximately ph , retaining the un-protonated imidazole form of his . after the nucleophilic attack, the c-terminal substrate fragment is released from the enzyme, leaving a covalently modified enzyme by thioester formation between the enzyme thiol group and the carbonyl group at the substrate scissile site. the thioester is then hydrolyzed by the nucleophilic attack of a deprotonated water molecule, and the corresponding c-terminal fragment of the substrate is released to generate the free enzyme. thus, compounds containing a functional group, a so-called "warhead" which interacts with the thiol group of cys , may be promising agents for inhibiting the catalytic potency of the cysteine protease. the first step of our inhibitor studies on sars-cov cl pro was the production of a mature protease on a scale of several milligrams per batch by a conventional expression procedure using e. coli to establish the assay protocol and crystallization procedure; however, the amount of the protease obtained from the initial trial was insufficient compared with the expected amount. careful examination of our expression procedure to yield mature sars-cov cl pro suggested that the mature cl pro was somehow susceptible to degradation, particularly considering arg located at the connecting loop between domains ii and iii. therefore, a mutated protease r i sars-cov cl pro , with an ile instead of an arg at position , was produced using e. coli to yield the expected amount with high homogeneity [ ] . of note, the mutation increased the stability and maintained almost the same three-dimensional structure (pdb code aw ) as that of native sars-cov cl pro . the use of the mutant r i sars-cov cl pro allowed the evaluation of enzymatic activity via conventional high-performance liquid chromatography (hplc) without the need to use any specific substrate with fluorescent substituents. after the sars-cov pandemic in , numerous studies have been conducted to identify inhibitors of sars-cov cl pro [ ] [ ] [ ] [ ] . these inhibitors are structurally classified into two types: peptide-mimetic inhibitors and nonpeptide small-molecule inhibitors. based on the inhibitory mechanism, these inhibitors can also be classified into irreversible (those that form a covalent bond with cl pro ), and reversible (noncovalent) inhibitors (those that compete with the substrate). a variety of first-generation inhibitors reported after the first outbreak of sars-cov provided valuable insights into further modifications through structure-based design. in the following sections, several typical inhibitory mechanisms, as well as our structural modification studies of peptide-mimetic to nonpeptide inhibitors, are described. a peptide-mimetic protease inhibitor generally contains a substrate-like sequence and a "warhead" interacting with the catalytic center of the target enzyme. in the inhibitors for sars-cov cl pro , the substrate-like sequence is designed by optimizing the specific interactions at the s ' to s sites of the substrate. the warhead interacting with the active center thiol of sars-cov cl pro should be an electrophilic functional group, considering the reaction mechanism of thiol protease described above. among the various electrophilic functional groups, a michael acceptor is one of the most commonly used warheads to effectively form a covalent bond by a nucleophilic attack with a thiol. a peptide-mimetic inhibitor containing a michael acceptor involves the replacement of a substrate's scissile amide bond with an appropriate michael acceptor. following the interaction with the active center of the sars-cov cl pro , the nucleophilic cys thiolate generated by a proton-withdrawing effect caused by his at the catalytic dyad promotes a typical , -addition to the α,β-unsaturated structure of the michael acceptor ( figure ). the resulting protonated his donates the proton to an unstable intermediate anion to form cl pro covalently bound to the inhibitor. thus, the michael acceptor type compound acts as a type of suicide substrate to abolish the catalytic activity of the enzyme by covalent modification. in a series of evaluations, using compound targeting rhinovirus cl pro as a starting compound, optimizations of the side-chain structures at p ' to p sites of the sars-cov substrate were conducted to develop sars-cov cl pro specific inhibitors [ ] [ ] [ ] (table ). the interactions of the sars-cov cl pro with some of these inhibitors were analyzed by x-ray crystallography (pdb codes zu and zu ), which confirmed that cys sulfur and the α-carbon of the michael acceptor at the p ' site formed a covalent bond of . Å. inhibitors with a five-membered lactam ring at the p site showed much stronger inhibitory activities than those with glutamine at the p site. for the p site substituent, a hydrophobic isopropyl substitute was preferred over a rigid and planar phenyl substitute (compounds vs ), indicating that the s pocket of sars-cov cl pro will accept rather large hydrophobic substituents. the p substituent is expected to be directed towards the bulk solvent and would have no interactions with the protease. unexpectedly strong inhibitory activities of compounds to were probably due to shifting of the n-terminal substituent toward the p site by a neighboring bulky tert-butyl group inducing hydrophobic interactions with cl pro . in another series of studies [ ] , the effect of methylene insertion between the reactive michael acceptor and the sessile site was investigated. the results clearly showed that no elongation of the michael acceptor structure toward the prime site was tolerated (table ) , which strongly suggests strict recognition of the prime-site structure. halomethyl ketone groups can form a covalent bond by an apparent alkylation caused by a thiolate anion, because the halomethyl group makes the adjacent ketone group more susceptible to a nucleophilic attack. the initial nucleophilic attack of a thiolate of cys of cl pro toward the carbonyl group of the halomethyl ketone leads to the reversible formation of a tetrahedral thiohemiketal that resembles the intermediate configuration in substrate cleavage ( figure ). the subsequent intramolecular rearrangement leads to the final product, a covalently modified inactive enzyme. a direct mechanism, in which the thiolate ion directly attacks the halomethyl carbonyl group to yield the alkylated enzyme, is less conceivable than the above intramolecular rearrangement pathway based on the experimental kinetics data. an initial study of a series of inhibitors containing p site n,n-dimethyl glutaminyl fluoromethyl ketone combined with different p site substituents revealed two possibilities. one was the effectiveness of a halogenated methyl group as a warhead, and another was the remarkable contribution of the p site substituent on the inhibitory activity, probably due to the specific interactions with cl pro (table a ) [ ] . the antiviral activity assessed by cytopathic effect (cpe) inhibition in sars-cov infected vero cell cultures, revealed that compound , with p -leu, can protect the cells against sars-cov infection with an ec of . µm, as well as the low toxicity in mice. the p leu of can be replaced by ile or val, resulting in slightly lower ec values (compounds and ) . in addition, these active compounds were inactive against rhinovirus type- in a cell-based assay, suggesting that they are specific for sars-cov. in contrast, removal of these p -site substituents abolished the inhibitory activity (compound ), indicating that the hydrophobic interactions at the p site were essential for potent inhibition. studies on another series of halomethyl ketone type inhibitors revealed additional possibilities regarding p site substituent and the inhibitory reaction mechanism (table b ) [ ] . the results indicated that hydrophobic p substituents, such as simple aromatic groups (phenyl and naphthyl) or an aliphatic bulky group, were tolerated as those of a rather complexed lactam ring, keto-glutamine analogs, or an α,β-unsaturated ester structure. these data also indicate that the corresponding s pocket of the sars-cov cl pro might accept a simple ring structure containing heteroatoms at this specific interaction site, which provides a clue to our design of a potent substrate-based inhibitor described later in this review. additional information obtained from these series of inhibitors is the effect of the halogen atom in the halomethyl ketone group. the rate of the final irreversible step of the inhibition pathway (k in figure ) is directly related to the acceptability of a nucleophilic attack for the eventual alkylation. the values of k of compound - ( . × − /s- . × − /s) indicated that the chloromethyl ketone was effective as a warhead, although the k value varied by two-fold depending on the bulkiness of the p site substituent. in contrast, the k value of bromomethyl ketone inhibitor was too small to be measured, indicating that the irreversible step was very slow. indeed, inhibitor behaved as a reversible inhibitor for several hours and irreversible inhibition of the enzyme activity was only noticed after a h incubation with inhibitor . thus, a time-dependent bimodal mode of inhibition for this inhibitor was suggested, in which the initial formation of a reversible complex (e-i*) occurred followed by rearrangement to an irreversible complex (e-i) after at least h incubation. in the crystal structure of sars-cov cl pro complexed with inhibitor (pdb code d ), a thioether bond ( . Å) between the carbon originally bound to bromine and the sulfur atom of cys was clearly detected supporting the above bimodal pathway. a trifluoromethyl group is another electron-withdrawing group that makes a neighboring carbonyl group susceptible to nucleophilic attack. initial studies of trifluoromethyl ketone type inhibitors ( table ) yielded in an n-protected tetrapeptide compound (ic = µm) [ ] . a lineweaver-burk plot obtained from the inhibition reaction by confirmed a competitive inhibition mode in the initial h reaction, whereas a time-dependent decrease in the enzymatic activity as a function of the inhibitor concentration was observed in the prolonged reaction. the slow formation of a covalent adduct caused by the nucleophilic attack of the thiol on the carbonyl carbon was assumed to provide a rational explanation of the kinetic results ( figure ). computational molecular modeling also rationalized the covalent bond formation, illustrating a transition state mimic in the substrate cleavage by sars-cov cl pro . table . inhibitory activity of substrate-based trifluoromethyl ketone compounds against sars-cov cl pro . further studies on the related groups using the trifluoromethyl equivalent revealed that a thiazolyl ketone group could increase the inhibitory activity ten-fold due to its high electrophilicity ( figure ). structural optimization at the p site combined with a benzothiazole warhead yielded inhibitors and , which both had ic values in the low nanomolar range [ , ] . a nitrile group used as a warhead in an anti-diabetes dpp inhibitor is another functional group that has been incorporated in the inhibitor for sars-cov cl pro (figure ) . among the nitrile-based tetrapeptide inhibitors with different n-protecting groups (mic: -methylisoxazole- -carboxyl, boc: tert-butyloxycarbonyl, and cbz: carboxybenzyl), cbz-avlq-cn was ten-times more potent (ic = . µm) than the other inhibitors [ ] . interestingly, the longer cbz-hexapeptide inhibitor, cbz-tsavlq-cn, was less active than the tetrapeptide inhibitor . the results suggest that the cbz group of might function as the p substituent interacting at the s pocket of sars-cov cl pro . the crystal structures of the sars-cov cl pro in complex with the nitrile-based inhibitor (pdb codes vb , vb , vb , and vb ) demonstrated that the inhibitor was covalently bonded to the thiol group of cys via the nitrile warhead ( figure ). in addition, the tetrapeptide inhibitor cbz-avlq-cn inhibited cl pro from human coronavirus strains such as e (ic = . µm), nl (ic = . µm), oc (ic = . µm), and hku (ic = . µm). in contrast, the same inhibitor had no observable inhibitory effect on caspases, which are common host cells proteins and effectors of apoptosis. these results suggest that the nitrile-based inhibitor is a specific and broad-spectrum inhibitor for coronavirus cl pro . peptide aldehyde with a substrate-like sequence has the potency to be an effective inhibitor for thiol proteases as an aldehyde group is another reactive electrophilic group involved in the nucleophilic addition of a thiol to yield hemithioacetal (figure ). initial studies on aldehyde-type inhibitors led to a potent inhibitor ( figure ; k i = nm) through the extensive structural optimization of a prototype inhibitor based on the structural evaluation of the sars-cov cl pro complexed with inhibitor (pdb code sn ) [ , ] . the analyses of the crystal structure of sars-cov cl pro complexed with the resulting inhibitor (pdb code gx ) revealed that the distance of the thiol sulfur atom of cys and the carbonyl carbon of the aldehyde was . Å, an equivalent distance to a covalent c-s bond. in addition, an oxyanion hole is expected to be formed by the coordination of the n-h of cys and gly at the s pocket, which would stabilize a tetrahedral intermediate of the nucleophilic addition reaction. the p and p substituents of inhibitor are held by hydrogen bonds at the corresponding s and s pockets of sars-cov cl pro , and the p site cyclohexyl group formed hydrophobic interactions at the s pocket of the protease. in our own studies on a series of substrate-based peptide aldehyde inhibitors, optimization of the p substituent was first conducted as the scissile site substituent generally shows the main influence on the enzyme specificity. as summarized in table , a series of replacement functional groups at the p site of the substrate-based inhibitor revealed imidazole was the most effective substituent (inhibitor , ic = . µm), which showed more than six-fold stronger inhibitory activity compared with (ic = µm). further structural analyses of the r i sars-cov cl pro complexed with inhibitor (pdb code aw ) revealed several noteworthy information regarding the interactions with cl pro : (i) the large hydrophobic s pocket was not fully occupied; (ii) the p substituent was directed outward of cl pro , resulting in no interactions with the protease; (iii) the s pocket was not fully occupied, and additional interactions via hydrogen bonds appeared feasible, and iv) the p substituent extended outside of cl pro and is not involved in the interactions with the cl pro . further structure optimization based on these inspections provided a potent tetra-peptide aldehyde inhibitor (ic = nm) [ ] . the x-ray crystal structure analysis of r i sars-cov cl pro complexed with (pdb code atw) confirmed the expected tight hydrophobic interactions formed by the p cyclohexyl group and hydrogen-bond interaction at the p site thr (figure ). table . optimization of a series of substrate-based peptide aldehyde inhibitors. in these x-ray structural analyses, the distance between the carbonyl carbon of the aldehyde and the thiol sulfur of the cys was . Å (figure ), and the electron density of the aldehyde group could be fitted to a carbonyl sp carbon. these findings suggest that the aldehyde of inhibitor interacts with the thiol of cl pro noncovalently. in addition, pre-incubation of inhibitor with cl pro prior to the addition of the substrate caused no change in the ic value compared with that obtained by simultaneous mixing of the inhibitor , cl pro , and the substrate, which suggests that no stable covalent bonds were formed between the inhibitor and cl pro . kinetic data obtained from lineweaver-burk plots confirmed that aldehyde type inhibitor functions as a competitive inhibitor without forming an irreversible covalent bond. figure . interactions of inhibitor with r i sars-cov cl pro . although the substrate-based inhibitors described above showed strong inhibitory potency, the in vivo instability of these peptide-based compound was expected to be a major drawback preventing use as an oral therapeutic agent. the development of nonpeptide small-molecular sars-cov cl pro inhibitors would be an approach to overcome these drawbacks. to this end, three different procedures have been generally employed: evaluation of natural products, high-throughput screening of synthetic compound libraries, and the rational design of a new scaffold of inhibitor. although a few examples of the inhibitors derived from natural products [ ] [ ] [ ] or virtual or high-throughput screening [ ] [ ] [ ] [ ] [ ] have been reported, examples of the structure-based rational design of a new scaffold are very limited, and the inhibitory activities are still moderate. nevertheless, the rational approach contributes largely to the design of a variety of novel scaffolds with high potential as a therapeutic agent for sars-cov -related diseases. in the following section, two rational approaches for the design of nonpeptide inhibitors derived from a peptide aldehyde are described. as an approach for nonpeptide inhibitor, serine was selected as an attractive scaffold as this commercially available proteinogenic amino acid has three functional groups available for modification: an alcohol, an amino, and a carboxylic acid group [ ] . each group of the serine can be orthogonally modified, which makes it feasible to introduce respective substituents corresponding to the p to p sites into the scaffold independently. as the parent inhibitor to be modified, the highly potent peptide aldehyde was used and substituents at the p , p , and p sites, as those are the sites that interact closely with the respective mode of sars-cov cl pro , were selected as the substituents to be introduced on the serine scaffold ( figure , compound ). an energetically favorable conformation mimicking the parent inhibitor was then sought by molecular mechanic's calculation performed with spartan combined with docking simulations by gold. the results of the initial trial suggested an unexpected positioning of the substituent, in which the cyclohexyl group at the serine amid carbonyl occupied the s pocket instead of the expected s pocket of cl pro . similarly, it was reported by bai et al. that a cinnamoyl derivative was expected to interact with cl pro at the s , s , and s pockets ( figure , compound ) based on simulations using autodock . . considering the contrasting results obtained from both simulations, a hybrid scaffold was designed combining bai's derivative and the serine derivative ( figure ). extensive sar studies of the hybrid scaffold focusing on the p ' and p substituents resulted in an optimized compound (ic = µm) as a novel small molecule inhibitor derived from serine. docking simulations of compound with sars-cov cl pro confirmed the expected interactions at the s ', s , and s pockets ( figure ). another approach starting from the same peptide inhibitor was based on closer inspection of the interactions with the sars cl pro [ ] . previous analyses of the crystal structure of the sars-cov cl pro complexed with inhibitor (pdb code atw) revealed that a cyclohexyl substituent of cyclohexylalanine (cha) at the p site was well packed in the hydrophobic s pocket and formed a critical interaction to make a highly potent inhibitor. detailed structural evaluation of this hydrophobic pocket revealed that the p site cyclohexyl ring was rather close to the peptide backbone. in the crystal structure, the distance of the position carbon (c ) of the cyclohexyl ring to the α-amide nitrogen of cha at the p site was estimated to be . Å. the distance is approximately equal to the sum of two covalent bonds, and the connection of the two atoms by a methylene linker appears to be feasible, yielding a novel fused ring structure, a decahydroisoquinoline scaffold, that acts as a hydrophobic substituent at the p position ( figure ). in addition, this fused ring scaffold can be a core scaffold to arrange the p site imidazole and active site functional aldehyde at the required positions. the acyl substituent on the nitrogen atom in the decahydroisoquinoline scaffold is expected to be a new substituent providing additional interactions with cl pro . to assess the validity of the above design, all possible configurations at the fused ring of the decahydroisoqunolin scaffold were separately synthesized by a combination of enantiomer resolution and pd-catalyzed stereoselective cyclization reaction. each synthesized derivative showed moderate but clear inhibitory activity, although the potency was different depending on the configuration. a specific configuration of inhibitor (figure ) was the most effective configuration, confirming the utility of the decahydroisoquinoline scaffold as a hydrophobic core. in addition, the acyl group on the nitrogen atom of the scaffold showed a limited effect on the inhibitory activity. x-ray crystal analyses of sars-cov cl pro complexed with inhibitor and related compounds (pdb codes tww, twy, and wy ) rationalized the effective interactions causing these differences. the distance between the carbonyl carbon of the aldehyde of and thiol sulfur of cys was . Å, suggesting that the decahydroisoquinoline inhibitor was a competitive inhibitor like the parent peptide inhibitor ( figure ) . the p site imidazole of located at the s pocket and the nitrogen atom of the imidazole formed hydrogen bonds, similar to the parent inhibitor . the decahydroisoquinoline scaffold of adopted a trans-fused configuration and occupied most of the s pocket of cl pro directing the p site imidazole and warhead aldehyde into the active center. in contrast, the acyl substituent bound to the nitrogen of the fused ring was located on the surface of cl pro , where an additional interaction with the protease might be possible. to improve the moderate inhibitory activity of above decahydroisoquinoline-type inhibitor , the overall interaction mode of was compared with that of the parent peptide inhibitor . an overlay of both interaction modes suggested that the nonprime site interactions of the parent inhibitor were missing in the interaction mode of the decahydroisoquinoline-type inhibitor ( figure ). a detailed evaluation of the overlay of above both inhibitors complexed with cl pro revealed that the distance between the α-nitrogen of cha in the peptide aldehyde and -position of the decahydroisoquinoline scaffold was estimated to be . Å, a distance equivalent to a covalent bond. thus, a dipeptide unit as a nonprime site substitute was introduced at the -position of the decahydroisoquinoline scaffold to yield a novel prototype inhibitor ( figure ) [ ] . the synthesized inhibitor showed . times greater inhibitory activity (ic = µm) than the initial decahydroisoquinoline-type inhibitor , which strongly suggested the positive effect of the -position substituent, probably through interaction with the nonprime site pocket of cl pro . structural optimization of this nonprime site substituent should be the next step to create a novel lead structure of nonpeptide small-molecule inhibitors based on a rational design. the inhibitor design briefly surveyed in this short review is a potential starting point for the development of anti-sars-cov agents; of note, most inhibitors described in this review have inhibitory potency against cov as well as good physicochemical and pharmacodynamics properties necessary for in vivo use. indeed, the inhibitory potencies against cov in cells were confirmed for several compounds, including peptide-based inhibitors and small-molecule inhibitors. the low toxicity for cells was also examined for a few compounds, although further in vivo studies are required. reevaluation of natural products such as flavonoids or indian medicinal plants is an alternative approach to design clinically useful inhibitors for sars-cov cl pro [ , ] . enzymatic evaluations of the cl protease of sars and mers is another base for the development of therapeutic agents for sars related respiratory diseases such as covid- . recent studies on the catalytic mechanism of the sars-cov cl pro and mers-cov cl pro revealed detailed insights regarding the difference in catalytic efficiencies between cl pro from sars-cov and mers-cov, and identified a potential allosteric site for inhibitor design. these findings should contribute to the development of therapeutic agents based on sars-cov- cl pro . in addition, the crystal structure of sars-cov- cl pro revealed the potential effectiveness of an inhibitor designed on the basis of sars-cov cl pro ; of notes, this may be another basis for the development of therapeutic agents for covid- [ ] . the rational design of a novel scaffold starting from a peptide-based inhibitor discussed in this review would be an alternative way to design novel cysteine protease inhibitors. although compounds showing antiviral activity can be discovered by screening a library composed of approved drugs or therapeutics in clinical development, as used in the present measures devised to combat covid , the development of novel and specific anti-sars cov inhibitors based on the achievements described in this review should be an alternative approach to consider, in the context of the treatment of sars-related infectious diseases. additionally, polymerase inhibitors and others should be considered, to target different vital routes, and effectively combat sars-cov- since multiple targets are useful to avoid resistance. author contributions: both authors (k.a. and h.k.) equally contributed to this article. all authors have read and agreed to the published version of the manuscript. funding: this work was supported, in part, by a grant-in-aid for scientific research h given to ka from the japan society for the promotion of science. the authors declare no conflict of interest. cultivation of viruses from a high proportion of patients with colds a new virus isolated from the human respiratory tract identification of a new human coronavirus human coronavirus nl new human coronavirus, hcov-nl , associated with severe lower respiratory tract disease in australia characterization and complete genome sequence of a novel coronavirus, coronavirus hku , from patients with pneumonia clinical and molecular epidemiological features of coronavirus hku -associated community-acquired pneumonia a major outbreak of severe acute respiratory syndrome in hong kong identification of a novel coronavirus in patients with severe acute respiratory syndrome a novel coronavirus associated with severe acute respiratory syndrome the newly emerged sars-like coronavirus hcov-emc also has an "achilles' heel": current effective inhibitor targeting a c-like protease assessing activity and inhibition of middle east respiratory syndrome coronavirus papain-like and c-like proteases using luciferase-based biosensors world health organization. covid- situation reprorts a pneumonia outbreak associated with a new coronavirus of probable bat origin a new coronavirus associated with human respiratory disease in china systematic comparison of two animal-to-human transmitted human coronaviruses: sars-cov- and sars-cov angiotensin-converting enzyme is a functional receptor for the sars coronavirus coronavirus main proteinase ( cl pro ) structure: basis for design of anti-sars drugs biosynthesis, purification, and substrate specificity of severe acute respiratory syndrome coronavirus c-like proteinase crystal structure of sars-cov- main protease provides a basis for design of improved α-ketoamide inhibitors an overview of severe acute respiratory syndrome-coronavirus (sars-cov) cl protease inhibitors: peptidomimetics and small molecule chemotherapy characterization of a novel coronavirus associated with severe acute respiratory syndrome the genome sequence of the sars-associated coronavirus cleavage of small peptides in vitro by human rhinovirus c protease expressed in escherichia coli mechanisms and enzymes involved in sars coronavirus genome expression dissection study on the severe acute respiratory syndrome c-like protease reveals the critical role of the extra domain in dimerization of the enzyme: defining the extra domain as a new target for design of highly specific protease inhibitors c-like proteinase from sars coronavirus catalyzes substrate hydrolysis by a general base mechanism comprehensive insights into the catalytic mechanism of middle east respiratory syndrome c-like protease and severe acute respiratory syndrome c-like protease processing of the sars-cov pp a/ab nsp - region evaluation of peptide-aldehyde inhibitors using r i mutant of sars cl protease as a proteolysis-resistant mutant synthesis and evaluation of keto-glutamine analogues as potent inhibitors of severe acute respiratory syndrome cl pro design and synthesis of peptidomimetic severe acute respiratory syndrome chymotrypsin-like protease inhibitors cinanserin is an inhibitor of the c-like proteinase of severe acute respiratory syndrome coronavirus and strongly reduces virus replication in vitro high-throughput screening identifies inhibitors of the sars coronavirus main proteinase inhibition of the severe acute respiratory syndrome cl protease by peptidomimetic alpha,beta-unsaturated esters synthesis, crystal structure, structure-activity relationships, and antiviral activity of a potent sars coronavirus cl protease inhibitor structural basis of inhibition specificities of c and c-like proteases by zinc-coordinating and peptidomimetic compounds structure-based design, synthesis, and evaluation of peptide-mimetic sars cl protease inhibitors design and synthesis of dipeptidyl glutaminyl fluoromethyl ketones as potent severe acute respiratory syndrome coronovirus (sars-cov) inhibitors development of broad-spectrum halomethyl ketone inhibitors against coronavirus main protease design, synthesis, and evaluation of trifluoromethyl ketones as inhibitors of sars-cov cl protease development of potent dipeptide-type sars-cov cl protease inhibitors with novel p scaffolds: design, synthesis, biological evaluation, and docking studies design, synthesis, and biological evaluation of novel dipeptide-type sars-cov cl protease inhibitors: structure-activity relationship study design, synthesis and crystallographic analysis of nitrile-based broad-spectrum peptidomimetic inhibitors for coronavirus c-like proteases peptide aldehyde inhibitors challenge the substrate specificity of the sars-coronavirus main protease synthesis, modification and docking studies of -sulfonyl isatin derivatives as sars-cov c-like protease inhibitors chalcones isolated from angelica keiskei inhibit cysteine proteases of sars-cov evaluation of polyphenols from broussonetia papyrifera as coronavirus protease inhibitors virtual screening identification of novel severe acute respiratory syndrome c-like protease inhibitors and in vitro confirmation structure-based virtual screening against sars- cl(pro) to identify novel non-peptidic hits identification of novel drug scaffolds for inhibition of sars-cov -chymotrypsin-like protease using virtual and high-throughput screenings discovery, synthesis, and structure-based optimization of a series of n-(tert-butyl)- -(n-arylamido)- -(pyridin- -yl) acetamides (ml ) as potent noncovalent small molecule inhibitors of the severe acute respiratory syndrome coronavirus (sars-cov) cl protease discovery of n-(benzo[ , , ]triazol- -yl)-n-(benzyl)acetamido)phenyl) carboxamides as severe acute respiratory syndrome coronavirus (sars-cov) cl pro inhibitors: identification of ml and noncovalent nanomolar inhibitors with an induced-fit binding design and synthesis of a series of serine derivatives as small molecule inhibitors of the sars coronavirus cl protease fused-ring structure of decahydroisoquinolin as a novel scaffold for sars cl protease inhibitors evaluation of a non-prime site substituent and warheads combined with a decahydroisoquinolin scaffold as a sars cl protease inhibitor inhibition of sars-cov cl protease by flavonoids covid- : a promising cure for the global panic structural plasticity of sars-cov- cl m pro active site cavity revealed by room temperature x-ray crystallography this article is an open access article distributed under the terms and conditions of the creative commons attribution (cc by) license key: cord- -dxl wvcx authors: liu, kelly y. p.; hu, shuiqing; chan, ben c. l.; wat, elaine c. l.; lau, clara b. s.; hon, kam l.; fung, kwok p.; leung, ping c.; hui, patrick c. l.; lam, christopher w. k.; wong, chun k. title: anti-inflammatory and anti-allergic activities of pentaherb formula, moutan cortex (danpi) and gallic acid date: - - journal: molecules doi: . /molecules sha: doc_id: cord_uid: dxl wvcx pentaherb formula (phf) has been proven to improve the quality of life of children with atopic dermatitis without side effects. the aim of this study was to elucidate the potential anti-inflammatory and anti-allergic activities of phf, moutan cortex (danpi/dp) and gallic acid (ga) using human basophils (ku cells), which are crucial effector cells in allergic inflammation. phf, dp and ga could significantly suppress the expression of allergic inflammatory cytokine il- -upregulated intercellular adhesion molecule (icam)- , and the release of chemokines ccl , ccl , cxcl and inflammatory cytokine il- from ku cells (all p < . ). with the combined use of dexamethasone ( . μg/ml) and ga ( μg/ml), the suppression of icam- expression and ccl and il- release of il- -activated ku cells were significantly greater than the use of ga alone (all p < . ). the suppression of the il- -induced activation of intracellular signalling molecules p mitogen activated protein kinase, nuclear factor-κb and c-jun amino-terminal kinase in ga-treated ku cells could be the underlying mechanism for the suppression on icam- , chemokines and cytokines. the combined use of dexamethasone with the natural products phf or dp or ga might therefore enhance the development of a novel therapeutic modality for allergic inflammatory diseases with high potency and fewer side effects. the prevalence of allergic diseases such as allergic rhinitis, asthma and atopic dermatitis (ad) has been increasing dramatically in both developed and developing countries [ ] [ ] [ ] . the world health organization (who) estimated that in , million people in the world population of . billion (about %) have allergic asthma, adversely affecting their quality of life and the socio-economic welfare of the society [ ] . ad is one of the most frequent chronic inflammatory skin diseases, affecting up to % of children and - % of adults worldwide [ ] . also named eczema, ad is the most common type of chronic allergic skin diseases. it can occur at any age, affecting infants, children and adults. the worldwide prevalence of ad is increasing with about % of cases occurring before the age of [ ] . ad infants are prone to subsequently develop allergic asthma and allergic rhinitis during childhood [ , , ] . ad is a long-lasting skin disorder; patients may suffer from episodic exacerbations and remissions during their life [ ] . typical symptoms of ad include extremely itchy, inflamed and dry skin, the inflamed area can be red, swollen, cracked, scaled, webby and crusted [ , ] . there is no definitive cure for ad and the current effective treatment involves topical application of immunosuppressive steroids with undesirable side effects. therefore, there has been a rising interest in the development of safer and non-steroid immunomodulatory formulas for the treatment of ad. traditional chinese medicine (tcm) has become a more accepted and increasingly used modality for immunomodulation, especially in asia. our previous clinical trial has shown that children with moderate-to-severe ad manifested significantly improved quality of life after a -week treatment with tcm pentaherb formula (phf) concurrently with reduced use of topical corticosteroids [ ] . such a finding suggested that phf could potentially be an alternative adjunct therapy for ad. phf contains five herbs: lonicerae flos (jinyinhua), menthae herba (bohe), moutan cortex (danpi/dp), atractylodis rhizoma (cangzhu), and phellodendri cortex (huangbai) in a ratio of : : : : [ ] . our group and others have previously shown that different herbs in phf possessed anti-inflammatory activities and exhibit potential therapeutic efficacy on inflammatory diseases such as rheumatoid arthritis [ ] [ ] [ ] . therefore, phf has been formulated to further evaluate its cellular mechanisms for anti-inflammatory activities. phf with an increased ratio of danpi exhibited a greater inhibitory effect on the release of the cytokines interleukin (il)- and il- from hmc- human mast cells [ ] . gallic acid (ga, , , trihydroxybenzoic acid), a potent anti-oxidant found in witch hazel and tea leaves, is one of the main ingredients in danpi [ ] . ga has been shown to possess anti-tumorigenic effect and anti-inflammatory activity in both nude mice and human cell lines [ ] [ ] [ ] [ ] [ ] . il- is a novel member of the il- cytokine family that is released passively during cell necrosis and tissue damage [ ] [ ] [ ] [ ] . it has been characterized as a potent pro-inflammatory th cytokine that acts on immune cells such as mast cells, eosinophils and basophils in allergic inflammation [ ] [ ] [ ] [ ] . in addition, the inflamed skin lesions of ad patients have both elevated protein and mrna expression of il- compared to normal subjects [ ] [ ] [ ] [ ] . tissue inflammation consists of two essential steps: the adherence of leukocytes to vascular endothelial cells before their migration to inflammatory sites via diapedesis, and release of allergic inflammatory chemokines and cytokines by the leukocytes [ ] [ ] [ ] . the aim of the present study was to elucidate the mechanisms of the in vitro anti-inflammatory and anti-allergic activities of phf, danpi and ga via their modulation of: (i) expression of cell surface adhesion molecules and (ii) the release of chemokines and cytokines from allergy-related alarmin il- -activated human basophils, which are crucial effector cells of allergic inflammation in allergic asthma and ad [ ] . circulating basophils can be recruited to the inflammatory tissues in allergic disorders including allergic asthma, ad and allergic rhinitis [ ] . during asthma exacerbation and in response to allergen inhalation challenge, basophils markedly infiltrate into allergic inflammatory sites [ ] . since human basophils represent % of peripheral blood leukocytes and only . × cells can be purified from × peripheral blood mononuclear cells, it is not feasible to obtain enough number of basophils for the present in vitro studies. according to our previous experiments, we found that the representative ku basophilic cells showed similar results for the expression of icam- and induction of chemokines and cytokines (data not shown). therefore, like in our previous publication, we adopted the representative ku basophilic cells for the present mechanistic study [ ] . il- -activated ku cells were treated with various concentrations of phf, dp and ga, followed by measurement of cell surface expression of adhesion molecule. as shown in figure , phf, dp and ga could significantly suppress the expression of il- -induced intercellular adhesion molecule (icam)- on ku cells in a dose-dependent manner (all p < . ). the reduced icam- expression on il- activated ku cells suggests that phf, dp and ga may inhibit the endothelial transmigration of basophiles to the inflamed sites, and dampen the subsequent allergic responses [ ] [ ] [ ] . chemokine ccl (monocyte chemotactic protein- /mcp- ), ccl (regulated and normal t cell expressed and secreted/rantes) and cxcl (il- ) are known to be associated with inflammation [ ] [ ] [ ] [ ] . ccl is known to be an ad-associated chemokine that recruits dendritic cells, monocytes and memory t cells from the circulation to inflammatory sites [ ] . cxcl is a potent chemo-attractant for immune cells, especially neutrophils, to the inflamed sites where ccl is chemotactic for t helper type (th ) cells, eosinophils and basophils [ ] [ ] [ ] [ ] . suppressive effect of icam- expression on il- -activated human basophilic ku cells treated with (a) phf ( , and , μg/ml), (b) dp ( , and , μg/ml), (c) ga ( , and μg/ml) for h. as shown in bar charts, surface expressions of icam- on , ku cells are expressed as the mean plus sem of mfi and normalized by subtracting appropriate isotypic control of three independent experiments. * p < . , ** p < . , *** p < . . p - : phf - , μg/ml, dp - : danpi - , μg/ml, ga - : ga - μg/ml. figure shows that the release of ccl , ccl and cxcl from il- -activated ku cells was significantly suppressed by the treatment with phf, dp and ga. there was a dose-dependent suppression of ccl release by phf and ga ( figure a ). the suppressed release of these inflammation-related chemokines from il- -activated ku cells may reduce the infiltration of immune cells such as th cells, eosinophils, basophils and macrophages to the inflamed sites, thereby lessening the subsequent inflammation and other allergic responses. th cells including th and th which play important roles for the cell-mediated immunity and humoral immunity, respectively [ ] . since th cells is responsible for the ige production for the sensitization and activation of mast cells in type i hypersensitivity, the suppression of the release of th chemokine ccl by phf, dp and ga can subsequently reduce the infiltration of th cells into the inflammatory site and hence the allergic inflammation [ , ] . suppressive effect on (a) ccl , (b) ccl and (c) cxcl release from il- activated human basophilic ku cells treated with phf ( , and , μg/ml), dp ( , and , μg/ml) or ga ( , and μg/ml) for h. release of chemokines in culture supernatants was determined by cba. * p < . , ** p < . , *** p < . . p - : phf - , μg/ml, dp - : danpi - , μg/ml, ga - : ga - μg/ml. (c) ga the proinflammatory cytokine il- is produced by t cells and macrophages at the inflammatory site. it plays an important role in the regulation of immune responses, immediate and late-phase allergic inflammation, and th cell activation [ ] [ ] [ ] . the combined effects of il- with soluble il- receptor-α can lead to the transition of acute to chronic inflammation by means of switching transmigration from neutrophils to monocytes/macrophages into the inflamed site [ ] [ ] [ ] . as shown in figure , the dose-dependent suppression of il- release by phf, dp and ga from il- -activated ku cells may reduce the progression from acute to chronic inflammation in allergic diseases. . suppressive effect of inflammatory cytokine il- release from il- -activated human basophilic ku cells treated with phf ( , and , μg/ml), dp ( , and , μg/ml) or ga ( and μg/ml) for h. release of il- in culture supernatants was determined by cba. * p < . , ** p < . , *** p < . . p - : phf - , μg/ml, dp - : dp - , μg/ml, ga - : ga - μg/ml. [ , , ] . both p mapk and jnk play crucial roles in regulating cell apoptosis, cell proliferation and inflammatory cytokines release [ ] [ ] [ ] [ ] . nf-κb, bound by inhibitory protein iκbα, is a key regulator of pro-inflammatory mediator expression and inflammatory cytokines induction in lymphocytes, granulocytes, macrophages and fibroblasts [ , [ ] [ ] [ ] [ ] [ ] . the level of the phosphorylation of iκbα is directly related to the release of active nf-κb and the subsequent translocation into the nucleus to activate the genes transcription for cytokines, chemokines and adhesion molecules. figure shows that the il- -upregulated phosphorylation of p mapk, iκbα and jnk, in ku cells was significantly inhibited by ga (all p < . ). however, ga did not exhibit any effect on the phosphorylation of extracellular signal regulated kinase (erk) (data not shown). therefore, the reduction of the phosphorylation of p mapk, iκbα and jnk by ga could be the underlying intracellular mechanisms accounting for the suppression of icam- expression and release of ccl , ccl , cxcl and il- . in figure a , since ga at dose m could potently suppress the il- -induced phosphorylation of p mapk, ga ( m) did not show any significant further suppression on the il- -induced phosphorylation of p mapk (p > . ). however, the mean of the phosphorylation of p mapk of ga ( m) is still lower than that of ga ( m). since phf and dp are complex heterogeneous mixtures but not single and purified compound, it could be difficult to interpret their effect on the individual intracellular signaling pathways. therefore, phf and danpi were not investigated for their effects on intracellular signaling molecules in the present study. results are shown as the mean plus sem of mfi and normalized by subtracting appropriate isotypic control of three independent experiments in bar charts. representative histograms of intracellular expression of phosphor-signaling molecules in ku cells were also shown. * p < . , ** p < . , *** p < . . p - : phf - , μg/ml, dp - : dp - , μg/ml, ga - : ga - μg/ml. dexamethasone, a potent synthetic member of the glucocorticoid class of steroid drugs, is usually prescribed for patients with ad for resolving the tissue inflammation. it has been reported that dexamethasone can promote eosinophilic apoptosis [ ] and inhibit basophilic migration [ ] . we investigated the expression of adhesion molecules, chemokines and cytokines in il- activated ku cells treated with the combined use of dexamethasone at various concentrations ( . , . and μg/ml) and the natural products (phf, dp and ga). as shown in figure , the combined use of a low concentration of dexamethasone ( . μg/ml) with ga ( μg/ml) could further suppress icam- , ccl and il- expression of ku cells compared to the use of ga ( μg/ml) or dexamethasone ( . μg/ml) alone. however, higher concentrations of dexamethasone ( . and μg/ml) together with ga ( μg/ml) did not exhibit any significant suppression of icam- , ccl and il- comparing with ga or dexamethasone alone (data not shown). in addition, phf and dp ( μg/ml) showed similar enhanced suppressive effect on the il- -induced expression of icam- , ccl and il- (data not shown). the above results suggest that phf, dp or ga could be used together with a lower dose of dexamethasone for the resolution of inflammation in patients with allergic diseases such as ad, thereby promoting the therapeutic efficacy and lessening the adverse side effect of steroidal drugs. phf and dp were supplied by the institute of chinese medicine, the chinese university of hong kong, from which water extract was prepared by the hong kong biotechnology institute, hong kong. for phf extract, lonicerae flos ( g), menthae herba ( g), moutan cortex ( g), atractylodis rhizoma ( g) and phellodendri cortex ( g) were refluxed in distilled water ( ml) of at °c for h and the whole process was repeated twice. the three batches of water extracts were mixed together and centrifuged to remove the herbal debris. finally, the combined extract was vacuum dried to form herbal powder which was stored in desiccators until use. these processes fulfilled the good manufacturing practice according to the australian therapeutic goods administration standard. ga was purchased from sigma-aldrich. the dosages of dp used in this study were - , µg/ml. based on our previous high-performance liquid chromatography (hplc) analysis, the amount of ga present in dp was . % w/w. hence, the dosages of ga used for the experiments were in the range of - µg/ml. the phf, danpi and ga used in this in vitro study were weighed and dissolved in pyrogen-free phosphate buffered saline (pbs) at °c for h, centrifuged at , rpm for min. the supernatants were filtered through . μm polyethersulphone filter. if there was any herbal debris, it was dried at °c overnight for weighing. the solutions were stored at °c. the concentration of the solution = (weighted mass before dissolving in pbs − mass of herbal debris) / volume of pbs used. hplc analyses were performed using hewlett packard agilent series hplc system, equipped with g a als autosampler and g a diode array detector (agilent technologies, santa clara, ca, usa). calibration curves for ga was prepared using standard solutions containing , , , , and μg/ml ga. danpi aqueous extract was prepared with double distilled water ( μg/ml). sample solution was injected onto an ultrasphere ods column ( × . mm i.d., particle size μm, beckman instrument inc., fullerton, ca, usa). all solvents were pre-filtered with . μm millipore filter disk (millipore corp., billerica, ma, usa) and de-gassed. a gradient elution was carried out using the following solvent systems: mobile phase a-acetonitrile; mobile phase b-double distilled water/phosphoric acid ( . / . ; v/v). the analyses were performed for min. the flow rate used was . ml/min and detection was performed at nm. each sample ( μl) was injected into the column after filtration through a . μm filter disk. the system was monitored by a computer equipped with the karat software (beckman) for data collection, integration and analysis of ga quantity. the human basophilic cell line ku cells (american type culture collection, manassas, va, usa) were maintained in rpmi- medium supplemented with % (v/v) fbs (rpmi- complete medium). cells were incubated at °c in atmospheric air enriched with % (v/v) co . at - % cell confluence, cells dispersed in rpmi- complete medium to a final cell concentration of × cells/ml for further treatment. ku cells were seeded in -well plate at a density of × cells in μl rpmi- complete medium per well. they were then pre-activated with il- ( ng) for h followed by treatment with various concentrations of phf, dp or ga in the presence of il- for further h. cells were then used for the detection of expression of adhesion molecules, while the supernatants collected was subjected to bd cytometric beads array (cba) assays for allergic inflammation-related chemokines and cytokine (see below) using flow cytometry. for the analysis of adhesion molecules, cells were washed and suspended in cold pbs followed by blocking with % human pooled serum for min at °c. they were incubated with fitc-conjugated mouse anti-human icam- or mouse igg isotype (r&d systems) for min at °c in the dark. after washing, the cells were re-suspended in pbs for flow cytometric analysis. for the analysis of chemokines and cytokines, supernatants obtained were subjected for the bead-based multiplex cba immunoassay with human inflammatory kit (cxcl , il- β, il- , il- , tnf-α and il- p ) and human chemokine kit (cxcl , ccl , cxcl , ccl and cxcl ). the capture beads contained monoclonal antibodies against individual cytokine and chemokine. supernatant ( μl) was incubated with different capture bead mixtures ( μl) and phycoerythrinconjugated detection antibodies ( μl) for three hours at room temperature with constant shaking. after incubation, the beads were washed and re-suspended in wash buffer. the samples were subsequently analyzed by bd facscalibur flow cytometer (bd biosciences, san jose, ca, usa) with bd cba analysis software. for the analysis of intracellular expression of phosphorylated signaling molecules, × cells seeded in -well culture plate were pre-activated with il- ( ng) for min or h followed by -min treatment with ga. after cells were fixed with % paraformaldehyde for min at °c and centrifugation, cells were permeabilized in ice-cold methanol for min and then stained with fitc-conjugated mouse anti-human phosphorylated erk / , phosphorylated jnk, phosphorylated p mapk, phosphorylated ibα or mouse igg antibodies (bd pharmingen) for min at °c in the dark. cells were then washed, resuspended and subjected to analysis. expressions of surface adhesion molecule, intracellular phosphorylated signaling molecules and chemokines and cytokines were analyzed by flow cytometry (facscalibur) as mean fluorescence intensity (mfi), which includes both the changes of target molecule expression in individual cells and the percentage of cells expressing the target molecules. all data are expressed as the means ± sem. differences between groups were assessed by students's t-test. a probability p < . was considered significantly different. when anova indicated a significant difference, the bonferroni post hoc test was then used to assess the difference between groups. all analyses were performed using the statistical package for the social sciences statistical software for windows, version . (spss inc., chicago, il, usa). since the immunopathological mechanism of allergic diseases such as ad is complex, involving both cellular and humoral components of the immune system [ ] , the above results suggest that tcm pentaherb formula and its herbal component danpi and bioactive compound gallic acid can suppress inflammation elicited by il- -activated allergic inflammation-related effector cells, namely, the basophils, by the inhibition of the expression of adhesion molecules and allergy-related cytokines and chemokine. our present study has also provided evidence for the intracellular mechanisms including the mpak and nf-b pathways by which ga could modulate the function of basophils. since corticosteroids are not present in pentaherb formula [ ] , the combined use of lower dose of dexamethasone with natural product pentaherb formula or danpi or gallic acid may enhance the development of the novel therapeutic modality for allergic inflammatory diseases such as ad with high potency and less side effects. advances in allergic skin disease, anaphylaxis, and hypersensitivity reactions to foods, drugs, and insects in halting the allergic march allergic rhinitis and its impact on asthma in asia pacific and the aria update clinical practice. atopic dermatitis epidemiology of atopic dermatitis and atopic march in children development of allergies and asthma in infants and young children with atopic dermatitis-a prospective follow-up to years of age phase three study group. worldwide time trends in the prevalence of symptoms of asthma, allergic rhinoconjunctivitis, and eczema in childhood: isaac phases one and three repeat multicountry cross-sectional surveys atopic dermatitis organizer (ado) guideline for children efficacy and tolerability of a chinese herbal medicine concoction for treatment of atopic dermatitis: a randomized, double-blind, placebo-controlled study one-year evaluation of radiographic progress in patients with rheumatoid arthritis treated by qingre huoxue decoction effects of salviae mitiorrhizae and cortex moutan extract on the rat heart after myocardial infarction: a proteomic study anti-inflammatory activities of chinese herbal medicine sinomenine and liang miao san on tumor necrosis factor-α-activated human fibroblast-like synoviocytes in rheumatoid arthritis traditional chinese medicine for atopic eczema: pentaherbs formula suppresses inflammatory mediators release from mast cells stupans, i. gallic acid and gallic acid derivatives: effects on drug metabolizing enzymes anticancer effects of gallic acid isolated from indonesian herbal medicine, phaleria macrocarpa gallic acid, an active constituent of grape seed extract, exhibits anti-proliferative, pro-apoptotic and anti-tumorigenic effects against prostate carcinoma xenograft growth in nude mice anti-tumour potential of a gallic acid-containing phenolic fraction from oenothera biennis anti-inflammatory activity of gallic acid anti-inflammatory gallic acid and wedelolactone are g protein-coupled receptor- agonists activation of eosinophils interacting with dermal fibroblasts by pruritogenic cytokine il- and alarmin il- : implications in atopic dermatitis il- amplifies both th -and th -type responses through its activity on human basophils, allergenreactive th cells, inkt and nk cells an il- cytokine member, il- , induces human basophil activation via its st receptor interleukin- : a regulator of basophils mechanisms of transendothelial migration of leukocytes plasma inflammatory cytokines and chemokines in severe acute respiratory syndrome t cell homing to epithelial barriers in allergic disease interleukin- a activation on bronchial epithelium and basophils: a novel inflammatory mechanism basophil recruitment and activation in inflammatory skin diseases basophils, eosinophils, and mast cells in atopic and nonatopic asthma and in late-phase allergic reactions in the lung and skin mechanical forces in endothelial cells during firm adhesion and early transmigration of human monocytes adhesion of human basophils, eosinophils and neutrophils to interleukin -activated human vascular endothelial cells: contribution of endothelial cell adhesion molecules hug tightly and say goodbye:role of endothelial icam- in leukocyte transmigration cytokines and chemokines orchestra atopic skin inflammation the biology of chemokines and their receptors clinical applications of cytokine assays bronchial epithelial cells in allergic reactions immunoglobulin d enhances interleukin- release from the ku human prebasophil cell line interleukin- and chronic inflammation inflammasome-il- -th response in allergic lung inflammation role of p mapk and nf-κb for chemokine release in coculture of human eosinophils and bronchial epithelial cells erk and p mapk-activated protein kinases: a family of protein kinases with diverse biological functions. microbiol role of jnk activation in apoptosis: a double-edged sword nf-κb regulation in the immune system signaling to nf-kappab role of caspases in dexamethasone-induced apoptosis and activation of c-jun nh -terminal kinase and p mitogen-activated protein kinase in human eosinophils dexamethasone inhibits basophil migration circulating immunoglobulins, leucocytes and complements in childhood-onset atopic eczema corticosteroids are not present in a traditional chinese medicine formulation for atopic dermatitis in children sample availability: samples of the pentaherb formula, danpi and gallic acid are available from the authors this study is financially supported by a hong kong itf grant titled -development of garments with traditional chinese single herbal medicine (tcshm) for treatment of atopic dermatitis (ad)‖ ref no: its/ / . the authors declare no conflict of interest. key: cord- -trt s wp authors: kuang, haixue; su, yang; yang, bingyou; xia, yonggang; wang, qiuhong; wang, zhibin; yu, zhengfan title: three new cycloartenol triterpenoid saponins from the roots of cimicifuga simplex wormsk date: - - journal: molecules doi: . /molecules sha: doc_id: cord_uid: trt s wp three new cycloartenol triterpene saponins, named shengmaxinsides a-c, have been isolated from the ethyl acetate soluble fraction of an ethanol extract of cimicifuga simplex wormsk roots. their structures were established by chemical tests and detailed spectroscopic analysis as -o-acetyl- , -didehydrocimigenol- -o-β-d-galactopyranoside ( ), , -didehydrocimigenol- -o-β-d-galactopyranoside ( ) and , -didehydro- s-o-acetylhydroshengmanol- -o-β-d-galactopyranoside ( ), respectively. the ranunculaceae is a small family with five genera and around species found throughout the world. currently, about nine cimicifuga species grow in china. c. simplex (shengma in chinese) is a deciduous perennial herb widely distributed in china. traditionally, the root of c. simplex has been used in oriental countries as an anti-inflammatory and anti-viral agent [ ] [ ] [ ] and the beneficial ingredients responsible for the anti-inflammatory effects are ferulic acid and isoferulic acid [ , ] . this herb has also been used for the treatment of human immunodeficiency virus (hiv), and its more general analgesic, antipyretic, antidiabetes, antimalaria and vasoactive properties [ ] [ ] [ ] . its chemical open access constituents have been extensively investigated and the main constituents are , -cyclolanostane triterpenoid glycosides, flavonoids, alkaloids, and chromones [ ] [ ] [ ] [ ] . more than uncommon cycloartane-type triterpenoid saponins have been isolated from cimicifuga plants [ ] . genjiro and his team have isolated more than fifty cycloartane-type triterpenoids from c. simplex grown in japan [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] . it was reported that , -cyclolanostane triterpene glycosides exhibited antiosteoporosis, anti-tumor and anti-complement activities [ ] [ ] [ ] . furthermore, triterpenoids may be useful candidates for the development of new drugs for cardiovascular disorders due to their antioxidant and anti-inflammatory activity [ ] . in continuation of our search for pharmacological and structurally interesting substances from chinese traditional herbal drugs, we investigated the chemical constituents of c. simplex. fractionation of the ethyl acetate soluble extract of the roots of c. simplex by column chromatography afforded three new cycloartane-type triterpenoid saponins ( figure ). we report here on the isolation and structural elucidation of these compounds by chemical and spectroscopic analysis. compound , named shengmaxinside a, was obtained as colorless needles and gave positive results for the liebermann-burchard reaction and molish reagents, indicating it to be a triterpenoid glycoside. . ) and a series of overlapped signals suggesting a cycloartane-type triterpene glycoside. the c-nmr spectrum (table ) displayed a total of thirty eight carbon signals due to the aglycon moiety, along with a sugar unit and an acetyl unit. the c-nmr spectrum exhibited anomeric carbons at δ c . . all the above evidence suggested that was a highly oxygenated , -cycloartane triterpene glycoside. moreover, δ c . suggests to be a cimigenol type saponin [ ] . after acid hydrolysis and derivatization as alditol acetates, the gas chromatography (gc) analysis revealed the presence of d-galacose. the presence of a galacose was further confirmed by its nmr data [ ] , and the galactose linkage was assigned as β from observation of the anomeric proton coupling constant at δ h . ( h, d, j = . hz). the residual three further signals at δ h . ( h, ddd, j = . , . , . hz), . ( h, d, j = . hz) and . ( h, d, j = . hz) in the region of aglycon moiety suggest three additional oxygen-bearing carbons on the aglycone. this hypothesis was confirmed by the hmbc spectrum, which showed cross-peaks between proton signal at δ h . ( h, d, j = . hz) with c- and c- , c- and c- , between proton signal at δ h . ( h, ddd, j = . , . , . hz) with c- and c- , and between proton signal at δ h . ( h, d, j = . hz) with c- and c- . this unambiguously iindicated that the oxygen-bearing carbons are c- , c- and c- . in the hmbc spectrum, significant correlations between δ h . (h- ') and . (c- ) suggested that the galactopyranosyl was located at the c- position. furthermore, the long-range correlations between an acetyl proton (δ h . ) with c- (δ c . ) indicated that the acetyl unit locating at c- . other key long-range correlations were observed for h- /c- , h- '/c- , h- /c- , h- /c- and c- , and an acetyl methyl proton and an acetyl carbon and c- . comparison of the c-nmr spectral data of with those of the known compound [ ] showed that the aglycone of was very similar to that of the known compound, except for the signals of the sugar moieties. this suggested that had the same aglycone as -o-acetyl- , -didehydrocimigenol- -o-β-d-xyloside. thus, from the above the h - h cosy, hsqc, dept and hmbc we concluded that the planar structure of corresponded to compound , named shengmaxinside b, was obtained as colorless needles and gave positive results for the liebermann-burchard reaction and molish reagent, which was considered evidence of a triterpenoid glycoside. its molecular formula was established as c in the c-nmr spectrum ( table ) a total of thirty six carbon signals due to the aglycon moiety were observed, along with a sugar unit. compared to , there is no acetyl unit signal. in the meantime, only the chemical shifts of c- , c- and c- , located at δ c . , . , . , respectively, were changed compared to . the comparison of the c-nmr data of to those of the moieties of the ether-linkage and ester-linkage sugar chains of suggested that possessed the same sugar chains as . this deduction was confirmed by the hmbc experiment. on the basis of these data, was elucidated as , compound , named shengmaxinside c, was obtained as a white amorphous powder, which was considered to be a triterpenoid glycoside due to the positive results with the liebermann-burchard reaction and molish reagents. its molecular formula was determined as c h o (table ) showed a total of thirty eight carbon signals due to the aglycon moiety, along with a sugar unit and an acetyl unit. the c-nmr spectrum exhibited anomeric carbons at δ c . . all the above evidence suggested that was a highly oxygenated , -cycloartane triterpene glycoside. moreover, δ c . indicated to be a hydroshengmanol type saponin [ ] . after acid hydrolysis and derivatization as alditol acetates, the gas chromatography (gc) analysis revealed the presence of d-galacose. this was further confirmed by its nmr data [ ] , and the galactose linkage was assigned as β form on the basis of the anomeric proton coupling constant at δ according to the literature, the configuration of c- is r when c- chemical shift in the c-nmr spectrum should be . ~ . , while for s it appears to be . ~ . [ ] in the case of , the c- chemical shift is . . the h-and c-nmr spectrum of were similar to those of , didehydro- s-o-acetylhydroshengmanol- -o-xyloside [ ] , respectively, except for the sugar moiety (table ) the optical rotations were recorded on a perkin-elmer polarimeter. ir spectra were taken on a shimadzu ftir- s. the nmr spectra were recorded on a bruker dpx instrument ( mhz for h-nmr and mhz for c-nmr). samples were prepared in pyridine-d with tms as an internal standard and coupling constants j are given in hz. the uv spectra were recorded on a shimadzu uv- instrument and gc analysis was carried out on an agilent hp n gas chromatograph using an hp- capillary column. the hresims was determined on an ionspec ultima . t fticr. preparative hplc (waters, delta - ) was performed on hypersil-ods ii ( μm, × mm, yilite, da lian, china). column chromatography was performed with silica gel ( - mesh, qingdao haiyang chemical group co. ltd, qingdao, p. r. china), ods-a ( a, μm, ymc co.) and sephadex lh- ( - μm, pharmacia). analytical tlc spots were detected on silica gel f (merck, germany) by spraying with % ethanolic h so reagent followed by heating. the roots of c. simplex ( . kg) was extracted under reflux conditions with % ethanol ( l× × h each). the ethanolic solution was concentrated in vacuo to yield a syrup-like extract ( g), which was dissolved in h o ( ml) and then partitioned with different solvents to give petroleum ether-soluble ( . g), ethyl acetate-soluble ( g) and n-butyl alcohol-soluble ( g) portions. the ethyl acetate-soluble portion was subjected to silica gel column chromatography (chcl /meoh, : → : ) to afford fractions a-h. fraction d ( g) was re-chromatographed on silica gel ( - mesh, g), eluted with chcl -meoh ( : ) as solvent, to afford three sub-fractions. sub-fraction d ( . g) was further separated by ods (meoh/h o, : → : ) to afford five fractions. fraction d - was followed by sephadex lh- and purified by preparative hplc with meoh/h o : to afford compound ( mg acid hydrolysis was performed by a previously described method [ ] . for this purpose, each compound ( mg) was heated in an ampule with aqueous % hcl ( ml) at °c for h. the aglycone was extracted with chloroform, and each aqueous residue was adjusted to ph . with % naoh and reduced with nabh ( mg), followed by acidification with dilute ch cooh, and then co-distilled with pure ch oh to remove excess boric acid. the reduced sugars were acetylated with : pyridine-ac o in a boiling water bath for h to give the corresponding alditol acetates, which were analyzed by glc on a hp n gas chromatograph (agilent) equipped with a flame ionization detector fid) using n as carrier gas. the instrument was fitted with a hp- capillary column ( m× . mm× . μm). the injector temperature was set at °c and the column temperature program was as follows: the initial temperature of °c was increased by °/min to the final temperature of °c, then was held min. the detector temperature was set at °c. the standard monosaccharides were subjected to the same reaction and gc analysis under the same conditions (d-galacose, t r , . min) it has been reported that , -cyclolanostane triterpene glycosides exhibit varied biological activities, including antiosteoporosis, antitumor, anti-complement, antioxidant and anti-inflammatory effects [ ] [ ] [ ] . as a part of our chemical investigation on c. simplex, three new cycloartenol triterpene saponins with galactopyranosyl moieties, shengmaxinsides a-c, were isolated. their structures were established on the basis of spectroscopic analysis and chemical evidence. their biological activities will be further researched in our laboratory. aqueous extracts of cimicifuga racemosa and phenolcarboxylic constituents inhibit production of proinflammatory cytokines in lps-stimulated human whole blood. can isoferulic acid as active principle from the rhizoma of cimicifuga dahurica to lower plasma glucose in diabetic rats in vitro inhibition of coronavirus replications by the traditionally used medicinal herbal extracts, cimicifuga rhizoma, meliae cortex, coptidis rhizoma, and phellodendron cortex inhibitory effect of ferulic acid and isoferulic acid on murine interleukin- production in response to influenza virus infections in vitro and in vivo inhibitory effect of ferulic acid and isoferulic acid on the production of macrophage inflammatory protein- in response to respiratory syncytial virus infection in raw . cells cimicifugae rhizoma: from origins, bioactive constituents to clinical outcomes chemical constituents from cimicifuga foetida a unusual cycloartane triterpenoid from cimicifuga foetida cimicifoetisides a and b, two cytotoxic cycloartane triterpenoid glycosides from the rhizomes of cimicifuga foetida, inhibit proliferation of cancer cells studies on the constituents of cimicifuga spp. xiii. structure of cimicifugoside studies on the constituents of cimicifuga species. xiv. a new xyloside from the aerial parts of cimicifuga simplex wormsk studies on the constituents of cimicifuga species. xv. two new diglycosides from the aerial parts of cimicifuga simplex wormsk studies on the constituents of cimicifuga species. xvi. three new cycloartane xylosides from the aerial parts of cimicifuga simplex wormskjord studies on the constituents of cimicifuga species. xvii. four new glycosides from the aerial parts of cimicifuga simplex wormsk studies on the constituents of cimicifuga species. xviii. four new xylosides from the aerial parts of cimicifuga simplex wormsk studies on the constituents of cimicifuga species. xix. eight new glycosides from cimicifuga simplex wormsk studies on the constituents of cimicifuga species. xx. absolute stereostructures of cimicifugoside and actein from cimicifuga simplex wormsk studies on the constituents of cimicifuga species. xxi. two new cyclolanostanol xylosides, bugbanosides a and b from cimicifuga simplex wormsk studies on the constituents of cimicifuga species. xxvi. twelve new cyclolanostanol glycosides from the underground parts of cimicifuga simplex wormsk cycloarta- , -dien- β-ol: revised structure of cimicifugenol, a cycloartane triterpenoid studies on the constituents of cimicifuga species. xxvii. malonyl cyclolanostanol glycosides from the underground parts of cimicifuga simplex wormsk studies on the constituents of cimicifuga species. xxviii. four new cycloart- -enol glycosides from the underground parts of cimicifuga simplex wormsk anticomplement activity of cycloartane glycosides from the rhizome of cimicifuga foetida cytotoxicity of cycloartane triterpenoids from aerial part of cimicifuga foetida cimicifuga foetida extract inhibits proliferation of hepatocellular cells via induction of cell cycle arrest and apoptosis the spectroscopic features of natural , -cyclolanstane triterpenic glycosides. chin constituents of cimicifugae rhizoma. i. isolation and characterization of ten new cycloartenol triterpenes from cimicifuga heracleifolia komarov leiyemudanosides a-c, three new bidesmosidic triterpenoid saponins from the roots of caulophyllum robustum this article is an open access article distributed under the terms and conditions of the creative commons attribution license we appreciate the kind help of weiguo zhu of zhengzhou university for measurement of nmr spectra. we are grateful to zhenyue wang in college of pharmacy, heilongjiang university of chinese medicine, for the plant identification. key: cord- -sbdtpsz authors: ramírez-pérez, sergio; hernández-palma, luis alexis; oregon-romero, edith; anaya-macías, brian uriel; garcía-arellano, samuel; gonzález-estevez, guillermo; muñoz-valle, josé francisco title: downregulation of inflammatory cytokine release from il- β and lps-stimulated pbmc orchestrated by st , a myd dimerisation inhibitor date: - - journal: molecules doi: . /molecules sha: doc_id: cord_uid: sbdtpsz the inflammatory process implicates homeostasis disruption and increased production of inflammatory mediators. myeloid differentiation primary response (myd ) is an essential protein recruited after lipopolysaccharide (lps) and interleukin (il)- β stimulation, a process that converges in nuclear factor kappa b (nf-κb) activation, as well as a transcription of several genes of both pro- and anti-inflammatory cytokines. the inhibition of myd has shown efficacy by decrease inflammatory response, and has demonstrated potential application as a therapeutic target in chronic diseases. in this study, we investigate the effect of myd dimerisation inhibitor st on cytokine production from rhil- β and lps-stimulated peripheral blood mononuclear cells (pbmc) from healthy blood donors (hbd). st significantly downregulates the production of ifn-γ, il- , il- , il- , il- , il- , vegf, il- ra, il- , il- , il- and il- (p < . ) in lps-stimulated pbmc. moreover, st had a relatively low impact on il- β signalling pathway inhibition, showing that only a few specific cytokines, such as ifn-γ and il- ra, are inhibited in rhil- β-stimulated pbmc (p < . ). in conclusion, myd dimerisation inhibitor st showed high efficacy by inhibiting pro- and anti-inflammatory cytokine production in lps-stimulated pbmc. moreover, although rhil- β induced a sustained cytokine production (p < . ), st did not show a significant effect in the secretion of neither pro- nor anti-inflammatory cytokines in rhil- β-stimulated pbmc. myeloid differentiation primary response (myd ) represents an important molecule associated with activation of several signalling pathways, which are implicated in the inflammatory immune response [ ] . interleukin (il)- β and toll-like receptors (tlr) signalling pathways are one of the most strongly studied mechanisms in which myd plays a primary role [ ] [ ] [ ] [ ] . the il- β activity implicates binding between this cytokine and the il- r type i (il- ri), this binding triggers the interaction of toll-il- -receptor (tir) domains followed by myd recruitment and downstream signalling cascades, which converge in the activation of several transcription factors and pro-inflammatory cytokine production [ , ] . regarding inflammation-associated tlr activation, molecules , , of tlr has been the best-studied molecule in both innate and adaptive immunity [ , ] . tlr signalling pathway can be activated myd -dependent or independent manner [ ] ; nevertheless, tlr -dependent lps-regulated signalling involves the recruitment and homodimerisation of myd and leads to pathways of intracellular signal transduction which converge in the production of inflammatory mediators implicated in the regulation of the inflammatory process [ , ] . overactivated myd -dependent lps and il- β inflammatory signalling pathways have displayed high expression of pro-inflammatory mediators not only in healthy blood donors (hbd), but also in patients with chronic, systemic and autoimmune diseases [ ] [ ] [ ] [ ] [ ] [ ] . due to this fact, the identification of new molecules that regulate these signalling pathways has taken high relevance [ , , [ ] [ ] [ ] . the chemical molecule st acts as an inhibitor of myd dimerisation and its activity has been demonstrated through the inhibition of tlr -dependent cpg-regulated signalling, and inhibition of il- , il- β, il- and tumor necrosis factor alpha (tnf-α) expression in lps-stimulated raw . cells [ ] [ ] [ ] [ ] . however, the effect of the st molecule on cytokine production mediated by il- β and lps-stimulated peripheral blood mononuclear cells (pbmc) has yet been clarified. in the present study, pbmc obtained from hbd were stimulated with both rhil- β and lps to identify pro-and anti-inflammatory cytokine profiles, as well as, the inhibitory activity of st molecule on the cytokine secretion. our results indicate that inhibition of myd dimerisation mediated by st molecule causes a decrease secretion of both pro-and anti-inflammatory cytokines in supernatants of lps-stimulated pbmc; however, st showed high impact neither pro-nor anti-inflammatory cytokine secretion in rhil- β-stimulated pbmc. to determine the specific concentration of st in which the secretion of cytokines was inhibited, curves of different concentrations of st were performed ( figure ). tnf-α quantification was taken as a positive control, and the concentration of this cytokine was determined in supernatants of lps-stimulated pbmc after h ( figure a ). the concentration of tnf-α in supernatants of pbmc without stimuli was . pg/ml; whereas, a high concentration of tnf-α in lps-stimulated pbmc was identified ( . pg/ml). regarding st stimuli three different concentrations were taken , and µm and tnf-α levels were determined, the medium values were . pg/ml (p = not significant [n.s.]), . pg/ml (p < . ), and . pg/ml (p < . ), respectively. similarly, the concentration of tnf-α was determined in supernatants of rhil- β-stimulated pbmc ( figure b ). tnf-α levels from rhil- β-stimulated pbmc were . pg/ml, for rhil- β plus µm of st were . pg/ml (p = n.s.), and after add and µm of st to rhil- β-stimulated pbmc, the tnf-α levels were pg/ml for both (p < . ) ( figure b ). lps has been implicated in the production of pro-inflammatory cytokines through tlr activation. our results indicate that lps is a potent inductor of several pro-inflammatory cytokines in pbmc. statistically significant differences were found between pbmc treated with rpmi alone and lps (p < . ). in addition, st molecule was used as a negative regulator of tlr -dependent lps-regulated signalling pathway. st in lps-stimulated pbmc decreased secretion of interferon gamma (ifn-γ) (p < . ), il- (p < . ), il- (p < . ), il- (p < . ), vascular endothelial growth factor (vegf) (p < . ), il- (p < . ) and il- (p < . ) ( figure ; table s ). since our study included males and females; in order to identify differential effects on cytokine production release a statistical analysis by gender was performed. however, our results showed non-statistically significant differences between males and females (data not shown). as a control for the effect of st alone, a statistical analysis was performed by comparing the production of inflammatory cytokines studied in the presence or absence of st . for ifnγ, tnfα, il- ra and il- , statistically significant differences were found (p < . ). the levels of these cytokines in the presence of st were significantly lower than in pbmc treated with rpmi alone; furthermore, a higher cytokine secretion as an effect of st than in the basal response of untreated pbmc was not observed (table s ) . molecules , , x of as a control for the effect of st alone, a statistical analysis was performed by comparing the production of inflammatory cytokines studied in the presence or absence of st . for ifnγ, tnfα, il- ra and il- , statistically significant differences were found (p < . ). the levels of these cytokines in the presence of st were significantly lower than in pbmc treated with rpmi alone; furthermore, a higher cytokine secretion as an effect of st than in the basal response of untreated pbmc was not observed (table s ) . the concentration of anti-inflammatory cytokines was determined in supernatants of lpsstimulated pbmc. interestingly and contrary to expectations, after h of stimulation with lps in pbmc, we observed anti-inflammatory cytokine production of il- ra, il- , il- , il- , il- and il- (p < . ). additionally, st inhibited the secretion of il- ra (p < . ), il- (p < . ), il- (p < . ), il- (p < . ) and il- (p < . ), but not il- ( . pg/ml, p = n.s.) ( figure ; table s ). moreover, it was observed that anti-inflammatory cytokine secretion from pbmc stimulated with st alone, have a similar response to unstimulated cells (table s ). based on these results, pbmc stimulated with lps can secrete pro-and anti-inflammatory cytokines and st can inhibit the observed response; this is a relevant finding that has not been reported till date. (b) the soluble levels of tnf-α in the supernatant of rhil- β-stimulated pbmc at ng/ml and rhil- β ( ng/ml) plus different concentrations of st ( , and µm) were determined. significant inhibition was identified at µm (p < . ) and µm (p < . ) of st for lps; while for rhil- β significant inhibition was identified at µm (p < . ) and µm (p < . ) of st . data provided in medians and interquartile ranges (n = ), Φ kruskal-wallis test was performed, and dunn's test obtained statistically significant differences. the concentration of anti-inflammatory cytokines was determined in supernatants of lps-stimulated pbmc. interestingly and contrary to expectations, after h of stimulation with lps in pbmc, we observed anti-inflammatory cytokine production of il- ra, il- , il- , il- , il- and il- (p < . ). additionally, st inhibited the secretion of il- ra (p < . ), il- (p < . ), il- (p < . ), il- (p < . ) and il- (p < . ), but not il- ( . pg/ml, p = n.s.) ( figure ; table s ). moreover, it was observed that anti-inflammatory cytokine secretion from pbmc stimulated with st alone, have a similar response to unstimulated cells (table s ). based on these results, pbmc stimulated with lps can secrete pro-and anti-inflammatory cytokines and st can inhibit the observed response; this is a relevant finding that has not been reported till date. the role of il- β has been widely described in several chronic conditions, as well as in several cell types. our results showed high secretion of pro-inflammatory cytokines in rhil- β-stimulated pbmc (table ). in an exciting way and as previously reported, il- β represents an important cytokine capable of inducing th -related cytokine profile. in this study, we observed high production of these cytokines: however, only il- a, granulocyte-monocyte colony-stimulating factor (gm-csf) and granulocyte colony-stimulating factor (g-csf) increased significantly after h of rhil- β stimulation (p < . ). furthermore, the concentration of cytokines, such as il- (p < . ), vegf (p < . ) and il- (p < . ), were found higher after rhil- β stimulation. on the other hand, only il- and il- significantly increased after rhil- β stimulation (p < . ). regarding the st effect on rhil- β-stimulated pbmc and contrary to our expectations; our results showed that this molecule had a relatively low impact on the il- β signalling pathway inhibition (table ) . st molecule only inhibited the secretion of ifn-γ (p < . ) and il- ra molecules , , of (p < . ). the present study shows that the specific inhibition of critical components in the il- signalling pathway is not enough to avoid the secretion of inflammatory mediators, the above suggests that various myd -independent mechanisms could regulate the production of cytokines in pbmc. the role of il- β has been widely described in several chronic conditions, as well as in several cell types. our results showed high secretion of pro-inflammatory cytokines in rhil- β-stimulated pbmc (table ). in an exciting way and as previously reported, il- β represents an important cytokine capable of inducing th -related cytokine profile. in this study, we observed high production of these cytokines: however, only il- a, granulocyte-monocyte colony-stimulating factor (gm-csf) and granulocyte colony-stimulating factor (g-csf) increased significantly after h of rhil- β stimulation (p < . ). furthermore, the concentration of cytokines, such as il- (p < . ), vegf (p < . ) and il- (p < . ), were found higher after rhil- β stimulation. on the other hand, only il- and il- significantly increased after rhil- β stimulation (p < . ). regarding the st effect on rhil- β-stimulated pbmc and contrary to our expectations; our results showed that this molecule had a relatively low impact on the il- β signalling pathway inhibition (table ) . st molecule only inhibited the secretion of ifn-γ (p < . ) and il- ra (p < . ). the present study shows that the specific inhibition of critical components in the il- signalling pathway is not enough to avoid the secretion of inflammatory mediators, the above suggests that various myd -independent mechanisms could regulate the production of cytokines in pbmc. the role of inflammatory key mediators, such as il- β and lps, in activating the innate immune response and subsequently leading to inflammation has been widely described in several studies [ , ] . il- β and lps-signalling pathways trigger cascades of intracellular activation mediated by myd recruitment, which converges in the activation of transcription factors, such as nf-κb and its translocation to the nucleus [ , , , ] . the expression of receptors associated with these pro-inflammatory mediators can be given in a variety of cells of the immune system, within the main ones are professional antigen-presenting cells, treg cells and effector t cells [ ] [ ] [ ] [ ] . our results show that after h, pro-inflammatory cytokine levels of il- β, tnf-α, ifn-γ, il- , il- , il- a, g-csf, gm-csf, il- , vegf, il- and il- significantly increased in lps-stimulated pbmc. recent studies performed in vitro have described that in both pbmc and thp- cells can be possible to carry out a differentiation towards m macrophages by stimulation with lps, ifn-γ or gm-csf; m macrophages have been characterised by the expression of pro-inflammatory cytokines, such as il- , il- , tnf-α and il- β [ , , ] . regarding cytokines, such as il- and tnf-α, previous studies performed in lps-stimulated pbmc from hbd reported increased levels of these cytokines in comparison with those levels found in unstimulated pbmc [ , ] . an important cytokine involved in the innate immune response mediated by nk and nkt cells is il- , which increased after lps stimulation; concerning this result, one study reported that lps-stimulated monocytes could produce il- [ ] . concerning the expression of gm-csf and vegf, previous studies conducted in lps-stimulated pbmc after h showed high levels of both cytokines, as we observed in this study [ ] . an earlier report indicated that gm-csf expression increased in lps-stimulated pbmc; nonetheless, its expression remains low compared with other pro-inflammatory cytokines, such as tnf-α or il- , which could suggest that this cytokine is produced only for a specific population of monocytes [ ] . the activation of several transcription factors after lps stimulation has been demonstrated in a large number of studies. rothfuchs et al. reported that ifn-γ mrna expression was strongly diminished in tlr -/macrophages compared with the wild type phenotype; moreover, tlr activation causes myd -dependent ifn-α production, which results in an autocrine effect that regulates ifn-γ mrna expression by stat activation [ ] . on the other hand, increased expression of ror-γt and the phosphorylated form of nf-κb were found after lps stimulation, and this effect was associated with the differentiation of th cells and high expression of il- a [ ] . peyssonnaux et al. reported that hif- α was highly expressed in lps-stimulated macrophages; interestingly, hif- α/rorγt/p complex can be linked to the promoter region of il a and can also promote its transcription [ , , ] . vegf is a cytokine produced in response to the hypoxia process and subsequently to hif- α activation which, as mentioned above, hif- α is a transcription factor induced in response to lps [ , ] . transcription factor activation followed by high production of pro-inflammatory cytokines in a tlr -lps-dependent pathway has been widely described. however, posttranscriptional mechanisms implicated in tlr activation have also recently been described. for example, arid a protein is a crucial factor for the production of il- ; this protein binds to the '-utr region of the il- mrna and leads to its stabilisation and subsequently, to its efficient expression in vivo [ ] . moreover, nyati et al. described an alternative lps signalling pathway that is independent on p activation, but dependent on the mitogen-activated protein kinase (mapk) phosphatase (mkp- ) [ ] . this phosphatase mkp- can induce the translocation from the nucleus to the cytoplasm of au-rich element rna-binding protein (auf- ), and auf- acts by stabilising the mrna of il- , tnf-α and il- [ ] . according to the present study, we observed that the production of anti-inflammatory cytokines significantly increases in lps-stimulated pbmc. several studies have reported that tlr -dependent lps-regulated signalling pathway causes a predominantly pro-inflammatory response in pbmc. however, some interesting studies have reported that the expression of irf could also be expressed after lps/ifn-γ or il- stimulation; irf is a key transcription factor involved in the differentiation of m macrophages [ , , ] . however, a significant limitation in our study is that the expression of cytokines was determined after h of stimulation. in this regard, several reports indicate that pro-inflammatory cytokines have an early maximum expression peak within the first eight hours of lps stimulation (il- , il- β, il- , tnf-α and il- ) and expression of cytokines, such as il- and il- ra, exhibit a maximum expression peak at and h, respectively [ ] . this behaviour on the cytokine profile could also be explained by a process known as "tlr tolerance", which is characterised by reducing expression of pro-inflammatory cytokines and high expression of m activation markers after sustained exposure to tlr ligands [ ] . additionally, a specific type of m polarisation in human mononuclear cells after induction of lps tolerance has been reported [ ] , and after lps tolerance recovering, macrophages can express both m and m polarisation states [ ] . stimulation with rhil- β triggered the production of several pro-inflammatory cytokines. interestingly, as previously described in other studies, this cytokine induces the release of th -related cytokine profile; il- a, gm-csf and g-csf were significantly expressed in our research [ ] [ ] [ ] . nevertheless, a new t helper cell subset characterised by high production of gm-csf has been currently described [ ] . this cell subset was identified as gm-csf producing cd + t cell (th-gm-csf), and its differentiation depends on il- β signalling pathway, and subsequent irak and nf-κb activation [ ] . furthermore, th-gm-csf cells are able to produce high concentrations of pro-inflammatory cytokines, such as il- , il- and tnf-α [ ] . our results also showed increased expression of il- and vegf; regarding il- production, langlet et al. reported that this cytokine could be expressed in an il- β-dependent activation [ ] . moreover, previous reports have shown that vegf expression requires activation of hif- α, and activation of hif- α occurs after il- β stimulation [ ] [ ] [ ] . concerning the results of the inhibition of myd dimerisation and according to expectations, st molecule significantly inhibits the pro-inflammatory and anti-inflammatory cytokine production mediated by the activation of lps-tlr pathway. about these results, long et al. reported a significant decrease in cytokine expression of il- , il- β, il- and tnf-α from raw . cells treated with lps plus st [ ] . several studies have reported that st causes decreased recruitment and activation of specific molecules and transcription factors involved in the activation of tlr and the lps-dependent immune response activation. the main inhibited molecules, due to the activity of st , are irak , irak , traf , p-ikk, p-ikbα, p-nf-κb and hif- α [ , ] . st has also been proposed as a novel drug targeting in diseases like lymphoma, leukaemia, human hepatocellular carcinoma, and traumatic brain injury [ ] [ ] [ ] . however, its role as a possible inhibitor in the production of cytokines produced after stimulation with lps remains undetermined. the decrease recruitment of molecules implicated in the myddosome formation may explain the inhibition of pro-and anti-inflammatory cytokines as a consequence of tlr -dependent lps-regulated signalling. at the same time, signalling pathway activation after il- -il- ri-il- racp complex formation has been widely described [ , , , , ] . this process implicates recruitment and homodimerisation of myd , as well as intracellular signalling cascades that converge in the transcription of genes of pro-inflammatory mediators [ , , , , ] . specific myd dimerisation inhibition has been tested in many studies where the role of this molecule in an il- β-dependent activation pathway was evident [ , , ] . another study performed in healthy human articular chondrocytes reported decreased map kinase (mapk) activation after myd dimerisation inhibition and il- β stimulation [ ] . an interesting study conducted by wang et al. ( ) previously reported that st decreases the expression of several molecules involved in the myddosome formation, such as phosphorylated btk and iκb, along with the decreased secretion of il- and ifn-β from b-cell lymphoma cell lines [ ] . in our study, il- release decreases on both il- β and lps-stimulated pbmc treated with st ; nonetheless, the effect was not statistically significant. in relation to the ifn response, a decreased secretion of ifn-γ on both il- β and lps-stimulated pbmc was observed our study as an effect of st . however, our results did not show a substantial inhibitory effect on cytokine production from pbmc treated with il- β plus st . in regard to this result, loiarro et al. ( ) previously described an inhibitory effect observed on nf-κb activity after stimulation with il- β stimulation ( ng/ml) plus st ( µm) on hek t cells [ ] . nevertheless, although nf-κb activity decreases, st did not deplete nf-κb activation, which could provide signalling activation enough to produce some of the inflammatory cytokines observed in our study [ ] . indeed, since this st chemical compound affects only the association of tir domains of myd and the disruption of this tir domain interaction inhibits the recruitment of irak . by extension, irak and the subsequent signalling cascade, dimerisation inhibition of myd might affect only one specific signalling pathway [ ] . these results might suggest that alternative il- β signalling pathways independent of myd homodimerisation and recruitment in pbmc could be active as well. this approach arises from previous studies in which new receptors associated with the recognition of il- β have been observed. il- racpb is a unique receptor expressed on neurons, and its expression implicates activation of certain signalling pathways, such as p mapk, but not nf-κb; it has even been observed that this alternative signalling pathway is independent on myd , irak and traf [ , ] . moreover, heinz et al. reported a new molecule known as unc cl; this protein contains death domains (dd) similarly to those found in myd [ ] . unc cl is considered as a pro-inflammatory signalling inducer and involves recruitment of irak , irak , traf and converges in nf-κb and jnk activation in a myd -independent manner [ ] . currently, there are not reports in which these new molecules have been reported in pbmc; however, the possibility to perform future studies that elucidate this observation remains open. moreover, in spite of the fact that st did not show a substantial inhibitory effect on downregulation of inflammatory cytokine from il- β-stimulated pbmc, it might be necessary to consider the use of other il- inhibitors, such as il- and il- [ , ] , which could be useful to compare a differential response orchestrated by st on il- β-stimulated pbmc and the inhibitory effect of both il- and il- . in this regard, conti et al. mentioned that il- suppresses the innate and acquired immune response and inhibits the inflammation through its binding with il- receptor-α chain (il- rα) [ ] . additionally, il- can activate the mtor signalling pathway and increase the adenosine monophosphate (amp) kinase [ ] ; therefore, il- and il- might represent cytokines of great interest in experimental models where the suppressive effect of several inflammatory mediators is required. furthermore, a weakness of this study was not to measure cell viability after stimulation with st . differences (that were not statistically significant) have been previously reported for st in other studies at , or µm on cell viability [ , , ] . our study used pbmc, reduction on specific pbmc subpopulations might contribute to the behaviour observed on these inflammatory cytokines. therefore, considering this limitation will be important in future studies to analyse the observed response of this molecule on cell viability and the percentage of apoptosis by the effect of st . the understanding of inflammatory mechanisms regarding activation and effector function on physiological processes in the first instance can provide us with an overview of the behaviour of specific factors involved in the regulation and maintenance of the inflammatory process and how it can lead to the development of chronic inflammation. this study provides information about the inhibitory activity of st on cytokine production (figure ) , possibly through the tlr signalling pathway regulation in lps-stimulated pbmc. moreover, a relatively low impact on il- β signalling pathway inhibition orchestrated by st in pbmc was observed ( figure ) ; possibly due to the various mechanisms of activation that remain unexplored on this signalling pathway, which could be cell subset-dependent. nevertheless, future studies focused on the identification of specific down-stream factors implicated in both il- β and lps signalling pathway activation to elucidate how the regulation of cytokine production takes place in distinct pbmc subpopulations will be required. therefore, our results not only provide valuable evidence about the potential use of the st chemical molecule to inhibit the inflammatory cytokine release in pbmc from hbd, but also leave open the possibility to study the effect of this molecule in chronic diseases, such as rheumatic and autoimmune diseases, in which the inflammatory process plays a critical role. regulation of cytokine production takes place in distinct pbmc subpopulations will be required. therefore, our results not only provide valuable evidence about the potential use of the st chemical molecule to inhibit the inflammatory cytokine release in pbmc from hbd, but also leave open the possibility to study the effect of this molecule in chronic diseases, such as rheumatic and autoimmune diseases, in which the inflammatory process plays a critical role. ten hdb over years of age were included in the study, five females and five males. a blood sample ( ml) was collected from each participant to obtain pbmc and perform cell culture experiments. the presence of infections and body mass index ≥ kg/m were taken as exclusion criteria. all subjects included in the present study signed the informed consent letter before their inclusion, and the present study was performed following the declaration of helsinki amendments. the pbmc were isolated from a blood sample following the density gradient separation method using histopaque ® - (sigma aldrich, st. louis, mo, usa; ρ . - . g/ml). the obtained pbmc were washed and resuspended in rpmi- medium supplemented with penicillin ( u/ml, sigma aldrich, st. louis, mo, usa) and streptomycin ( µg/ml, sigma aldrich, st. louis, mo, usa) at a concentration of % for both. the trypan blue test analysed the cell viability, and the total of separated cells per ml was quantified directly in a neubauer chamber. the pbmc were placed in -well plates after the density adjustment at × cells/ml (final volume of µl). cells were cultured in rpmi- medium without serum and supplemented with antibiotic and antifungal. untreated cells were taken as the control group for each experiment. the effect of lps ( ng/ml) in the secretion of tnf-α was studied in samples from four hbd to demonstrate the positive stimulation of the cells. the concentration of st necessary to inhibit the rhil- β and lps response in pbmc was determined at , , and µm. subsequently, pbmc culture was performed with lps ( ng/ml), lps ( ng/ml) plus st ( µm) or without stimulation (control group). similarly, pbmc were stimulated with rhil- β ( ng/ml), rhil- β ( ng/ml) plus st ( µm), st ( µm) alone or without stimulation (control group). st molecule was added to the corresponding well min before the stimuli with rhil- β or lps. each experiment was done with two biological replicates and was incubated for h at • c in a humidified % co atmosphere. once the incubation time had elapsed, supernatants were collected and stored at − • c for the subsequent quantification of pro-inflammatory and anti-inflammatory cytokine secretion. the concentration of il- β, tnf-α, ifn-γ, il- , il- , il- a, m-csf, gm-csf, il- , vegf, il- , il- , il- ra, il- , il- , il- , il- and il- and were quantified from culture supernatants by multiplex immunoassay method using the bio-plex pro tm human cytokine -plex assay (bio-rad laboratories, inc., hercules, ca, usa) according to the manufacturer's instructions. luminex magpix ® (luminex corporation, austin, tx, usa) instrument was used for xmap assays. all data were shown as medians and interquartile ranges. the differences between the non-parametric quantitative variables were analysed by u of mann-whitney test to compare two groups and both the kruskal-wallis test and the dunn's post hoc test for multiple comparisons. data analysis was performed using graphpad prism v . software, and a p-value < . was considered statistically significant. myd : a central player in innate immune signaling understanding early tlr signaling through the myddosome the il- family of cytokines and receptors in rheumatic diseases signaling in innate immunity and inflammation future potential therapeutic targets for ra the interleukin- receptor family toll-like receptors: critical proteins linking innate and acquired immunity toll-like receptors in immunity and inflammatory diseases: past, present, and future lps-induced cytokine production in human monocytes and macrophages lipopolysaccharide and il- β coordinate a synergy on cytokine production by upregulating myd expression in human gingival fibroblasts the il- β signalling pathway and its role in regulating pro-inflammatory and pro-labour mediators in human primary myometrial cells effect of pro-inflammatory/anti-inflammatory agents on cytokine secretion by peripheral blood mononuclear cells in rheumatoid arthritis and systemic lupus erythematosus elispot analysis of lps-stimulated leukocytes: human granulocytes selectively secrete il- , mip- beta and tnf-alpha increased production of il- and il- in lipopolysaccharide-stimulated peripheral mononuclears from patients with rheumatoid arthritis analysis of cytokines and chemokines produced by whole blood, peripheral mononuclear and polymorphonuclear cells discovery of small molecule inhibitors of myd -dependent signaling pathways using a computational screen the role of intermediary domain of myd in cell activation and therapeutic inhibition of tlrs toll-like receptors as therapeutic targets for autoimmune connective tissue diseases advance: inhibition of myd dimerisation and recruitment of irak and irak by a novel peptidomimetic compound pharmacological inhibition of tlr activation blocks autoantibody production in human b cells from sle patients targeting the toll-like receptor/interleukin receptor pathway in human diseases: rational design of myd inhibitors polygonatum sibiricum polysaccharides play anti-cancer effect through tlr -mapk/nf-κb signaling pathways interleukin- (il- ) pathway the regulatory roles of toll-like receptor in secretions of type /type relative cytokines by splenocytes and dendritic cells exposed to clonorchis sinensis excretory/secretory products expression of interleukin receptors on human peripheral t cells the interleukin- family: back to the future optimised flow cytometry protocol for analysis of surface expression of interleukin- receptor types i and ii thp- and human peripheral blood mononuclear cell-derived macrophages differ in their capacity to polarise in vitro m macrophages and their role in rheumatic diseases peripheral blood mononuclear cells from neovascular age-related macular degeneration patients produce higher levels of chemokines ccl (mcp- ) and cxcl (il- ) intracellular bacterial infection-induced ifn-gamma is critically but not solely dependent on toll-like receptor -myeloid differentiation factor -ifn-alpha beta-stat signaling lipopolysaccharide directly stimulates th differentiation in vitro modulating phosphorylation of relb and nf-κb cutting edge: essential role of hypoxia inducible factor- alpha in development of lipopolysaccharide-induced sepsis e ubiquitin ligases siah / regulate hypoxia-inducible factor- (hif- )-mediated th cell differentiation tlr -induced nf-κb and mapk signaling regulate the il- mrna stabilising protein arid a interleukin- induced interferon regulatory factor (irf) participates in the regulation of alternative macrophage priming negative regulation of toll-like-receptor signaling by irf- endotoxin tolerance represents a distinctive state of alternative polarisation (m ) in human mononuclear cells identification of a unique hybrid macrophage-polarisation state following recovery from lipopolysaccharide tolerance interleukin- and il- induce innate il- production from gammadelta t cells, amplifying th responses and autoimmunity pivotal role of dermal il- -producing γδ t cells in skin inflammation critical regulation of early th cell differentiation by interleukin- signaling interleukin- β-induced irak ubiquitination is required for th-gm-csf cell differentiation in t cell-mediated inflammation pkc-alpha controls myd -dependent tlr/il- r signaling and cytokine production in mouse and human dendritic cells interleukin induces hypoxia-inducible factor in human gingival and synovial fibroblasts il- beta-mediated up-regulation of hif- alpha via an nfkappab/cox- pathway identifies hif- as a critical link between inflammation and oncogenesis regulation of hypoxia-inducible factor- α (hif- α) expression by interleukin- β (il- β), insulin-like growth factors i (igf-i) and ii (igf-ii) in human osteoarthritic chondrocytes dioscin reduces lipopolysaccharide-induced inflammatory liver injury via regulating tlr /myd signal pathway tlr promotes the expression of hif- α by triggering reactive oxygen species in cervical cancer cells in vitro-implications for therapeutic intervention effect of st on the proliferation and apoptosis of human hepatocellular carcinoma cells inhibition of myeloid differentiation factor (myd ) by st provides neuroprotection after experimental traumatic brain injury in mice myd inhibitor st suppresses the growth of lymphoma and leukaemia cells overview of the interleukin- family of ligands and receptors the interleukin (il)- cytokine family-balance between agonists and antagonists in inflammatory diseases interactive sites in the myd toll/interleukin (il) receptor domain responsible for coupling to the il beta signaling pathway peptide-mediated interference of tir domain dimerisation in myd inhibits interleukin- -dependent activation of nf-{kappa}b irak and traf knockdown in human chondrocytes inhibits interleukin- -induced matrix metalloproteinase- gene expression and promoter activity by impairing map kinase activation disrupting myddosome assembly in diffuse large b-cell lymphoma cells using the myd dimerisation inhibitor st neuron-specific effects of interleukin- β are mediated by a novel isoform of the il- receptor accessory protein a central nervous system-restricted isoform of the interleukin- receptor accessory protein modulates neuronal responses to interleukin- the death domain-containing protein unc cl is a novel myd -independent activator of the pro-inflammatory irak signaling cascade interleukin- family cytokines and mast cells: activation and inhibition car-t cell therapy causes inflammation by il- which activates inflammatory cytokine mast cells: anti-inflammatory role of il- induction of pro-inflammatory cytokines (il- and il- ) and lung inflammation by coronavirus- (covi- or sars-cov- ): anti-inflammatory strategies this article is an open access article distributed under the terms and conditions of the creative commons attribution the authors declare no conflict of interest.molecules , , key: cord- -bvun eh authors: diaconu, dumitrela; mangalagiu, violeta; amariucai-mantu, dorina; antoci, vasilichia; giuroiu, cristian levente; mangalagiu, ionel i. title: hybrid quinoline-sulfonamide complexes (m( +)) derivatives with antimicrobial activity date: - - journal: molecules doi: . /molecules sha: doc_id: cord_uid: bvun eh two new series of hybrid quinoline-sulfonamide complexes (m( +): zn( +), cu( +), co( +) and cd( +)) derivatives (qsc) were designed, synthesized and tested for their antimicrobial activity. the synthesis is straightforward and efficient, involving two steps: acylation of aminoquinoline followed by complexation with metal acetate (cu( +), co( +) and cd( +)) or chloride (zn( +)). the synthesized qsc compounds were characterized by ftir and nmr spectroscopy and by x-ray diffraction on single crystal. the qsc compounds were preliminary screened for their antibacterial and antifungal activity and the obtained results are very promising. in this respect, the hybrid n-(quinolin- -yl)- -chloro-benzenesulfonamide cadmium (ii), considered as leading structure for further studies, has an excellent antibacterial activity against staphylococcus aureus atcc (with a diameters of inhibition zones of mm and a minimum inhibitory concentration (mic) of . × (− ) mg/ml), a very good antibacterial activity against escherichia coli atcc (with a diameters of inhibition zones of mm and a mic of × (− ) mg/ml), and again an excellent antifungal activity against candida albicans atcc (with a diameters of inhibition zones of mm and a mic of . × (− ) mg/ml). despite of the significant advances in the antimicrobial therapy accomplished in the last few decades, infectious diseases caused by microorganisms (bacteria, fungus, viruses, mycobacterium tuberculosis, etc.) represent seriously threaten of modern medicine and global public health. drug resistance, multi-drug resistance and extensively-drug-resistance are the leading cause of these drawbacks, but some other causes could be also taken into consideration [ ] [ ] [ ] . quinoline based compounds are small molecules of huge importance from pharmacological point of view, having a wide range of biological activities such as antiplasmodial and antimalarial, antibacterial, antifungal, antitubercular, anti-hiv, antiviral (including against , etc. [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] . a special class of quinoline derivatives which pay a particularly attention on scientific community, are quinoline-sulfonamide complexes (qsc), studied especially for their fotoluminiscent (mostly fluorescent) properties [ ] [ ] [ ] [ ] [ ] [ ] [ ] . as far for biological activity, these compounds were tested mostly as antiprotozoals [ , ] and very antiprotozoals [ , ] and very few data was found for their antibacterial and antifungal activity [ ] . the antibacterial activity of azaheterocycles sulfonamides is well known [ , ] . encouraged by our recent results in the field of (di)azine with antimicrobial activity [ , , [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] , we report here the design, synthesis, antibacterial and antifungal evaluation of some newly hybrid quinoline-sulfonamide complexes. having in view the biological potential of quinoline and sulfonamide scaffolds (especially antimicrobial) [ ] [ ] [ ] [ ] [ ] [ ] [ ] , as well as those one of quinoline-sulfonamide combined scaffold (especially anti-hiv- ) [ ] , we decide to combine the pharmacophoric properties of these core scaffolds with the complementary biological properties of counter cation m + (m + : zn + , cu + , co + and cd + ), with the final goal of obtaining better biological activity and better pharmacokinetic properties for our compounds. in this respect, we design two new classes of hybrid quinoline-sulfonamide complexes, namely n-(quinolin- -yl)- -r-benzene sulfonamide metal (ii) (qbsc) and n-(quinolin- -yl)-quinoline - -sulfonamide metal (ii) (qqsc), scheme . design in the class of hybrid quinoline-sulfonamide complexes derivatives. as in related cases [ ] [ ] [ ] [ ] , the general route to obtain the desired compounds involves a simple and efficient two-step procedure. the first step consist in the acylation of ( , or )amino-quinoline with variously -r-benzenesulfonyl chlorides (r = cl, no , ome) and quinolylsulfonyl chlorides, when the corresponding ligands quinoline-sulfonamide type are obtained. the second step consist in the complexation of ligands with metal acetate (cu + , co + , cd + ) or chloride (zn + ). in this way, we obtained the two classes of compounds desired: qbsc type - and qqsc type . the procedure is depicted in scheme for the complexes derived from -aminoquinoline. reaction pathways to obtain hybrid quinoline-sulfonamide complexes derivatives. the structures of qsc compounds were proved by elemental and spectral analysis (ft-ir, h-nmr, as in related cases [ ] [ ] [ ] [ ] , the general route to obtain the desired compounds involves a simple and efficient two-step procedure. the first step consist in the acylation of ( , or )amino-quinoline with variously -r-benzenesulfonyl chlorides (r = cl, no , ome) and quinolylsulfonyl chlorides, when the corresponding ligands quinoline-sulfonamide type are obtained. the second step consist in the complexation of ligands with metal acetate (cu + , co + , cd + ) or chloride (zn + ). in this way, we obtained the two classes of compounds desired: qbsc type - and qqsc type . the procedure is depicted in scheme for the complexes derived from -aminoquinoline. molecules , , x for peer review of antiprotozoals [ , ] and very few data was found for their antibacterial and antifungal activity [ ] . the antibacterial activity of azaheterocycles sulfonamides is well known [ , ] . encouraged by our recent results in the field of (di)azine with antimicrobial activity [ , , [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] , we report here the design, synthesis, antibacterial and antifungal evaluation of some newly hybrid quinoline-sulfonamide complexes. having in view the biological potential of quinoline and sulfonamide scaffolds (especially antimicrobial) [ ] [ ] [ ] [ ] [ ] [ ] [ ] , as well as those one of quinoline-sulfonamide combined scaffold (especially anti-hiv- ) [ ] , we decide to combine the pharmacophoric properties of these core scaffolds with the complementary biological properties of counter cation m + (m + : zn + , cu + , co + and cd + ), with the final goal of obtaining better biological activity and better pharmacokinetic properties for our compounds. in this respect, we design two new classes of hybrid quinoline-sulfonamide complexes, namely n-(quinolin- -yl)- -r-benzene sulfonamide metal (ii) (qbsc) and n-(quinolin- -yl)-quinoline - -sulfonamide metal (ii) (qqsc), scheme . scheme . design in the class of hybrid quinoline-sulfonamide complexes derivatives. as in related cases [ ] [ ] [ ] [ ] , the general route to obtain the desired compounds involves a simple and efficient two-step procedure. the first step consist in the acylation of ( , or )amino-quinoline with variously -r-benzenesulfonyl chlorides (r = cl, no , ome) and quinolylsulfonyl chlorides, when the corresponding ligands quinoline-sulfonamide type are obtained. the second step consist in the complexation of ligands with metal acetate (cu + , co + , cd + ) or chloride (zn + ). in this way, we obtained the two classes of compounds desired: qbsc type - and qqsc type . the procedure is depicted in scheme for the complexes derived from -aminoquinoline. scheme . reaction pathways to obtain hybrid quinoline-sulfonamide complexes derivatives. the structures of qsc compounds were proved by elemental and spectral analysis (ft-ir, h-nmr, the structures of qsc compounds were proved by elemental and spectral analysis (ft-ir, h-nmr, c{ h}-nmr, and two-dimensional experiments d-cosy, hmqc, hmbc), and single crystal x-ray structure determination. if we consider the m + [n-(quinolin- -yl)- -chloro-benzenesulfonamide] derivatives qbsc a-d as representative for the hybrid qsc, the spectral analysis reveal strong evidence for the proposed structures. in the ft-ir spectra of sulfonamide ligand a and its complexes qbsc a-d (figure ), the most representative bands, together with their assignments, are given in table . m + [n-(quinolin- -yl)- -chloro-benzenesulfonamide] derivatives qbsc a-d as representative for the hybrid qsc, the spectral analysis reveal strong evidence for the proposed structures. in the ft-ir spectra of sulfonamide ligand a and its complexes qbsc a-d (figure ), the most representative bands, together with their assignments, are given in table . -stretching; s-strong; m-medium; w-weak; as-asymmetric; sym-symmetric. the band from cm − (medium intensity), which is corresponding to the n-h stretching vibration in the free ligand, is missing in the spectra of the complexes, confirming deprotonation of the nitrogen atom of the sulfonamide and its coordination to the metal ion. the bands corresponding to the asymmetric and symmetric stretching vibration of the sulfonyl group in ligand appear at cm − respectively at cm − , while in spectra of the complexes are shifted with ≈ cm − to higher wave numbers respectively ≈ cm − to lower wave numbers; this is because in the free ligand a larger double bond character of the so bonds could be incriminated. the band due to the sulfonyl group is split in the spectra of the complexes, because of the different spatial orientation of these groups, as the x-ray show. we also notice a similar shift with ≈ cm − to higher wave numbers for the s-n stretching vibration in the spectra of the complexes, probably because the n atom is involved in the bonding to the metal atom. the remaining bands appear at wave numbers in accordance with the proposed structures both in the free ligand and complexes. the h-nmr and c{ h}-nmr spectra of the sulfonamide ligand and its complexes shows characteristic chemical shifts that are in agreement to the proposed structure. the most significant signal in the h-nmr spectra (figure ), corresponding of the hydrogen atom from sulfonamide group h and, appear in the free ligand at a chemical shift of . ppm. this signal is missing in the spectra of qbsc complexes, which is a solid prove for the complexation with the metal ion. we could also notice that the signals of hydrogen atoms near the metal ion h , h , h (from pyridine ring), are deshielded in qbsc complexes with about . ppm due to the powerful deshielding the band from cm − (medium intensity), which is corresponding to the n-h stretching vibration in the free ligand, is missing in the spectra of the complexes, confirming deprotonation of the nitrogen atom of the sulfonamide and its coordination to the metal ion. the bands corresponding to the asymmetric and symmetric stretching vibration of the sulfonyl group in ligand appear at cm − respectively at cm − , while in spectra of the complexes are shifted with ≈ cm − to higher wave numbers respectively ≈ cm − to lower wave numbers; this is because in the free ligand a larger double bond character of the so bonds could be incriminated. the band due to the sulfonyl group is split in the spectra of the complexes, because of the different spatial orientation of these groups, as the x-ray show. we also notice a similar shift with ≈ cm − to higher wave numbers for the s-n stretching vibration in the spectra of the complexes, probably because the n atom is involved in the bonding to the metal atom. the remaining bands appear at wave numbers in accordance with the proposed structures both in the free ligand and complexes. the h-nmr and c{ h}-nmr spectra of the sulfonamide ligand and its complexes shows characteristic chemical shifts that are in agreement to the proposed structure. the most significant signal in the h-nmr spectra (figure ), corresponding of the hydrogen atom from sulfonamide group h and, appear in the free ligand at a chemical shift of . ppm. this signal is missing in the spectra of qbsc complexes, which is a solid prove for the complexation with the metal ion. we could also notice that the signals of hydrogen atoms near the metal ion h , h , h (from pyridine ring), are deshielded in qbsc complexes with about . ppm due to the powerful deshielding effect of the metal ion, a electron density transfer from the ligand to the metal ion being responsible for this. molecules , , x for peer review of effect of the metal ion, a electron density transfer from the ligand to the metal ion being responsible for this. similar considerations could be done for c{ h}-nmr spectra, figure . the most relevant signals in the c{ h}-nmr spectra, correspond to the carbon atoms c ′, c , c and c . in qbsc complexes, the c ′ carbons (ipso-sulfonamide) appear to a chemical shift of about ppm due to the powerful deshielding effect of sulfonamide group and the metal ion, while c (carbon from attached benzene ring of quinoline) appear to a chemical shift of about ppm, the same reasons being incriminated. comparative with the free ligand, in qbsc complexes, these carbons are deshielded with about ppm for c , respectively, with about ppm for c ′, due to the powerful deshielding effect of the metal ion. the most shielded carbons are c and c . in qbsc complexes, these carbons appear to a chemical shift of about ppm for c , respectively, ppm for c , being shielded comparative with the free ligand with about ppm for c respectively with about ppm for c . as far for the nmr spectra of cu + and co + complexes, the spectra are broad because of their different magnetic properties (paramagnetic). similar considerations could be done for c{ h}-nmr spectra, figure . the most relevant signals in the c{ h}-nmr spectra, correspond to the carbon atoms c , c , c and c . in qbsc complexes, the c carbons (ipso-sulfonamide) appear to a chemical shift of about ppm due to the powerful deshielding effect of sulfonamide group and the metal ion, while c (carbon from attached benzene ring of quinoline) appear to a chemical shift of about ppm, the same reasons being incriminated. comparative with the free ligand, in qbsc complexes, these carbons are deshielded with about ppm for c , respectively, with about ppm for c , due to the powerful deshielding effect of the metal ion. the most shielded carbons are c and c . in qbsc complexes, these carbons appear to a chemical shift of about ppm for c , respectively, ppm for c , being shielded comparative with the free ligand with about ppm for c respectively with about ppm for c . as far for the nmr spectra of cu + and co + complexes, the spectra are broad because of their different magnetic properties (paramagnetic). in the case of compounds b and c, the structure of compounds was assigned unambiguously by single-crystal x-ray investigation (for a and d, we did not obtained yet proper crystals), figure a , b. in the case of compounds b and c, the structure of compounds was assigned unambiguously by single-crystal x-ray investigation (for a and d, we did not obtained yet proper crystals), figure a ,b. the structure of qbsc cobalt complex c was resolved by direct methods and refined in the monoclinic p /n space group type. molecular information's shows a tetrahedral coordination of cobalt with the bidentate ligand trough co-nquinoline and co-nsulfonamide bonds. the co-nquinoline bond lengths ( . and . Å ) are slightly larger than co-nsulfonamide bonds ( . and . Å ) but in an acceptable range as reported by other papers for similar complexes. the structure of qbsc copper complex b was resolved by direct methods and refined in the monoclinic i /a space group type. molecular information's shows a tetrahedral coordination of copper with the bidentate ligand trough cu-nquinoline and cu-nsulfonamide bonds. the cu-nquinoline bond lengths ( . and . Å ) are slightly larger than cu-nsulfonamide bonds ( . and . Å ) but in an acceptable range as reported by other papers for similar complexes. copies of nmr spectra of ligand and qbsc complexes and checkcif files for x-ray data of qbsc complex b and c are presented to supplementary materials. a b the structure of qbsc cobalt complex c was resolved by direct methods and refined in the monoclinic p /n space group type. molecular information's shows a tetrahedral coordination of cobalt with the bidentate ligand trough co-nquinoline and co-nsulfonamide bonds. the co-nquinoline bond lengths ( . and . Å) are slightly larger than co-nsulfonamide bonds ( . and . Å) but in an acceptable range as reported by other papers for similar complexes. the structure of qbsc copper complex b was resolved by direct methods and refined in the monoclinic i /a space group type. molecular information's shows a tetrahedral coordination of copper with the bidentate ligand trough cu-nquinoline and cu-nsulfonamide bonds. the cu-nquinoline bond lengths ( . and . Å) are slightly larger than cu-nsulfonamide bonds ( . and . Å) but in an acceptable range as reported by other papers for similar complexes. copies of nmr spectra of ligand and qbsc complexes and checkcif files for x-ray data of qbsc complex b and c are presented to supplementary materials. the in vitro antimicrobial activity of ligand and qsc compounds a-d was determined by the kirby-bauer disk diffusion method [ ] using nutrient agar medium (mueller hinton agar for antibacterial tests and sabouraud agar for antifungal tests). the antibacterial activity was evaluated against two strains bacteria (gram-positive staphylococcus aureus atcc and gram-negative escherichia coli atcc ) and the antifungal activity against fungus candida albicans atcc . as positive control (c+) was used, penicillin iu for staphylococcus aureus, carbenicillin µg/ml for escherichia coli and nystatin , iu for candida albicans; the negative control (c−) consist in sterile filter paper disks with no antimicrobial compounds. the more susceptible were the germs, the larger the diameter (mm) of the inhibition zones is. the obtained results are expressed as diameters of inhibition zones (mm) and, for ligand , and qbsc a-d compounds, are presented in table and figure a -c. underline means active and bold and underline means very active. a diameter of inhibition zone (mm), a x ± sd, mean of five measurements ± standard deviation. from the data presented in table and figure a -c, we may notice that some of our qbsc compounds demonstrated to be effective against the tested strain. against bacterial strain staphylococcus aureus one compound qbsc d is very active (having a diameter of inhibition zone of mm) and three others compounds are active: qbsc c, qbsc b and the ligand a (with the diameter of inhibition zone of , and mm). against bacteria escherichia coli one compound qbsc d is active ( mm) while against fungus candida albicans two compounds are very active: qbsc d and the ligand a ( respectively . mm). the compounds which exhibited significant antimicrobial activity were later tested using the standardized broth microdilution assay procedure to determine the minimum inhibitory concentration (mic) of the compounds under investigation against the reference microorganisms [ ] . the resulted mic value is defined as the lowest concentration of the antimicrobial agent under investigation, which prevents visible growth of the tested microorganism. the obtained results are listed in table . from the data presented in table and figure a -c, we may notice that some of our qbsc compounds demonstrated to be effective against the tested strain. against bacterial strain staphylococcus aureus one compound qbsc d is very active (having a diameter of inhibition zone of mm) and three others compounds are active: qbsc c, qbsc b and the ligand a (with the diameter of inhibition zone of , and mm). against bacteria escherichia coli one compound qbsc d is active ( mm) while against fungus candida albicans two compounds are very active: qbsc d and the ligand a ( respectively . mm). the compounds which exhibited significant antimicrobial activity were later tested using the standardized broth microdilution assay procedure to determine the minimum inhibitory concentration (mic) of the compounds under investigation against the reference microorganisms [ ] . the resulted mic value is defined as the lowest concentration of the antimicrobial agent under investigation, which prevents visible growth of the tested microorganism. the obtained results are listed in table . as we may notice from table , cd complex (qbsc d) is active to a very low concentration, having a mic of . × − mg/ml in the case of staphylococcus aureus and candida albicans, respectively × − mg/ml in the case of escherichia coli. significant results was obtained also for cu complex (qbsc b) which have mic value of × − mg/ml in the case of bacterial streams staphylococcus aureus and escherichia coli. the diameter of inhibition zone and mic data reveal that our qbsc compounds (except the zn complexes) have a quasi-nonselective activity against bacteria staphylococcus aureus. against escherichia coli only cd complex (qbsc d) have a significant activity, while against fungus candida albicans cd complex (qbsc d) and quinoline sulfonamide ligand ( a) present a strong activity. the above presented results also reveal a clear influence of the metal cation, the cd complex (qbsc d) being far away the most active tested compound, having a quasi-nonselective antibacterial and antifungal activity. the activity of cadmium complexes are higher the most probable because of a better synergism ligand cadmium. as to the mechanism of action of our qbsc compounds, we may presume that they could act as carbonic anhydrase inhibitors, as the literature describe for related compounds [ , ] . all the reagents and solvents were purchased from commercial sources and used without further purification. melting points were recorded on a electrothermal mel-temp ii (barnstead international, dubuque, ia, usa) apparatus in open capillary tubes and are uncorrected. analytical thin-layer chromatography (tlc) was performed with commercial merck silica gel f plates and visualized with uv light (λ max = or nm). the nmr spectra were recorded on a bruker avance iii mhz spectrometer (bruker vienna, austria) operating at mhz for h and mhz for c. chemical shifts were reported in delta (δ) units, part per million (ppm) and coupling constants (j) in hz. the following abbreviations were used to designate chemical shift multiplicities: s = singlet, d = doublet, ad = apparent doublet, t = triplet, q = quartet, aq = apparent quartet, m = multiplet. infrared (ir) data were recorded as films on potassium bromide (kbr) pellets on a ft-ir vertex bruker spectrophotometer. in order to determine the structure of complexes, a crystal of each complex was selected, mounted on a hair thread and inserted into the four-circle supernova single crystal diffraction instrument. data acquisition was made at • k (− . • c) using cuka radiation. a number of reflections from a total of acquired in . < θ < . range were used for refinement and structure solution. compound a, which have already been reported in literature, showed spectral data in agreement to the reported data [ , , ] . synthesis of the ligand -chloro-n-(quinolin- -yl)benzenesulfonamide ( a), mmol of -aminoquinoline was dissolved in minimum volume of dichloromethane ch cl , then was added to a stirred solution of -chlorobenzenesulfonyl chloride ( . mmol) and pyridine. the crude product was washed with hcl ( m), then with a solution of saturated nahco . the organic extract was dried on na so and the solvent was removed in vacuum. the solid obtained was purified on column chromatography (ch cl /acoet = / ) giving the white solid -chloro-n-(quinolin- -yl)benzenesulfonamide. the complexes were prepared by direct reaction between the sulfonamide ligand and zn(ii), cu(ii), co(ii) and cd(ii) salts. [zn(n-(quinoline- -yl)- -chloro-benzenesulfonamide) ] ( a), mmol of sulfonamide ligand a were dissolved in ml meoh and ml nh oh were added. mmol of zncl dissolved in ml methanol were added dropwise while solution was magnetically stirred. when addition is completed, a yellow precipitate is formed, which is separated by filtration. addition of a base for deprotonating the nitrogen from amine was necessary to prepare the zinc complex. [ ] [cu(n-(quinoline- -yl)- -chloro-benzenesulfonamide) for inoculum preparation, reference microbial cultures of bacteria (staphylococcus aureus atcc , escherichia coli atcc ) and fungi (candida albicans atcc ) were employed. a number of approximately colonies from each type of culture were used to inoculate ml of mueller hinton (mh) agar (for antibacterial tests) and sabouraud agar (for antifungal tests). using a beckman coulter du spectrophotometer (λ = nm), the turbidity of the inoculum was adjusted to a . mcfarland standard ( - × cfu/ml for bacteria and - × cfu/ml for candida), and the inoculum was transferred, in a ml volume, onto the surface of the growth media specific for bacteria (mh) and fungi (sabouraud). once the inoculum was absorbed, sterile paper disks of approximately mm in diameter and impregnated with µl of antibacterial compound (dissolved in dmso %) were placed on the surface of the culture media; for all the tested compounds, the concentration used was mg/ml. following incubation at the optimal temperatures for bacteria and fungi, of • c and • c, respectively, for h (bacteria) and h (fungi), the diameters of the inhibition zones were measured using a ruler. the controls were prepared in the same growth conditions (i.e., c+: sterile filter paper disks impregnated with antibiotics inducing sensitivity in the organisms under investigation, namely penicillin iu for staphylococcus aureus, carbenicillin µg/ml for escherichia coli and nystatin , iu for candida albicans, and c−: sterile filter paper disks with no antimicrobial compounds). the working technique involves the use of a -well microtiter plate (microdilution). in each well of the plate, µl of growth medium mh, µl of microbial inoculum (staphylococcus aureus atcc , escherichia coli atcc , or candida albicans atcc ) prepared in the same manner as in the diffusion test (i.e., by diluting the standardized microbial suspension adjusted to a . mcfarland standard), and µl of antimicrobial substance to be tested were transferred by pipetting, in different concentrations. to this purpose, double dilutions of the antimicrobial agent were made in the dmso %, starting with the mg/ml dilution (e.g., . mg/ml, . mg/ml, . mg/ml, . mg/ml, . mg/ml and so on). for each tested microorganism, a positive control c+ (containing µl of mh growth medium and µl of antimicrobial compound) and a negative one c-(containing µl of mh growth medium and µl of diluted microbial culture) were prepared. following the incubation of the microplates at • c for h (for staphylococcus aureus atcc and escherichia coli atcc ) and at • c for h (for candida albicans atcc ), µl of resazurin were added in each well. the samples were incubated once again at the temperature optimal for each microorganism for one hour. the color of the indicator turned from purple to pink. resazurin is a colorimetric indicator for cell viability widely applied for monitoring cell proliferation. the redox dye, resazurin, enters the cytosol in the oxidized form (purple-blue) and is converted to the reduced form, resorufin (pink). two new series of hybrid quinoline-sulfonamide complexes (m + : zn + , cu + , co + and cd + ) derivatives were designed, synthesized and tested for their antimicrobial activity. the synthesis is straight and efficient, involving two steps: acylation of aminoquinoline followed by complexation with metal acetate (cu + , co + and cd + ) or chloride (zn + ). the synthesized compounds were characterized by ftir, nmr spectroscopy and by x-ray diffraction on single crystal. the co (ii) complex crystallize in the monoclinic p /n space group type, with a tetrahedral coordination of cobalt with the bidentate ligand trough co-n quinoline and co-n sulfonamide bonds. the qsc compounds were preliminary in vitro screened for their antibacterial and antifungal activity and the obtained results are very promising. against bacterial strain staphylococcus aureus one compound have an excellent antibacterial activity qbsc d (Φ = mm, mic = . × − mg/ml) and three others compounds are active: qbsc c, qbsc b and the quinoline sulfonamide ligand a (Φ of , and mm). against bacteria escherichia coli only one compound qbsc d present activity ( mm, mic of × − mg/ml). against fungus candida albicans again the qbsc d complex have an excellent antibacterial activity (Φ = mm, mic = . × − mg/ml) and also the ligand a have an excellent antibacterial activity (Φ = . mm) but to a relatively high concentration (mic = . mg/ml). these results reveal a clear influence of the metal cation, the cd complex (qbsc d) being far away the most active tested compound, having a quasi-nonselective antibacterial and antifungal activity. the activity of cadmium complexes are higher the most probable because of a better synergism ligand cadmium. gilman's the pharmacological basis of therapeutics the organic chemistry of drug design and drug action who global strategy for containment of antimicrobial resistance three emerging coronaviruses in two decades the story of sars, mers, and now covid- hiv- did not contribute to the -ncov genome virtual screening and repurposing of fda approved drugs against covid- main protease covid- : a promising cure for the global panic structural basis for the recognition of sars-cov- by full-length human ace synthesis and biological potentials of quinoline analogues: a review of literature , , -triazole-quinoline/quinolone hybrids as potential anti-bacterial agents synthesis and antibacterial evaluation of new pyrrolo a review on diverse heterocyclic compounds as the privileged scaffolds in antimalarial drug discovery microwave assisted synthesis and antimicrobial potential of quinoline-based -hydrazide-hydrazone derivatives quinoline hybrids and their antiplasmodial and antimalarial activities mangalagiu, i.i. hybrid imidazole (benzimidazole)/pyridine (quinoline) derivatives and evaluation of their anticancer and antimycobacterial activity identification of benzenesulfonamide quinoline derivatives as potent hiv- replication inhibitors targeting rev protein visible light-promoted photocatalytic c- carboxylation of -aminoquinoline amides and sulfonamides via a single electron transfer pathway alzuet-piña, g. photoinduced and self-activated nuclease activity of copper(ii) complexes with n-(quinolin- -yl)quinolin- -sulfonamide-dna and bovine serum albumin binding -methoxy- -p-toluenesulfonamidoquinoline), a common fluorescent sensor for cellular zinc, images zinc proteins from sensors to silencers: quinoline-and benzimidazole-sulfonamides as inhibitors for zinc proteases synthesis and structural characterization of zinc complexes with sulfonamide containing -aminoquinoline aqueous coordination chemistry of quinoline-based fluorescence probes for the biological chemistry of zinc synthesis, structure and physicochemical properties of zinc and copper complexes based on sulfonamides containing -aminoquinoline ligands synthesis and in vitro evaluation of leishmanicidal and trypanocidal activities of n-quinolin- -yl arylsulfonamides in vitro antiprotozoal evaluation of zinc and copper complexes based on sulfonamides containing -aminoquinoline ligands cu (ii), and zn (ii) complexes as potential antifungal agents mangalagiu, i.i. bis-(imidazole/benzimidazole)-pyridine derivatives: synthesis, structure and antimycobacterial activity antimycobacterial activity of nitrogen heterocycles derivatives: -(pyridine- -yl)-indolizine derivatives. part vii synthesis, stereochemical studies and antimycobacterial activity of new acetyl-hydrazines pyridazinone synthesis, structure, antimycobacterial and anticancer evaluation of new pyrrolo-(phenanthroline) derivatives synthesis of new antibacterial and antifungal drimane sesquiterpenoids with azaheterocyclic units design, synthesis and antimycobacterial activity of some new azaheterocycles: , -phenanthroline with p-halogeno-benzoyl skeleton. part vi new indolizines with phenanthroline skeleton: synthesis, structure, antimycobacterial and anticancer evaluation mangalagiu, i.i. design, synthesis and antimycobacterial activity of some new azaheterocycles: phenanthroline with p-halo-benzoyl skeleton synthesis and in vitro analysis of novel dihydroxyacetophenone derivatives with antimicrobial and antitumor activities mangalagiu, i.i. design, syntheses and antimicrobial activity of some novel homodrimane sesquiterpenoids with diazine skeleton new pyridazine-fluorine derivatives: synthesis, chemistry and biological activity. part ii mangalagiu, i.i. design, synthesis and antituberculosis activity of some new pyridazine derivatives: bis-pyridazine. part iv synthesis and antituberculosis activity of some new pyridazine derivatives. part ii mangalagiu, i.i. diazinium salts with dihydroxyacetophenone skeleton: syntheses and antimicrobial activity clsi document m -a . methods for dilution antimicrobial susceptibility tests for bacteria that grow aerobically; approved standard effects of microplate type and broth additives on microdilution mic susceptibility assays carbonic anhydrases: novel therapeutic applications for inhibitors and activators this article is an open access article distributed under the terms and conditions of the creative commons attribution acknowledgments: authors are thankful to cernesim, for nmr and x-ray experiments. the authors gratefully acknowledge simona dunca for her help in biological assay and senior researcher tiberiu roman in x-ray experiments. the authors declare no conflict of interest. key: cord- - i kjk authors: daescu, monica; matea, adelina; negrila, catalin; serbschi, constantin; ion, alina c.; baibarac, mihaela title: photoluminescence as a valuable tool in the optical characterization of acetaminophen and the monitoring of its photodegradation reactions date: - - journal: molecules doi: . /molecules sha: doc_id: cord_uid: i kjk in this work, new evidence for the photodegradation reactions of acetaminophen (ac) is reported by photoluminescence (pl), raman scattering and ftir spectroscopy. under excitation wavelength of nm, ac shows a pl band in the spectral range of – nm, whose intensity decreases by exposure to uv light. the chemical interaction of ac with the naoh solutions, having the concentration ranging between . and . m, induces a gradual enhancement of the photoluminescence excitation (ple) and pl spectra, when the exposure time of samples at the uv light increases until min, as a result of the formation of p-aminophenol and sodium acetate. this behavior is not influenced by the excipients or other active compounds in pharmaceutical products as demonstrated by ple and pl studies. experimental arguments for the obtaining of p-aminophenol and sodium acetate, when ac has interacted with naoh, are shown by raman scattering and ftir spectroscopy. acetaminophen (ac) is an active compound of different drugs marketed under the name of paracetamol, theraflu, coldrex, parasinus, algopirin, and so on. the therapeutic effects for which ac is administered are: (i) the reduction of headaches during migraines [ ] ; (ii) the diminution of kidney stone-induced pain [ ] ; (iii) osteoarthritis which involves the pain at the hip, knee and hands [ ] ; (iv) the cutting down of the pain resulting after dental procedures [ ] ; (v) fighting fever [ ] and recently in coronavirus disease (covid- ) [ ] . the most important adverse effects of ac reported until now consist of the generation of liver damage [ , ] . the use of a dose of ac higher than mg per day induces the appearance of acute liver failure, which then leads to the death of the un-treated patients [ ] . for the quantitative determination of paracetamol in biological fluids and tablets, a sustained effort was carried out in the development of analytical techniques such as high-performance liquid chromatography (hplc) [ ] , uv-vis absorption spectroscopy [ ] , surface enhanced raman scattering [ ] , cyclic voltammetry [ ] , square-wave adsorptive anodic stripping voltammetry [ ] , to study the behavior of ac in the absence of excipients, an aqueous solution of ac . m was prepared. the influence of the alkaline solutions on ac was analyzed using aqueous solutions of naoh with various concentrations. thus, in all experiments, ml of ac . m interacted with ml naoh − , − , − or . m. a tablet of each drug was dispersed into ml distilled water, under ultrasonication, for min. in order to obtain a clear solution, a successively filtration was carried out. for each drug containing ac, i.e., paracetamol, parasinus and pararemin, ml of pharmaceutical compounds interacted with ml naoh . m. the solid-state interaction of ac with naoh, at a non-hydrostatic pressure equal to . gpa for min has involved: (a) the preparation of three mixtures of ac and naoh as follows: (i) . g ac and . g naoh; (ii) . g ac and . g naoh; and (iii) . g ac and . g naoh. the weight ratios of the three mixtures of ac and naoh are equal to . , . and . . homogenization of each mixture of ac and naoh was performed by grinding for min. the samples were exposed successively at uv light, for min, using a mercury-vapors lamp with the power of w, by the selecting with an ug filter of the spectral range - nm, that contains the hg spectrum line of high intensity at nm. pl and ple spectra of the ac aqueous solution in the absence and in the presence of naoh were recorded, in the right-angle geometry, with a fluorolog- spectrophotometer, fl - . . model, from horiba jobin yvon, endowed with a w xe lamp. raman spectra of ac before and after the interaction with naoh in the solid state were recorded with a ft raman spectrophotometer, rfs s model, from bruker, endowed with a yag:nd laser (excitation wavelength nm). ir spectra of ac in the initial state and after the interaction with naoh in the solid state were recorded with an ftir spectrophotometer, vertex model, from bruker, in the attenuated total reflection geometry. additional information concerning the products resulting from the interaction of ac with naoh are shown by x-ray photoelectron spectroscopy (xps), using a specs spectrometer endowed with a phobios analyzer and an al kα source. according to figure a , the ple spectrum of the ac aqueous solution shows an intense band with the maximum at nm, which is accompanied of another of low intensity at nm. an important decrease in the intensity of the band at nm is observed to occur from . × to . × counts/sec when ac is exposed to uv light for min ( figure highlights changes in the pl band position and intensity of ac when an interaction of this with the naoh solutions having concentrations − , − , − and . m occurs. the pl spectrum of ac shows two emission bands at and nm, the last one having the intensity equal to . × counts/sec ( figure a ). as observed in figure a , the exposure of the ac aqueous solution to uv light, for min, leads to a shift of the maximum of emission band from to nm simultaneously with a decrease in the intensity of this band from . × to . × counts/sec. the interaction of the ac with naoh solutions having concentrations − , − , − and . m induces a variation in the intensity of the pl spectrum at cca. . × , . × , . × and × counts/sec, respectively. this result can be explained considering the decrease in ac concentration in the reaction mixture as a consequence of the chemical interaction of ac with naoh, according to reaction ( ) in scheme . regardless of the concentration of the naoh solution that interacted with the ac, after min of exposure to uv light, it is noticed that the pl spectra shown in figure b -e have maxima at approx. - nm. according to figure b -e, after min of exposure to uv light, the ac interacted with the naoh solutions having the concentrations equal to − , − , − and . m, the intensity of the pl spectra is equal to . × , . × , . × and . × counts/sec, respectively. a similar behavior with that reported in the case of ac (figure a ) takes place by the interaction of ac with naoh − m (figure b) . in this last case, one observes for the ple band at nm, a decrease in intensity from . × to . × counts/sec. for higher naoh concentrations such as those in the . - . m range, an inverse behavior is reported by exposure to uv light of ac solutions which interact with naoh. by increasing the exposure time at uv light up to min, according to figure c -e, an intensity increase of the ple band of ac interacted with − , − and . m naoh solutions from . × , . × and . × counts/sec to . × , . × and . × counts/sec, respectively, takes place. . all ple spectra were recorded at the emission wavelength of nm. blue and red curves correspond to the first and last spectrum, respectively, of the samples exposed to uv light for and min. black curves correspond to the intermediate spectra of above samples, each ple spectrum being collected at s, respectively. the photoluminescence (pl) spectra of the aqueous solution of ac before (a) and after the interaction with the aqueous solution of naoh − (b), − (c), − (d) and × − m (e). all pl spectra were recorded at the excitation wavelength of nm. blue and red curves correspond to the first and last spectrum, respectively, of the samples exposed to uv light for and min. black curves correspond to the intermediate spectra of above samples, each pl spectrum being collected at s, respectively. a similar behavior with that reported in the case of ac (figure a ) takes place by the interaction of ac with naoh − m (figure b) . in this last case, one observes for the ple band at nm, a decrease in intensity from . × to . × counts/sec. for higher naoh concentrations such as those in the . - . m range, an inverse behavior is reported by exposure to uv light of ac solutions which interact with naoh. by increasing the exposure time at uv light up to min., according to figure c -e, an intensity increase of the ple band of ac interacted with − , − and . m naoh solutions from . × , . × and . × counts/sec to . × , . × and . × counts/sec, respectively, takes place. these variations in the ple and pl spectra of ac are also found in the case of pharmaceutical compounds, after the filtering process. in order to support this statement, figure , figure and all pl spectra were recorded at the excitation wavelength of nm. blue and red curves correspond to the first and last spectrum, respectively, of the samples exposed to uv light for and min. black curves correspond to the intermediate spectra of above samples, each pl spectrum being collected at s, respectively. these variations in the ple and pl spectra of ac are also found in the case of pharmaceutical compounds, after the filtering process. in order to support this statement, ( )) and naoh (equation ( )). the interaction of ac with an excess of naoh is described by equation ( ). ( )) and naoh (equation ( )). the interaction of ac with an excess of naoh is described by equation ( ). intensity (counts/sec) intensity (counts/sec) paracetamol magistra + naoh paracetamol magistra + naoh parasinus + naoh in the presence of excipients, the band of the ac ple spectrum is shifted to nm. by exposure to uv light of the aqueous solution of paracetamol, containing ac and excipients, for min, a variation in the intensity of the ple band is observed from . × to . × counts/sec (figure a ). the interaction of the aqueous solution of paracetamol with naoh leads to a shift of the ple band from nm (figure a ) to nm (figure c ), the subsequent exposure at uv light for min inducing an intensity increase of the ple band from . × to . × counts/sec. regarding the pl spectrum of paracetamol, it is observed that the most intense emission band from nm (figure a ) is shifted at nm (figure b) , while the lowest intensity band has the maximum at nm (figure b) , i.e., at the same wavelength as the pl spectrum of ac in the absence of excipients (figure a) . by exposure to uv light of the aqueous solution of paracetamol, for min, a variation of the intensity of the pl band is noticed from . × to . × counts/sec. the interaction of paracetamol with naoh induces a down-shift of emission band from nm (figure b ) to nm ( figure d ) and subsequent exposure to uv light induces a gradual up-shift of the band from at nm simultaneous with an intensity increase of this emission band from . × to . × counts/sec (figure d) . these results confirm that the excipients do not influence the evolution of ple and pl spectra under uv light. in the presence of excipients, the band of the ac ple spectrum is shifted to nm. by exposure to uv light of the aqueous solution of paracetamol, containing ac and excipients, for min, a variation in the intensity of the ple band is observed from . × to . × counts/sec (figure a ). the interaction of the aqueous solution of paracetamol with naoh leads to a shift of the ple band from nm (figure a the increase in the intensity of the ple and pl spectra of ac in the presence of other active compounds and excipients (figure ; figure ) is similar to that reported in the case of pure active compounds ( figure ; figure ). to quantify the impact of additional active compounds on ac in the particular case of parasinus and pararemin, a careful analysis of figures and is necessary. in this context, one remarks that: (i) in the initial state, for the two pharmaceutical formulations the maximum of the ple band is situated at nm, the variation in the intensity of the ple band, when the samples were exposed to uv light for min, being only of . % and . % in the case of parasinus (figure a ) and pararemin (figure a) , respectively. these values are lower than those reported in the case of ac and paracetamol, when one remarks a variation in the intensity of the ple bands with maxima at and nm of . % (figure a ) and . % (figure a) . changes in the intensities of the pl bands of parasinus and pararemin, with maxima located at and nm are reported to be equal to . % (figure b ) and . % (figure b) , respectively. these variations are smaller compared to those of ac and paracetamol, for which variations of~ . % and~ . % in intensity of the pl bands with maxima at nm ( figure a) and nm (figura b), respectively, are reported; ii) after min of exposure to uv light of the two pharmaceutical products, which were interacted with ml naoh . m, an increase in intensity of ple bands of parasinus and pararemin of~ . % (figure c ) and . % (figure c ) was reported. these values demonstrate that the presence of chlorpheniramine maleate and pseudoephedrine hydrochloride does not induce additional changes in comparison with those of ac interacting with naoh, and by successive exposure at uv light for min, an increase in the intensity of the ple band of . % (figure e ) was noticed. referring to ac (figure e) , the presence of propyphenazone and caffeine induces a slight inhibition of the photodegradation process of ac according to figure c . as increasing the weight of the additional active compounds to ac from . and wt.%, a reduced variation of the intensity of pl spectra of paracetamol, parasinus and pararemin, from % to . % and % respectively, is reported. this result confirms that the active compounds contained in parasinus (chlorpheniramine maleate and pseudoephedrine hydrochloride) and pararemin, (propyphenazone and caffeine) have the role to inhibit the photodegradation of ac in the alkaline environment. these changes reported in figures - can be explained if we take into account the chemical reactions shown in scheme . according to scheme , depending on the molar ratio between ac and naoh, i.e., : (equation ( )) or : (equation ( )), the reaction products correspond to sodium acetate and p-aminophenol or sodium p-aminophenoxide, respectively. figures and prove that the chemical reactions of ac with naoh, shown in scheme , lead to the generation of p-aminophenol and sodium acetate. as shown in table , the main drawback of the ir spectroscopy and raman scattering in comparison with pl is that these methods do not allow acquiring information in dilute analytes solutions such as those used in the pl studies reported above. therefore, in the future experiments solid state samples were used to illustrate the interaction of ac with naoh. in figure , the black curve shows the raman spectrum of ac, which is characterized by the lines peaked at , , , , , , , , , , , and cm − , that are attributed to the vibrational modes of the deformation of phenyl ring + c-h twisting, deformation of phenyl ring, deformation of phenyl ring + c-c rocking in amide group, breathing of phenyl ring, c-o-h bending + c-c-h bending in aryl group, c-n stretching + c-c stretching in amide group, c-o-h bending + c-c-h bending in aryl group + c-n rocking in amide group, c-o-h bending + c-h rocking + c-c stretching in aryl group, c-n-h bending in amide group, c-n stretching + c-o stretching +c-c stretching in aryl group, c=o stretching in amide group, symmetrical c-h stretching in amide group and asymmetrical c-h stretching in amide group, respectively. [ ] the interaction of ac with naoh leads to the variations in raman spectrum of ac shown in figure as follows: (i) an up-shift or the raman line assigned to the vibrational mode of breathing of the phenyl ring from to cm − simultaneous with its increase in the intensity; (ii) a gradual decrease in the intensity of the raman lines situated in the spectral range - cm − as well as the raman lines assigned to the vibrational modes of c-n-h bending in the amide group and c=o stretching in the amide group peaked at and cm − , respectively; (iii) a down-shift of the raman line from to cm − ; (iv) a gradual increase in the intensity of the raman line, assigned to the vibrational mode of c-n stretching + c-o stretching + c-c stretching in aryl group, which is peaked at cm − ; and v) a change of the ratio between the intensities of the raman lines peaked at and cm − from . (value reported in the case of ac) to . and . , when samples contain the ac: naoh mass ratios equal to . and . , respectively. a puzzling fact is that the aniline-derived compounds and carboxylic acids show in the raman spectra, lines at [ , ] and cm − [ ] , respectively, very close from those reported in figure . taking into account these facts, in our opinion, the increase in the relative intensity of the raman lines at and cm − originates in the formation of p-aminophenol and sodium acetate, respectively. molecules , , x for peer review of figure . the raman spectrum of ac (black curve). red, green and blue curves correspond to the raman spectra of ac interacted with naoh, and subsequently exposed to uv light for min., when the ac: naoh weight ratio is equal to . , . and . , respectively. magenta curve corresponds to the raman spectrum of naoh. figure . the ir spectra of ac before (black curve) and after the interaction with naoh, when the ac: naoh weight ratio is equal to . (red curve) and . (green curve), the samples being subsequently exposed to uv light for min. blue curve corresponds to the ir spectrum of naoh. c c c c figure . the raman spectrum of ac (black curve). red, green and blue curves correspond to the raman spectra of ac interacted with naoh, and subsequently exposed to uv light for min, when the ac: naoh weight ratio is equal to . , . and . , respectively. magenta curve corresponds to the raman spectrum of naoh. molecules , , x for peer review of figure . the raman spectrum of ac (black curve). red, green and blue curves correspond to the raman spectra of ac interacted with naoh, and subsequently exposed to uv light for min., when the ac: naoh weight ratio is equal to . , . and . , respectively. magenta curve corresponds to the raman spectrum of naoh. figure . the ir spectra of ac before (black curve) and after the interaction with naoh, when the ac: naoh weight ratio is equal to . (red curve) and . (green curve), the samples being subsequently exposed to uv light for min. blue curve corresponds to the ir spectrum of naoh. c c c c figure . the ir spectra of ac before (black curve) and after the interaction with naoh, when the ac: naoh weight ratio is equal to . (red curve) and . (green curve), the samples being subsequently exposed to uv light for min. blue curve corresponds to the ir spectrum of naoh. table . the most important advantages and drawbacks of the pl in comparison with other methods to assess the photodegradation processes of the pharmaceutical compounds. shorter time of collecting results in the case of the samples in powder state or as solutions high cost of infrastructure uv-vis spectroscopy this work; [ ] shorter time of collecting results; no need additional reagents; the photodegradation process can be carried both in-situ and ex-situ unseparated products from the reaction mixture hplc this work; [ ] in pl studies, pharmaceutical products can be both as powder, tablet or dilute solutions; the photodegradation process can be analyzed both in-situ and ex-situ. [ ] as increasing naoh weight that interacts with ac, the following variations are induced in figure : (i) an up-shift of the ir band assigned to the vibrational mode c-c-h bending in aryl group from to cm − , simultaneous with the decrease of the absorbance of ir band attributed to the phenyl ring deformation + c-c rocking in amide group vibrational mode; (ii) the decrease in the absorbance of the ir bands localized in the spectral ranges - and - cm − and the down-shift of the ir bands from , and cm − to , , cm − , respectively; (iii) the increase in the absorbance of the ir bands situated in the spectral range - cm − and the down-shift of the ir bands from and cm − to and cm − ; and (iv) the gradual decrease in the absorbance of the ir bands localized in the spectral ranges - and - cm − . the absence of the ir bands of , and cm − as well as the diminution of the absorbance of ir bands localized in the spectral ranges - and - cm − simultaneous with the increase of the absorbance of ir band at cm − attributed to the c=c stretching vibrational mode in compounds of the type aniline [ , ] indicate a decrease in the amide group weight as a consequence of the appearance of the amine and carboxylic groups, as shown in scheme . a decrease in the amide group weight after the interaction of ac with naoh is also observed by xps spectroscopy. according to figure a , the xps c s spectrum of ac is characterized by the five peaks at , , , . and ev, assigned to the following bonds: c=c, c-n, c-o in phenolic compounds, c=o in amide and c=o/c=c in aromatic ring, respectively. [ ] two peaks are observed in the n s spectrum of ac (figure b ) at . and . ev which were attributed to the -nhco and -nh-bonds. [ ] in contrast with ac, the following variations are highlighted in figure , as a consequence of the interaction of ac with naoh: (i) an increase of the intensity of the peak at ev; (ii) a decrease of the intensity ratio of the peaks at . and . ev from . to . (figure b ,b ) , and iii) the presence of the na s peaks at . and ev in figure c , which were attributed to ch coona and un-reacted naoh [ , ] , respectively. the na s spectrum was also fitted with two components (figure c ) -the peak at ev attributed to naoh and another one at ev attributed to ch coona. due to the large difference in kinetic energy of the photoelectrons from na s and na s orbitals, it is proper to assume that the na s photoelectrons describe the first layers of the sample's surface while the na s photoelectrons describe the deeper layers. comparing the peak intensities in the two spectra we can deduce that the unreacted naoh is only on the surface of the sample (figure c ,c ). all these changes plead for the formation of p-aminophenol and sodium acetate. based on these results, the most important advantages and drawbacks of the pl in comparison with other methods used in the assessing of the photodegradation processes of the pharmaceutical compounds are shown in table . molecules , , x for peer review of figure . xps c s (a) and n s (b) spectra of ac (a , b ) as well as ac interacted with naoh (a , b ). figures c and c show xps na s and na s spectra of ac interacted with naoh. using photoluminescence (pl), raman scattering and ftir spectroscopy, new evidence concerning the interaction of ac with naoh was reported in this work. our results lead to the (c ,c ) show xps na s and na s spectra of ac interacted with naoh. using photoluminescence (pl), raman scattering and ftir spectroscopy, new evidence concerning the interaction of ac with naoh was reported in this work. our results lead to the following conclusions: (i) photoluminescence represents an alternative method to uv-vis spectroscopy to monitor the reaction of ac with naoh under uv light; this reaction is highlighted by the up-shift of the ple and pl bands from and nm to and nm, respectively, simultaneous with an intensity increase of these spectra; (ii) the ple and pl studies have demonstrated that this photochemical reaction is not influenced by the excipients existing in pharmaceutical compounds or the other active compounds such as those used in antipyretic drugs; (iii) the changes invoked in the emission spectra are a consequence of the fact that the interaction of ac with naoh results in the formation of p-aminophenol and sodium acetate; (iv) the main evidence shown in raman spectra for the formation of p-aminophenol and sodium acetate consist of the presence of the line at cm − and the change of the ratio between the relative intensities of the raman lines at and cm − in the favor of the last one; the raman lines at and cm − correspond to the c=c stretching vibrational mode in the aniline-derive compound and carboxylic group, respectively; and (v) diminution of the amide group weight in ac as a consequence of the interaction with naoh was highlighted in ir spectra by the absence of the bands at , and cm − , the decrease in the absorbance of the ir bands situated in the spectral ranges - and - cm − and the presence of the ir band at cm − attributed to the c=c stretching vibrational mode in aniline-derive compounds. a diminution of the amide group simultaneous with the formation of the amine and carboxylic groups, as a consequence of the interaction of ac with naoh, was also highlighted by xps spectroscopy. self-medication of migraine and tension type headache summary of the evidence based recommendations of the deutsche migrane und kopbschmerzgesellschaft (dmkg) abdel shaheed, c. paracetamol for paint in adults american college of rheumantology recommendations for the use of nonpharmacology and pharmacologic therapies in osteoarthritis of the hand, hip and knee efficacy of otc analgesics efficacy and safety of acetaminophen vs. ibuprofen for treating children's pain or fever: a meta-analysis medications on covid- patients: summarizing the current literature from an orthopedic perspective acetaminophen (apap) hepatotoxicity isn't it time for apap to go away? the acute live failure study group, acetaminophen induced acute liver failure: results of a united states multicenter, perspective study acetaminophen poisoning and toxicity determination of paracetamol: historical evolution a flow-through solid phase uv spectrophotometer biparameter sensor for the sequential determination of ascorbic acid and paracetamol fast detection of paracetamol on a fold nanoparticle-chitosan substrate by sers carbon paste electrode modified with poly( -aminophenaol) for voltammetric determination of paracetamol an electrochemical sensor prepared by sonochemical one-pot synthesis of multi-walled carbon nanotube-supported cobalt nanoparticles for the simultaneous determination of paracetamol and dopamine sensitive detection of acetaminophen with graphene based electrochemical sensor simultaneous voltametric determination of tyrosine and paracetamol using a carbon nanotube-graphene nanosheet nanocomposite modified electrode in human blood serum and pharmaceuticals determination of the paracetamol degradation process with online uv spectroscopic and multivariate curve resolution-alternating least squares methods comparative validation by hplc the possibility of using x-ray powder diffraction, infrared and raman spectroscopy in the study of the identification of structural polymorphs of acetaminophen vibrational spectra of n,n-dimethylaniline and its radical cation. an interpretation based on quantum chemical calculations infrared and raman spectra of non-vicinally trisubstituted , -dimethylaniline and -methyl- -chloroaniline infrared and raman studies of the solids in the mg(ch coo) -zn(ch coo) -h o system aniline oligomer-modified graphene for enhanced electrochemical performances the influence of activated carbon surface chemical composition of the adsorption of acetaminophen (paracetamol) in vitro part ii. tg, ftir, and xps analysis of carbons and the temperature dependence of adsorption kinetics at the neutral ph reversibly physisorbed and chemisorbed water on carboxylic salt surfaces under atmospheric conditions high-resolution x-ray photoemission from sodium metal and its hydroxide this article is an open access article distributed under the terms and conditions of the creative commons attribution the authors declare no conflict of interest. the funders had no role in the design of the study; in the collection, analyses, or interpretation of data; in the writing of the manuscript, or in the decision to publish the results. key: cord- - rg y authors: yadav, yogesh; sharma, deepti; kaushik, kumar; kumar, vineet; jha, amitabh; prasad, ashok k.; len, christophe; malhotra, sanjay v.; wengel, jesper; parmar, virinder s. title: synthetic, structural, and anticancer activity evaluation studies on novel pyrazolylnucleosides date: - - journal: molecules doi: . /molecules sha: doc_id: cord_uid: rg y the synthesis of novel pyrazolylnucleosides a–e, a–e, a–e, and a–e are described. the structures of the regioisomers were elucidated by using extensive nmr studies. the pyrazolylnucleosides a–e and a–e were screened for anticancer activities on sixty human tumor cell lines. the compound e showed good activity against cancer cell lines. in particular, it showed significant inhibition against the lung cancer cell line hop- (gi( ) . µm) and breast cancer cell line hs t (gi( ) . µm). the chemistry of nucleosides has been extensively studied and several analogs have been found to exhibit potential as fungicidal, antitumor, and antiviral agents [ ] [ ] [ ] [ ] [ ] [ ] [ ] . modifications in both the heterocyclic bases and the sugar moieties have led to active and safer nucleoside analogues that have found applications as agents effective against human immunodeficiency virus (hiv), the causative agent of acquired immune deficiency syndrome (aids), and also against viral infections caused by the herpes simplex virus (hsv types and ), varicella zoster virus (vzv), hepatitis c virus (hcv), human cytomegalovirus (hcmv), and epstein-barr virus (ebv) [ , ] . nucleoside and nucleotide modifications resulted in an increased interest in the regio-and stereoselective synthesis of nucleosides [ , ] . moreover, modified nucleosides and nucleotides with a restricted biomacromolecule for its natural ligand as well as the molecular recognition in an oligonucleotide chain (rna/dna) [ ] [ ] [ ] [ ] . similar studies of anti-sense and anti-gene oligonucleotides (ons) as potential and selective inhibitors of gene expression [ ] [ ] [ ] [ ] and their use as anti-tumor or anti-viral agents [ ] [ ] [ ] [ ] have also influenced the developments in the field of nucleic acid-based drugs. among the nucleoside analogues with significant biological activities, dideoxynucleoside-based compounds such as ′, ′-dideoxycytidine (ddc) [ ] , ′, ′-dideoxyinosine (ddi) [ ] , and ′-azidothymidine (azt) [ ] are effective therapeutic agents for the treatment of aids, while ribavirin (virazole) [ , ] is an antiviral drug (figure ) . similarly, other dideoxynucleosides such as d t ( ′, ′-didehydro- ′-deoxythymidine, stavudine) [ , ] and azddu ( ′-azido- ′, ′-dideoxyuridine) [ ] have gone through clinical studies. different nucleosides isolated from nature such as oxazinomycin [ ] , pyrazofurin [ , ] , showdomycin [ , ] , formycin a, and formycin b [ , ] have shown antibiotic properties and have also been found to exhibit anticancer and/or antiviral activities ( figure ) . these examples and new developments in the chemistry and biology of these compounds and their analogs [ ] [ ] [ ] [ ] [ ] have motivated us to work in this area and have led us to investigate the interesting chemical and biological properties of novel nucleoside-based compounds. despite the developments in nucleoside chemistry, the clinical use of nucleosides has some drawbacks due to their side-effects and primary or acquired drug resistance [ ] . therefore, the search for the design of new and effective nucleoside-based analogues continues to motivate studies in this field. our efforts toward this goal have led to the synthesis of twenty novel pyrazolylnucleosides with new structures and potent antiviral and antitumoral behaviors. in this regard, two regioisomers for each pyrazole derivative have been produced and characterized using spectroscopic techniques such as h nmr, c nmr, noesy, hmbc, ir, and mass spectroscopy. all compounds were evaluated against the national cancer institute (nci)'s panel of human tumor cell lines for their anticancer activities, and for their antiviral activities against representative viruses. in our approach, the synthesis of the target compounds a-e and a-e was achieved in two steps starting from the already reported -cyanomethyl- -aryl- h-pyrazoles a-e [ ] and the well-known -deoxy- , -di-o-p-toluoyl-α-d-ribofuranosyl chloride ( ) [ ] (scheme ). treatment of pyrazoles a-e with sodium hydride in acetonitrile and the subsequent addition of chlorosugar gave two regioisomers of the modified pyrazolyl nucleosides a-e (by coupling with the n- nitrogen of the pyrazole derivatives) and a-e (by coupling chlorosugar with the n- nitrogen of the pyrazole derivatives) in - % yields. the products a-e and a-e were de-toluoylated by despite the developments in nucleoside chemistry, the clinical use of nucleosides has some drawbacks due to their side-effects and primary or acquired drug resistance [ ] . therefore, the search for the design of new and effective nucleoside-based analogues continues to motivate studies in this field. our efforts toward this goal have led to the synthesis of twenty novel pyrazolylnucleosides with new structures and potent antiviral and antitumoral behaviors. in this regard, two regioisomers for each pyrazole derivative have been produced and characterized using spectroscopic techniques such as h nmr, c nmr, noesy, hmbc, ir, and mass spectroscopy. all compounds were evaluated against the national cancer institute (nci)'s panel of human tumor cell lines for their anticancer activities, and for their antiviral activities against representative viruses. in our approach, the synthesis of the target compounds a-e and a-e was achieved in two steps starting from the already reported -cyanomethyl- -aryl- h-pyrazoles a-e [ ] and the well-known -deoxy- , -di-o-p-toluoyl-α-d-ribofuranosyl chloride ( ) [ ] (scheme ). treatment of pyrazoles a-e with sodium hydride in acetonitrile and the subsequent addition of chlorosugar gave two regioisomers of the modified pyrazolyl nucleosides a-e (by coupling with the n- nitrogen of the pyrazole derivatives) and a-e (by coupling chlorosugar with the n- nitrogen of the pyrazole derivatives) in - % yields. the products a-e and a-e were de-toluoylated by using sodium methoxide in methanol, resulting in the formation of the corresponding modified nucleosides a-e and a-e in - % yields, respectively. using sodium methoxide in methanol, resulting in the formation of the corresponding modified nucleosides a-e and a-e in - % yields, respectively. scheme . synthesis of pyrazolylnucleoside analogues a-e and a-e. pyrazoles with no substitution on either of the two n ring atoms can be alkylated to produce two regioisomers under strongly basic conditions. the anion generated produces the two resonance forms that react with chlorosugar in the glycosidation step, leading to mixtures of two regioisomeric pyrazolyl nucleosides (i.e., a-e and a-e) (scheme ). mechanistic explanation for the regioisomeric alkylation of pyrazole derivatives a-e. the iupac numbering scheme of a-e is retained in a-e (and in a-e and a-e) for easy comparison. the structural identification of the isomeric disubstituted pyrazolyl nucleosides, based on their h nmr spectral data (see supplementary materials), has been reported in the literature [ ] [ ] [ ] . it has been observed that the anomeric protons in the isomeric pyrazolyl nucleosides exhibit different proton chemical shifts. the anomeric proton adjacent to n- of the pyrazole compounds a-e and a-e would appear downfield when compared to the anomeric proton adjacent to n- of the pyrazole compounds a-e and a-e [ ] [ ] [ ] . in addition to using h nmr chemical shifts, extensive noe and d nmr experiments have been employed to confirm the structures of the isomeric pyrazolyl nucleosides. using h and c nmr studies, we confirmed the positional assignments of the isomeric pyrazolyl nucleosides synthesized in the present work (tables - ). the anomeric proton of the analogues a-e generally appeared at . - . ppm upfield when compared to the corresponding proton in the h nmr spectra of its corresponding series isomers. this is in agreement with observations for other , -and , -disubstituted pyrazole nucleosides [ ] [ ] [ ] . we pyrazoles with no substitution on either of the two n ring atoms can be alkylated to produce two regioisomers under strongly basic conditions. the anion generated produces the two resonance forms that react with chlorosugar in the glycosidation step, leading to mixtures of two regioisomeric pyrazolyl nucleosides (i.e., a-e and a-e) (scheme ). pyrazoles with no substitution on either of the two n ring atoms can be alkylated to produce two regioisomers under strongly basic conditions. the anion generated produces the two resonance forms that react with chlorosugar in the glycosidation step, leading to mixtures of two regioisomeric pyrazolyl nucleosides (i.e., a-e and a-e) (scheme ). mechanistic explanation for the regioisomeric alkylation of pyrazole derivatives a-e. the iupac numbering scheme of a-e is retained in a-e (and in a-e and a-e) for easy comparison. the structural identification of the isomeric disubstituted pyrazolyl nucleosides, based on their h nmr spectral data (see supplementary materials), has been reported in the literature [ ] [ ] [ ] . it has been observed that the anomeric protons in the isomeric pyrazolyl nucleosides exhibit different proton chemical shifts. the anomeric proton adjacent to n- of the pyrazole compounds a-e and a-e would appear downfield when compared to the anomeric proton adjacent to n- of the pyrazole compounds a-e and a-e [ ] [ ] [ ] . in addition to using h nmr chemical shifts, extensive noe and d nmr experiments have been employed to confirm the structures of the isomeric pyrazolyl nucleosides. using h and c nmr studies, we confirmed the positional assignments of the isomeric pyrazolyl nucleosides synthesized in the present work (tables - ). the anomeric proton of the analogues a-e generally appeared at . - . ppm upfield when compared to the corresponding proton in the h nmr spectra of its corresponding series isomers. this is in agreement with observations for other , -and , -disubstituted pyrazole nucleosides [ ] [ ] [ ] . we scheme . mechanistic explanation for the regioisomeric alkylation of pyrazole derivatives a-e. the iupac numbering scheme of a-e is retained in a-e (and in a-e and a-e) for easy comparison. the structural identification of the isomeric disubstituted pyrazolyl nucleosides, based on their h nmr spectral data (see supplementary materials), has been reported in the literature [ ] [ ] [ ] . it has been observed that the anomeric protons in the isomeric pyrazolyl nucleosides exhibit different proton chemical shifts. the anomeric proton adjacent to n- of the pyrazole compounds a-e and a-e would appear downfield when compared to the anomeric proton adjacent to n- of the pyrazole compounds a-e and a-e [ ] [ ] [ ] . in addition to using h nmr chemical shifts, extensive noe and d nmr experiments have been employed to confirm the structures of the isomeric pyrazolyl nucleosides. using h and c nmr studies, we confirmed the positional assignments of the isomeric pyrazolyl nucleosides synthesized in the present work (tables - ). the anomeric proton of the analogues a-e generally appeared at . - . ppm upfield when compared to the corresponding proton in the h nmr spectra of its corresponding series isomers. this is in agreement with observations for other , -and , -disubstituted pyrazole nucleosides [ ] [ ] [ ] . we found that the effect was less pronounced in the series of nucleosides as compared to the series nucleosides where this difference was in the range of . - . ppm. the c chemical shift of the -ch cn bearing pyrazole carbon atom can help distinguish between positional isomers. in the and series of nucleosides, this carbon signal was - ppm downfield relative to the corresponding signal in the and series nucleosides. in most cases, the aryl bearing pyrazole carbon in the and series was - ppm upfield relative to the corresponding carbon in the and series. however, in the case of d versus d, the difference was small and reversed, making the aryl bearing pyrazole carbon less reliable for use in positional assignments. table . chemical shift values of the anomeric protons in the h nmr spectra and chemical shift values of the ar-and ch cn bearing carbons of the pyrazole ring in the c nmr spectra of the isomeric pyrazolyl nucleosides a-e, a-e, a-e, and a-e in cdcl on a bruker avance spectrometer. ar-bearing c shifts in the table . chemical shift and coupling constants from the h-nmr of compounds d, d, d, and d in acetone-d . a please refer to structures in table for skeleton numbering. table were all measured in acetone-d (on an bruker avance instrument) and may therefore differ from those in table , which were measured in cdcl (on bruker avance instrument). * due to the spatial interactions between different protons as explicitly shown in table , these protons exhibited multiplicities as shown here when the spectra were recorded in acetone-d on a bruker avance instrument as against those recorded in cdcl , cd cn or dmso-d as given in the experimental section for the corresponding protons where they appeared as broad singlets or ill resolved multiplets when their spectra were recorded on a bruker avance instrument. table were all measured in acetone-d (on an bruker avance instrument) and may therefore differ from those in table , which were measured in cdcl (on bruker avance instrument). in order to confirm the above positional assignments, we performed d nmr experiments (noesy and hmbc) on the two pairs of compounds (i.e., one pair consisting of the ditoluoyl protected nucleosides d and d and another pair consisting of the deprotected nucleosides d and d) ( table ). the results of the hmbc experiments rely on the fact that the anomeric proton may see the aryl bearing pyrazole carbon in d and d, but not in d and d, while the noesy results would explain the spatial proximity of the anomeric proton to either the protons in the -ch cn group or to the ortho protons of the aryl group. our results verified the positional properties at the pyrazole ring in the four compounds and are summarized in table . the isomers of the series nucleosides generally had higher r f values on the tlc than the corresponding isomer of the series of nucleosides. the same was observed for the isomers of the series relative to those in the series of nucleosides. table . results from the noesy and hmbc spectra of the pairs d- d and d- d *. blue and red arrows indicate the noesy and hmbc cross peaks, respectively. * the presence of a cross peak is indicated by (x). when it contributes to the positional verification, it is underlined (x). see tables - for positions of the relevant protons and carbons in the nmr spectra of the compounds d, d, d and d. the anticancer and toxicity of compounds a-e and a-e were evaluated by using the national cancer institute′s human cancer cell lines. compounds a-e, where the sugar is attached to n- of the pyrazole ring, were found to be inactive against all the cell lines irrespective of the substituent on the aromatic ring. in the other series, compounds a, b, and c with -methyl, -methoxy, and -fluoro substituents, respectively, at the aromatic ring were also inactive. however, compounds d and e with -chloro and -bromo substituents at the aromatic ring, respectively, showed inhibition against multiple anticancer cell lines. the gi (cytostatic parameter) and lc (toxicity parameter) values of d and e for the selected cell lines are given in table . compound d showed inhibition in cell lines, and was most active against the renal cancer cell line uo- and breast cancer cell line hs t with gi < µm in both cases. the most active compound was e, which showed moderate inhibition in cell lines. it showed significant inhibition against lung cancer cell line hop- with a gi of . µm and breast cancer cell line hs t with a gi of . µm. most importantly, both compounds ( d and e) did not show any toxicity, even at the highest concentration tested, as indicated by their high lc values. the pyrazolyl nucleosides a-e and a-e were also examined for their antiviral activities against a number of viruses such as simplex virus type (hsv- ) and type (hsv- ), thymidine kinase-deficient (tk -) strains of hsv- , vaccinia virus, para-influenza- virus, sindbis virus, coxsackie virus, punta toro virus, vesicular stomatitis virus (vsv), coxsackie virus b (cv-b ), respiratory syncytial virus (rsv), feline corona virus, and feline herpes virus. however, none of these compounds showed any significant activity against any of these viruses. blue and red arrows indicate the noesy and hmbc cross peaks, respectively. the anticancer and toxicity of compounds a-e and a-e were evaluated by using the national cancer institute s human cancer cell lines. compounds a-e, where the sugar is attached to n- of the pyrazole ring, were found to be inactive against all the cell lines irrespective of the substituent on the aromatic ring. in the other series, compounds a, b, and c with -methyl, -methoxy, and -fluoro substituents, respectively, at the aromatic ring were also inactive. however, compounds d and e with -chloro and -bromo substituents at the aromatic ring, respectively, showed inhibition against multiple anticancer cell lines. the gi (cytostatic parameter) and lc (toxicity parameter) values of d and e for the selected cell lines are given in table . compound d showed inhibition in cell lines, and was most active against the renal cancer cell line uo- and breast cancer cell line hs t with gi < µm in both cases. the most active compound was e, which showed moderate inhibition in cell lines. it showed significant inhibition against lung cancer cell line hop- with a gi of . µm and breast cancer cell line hs t with a gi of . µm. most importantly, both compounds ( d and e) did not show any toxicity, even at the highest concentration tested, as indicated by their high lc values. the pyrazolyl nucleosides a-e and a-e were also examined for their antiviral activities against a number of viruses such as simplex virus type (hsv- ) and type (hsv- ), thymidine kinase-deficient (tk -) strains of hsv- , vaccinia virus, para-influenza- virus, sindbis virus, coxsackie virus, punta toro virus, vesicular stomatitis virus (vsv), coxsackie virus b (cv-b ), respiratory syncytial virus (rsv), feline corona virus, and feline herpes virus. however, none of these compounds showed any significant activity against any of these viruses. reactions were conducted under an atmosphere of nitrogen in anhydrous solvents. column chromatography was carried out by using silica gel ( - mesh). melting points were determined in a concentrated h so acid bath and were uncorrected. analytical tlcs were performed on pre-coated merck silica gel f plates; the spots were detected either by using uv light or by charring with % alcoholic sulfuric acid. the ir spectra were recorded on a perkin-elmer ft-ir spectrophotometer (waltham, ma, usa). the optical rotations were measured with a bellingham-stanley ad polarimeter and the concentrations expressed as g/ml. the h-nmr and c-nmr spectra were recorded on a bruker avance spectrometer (billerica, ma, usa) at and . mhz, respectively in cdcl , dmso-d , or cd cn. all d measurements were performed in acetone-d on a bruker avance spectrometer (billerica, ma, usa). chemical shifts are relative to internal tms. assignments were based on cosy, noesy, hmbc (by using bruker's microprogram inv gslplrnd, which includes the low-pass j-filter to suppress one-bond correlations), hsqc, and jres spectra. the chemical shift values were reported as δ ppm relative to tms used as the internal standard and the coupling constants (j) were measured in hz. the esi-hrms spectra of all compounds were recorded on a jeol jms-ax w high-resolution mass spectrometer (tokyo, japan) in positive ion mode by using the matrix heds (bis-hydroxyethylsulfide) doped with sodium acetate. acetonitrile was used after distillation over freshly ignited potassium carbonate. a solution of -cyanomethyl- -arylpyrazole ( a-e, mmol) in acetonitrile ( ml) was added into a stirred mixture of sodium hydride ( . mmol) in acetonitrile ( ml) under a nitrogen atmosphere at - • c, and continuously stirred for min. the reaction mixture was cooled to • c and -α-chloro- , -di-o-toluoyl- , -dideoxyribose ( , mmol) was added and the contents stirred at • c for . to h. the progress of the reaction was monitored by using silica gel tlc. on completion, the reaction mixture was poured over ice-cold water and extracted with ethyl acetate ( × ml). the combined organic layer was washed with water ( × ml), dried over anhydrous sodium sulfate, the solvent was removed under reduced pressure, and the residue thus obtained was column chromatographed over silica gel with ethyl acetate ( - %) in petroleum ether as the eluent to afford the pyrazolyl nucleosides a-e and a-e in to % and to % yields, respectively. was suspended in dry methanol ( ml), then a mixture of naome and meoh ( ml, : , v/v) was added to the resulting solution, and the contents stirred at room temperature for - h. the progress of the reaction was monitored by using silica gel tlc. on completion, nh cl was added to the reaction mixture to adjust the ph to . one-third of the solvent was removed under reduced pressure and the reaction mixture was poured in water and extracted with ethyl acetate ( × ml), the organic layer was dried over anhydrous sodium sulfate, the solvent was removed under reduced pressure, and the residue thus obtained was subjected to column chromatography over silica gel with methanol ( - . %) in chloroform as the eluent to afford -deoxy- -( -cyanomethyl- -aryl)pyrazolyl-β-d-ribofuranose ( a-e) and -deoxy- -( -aryl- -cyanomethyl)pyrazolyl-β-d-ribofuranose ( a-e). details of the methodology are described at http://dtp.nci.nih.gov/branches/btb/ivclsp.html. briefly, the panel was organized into nine subpanels representing a diverse histology: leukemia, melanoma, and cancers of the lung, colon, kidney, ovary, breast, prostate, and central nervous system. the cells were grown in supplemented rpm medium for h. for the five dose study, the test compounds were dissolved in dmso and incubated with cells at five concentrations with -fold dilutions, the highest being − m and the others being − , − , − , and − m. the assay was terminated by the addition of cold trichloroacetic acid, and the cells were fixed and stained with sulforhodamine b. the bound stain was solubilized, and the absorbance was read on an automated plate reader. the cytostatic parameter, which determines the % growth inhibition (gi ) of the tumor cells, was calculated from time zero, control growth, and the absorbance at the five concentration levels. the cytotoxic parameter, lethal concentration (lc is the concentration of a drug resulting in a % reduction in the measured protein at the end of the drug treatment when compared to that at the beginning), indicating a net loss of cells following the treatment, was calculated as the average of two independent experiments. we successfully achieved the synthesis of twenty novel pyrazolyl nucleosides a-e, a-e, a-e, and a-e, which have been characterized in detail by using various nmr spectroscopic techniques such as h nmr, c nmr, noesy, hmbc, etc. the pyrazolyl nucleosides a-e and a-e were screened for anticancer activities on human tumor cell lines, and we identified one compound e, which showed good activity against cancer cell lines and showed significant inhibition against lung cancer cell line hop- (gi . µm) and breast cancer cell line hs t (gi . µm). our studies have demonstrated the potential of newly synthesized pyrazolyl-nucleosides that will be pursued further. supplementary materials: the following are available online at http://www.mdpi.com/ - / / / /s , scanned copies of the nmr and ir spectra of selected compounds, as appropriate are given in the "supplementary information". author contributions: y.y., k.k. and d.s. synthesized the compounds; v.k. and s.v.m. compiled the nci- activity; a.j. interpreted the nmr data and drafted the manuscript; j.w., c.l., a.k.p. and v.s.p. planned and designed the whole study and finalized the manuscript. funding: please add: this research received no external funding; see the "acknowledgements section" for details. ( h, m, c- h β ), . ( h, brs, ch cn), . ( h, brs, c- h), . ( h, brs, c- h & oh ), . ( h, brs, c- oh), . ( h, t, j = . hz, c- h), . ( h, s, c- h), . ( h, d ( h, s, ph-ch ), . - . ( h, m, c- h β ), . - . ( h, m, c- h α ), . - . ( h, m, c h β ) calculated for c h n o [m + na] + . , found [m + na] + . . -deoxy- -( -cyanomethyl- -p-methoxyphenyl)pyrazolyl-β-d-ribofuranose ( b): obtained as a semisolid in a % yield - . ( h, m, c- h α ), . - . ( h, m, c- h β ), . ( h, s deoxy- -( -cyanomethyl- -p-fluorophenyl)pyrazolyl-β-d-ribofuranose ( c): obtained as a semisolid in a % yield cdcl ): δ . - . ( h, m, c- h α ), . - . ( h, m, c- h β ), . - . ( h, m, c- h α ), . - . ( h, m, c- h β ) calculated for c h n o f [m + na] + . , found [m + na] + . . -deoxy- -( -p-chlorophenyl- -cyanomethyl-)pyrazolyl-β-d-ribofuranose ( d): obtained as a semisolid in a % yield cd cn): δ . - . ( h, m, c- h α ), . - . ( h, m, c- h β ), . ( h, brs, c- h), . - . ( h, m, c- h α calculated for c h n o cl mp = - • c; r f = . ( % methanol in chloroform) cm − ; h nmr ( mhz, dmso-d ): δ . - . ( h, m, c- h α ), . - . ( h, m, c- h β the evolution of antiviral nucleoside analogues: a review for chemists and non-chemists. part ii: complex modifications to the nucleoside scaffold nucleosides with modified sugar ring: synthesis and biological activities synthesis and evaluation of , -modified purine -c-methyl ribonucleosides as inhibitors of hcv replication milestones in the discovery of antiviral agents: nucleosides and nucleotides the synthesis of therapeutic locked nucleos(t)ides discovery of novel arylethynyltriazole ribonucleosides with selective and effective antiviral and antiproloferative activity nucleosides and nucleotides as antitumor and antiviral agents an overview of sugar-modified oligonucleosides for antisense therapeutics clinical pharmacokinetics of second generation antisense oligonucleotides locked nucleic acid: tighter is different inhibition of the in vitro infectivity and cytopathic effect of human t-lymphotrophic virus type iii/lymphadenopathy-associated virus (htlv-iii/lav) by ', '-dideoxynucleosides synthesis of ', '-didehydro- ', '-dideoxynucleosides via nucleoside route synthesis of ', '-didehydro- ', '-dideoxynucleosides having variations at either or both of the '-and '-positions synthesis of cyclonucleosides having a c-c bridge synthesis of '-c-and '-c-branched oligodeoxynucleotides and the development of locked nucleic acid (lna) lna: a versatile tool for therapeutics and genomics conformationally controlled high-affinity targeting of rna or dna by novel '-amino-dna/lna mixmers and pyrenyl-functionalized '-amino-dna optimizing dna targeting using n,n-bis( -pyridylmethyl)-|?-alanyl '-amino-lna synthesis and biological evaluation of nucleobase-modified analogs of the anticancer compounds '-c-ethynyluridine (eurd) and '-c-ethynylcytidine synthesis and evaluation of s-acyl- -thioethyl esters of modified nucleoside '-monophosphates as inhibitors of hepatitis c virus rna replication oligonucleotide therapeutics: chemistry, delivery and clinical progress characterization of the transport of nucleoside analog drugs by the human multidrug resistance proteins mrp and mrp synthetic and mass spectral fragmentation studies on trisubstituted h-pyran- -ones and comparative eims behavior of biologically active , -disubstituted pyrazoles and isoxazoles an engrossing history of azidothymidine broad-spectrum antiviral activity of virazole: -β-d-ribofuranosyl- , , -triazole- -carboxamide the synthesis and antiviral activity of -fluoro- -β-d-ribofuranosyl- h-pyrazole- -carboxamide the anti-htlv-iii (anti-hiv) and cytotoxic activity of ', '-didehydro- ', '-dideoxynucleosides: a comparison with their parental ', '-dideoxyribonucleosides inhibitory effect of ', '-didehydro- ', '-dideoxynucleosides on infectivity, cytopathic effects, and replication of human immunodeficiency vírus synthesis and antiviral activity of various 'azido analogues of pyrimidine deoxyribonucleosides against human immunodeficiency virus (hiv- , htlv-iii/lav) oxazinomycin, a new carbon-linked nucleoside antibiotic experimental antitumor activity of pyrazomycin efficient and β-steroselective synthesis of pyrazole c-nucleosides syntheses of pyrazole iso-c-nucleosides a new antibiotics, formycin carbocyclic '-epi-formycin effects of introduction of hydrophobic group on ribavirin base on mutation induction and anti-rna viral activity pyrazole related nucleosides . synthesis and biological activity of '-deoxy- ', '-dideoxy-and acyclo-analogues of -iodo- -β-d-ribofuranosyl- -carboxymethyl pyrazole (ipcar) synthesis of new pseudo-c-nucleosides containing pyrazole rings in their structure the role of nucleoside transporters in cancer chemotherapy with nucleoside drugs recent advances in studies on biochemical and structural properties of equilibrative and concentrative nucleoside transporter antisense oligonucleotides: a new therapeutic principle efficient preparation of -deoxy- , -di-o-p-toluoyl-α-d-ribofuranosyl chloride carbon- magnetic resonance study of solvent stabilized tautomerism in pyrazoles design and synthesis of heterocyclic carboxamides as natural nucleic acid base mimics carbocyclic nucleosides (carbanucleosides) as new therapeutic leads synthesis of triazenopyrazole derivatives as potential inhibitors of hiv- synthesis and reactions of -aryl-and -styryl- -cyano- -methylthio- h-pyran- -ones this article is an open access article distributed under the terms and conditions of the creative commons attribution we would like to thank the department of biotechnology (dbt, govt. of india, new delhi), the university of delhi (du, india), and the nucleic acid center (nac, university of southern denmark, odense, denmark) for their financial assistance. the authors are grateful to carl-erik olsen, university of copenhagen, denmark for carrying out extensive nmr spectral studies and to eric de clercq, rega institute for medical research, k.u. leuven, belgium for carrying out the antiviral evaluation studies. the authors declare no conflict of interest. key: cord- -npug c p authors: liu, yang; liu, jianying; pang, xiaojing; liu, tao; ning, zhijie; cheng, gong title: the roles of direct recognition by animal lectins in antiviral immunity and viral pathogenesis date: - - journal: molecules doi: . /molecules sha: doc_id: cord_uid: npug c p lectins are a group of proteins with carbohydrate recognition activity. lectins are categorized into many families based on their different cellular locations as well as their specificities for a variety of carbohydrate structures due to the features of their carbohydrate recognition domain (crd) modules. many studies have indicated that the direct recognition of particular oligosaccharides on viral components by lectins is important for interactions between hosts and viruses. herein, we aim to globally review the roles of this recognition by animal lectins in antiviral immune responses and viral pathogenesis. the different classes of mammalian lectins can either recognize carbohydrates to activate host immunity for viral elimination or can exploit those carbohydrates as susceptibility factors to facilitate viral entry, replication or assembly. additionally, some arthropod c-type lectins were recently identified as key susceptibility factors that directly interact with multiple viruses and then facilitate infection. summarization of the pleiotropic roles of direct viral recognition by animal lectins will benefit our understanding of host-virus interactions and could provide insight into the role of lectins in antiviral drug and vaccine development. animal lectins will benefit our understanding of host-virus interactions and could provide insight into the role of lectins in antiviral drug and vaccine development. keywords: lectin; virus; direct interaction; antiviral immunity; viral pathogenesis lectins, a highly diverse group of proteins that recognize carbohydrates, have been demonstrated to play a vital role in numerous life processes and to be critical for several viral infections and pathogeneses in a variety of organisms [ , ] . based on their conserved structure of sequence motifs for sugar binding and carbohydrate specificities, lectins have been categorized into many families conventionally designated as calnexin, c-type, l-type, p-type/mannose- -phosphate receptors (mprs), i-type/siglecs, m-type, f-type (absent in mammals), r-type, f-box, chitinase-like lectins, galectins and intelectins (table ) [ ] . the features of carbohydrate recognition domains (crds), such as structure peculiarity, carbohydrate binding selectivity and geometrical arrangement of multiple crds, determine the different properties of lectins [ ] [ ] [ ] . furthermore, the diversity of locations and functions indicates the importance of lectins in the basic life processes of organisms. lectins are exploited as susceptibility factors to facilitate viral entry, replication or assembly ( figure ). insight into direct lectin-based viral recognition will provide a deep understanding of host-virus interactions. mammalian lectins have been categorized into multiple classes according to the features of their crds, as well as their sugar recognition specificity. some lectins generally play a role outside the cell, whereas others are predominantly intracellular and located on cytoplasmic organelles. extracellular lectins, including c-type, r-type, i-type/siglecs lectins and galectins, are secreted into the extracellular milieu or are localized to the plasma membrane and are capable of mediating cell adhesion, immune signaling and pattern recognition activities for host-pathogen interactions. however, intracellular lectins, such as the calnexin family, m-type, l-type and p-type lectins, are located in luminal compartments of the secretory pathway and function in the trafficking, sorting and maturation of glycoproteins [ ] [ ] [ ] . as lectins play diverse roles in mammalian physiological processes, we only focus herein on a portion of lectins that directly interact with viral components and describe their functions in viral propagation and pathogen-host immune responses. c-type lectins (ctls) are a large group of proteins in metazoans that were originally named according to their property of ca + (c-type)-dependent carbohydrate binding. sequence and structural comparisons of c-type lectins have suggested that their carbohydrate-binding activity is mediated by a specific crd that is conserved in a variety of organisms. although some c-type lectins do not possess carbohydrate-binding activity, all of them show distinct sequence similarity and are believed to descend from a common ancestor during evolution [ ] [ ] [ ] . to date, c-type lectins have been divided into subgroups according to their domain architecture and the phylogenetic relationship between their crd sequences [ ] . in general, c-type lectins can be separated into two groups, mannose-binding and galactose-binding c-type lectins, based on the specificity of their carbohydrate-binding activity. the binding specificity of these two groups is mediated by diverse residues flanking the conserved cis-proline in the long loop region, in which the sequence of the core motif is e-p-n for mannose-binding and q-p-d for galactose-binding specificity [ , ] . previous studies have demonstrated that interchange of the e-p-n and q-p-d sequences is sufficient to switch the mannose-and galactose-binding specificity ( figure ) [ ] . however, several lectins are exceptions to this rule. for example, surfactant protein a, possessing an e-p-k but not an e-p-n motif, binds to mannose sugars [ , ] . although human tetranectin contains a galactose-type q-p-d motif, it is not responsible to the lectin-carbohydrate interaction [ ] . therefore, other determinants, including modifications around the binding sites and stereochemical factors, should be taken into consideration when examining binding specificity [ ] [ ] [ ] . robust investigations have shown that multiple mannose-/galactose-binding c-type lectins play important roles in viral infections in mammals. mbl, one of the most intensively studied lectins, is a member of the collectin family, a subgroup of c-type lectins ( figure a ). the mbl molecule contains four domains that are standard for collectin family proteins: a cysteine-rich region, a collagen-like domain, a neck region and a carbohydrate recognition domain. the native functional form of mbl is a hexamer; however, although mbl can form several oligomeric forms, the dimers and trimers do not have biological activity, and at least a tetramer form is needed to activate the complement cascade [ ] . mbl functions as a soluble pattern recognition receptor in the host complement system and is involved in resistance to many viral infections [ ] . the crds of mbl multimers recognize carbohydrate patterns on the virus surface, and consequently, the binding of mbl and viral particles results in activation of the lectin pathway of the complement system. masps (mannose-binding lectin-associated serine proteases), which are protease zymogens (an inactive form of an enzyme) similar to the c r and c s molecules of the classical complement pathway, are activated to cleave complement components c and c into c a/c b and c a/c b, respectively. interactions between c b and c b produce the c convertase, which continuously activates c further downstream in the cascade to eliminate viruses ( figure a ) [ ] . current investigations have reported a resistance role of mbl in infections of multiple human viruses, including human immunodeficiency virus (hiv) [ ] , hepatitis b virus (hbv) [ , ] , hepatitis c virus (hcv) [ ] , west nile virus (wnv) [ ] and dengue virus (denv) [ ] . specific recognition of these viral particles by mbl is a central event for activation of the lectin-based complement cascade. the mechanism of viral elimination by the mbl-based complement cascade is still unclear. unlike bacterial elimination by the complement system, mbl-based complement components do not appear to form a membrane-attacking complex on the viral surface; therefore, viral eradication may not be mediated by known complement mechanisms. three possible mechanisms have been proposed for mbl-mediated viral elimination. ( ) mbl-mediated complement c /c deposition onto the viral surface. viral neutralization can be processed in cell-free serum and is completely dependent on c and c activation, but not on c q and c , suggesting that neither opsonization nor the classical/alternative complement pathway is sufficient for viral neutralization [ ] . ( ) mbl can directly neutralize viruses. pre-incubation of serial concentrations of recombinant mbl with hiv cell-derived living particles was found to dramatically neutralize hiv infection [ , ] . however, other independent investigations have suggested that some primary hiv isolates resist direct neutralization by mbl [ ] , indicating that the possible neutralizing activity depends highly on the different carbohydrate structures on the surfaces of various viral strains. ( ) mbl can block the recognition of viruses and receptors. dendritic cell-specific intercellular adhesion molecule -grabbing nonintegrin (dc-sign), which is present on the surface of dendritic cells, functions as a key attachment factor used for the recognition and uptake of multiple viruses [ ] [ ] [ ] [ ] [ ] . a study reported that mbl can prevent interaction between hiv and dc-sign, thereby inhibiting the hiv infection of t cells, which is mediated by dc-sign [ ] . furthermore, mbl interacts with the viral envelope glycoproteins of ebola and marburg viruses (marv), resulting in the impairment of viral internalization by blocking virus-dc-sign interaction ( figure b ) [ ] . two soluble collectins, designated sp-a and sp-d ( figure b ,c), have been found to be involved in the recognition of viral particles for limiting infection in humans. sp-a is produced within the respiratory tract, gastrointestinal tract, and possibly other sites; conversely, sp-d is primarily synthesized in the respiratory tract [ ] . these factors are constitutively secreted into the lungs by alveolar type ii cells, unciliated bronchial epithelial cells and other mucosal tissue cells [ , ] . the specific location of surfactant proteins suggests a defensive role against viral invasion of the respiratory system. both sp-a and sp-d interact with different strains of influenza a virus (iav) via glycosylated hemagglutinin (ha) and neuraminidase (na) on the viral surfaces. the binding of iav to sp-a leads to agglutination of the virions, inhibition of iav infectivity and dissemination and also facilitates clearance by macrophages and neutrophils ( figure a ,b) [ ] . sp-d binds to iavs and thereby inhibits virus attachment and entry by viral aggregation ( figure b ) [ ] [ ] [ ] [ ] [ ] and also controls iav infection in human by activating neutrophil chemoattraction ( figure a ) [ , ] . respiratory syncytial virus (rsv) infects humans via the respiratory tract. sp-a has been reported to bind fusion (f) and adherence (g) glycoproteins on the surfaces of rsv virions, resulting in opsonization to reduce infection by enhancing viral uptake by peripheral blood mononuclear cells (pbmcs) and alveolar macrophages ( figure a ) [ , ] . sp-d also directly interacts with rsv surface g protein to modulate host immune responses to control rsv infection [ ] . dc-sign (cd ) and its homolog l-sign (also called dc-signr, cd l) are one of the most investigated c-type lectins involved in viral infection ( figure f ). unlike the main role of collectins in host defense, dc-sign and l-sign have been widely reported to be susceptibility factors that facilitate viral entry into host immune cells [ , , , ] . both dc-sign and l-sign are trans-membrane proteins that are composed of a short cytoplasmic tail, which is responsible for signaling and internalization, a transmembrane region, a neck domain, which consists of eight repeat regions of amino acids, and a carbohydrate recognition domain [ ] . the roles of dc-sign/l-sign in viral infections have been summarized and reviewed previously [ , ] . studies have shown that dc-sign/l-sign are capable of binding to the surface proteins of hiv [ ] , cytomegalovirus (cmv) [ ] , denv [ ] , wnv [ , ] , severe acute respiratory syndrome coronavirus (sars-cov) [ ] [ ] [ ] , hcv [ , ] , ebola virus [ ] and marv [ ] and consequently facilitating viral entry ( figure c ). differential glycosylation patterns of viral surface proteins strongly influence the efficiency of viral recognition by dc-sign/l-sign [ , ] . for example, only mannosylated envelope (e) glycoproteins on denv, but not e proteins with complex glycosylation, have been shown to interact with dc-sign-expressing cells [ ] . the mannose receptor (mr, cd ) is another c-type lectin that functions as a viral recognition receptor on the cell membrane ( figure g ). mr is mainly expressed in multiple immune cells, including macrophages and dendritic cells, and is a key susceptibility factor for denv infection of human macrophages. binding of mr to denv e glycoproteins enhances viral attachment, thus facilitating denv internalization into macrophages, and deglycosylation of the denv e glycoprotein enables the abrogation of this binding, and denv infection of primary human macrophages can be blocked by anti-mr antibodies [ ] . moreover, the interaction between mr and hbv surface antigen (hbsag) enhances viral uptake by dendritic cells (dcs), resulting in the impairment of dc function and the ineffective antiviral response of chronic hbv [ ] . the recognition of viral surface glycoproteins by mr is also beneficial to influenza virus [ , ] and hiv [ ] invasion into host cells ( figure c ). overall, dc-sign/l-sign and mr function as receptors/attachment factors for viral entry into particular cell types. clec a/mdl- (myeloid dap -associating lectin) is a c-type lectin associated with dap ( -kda dnax-activating protein) on myeloid cells such as monocytes, macrophages and neutrophils ( figure h ) [ ] . a recent study has found that clec a binds to dengue glycoproteins. however, in contrast to other c-type lectin receptors, the association between clec a and denv does not result in viral entry, but rather induces dap -mediated immune signaling to stimulate the release of pro-inflammatory cytokines that potentially contribute to the pathogenesis of dengue hemorrhagic fever [ , ] . clec a also directly interacts with japanese encephalitis virus (jev) to induce dap phosphorylation in macrophages and therefore plays a role in jev-induced neuro-inflammation and lethality ( figure c ) [ ] . the blocking of clec a in mice can significantly reduce the infiltration of jev-harboring leukocytes into the central nervous system, thus attenuating neuro-inflammation and protecting the animals from jev-induced lethality [ ] . the discovery of a role of clec a in flaviviral pathogenesis suggests that the extracellular crd modules are generally responsible for the recognition of viral glycoproteins; nonetheless, the intracellular modules determine the role of c-type lectins in viral infection. langerin (also known as cd ), containing a single ca + -dependent crd domain, is a type ii transmembrane c-type lectin that is specifically expressed on langerhans cells ( figure i ). the physiological function of langerin is to trigger the cellular membrane superimposition and zippering that benefit birbeck granule (bg) formation [ ] . langerin is capable of directly binding to hiv- envelope protein gp and thus serves as a potential receptor for hiv- infection in langerhans cells ( figure c ) [ , ] . however, a recent study reported that langerin is a natural barrier for hiv- transmission among langerhans cells. langerin is capable of directly capturing hiv- and sequentially degrading it in bgs to promote t cell elimination of hiv- infection [ ] , suggesting that langerin plays a pleiotropic role in hiv infection. furthermore, langerin functions as an attachment factor to facilitate measles virus (mv) infection in langerhans cells [ ] . a large number of host proteins are abundantly glycosylated. therefore, microbial recognition by c-type lectins relies on the mechanism for distinguishing carbohydrate structures between self and non-self, and the c-type lectin structure largely influences binding avidity and selectivity in the recognition of self and non-self carbohydrate structures [ , ] . based on the x-ray crystal structures of mannose-binding lectin (mbl), the mbl crd sites in the trimer form are too far apart to spatially interact efficiently with common mammalian high-mannose oligosaccharides. however, the dense and repeated arrays of carbohydrates present on the microbial surface can span the distance between the binding sites in mbl, resulting in highly avid multivalent interaction [ , ] . moreover, the number of crds is another determinant for the avidity and strength of differential binding by c-type lectins. the eight different c-type crds of mr contribute to its high binding affinity for single sugars, even though each individual crd motif only displays weak affinity. the crd domain organization also confers mr with the ability to recognize the wide range of different carbohydrates found on the pathogen surface and to distinguish between self and foreign glycoproteins [ ] . galectins are a group of secreted proteins that associate with specific cell surface glycans containing beta-galactosides ( figure d ,e) [ ] . although mammalian galectins lack conventional signal sequences, they reach the cell surface via a particular mechanism. galectins accumulate directly beneath the plasma membrane and are subsequently involved in the establishment of membrane-bound vesicles that pinch off before release outside the cell; galectins then bind to glycoconjugates on the plasma membrane or remain in the extracellular matrix [ ] [ ] [ ] . fifteen galectins have been identified in mammals and are categorized into three structural forms: dimeric, tandem or chimeric. dimeric galectins, also called prototypical galectins, are homodimers and include galectin- , - , - , - , - , - , - , - and - . tandem galectins contain at least two distinct crds within one polypeptide and include galectin subtype- , - , - , - and - . galectin- is specific to mammals, has one crd and a long non-lectin domain, and exists in either a monomeric form or a multivalent complex associated via the non-lectin domains of monomers [ ] ; this property allows galectin- to effectively bridge different ligands to form adhesive networks. current investigations have indicated that direct recognition between galectin- /- and viral-surface glycoproteins is important for host-virus interaction. [ ] . because of its particular binding specificity for galactosides, galectin- recognizes the surface envelope proteins of many human viruses and therefore is involved in viral infection. galectin- binds to niv-f, a viral envelope glycoprotein of nipah virus (niv), to reduce the niv-f-mediated fusion of endothelial cells and thereby inhibit niv-induced syncytium formation [ ] . galectin- directly interacts with the envelope glycoproteins of influenza a/wsn/ virus and inhibits its hemagglutination activity, resulting in the reduction of influenza virus infectivity ( figure b) [ ] . however, galectin- has also been reported to be a susceptibility factor for viral entry. hiv- exploits galectin- to enhance gp -cd interaction, leading to faster viral entry and more robust viral replication ( figure c ) [ ] [ ] [ ] [ ] . in addition to galectin- , the role of galectin- in viral infection has been elucidated by several studies. galectin- has been shown to interact with herpes simplex virus- (hsv- ). rnai-mediated knockdown of galectin- in human corneal keratinocytes significantly impaired hsv- infection, suggesting that hsv- exploits galectin- to enhance its attachment to host cells [ ] . recently, proteomic-based studies have identified galectin- as a host-binding partner of parvovirus minute virus of mice (mvm). the authors proposed that galectin- binding facilitates the access of mvm to its receptor(s) at the plasma membrane and thus promotes mvm endocytosis ( figure c ) [ ] . the above-mentioned evidence indicates a pleiotropic role of galectins during viral infections. the endoplasmic reticulum (er) of mammalian cells contains molecular chaperones and foldases, which are required for forming the active structures of newly synthesized peptides and thus serve as components of the er quality control system. the er-resident chaperones include bip, calnexin ( figure l ) and calreticulin (a calnexin-like soluble form without the transmembrane region) [ , ] . both calnexin and calreticulin are lectin-like, membrane-bound molecular chaperones that associate with newly synthesized proteins in the er. in addition, several studies have indicated that calnexin and calreticulin preferentially interact with glycoproteins that carry monoglucosylated n-linked oligosaccharides [ ] [ ] [ ] [ ] . the maturation of virus-encoded proteins occurs in the er, and calnexin family proteins have been shown to transiently interact with multiple viral proteins that consequently undergo rapid maturation ( figures d and a) . both calnexin and calreticulin can transiently interface with envelope glycoproteins f and hn of sendai virus (sev) [ ] and glycoproteins g /g of uukuniemi virus (uukv) (bunyaviridae family) [ ] to facilitate the rapid maturation of these proteins. during sars-cov infection, maturation of the viral s protein due to its interaction with calnexin is essential for the formation of infective virions [ ] . calnexin/calreticulin also plays a role in the assembly and secretion of hbv middle (m) envelope protein [ ] , hiv- envelope protein gp [ ] and gp [ ] . furthermore, during the rotavirus life cycle, calnexin binds to the er-associated viral transmembrane protein nsp , a nonstructural glycoprotein that acts as a toxin capable of inducing diarrhea in animals [ ] [ ] [ ] . calnexin/calreticulin is also associated with the glycosylation of hantaan virus (htnv, also known as hantavirus) envelope proteins gn and gc and plays a crucial role in the folding of htnv glycoproteins with a high content of high-mannose oligosaccharides [ ] . accumulated evidence suggests that the lectin-like calnexin proteins interact with viral components to largely facilitate viral assembly and protein maturation. the p-type lectin/mannose -phosphate receptors (mprs) are transmembrane glycoproteins that target lysosomal enzymes located in either intracellular organelles or the plasma membrane ( figure j ,k). mprs can bind newly synthesized lysosomal hydrolases in the trans-golgi network (tgn) and deliver them to pre-lysosomal compartments. the mpr crd was originally identified in two types of proteins, cation-independent and cation-dependent mannose -phosphate receptors (ci-mpr and cd-mpr, respectively), both of which recognize mannose- -phosphate (m- -p) to identify and route lysosomal enzymes to the lysosomal compartment [ ] . a previous study demonstrated that human herpes simplex virus (hsv) glycoprotein d (gd) binds to both ci-mpr and cd-mpr. these mprs sort glycoproteins modified with m- -p to lysosomes in the trans-golgi compartment and divert them to the endosomal pathway ( figures e and b) [ , ] . mprs were also found on the surfaces of mammalian cells as serving as putative cellular receptors for hsv entry and cell-cell viral spread; furthermore, chemo-or immuno-blocking mprs was shown to inhibit hsv entry and the production of hsv plaques in monkey cells ( figure c ). mouse cells lacking both ci-mpr and cd-mpr remain sensitive to hsv infection [ ] , suggesting that the expression of mprs is not essential for hsv invasion. varicella zoster virus (vzv) is known as a highly infectious human pathogen, and multiple vzv envelope glycoproteins are modified by m- -p; therefore, ci-mprs appear to be important for vzv infection. intracellular ci-mpr contributes to the transport of enveloped vzv to late endosomes, and the plasmalemmal form is necessary for cellular entry through cell-free vzv particles [ ] . l-type lectins are widely distributed in plants and animals. animal l-type lectins are intracellular luminal proteins that are involved in protein sorting in the luminal er-golgi compartments of animal cells. there are four l-type lectins in mammals: ergic- ( figure m) , ergl, vip , and vipl [ , ] . a recent study has shown that intracellular cargo receptor ergic- interacts with the glycoproteins of arenavirus, hantavirus, coronavirus, orthomyxovirus, and filovirus particles. ergic- is also essential for the propagation of arenavirus, coronavirus, and filovirus; in the absence of ergic- , viral particles can be formed but are noninfectious (figures e and c ) [ ] . in addition to the above-mentioned mammalian lectins, there are some other lectin classes in animals, e.g., m-type, r-type, i-type, chitinase-like, f-box lectins and intelectins. as we have not found reports on direct interactions between these lectins and viral components, we cannot determine the role of these lectins in viral infection based on the current knowledge. lectin crds are conserved throughout evolution, and many lectin homologues have been identified and reported in invertebrates. a homolog of galectin plays a role in opsonization for bacterial clearance in marsupenaeus japonicus [ ] . similarly, a galectin-like factor is expressed on the surface of oyster hemocytes and plays a role in oyster physiology through the recognition of oligosaccharides [ , ] . several proteins identified in crassostrea hongkongensis [ ] , venerupis philippinarum [ ] and trypanosoma cruzi [ ] have been categorized as homologs of mammalian i-type lectins/siglecs with high sialic acid-binding activity. in arthropods, multiple lectins identified in shrimp, such as l-type, p-type/mprs, m-type, and calnexin family factors, have been proposed to be important in shrimp innate immunity [ ] . many c-type lectin homologues in aedes and anopheles mosquitoes have been found to be involved in insect immune responses and pathogenesis [ ] [ ] [ ] . the current investigations of the immune roles of arthropod lectins mainly focus on their anti-bacterial or anti-parasite functions, including microorganism-induced lectin up-regulation, lectin-mediated microorganism recognition and opsonization [ , ] . however, little is known about the molecular details of lectins in arthropod immunity and pathogenesis, especially with regard to the function in arthropod-virus interactions. a recent study on c-type lectins in aedes aegypti initially assessed lectin functions in viral infections of arthropods. tens of c-type lectins were identified in aedes [ , ] and anopheles [ ] mosquitoes, and most are soluble forms [ ] . previous studies have shown that an aedes aegypti c-type lectin, mosquito galactose-specific c-type lectin- (mosgctl- ), interacts with wnv in a calcium-dependent manner to form a mosgctl-virus complex. this complex consequently interacts with mosquito protein tyrosine phosphatase- (mosptp- ), a mosquito homolog of human cd in a. aegypti, to enable viral attachment to the plasma membrane and enhance viral entry. in vivo experiments showed that mosgctl- and mosptp- function as part of the same pathway and are critical for wnv infection of mosquitoes [ ] . further investigations identified that another mosgctl paralogs facilitate dengue infection of mosquitoes. these mosgctls interact with denv- surface e protein and virions, functioning as susceptibility factors for dengue viral entry into mosquito cells. however, mosptp- did not influence dengue infection in mosquitoes, suggesting that other membrane receptors may recruit the denv-mosgctl complex onto the cell membrane for viral entry [ ] . in agreement with the findings in mosquitoes, a recent study has identified a c-type lectin in the shrimp marsupenaeus japonicus that interacts with an envelope protein of white spot syndrome virus (wssv) and consequently associates with a cell-surface calreticulin, which serves as a membrane receptor that facilitates viral entry in a cholesterol-dependent manner [ ] . the study therefore suggested that c-type lectins might play a broad role in expediting many viral infections of arthropods. the role might not be limited to wnv/denv in mosquito and wssv in shrimp but might extend to other virus infections in arthropods. lectins are potential targets for the development of antiviral drugs and vaccines. such lectin-based antiviral strategies are divided into two parts: ( ) lectin-based immune activation and ( ) blockade of lectin receptors against viral entry [ ] . many envelope viruses are protected by their dense carbohydrate shield against efficient recognition and persistent neutralization by the host immune system. various natural and synthetic carbohydrate-binding agents have been screened to refine candidates that can reinforce the recognition of specific pathogens, enhance the cascade amplification of the innate immune response and interrupt virus attachment to receptors. in fact, lectins have been considered as drug targets for many years. several heterologous lectins derived from various organisms have been already selected and introduced into pre-clinic trials for hiv therapy, including svn (scytovirin), a . -kd lectin isolated from aqueous extracts of the cyanobacterium scytonema varium [ ] , and uda (stinging nettle lectin), a . -kd plant lectin isolated from urtica dioica [ , ] . furthermore, the combined usage of uda with hha (amaryllis lectin, from a hippeastrum hybrid) and gna (snowdrop lectin from galanthus nivalis), another two carbohydrate-binding agents, showed broad anti-viral activity against four serotypes of denv in monocyte-derived dendritic cells by preventing virus attachment [ ] . additionally, an interesting monoclonal antibody, g , which interacts with specific, highly conserved glycosylation sites on hiv envelop protein gp shows a broad anti-hiv neutralizing activity. the mechanism of this antibody specifically targeting n-linked glycans is very similar to that of lectins [ ] [ ] [ ] . with regard to arthropod-borne viruses, vector ligands that interact with pathogens are ideal targets for interfering with the successful acquisition of the virus from the vertebrate host. due to the importance of c-type lectins in dengue infection of mosquitoes, these lectin factors may be proposed as targets for the development of vaccines or antiviral drugs. studies show that treatment with mosgctls antisera dramatically interrupted denv- infection of mosquitoes through blood feeding. therefore, the humoral response against mosgctls in mammals could feasibly impair dengue infection of mosquitoes. the approach to blocking mosquito c-type lectins may direct a future avenue for the development of a transmission-blocking vaccine that interrupts the mosquito-borne viral life cycle and reduces disease burden [ ] . lectins comprise highly diverse proteins with different carbohydrate recognition activities and play pleiotropic roles in the immune responses and pathogenesis of many viral infections (summarized in table ). the interaction between lectins and viral glycoproteins may lead to the three following consequences: ( ) lectins, such as mbl and sps, function as pattern recognition molecules that bind a repertoire of viruses and activate antiviral immune responses; ( ) lectins are employed as attachment factors that recruit viral particles to the cell membrane to enhance viral entry, e.g., some mammalian lectins (dc-sign, l-sign, mr and mprs) or their homologs in arthropods (mosgctls); and ( ) some intracellular lectins, such as calnexin and ergic- , function as susceptibility factors associated with virus-encoded proteins to facilitate viral replication or assembly (please refer to figures and ) . interestingly, the same lectin may show opposing roles in different virus infections. for example, galectin- binds to niv to inhibit syncytium formation and recognizes iav to reduce its infectivity [ , ] ; however, galectin- was also reported to be a susceptibility factor that enhanced gp -cd interactions, thus facilitating hiv entry [ ] [ ] [ ] [ ] . the current accumulated knowledge indicates that lectins are crucial host factors with complex and profound roles in the process of viral infection. lectins for investigation of proteins and carbohydrates molecular structure animal lectins animal lectins: a historical introduction and overview demonstration of carbohydrate recognition activity in diverse proteins which share a common primary structure motif two distinct classes of carbohydrate-recognition domains in animal lectins mannose-binding proteins isolated from rat liver contain carbohydrate-recognition domains linked to collagenous tails. complete primary structures and homology with pulmonary surfactant apoprotein the c-type lectin-like domain superfamily engineering galactose-binding activity into a c-type mannose-binding protein binding of sugar ligands to ca( +)-dependent animal lectins. ii. generation of high-affinity galactose binding by site-directed mutagenesis studies on the carbohydrate-binding characteristics of human pulmonary surfactant-associated protein a and comparison with two other collectins: mannan-binding protein and conglutinin the major lung surfactant protein, sp - , is a calciumdependent, carbohydrate-binding protein interaction of tetranectin with sulphated polysaccharides and trypan blue. scand lectin-carbohydrate interactions: different folds, common recognition principles mechanism of n-acetylgalactosamine binding to a c-type animal lectin carbohydrate-recognition domain mechanism of ph-dependent n-acetylgalactosamine binding by a functional mimic of the hepatocyte asialoglycoprotein receptor human mannose-binding protein carbohydrate recognition domain trimerizes through a triple alpha-helical coiled-coil collections and ficolins: humoral lectins of the innate immune defense two mechanisms for mannose-binding protein modulation of the activity of its associated serine proteases interaction of mannose-binding lectin with hiv- is sufficient for virus opsonization but not neutralization high frequency of variant alleles of the mannose-binding lectin (mbl ) gene are associated with patients infected by hepatitis b virus mannose-binding lectin in chronic hepatitis b virus infection mannose-binding lectin mbl gene polymorphisms and outcome of hepatitis c virus-infected patients direct complement restriction of flavivirus infection requires glycan recognition by mannose-binding lectin a human serum mannose-binding protein inhibits in vitro infection by the human immunodeficiency virus glycosylation inhibitors and neuraminidase enhance human immunodeficiency virus type binding and neutralization by mannose-binding lectin dc-sign, a dendritic cell-specific hiv- -binding protein that enhances transinfection of t cells c-type lectins dc-sign and l-sign mediate cellular entry by ebola virus in cis and in trans human cytomegalovirus binding to dc-sign is required for dendritic cell infection and target cell trans-infection dc-sign and l-sign can act as attachment receptors for alphaviruses and distinguish between mosquito cell-and mammalian cell-derived viruses dc-sign (cd ) mediates dengue virus infection of human dendritic cells inhibition of dc-sign-mediated trans infection of t cells by mannose-binding lectin mannose-binding lectin binds to ebola and marburg envelope glycoproteins, resulting in blocking of virus interaction with dc-sign and complement-mediated virus neutralization localization of lung surfactant protein d on mucosal surfaces in human tissues immunocytochemical localization of surfactant protein d (sp-d) in type ii cells, clara cells, and alveolar macrophages of rat lung expression sites of the collectin sp-d suggest its importance in first line host defence: power of combining in situ hybridisation, rt-pcr and immunohistochemistry mechanisms of anti-influenza activity of surfactant proteins a and d: comparison with serum collectins pulmonary surfactant protein d in first-line innate defence against influenza a virusinfections site-directed mutagenesis of cys- and cys- of pulmonary surfactant protein d. expression of a trimeric protein with altered anti-viral properties mechanism of binding of surfactant protein d to influenza a viruses: importance of binding to haemagglutinin to antiviral activity assessment of the antiviral properties of recombinant porcine sp-d against various influenza a viruses in vitro inhibition of influenza viral neuraminidase activity by collectins evidence for a protective role of pulmonary surfactant protein d (sp-d) against influenza a viruses recombinant sp-d carbohydrate recognition domain is a chemoattractant for human neutrophils surfactant protein a binds to the fusion glycoprotein of respiratory syncytial virus and neutralizes virion infectivity surfactant protein a enhances uptake of respiratory syncytial virus by monocytes and u macrophages respiratory syncytial virus and pulmonary surfactant c-type lectin receptors on dendritic cells and langerhans cells dc-sign: escape mechanism for pathogens the c type lectins dc-sign and l-sign: receptors for viral glycoproteins nile virus discriminates between dc-sign and dc-signr for cellular attachment and infection dc-sign enhances infection of cells with glycosylated west nile virus in vitro and virus replication in human dendritic cells induces production of ifn-alpha and tnf-alpha dc-sign and dc-signr interact with the glycoprotein of marburg virus and the s protein of severe acute respiratory syndrome coronavirus ph-dependent entry of severe acute respiratory syndrome coronavirus is mediated by the spike glycoprotein and enhanced by dendritic cell transfer through dc-sign -sign) is a receptor for severe acute respiratory syndrome coronavirus l-sign (cd l) is a liver-specific capture receptor for hepatitis c virus hepatitis c virus glycoproteins interact with dc-sign and dc-signr dendritic-cell-specific icam -grabbing non-integrin is essential for the productive infection of human dendritic cells by mosquito-cell-derived dengue viruses dendritic cell-specific intercellular adhesion molecule -grabbing non-integrin (dc-sign)-mediated enhancement of dengue virus infection is independent of dc-sign internalization signals the mannose receptor mediates dengue virus infection of macrophages the mannose receptor acts as hepatitis b virus surface antigen receptor mediating interaction with intrahepaticdendritic cells involvement of themannose receptor in infection of macrophages by influenza virus reading pc macrophage receptors for influenza a virus: role of the macrophage galactose-type lectin and mannose receptorin viral entry oligomerization of the macrophage mannose receptor enhances gp -mediated binding of hiv- immunoreceptor dap bearing a tyrosine-based activation motif is involved in activating nk cells clec a is critical for dengue-virus-induced lethal disease clec a is critical for dengue virus-induced inflammasome activation in human macrophages clec a regulates japanese encephalitis virus-induced neuroinflammation and lethality langerin, a novel c-type lectin specific to langerhans cells, is an endocytic receptor that induces the formation of birbeck granules the role of dendritic cell c-type lectin receptors in hiv pathogenesis diversity of receptors binding hiv on dendritic cell subsets langerin is a natural barrier to hiv- transmission by langerhans cells human langerhans cells capture measles virus through langerin and present viral antigens to cd + t cells but are incapable of cross-presentation the c-type lectin superfamily in the immune system self-and nonself-recognition by c-type lectins on dendritic cells trimeric structure of a c-type mannose-binding protein galectins: a family of animal beta-galactosidebinding lectins secretion of the galectin family of mammalian carbohydrate-binding proteins secretion of the baby hamster kidney -kda galactose-binding lectin from polarized and nonpolarized cells: a pathway independent of the endoplasmic reticulum-golgi complex plasma membrane targetting, vesicular budding and release of galectin from the cytoplasm of mammalian cells during secretion galectins: regulators of acute and chronic inflammation galectins: structure, function and therapeutic potential endothelial galectin- binds to specific glycans on nipah virus fusion protein and inhibits maturation, mobility, and function to block syncytia formation galectin- binds to influenza virus and ameliorates influenza virus pathogenesis galectin- promotes hiv- infectivity in macrophages through stabilization of viral adsorption galectin- acts as a soluble host factor that promotes hiv- infectivity through stabilization of virus attachment to host cells galectin- and hiv- infection host-soluble galectin- promotes hiv- replication through a direct interaction with glycans of viral gp andhost cd binding of transmembrane mucins to galectin- limits herpesvirus infection of human corneal keratinocytes proteomic analysis identifies a novel function for galectin- in the cell entry of parvovirus role of n-oligosaccharides endoplasmic reticulum processing reactions in glycoprotein folding and degradation lectins as chaperones in glycoprotein folding association of folding intermediates of glycoproteins with calnexin during protein maturation role of n-linked oligosaccharide recognition, glucose trimming, and calnexin in glycoprotein folding and quality control transient, lectin-like association of calreticulin with folding intermediates of cellular and viral glycoproteins glycan-dependent and -independent association of vesicular stomatitis virus g protein with calnexin kinetics of interactions of sendai virus envelope glycoproteins, f and hn, with endoplasmic reticulum-residentmolecular chaperones, bip, calnexin, and calreticulin transient association of calnexin and calreticulin with newly synthesized g and g glycoproteins of uukuniemivirus (family bunyaviridae) monitoring of s protein maturation in the endoplasmic reticulum by calnexin is important for the infectivity of severe acute respiratory syndrome coronavirus role for calnexin and n-linked glycosylation in the assembly and secretion of hepatitis b virus middle envelopeprotein particles calreticulin interacts with newly synthesized human immunodeficiency virus type envelope glycoprotein, suggesting a chaperone function similar to that of calnexin effects of inefficient cleavage of the signal sequence of hiv- gp on its association with calnexin, folding, and intracellular transport the rotavirus nonstructural glycoprotein nsp possesses membrane destabilization activity rotavirus nonstructural glycoprotein nsp alters plasma membrane permeability in mammalian cells the molecular chaperone calnexin interacts with the nsp enterotoxin of rotavirus in vivo and in vitro analysis of n-linked glycosylation of hantaan virus glycoproteins and the role of oligosaccharide side chains in protein folding and intracellular trafficking lysosomal enzyme binding to mouse p d macrophage membranes lacking the -kda mannose -phosphate receptor: evidence for the existence of a second mannose -phosphate receptor herpes simplex virus glycoprotein d acquires mannose -phosphate residues and binds to mannose -phosphate receptors role of mannose- -phosphate receptors in herpes simplex virus entry into cells and cell-to-cell transmission mannose -phosphate receptor dependence of varicella zoster virus infection in vitro and in the epidermis during varicella and zoster a putative novel class of animal lectins in the secretory pathway homologous to leguminous lectins structures of the carbohydrate recognition domain of ca + -independent cargo receptors emp p and emp p the intracellular cargo receptor ergic- is required for the production of infectious arenavirus, coronavirus, and filovirus particles a galectin from the kuruma shrimp (marsupenaeus japonicus) functions as an opsonin and promotes bacterial clearance from hemolymph hemocytes and plasma of the eastern oyster (crassostrea virginica) display a diverse repertoire of sulfated and blood group a-modified n-glycans the galectin cvgal from the eastern oyster (crassostrea virginica) binds to blood group a oligosaccharides on the hemocyte surface a novel sialic acid binding lectin with anti-bacterial activity from the hong kong oyster (crassostrea hongkongensis) cloning and characterization of a sialic acid binding lectins (sabl) from manila clam molecular interaction of siglecs (sialic acid-binding ig-like lectins) with sialylated ligands on trypanosoma cruzi diversity and multiple functions of lectins in shrimp immunity transmission-blocking antibodies against mosquito c-type lectins for dengue prevention cloning and characterization of a mannose binding c-type lectin gene from salivary gland of aedes albopictus the genome sequence of the malaria mosquito anopheles gambiae purification, characterization and cdna cloning of a novel lipopolysaccharide-binding lectin from the shrimp penaeus monodon a novel c-type lectin (fclec ) facilitates the clearance of vibrio anguillarumin in vivo in chinese white shrimp a c-type lectin collaborates with a cd phosphatase homolog to facilitate west nile virus infection of mosquitoes collaboration between a soluble c-type lectin and calreticulin facilitates white spot syndrome virus infection in shrimp targeting the c-type lectins-mediated host-pathogen interactions with dextran overexpression and purification of scytovirin, a potent, novel anti-hiv protein from the cultured cyanobacterium scytonema varium the mannose-specific plant lectins from cymbidium hybrid and epipactis helleborineand the (n-acetylglucosamine)n-specific plant lectin from urtica dioicaare potent and selective inhibitors of human immunodeficiency virus and cytomegalovirus replication in vitro carbohydrate-binding agents cause deletions of highly conserved glycosylation sites in hiv gp a new therapeutic concept to hit the achilles heel of hiv broad antiviral activity of carbohydrate-binding agents against the four serotypes of dengue virus in monocyte-derived dendritic cells the broadly neutralizing anti-human immunodeficiency virus type antibody g recognizes a cluster of alpha → mannose residues on the outer face of gp antibody domain exchange is an immunological solution to carbohydrate cluster recognition dissection of the carbohydrate specificity of the broadly neutralizing anti-hiv- antibody g key: cord- -pq obtrc authors: capasso, domenica; del gatto, annarita; comegna, daniela; russo, luigi; fattorusso, roberto; saviano, michele; di gaetano, sonia; zaccaro, laura title: selective targeting of αvβ integrin in hepg cell line by rgdechi d peptide date: - - journal: molecules doi: . /molecules sha: doc_id: cord_uid: pq obtrc recently, the research community has become increasingly concerned with the receptor αvβ , a member of the well-known integrin family. different ongoing studies have evidenced that αvβ integrin regulates not only physiological processes but also a wide array of pathological events, suggesting the receptor as a valuable biomarker to specifically target for therapeutic/diagnostic purposes. remarkably, in some tumors the involvement of the receptor in cell proliferation, tumor dissemination and angiogenesis is well-documented. in this scenario, the availability of a selective αvβ antagonist without ‘off-target’ protein effects may improve survival rate in patients with highly aggressive tumors, such as hepatocellular carcinoma. we recently reported a cyclic peptide, rgdechi d, obtained by structure-activity studies. to our knowledge it represents the first peptide-based molecule reported in the literature able to specifically bind αvβ integrin and not cross react with αvβ . here we demonstrated the ability of the peptide to diminish both adhesion and invasion of hepg cells, an in vitro model system for hepatocellular carcinoma, to reduce the cell proliferation through an apoptotic process, and to interfere with the pi k pathway. the peptide, also decreases the formation of new vessels in endothelial cells. taken together these results indicate that the peptide can be considered a promising molecule with properties suited to be assessed in the future for its validation as a selective therapeutic/diagnostic weapon in hepatocarcinoma. integrins are transmembrane receptors able to dictate cellular responses to a variety of inputs thanks to their capacity to differentially recognize distinct environments. to allow for this flexibility, integrins are comprised of α and β subunits that pair to form at least different functional heterodimeric receptors. they can transduce extracellular stimuli resulting in an extensive range of downstream effects on cell adhesion, migration, proliferation, differentiation and apoptosis [ ] . in vivo studies have shown that in various types of cancers the expression of some integrins on the surface of neoplastic cells is frequently up-regulated. the αv subunit, which forms heterodimers with β , β , β , β or β subunits, is able to recognize more extracellular matrix (ecm) ligands and growth factors thanks to the rgd sequence. the availability of ligands able to discriminate among different integrin subtypes, sharing sequence and structure homology but different biological effects, is a desirable aim in the biomedical field. over several decades the scientific community has focused on different integrins such as αvβ , α β and αvβ and little attention has been paid to integrin αvβ whose relevance has been assessed from several ongoing studies. αvβ integrin binds mainly the ecm protein vitronectin [ , ] and has the ability to control different biological and pathological events, thus representing one of the more intriguing integrins. it has been known for years that integrins are commonly used as receptors by many human viruses [ ] , and αvβ actually seems to play a critical role in multiple aspects of infection pathogenesis [ , ] . viral proteins with the rgd motif promote infection by binding integrin heterodimers [ , ] , thus activating pi k/akt or mapk signaling pathways which promote virus entry and infection of the host cell [ ] . moreover, in addition to the well-established contribution of αvβ integrin to angiogenesis [ ] [ ] [ ] , much evidence supports the crucial role of this integrin in promoting cancer cell migration and invasion. it seems that αvβ mediates the early steps of liver metastasis formation of colon carcinomas through different mechanisms [ , ] . additionally, there is substantial evidence for interplay between αv integrins and tissue growth factor (tgf-beta) during the pathological epithelial-to-mesenchymal transition that occurs in many types of cancer [ ] [ ] [ ] [ ] [ ] [ ] , which is the reason why anti-integrin therapeutics are indeed under development as treatment for tgf-beta-related disorders [ ] . hepatocellular carcinoma (hcc) is a primary malignancy of the liver that usually develops from a background of liver fibrosis and inflammation. it is associated with a high propensity for vascular invasion and metastasis, which accounts for its poor prognosis [ , ] . the high mortality rate is due to the advanced stage and frequent recurrence after surgical resection, and most patients are limited to surgical treatment or chemotherapy [ ] . consequently, to improve the survival rate, novel and effective therapeutic strategies are needed. recently, hoshino et al. proved that exosomal β integrin regulates liver tropism associated with liver metastasis in several tumors [ , , ] . in a more recent study, lin et al. demonstrated that β integrin is highly expressed in hcc tissues, facilitating cancer cell growth and promoting cell migration [ ] . in human hcc patients, fibrinogen [ ] activates hepatic stellate cells (hsc) [ ] and liver specific mesenchymal cells by binding αvβ integrin [ ] widely expressed by neoplastic cells to promote liver disease progression and tumor metastasis [ ] . recently yan et al. stated that treatment with cilengitide peptide, an αvβ /αvβ integrin antagonist, significantly blocked hsc activation and function and reduced proliferation of oncogenic hepatocytes and progression of liver fibrosis [ ] . thus, it is definitively clear that β integrin can be considered a diagnostic biomarker and a potential therapeutic target in hcc. to date only αvβ /αvβ and αvβ /α β antagonists have been reported in the literature, whilst selective αvβ peptide antagonists are still missing. selective modulation of αvβ integrin is highly desirable to allow targeted specific therapy and potentially to limit side-effects. this aspect, despite the availability of structural model of the head group of αvβ integrin constructed by homology modeling, is further complicated by the lack of the high-resolution d structure [ ] . in the last few years, our research activities have mainly focused on the identification of integrin selective peptide ligands by rational design [ ] [ ] [ ] [ ] . in we reported nmr and computational studies on the αvβ selective peptide, rgdechi, aiming to identify the molecular basis that regulates receptor recognition mechanism. by this combined approach we also demonstrated that the substitution of the key homocitrulline residue with aspartic acid shifts the receptor binding selectivity from αvβ to αvβ integrin, thus identifying a novel and selective αvβ ligand, named rgdechi d [ ] . here we reported the in vitro characterization of this peptide on the hepg cell line, one of the well-known model systems for hcc [ ] , evaluating its ability to selectively bind αvβ integrin and to interfere with angiogenesis, cell migration, invasion and proliferation. the expression levels of αvβ and αvβ integrins on surface of hepg were determined by means of quantitative cytofluorimetric assay using phycoerythrin (pe) conjugated antibodies. to maintain surface marker integrity, cells were detached using . mm edta in pbs. data indicate that hepg cells show an average surface expression of . × ± αvβ (figure ), while they do not present a significant amount of αvβ receptors. the good level of αvβ expression together with the absence of αvβ make this cell line a suitable model to study the biological behavior of a selective αvβ molecule. molecules , , x of the expression levels of αvβ and αvβ integrins on surface of hepg were determined by means of quantitative cytofluorimetric assay using phycoerythrin (pe) conjugated antibodies. to maintain surface marker integrity, cells were detached using . mm edta in pbs. data indicate that hepg cells show an average surface expression of . × ± αvβ (figure ), while they do not present a significant amount of αvβ receptors. the good level of αvβ expression together with the absence of αvβ make this cell line a suitable model to study the biological behavior of a selective αvβ molecule. figure . quantification of αvβ and αvβ receptors on hepg cell surface. cells were stained with phycoerythrin (pe)-anti-αvβ antibody (magenta curve) or pe-conjugated anti-αvβ antibody (green curve) or pe-control isotype (black curve). the histogram is representative of three independent experiments. cell adhesion assays were performed in order to confirm the capability of the peptide to bind only αvβ and not to cross react with αvβ or α β , unlike the current peptide-based molecules reported the in literature which are not able to discriminate between these integrins. rgdechi d, rgdechi and scrambled peptides were synthesized as previously reported [ , ] . the effect of rgdechi d on hepg adhesion to vitronectin, a matrix able to recognize both αvβ and αvβ integrins [ ] [ ] [ ] , was analyzed by crystal violet assay. to prevent receptor internalization, the cells were pre-incubated at °c with peptides or an anti αvβ integrin antibody; the treatment occurred in an appropriate adhesion buffer containing divalent cations essentials for receptor binding [ ] . then, cells were seeded onto vitronectin. as shown in figure a , rgdechi d greatly reduces the adhesion of hepg plated onto vitronectin of %; this result is even better than that obtained from the monoclonal anti-αvβ antibody ( %), used as positive control. incubation of cells with the scrambled peptide (negative control) does not decrease cell adhesion. considering the already reported rgdechi d binding specificity towards αvβ with respect to αvβ [ ] , this result confirms the specificity of action of rgdechi d towards αvβ on the hepg cell surface and indicates the ability of the peptide to compete with vitronectin and to prevent hepg cell adhesion. furthermore, it is worth noting that the inhibition occurred in a concentration-dependent manner cell adhesion assays were performed in order to confirm the capability of the peptide to bind only αvβ and not to cross react with αvβ or α β , unlike the current peptide-based molecules reported the in literature which are not able to discriminate between these integrins. rgdechi d, rgdechi and scrambled peptides were synthesized as previously reported [ , ] . the effect of rgdechi d on hepg adhesion to vitronectin, a matrix able to recognize both αvβ and αvβ integrins [ ] [ ] [ ] , was analyzed by crystal violet assay. to prevent receptor internalization, the cells were pre-incubated at • c with peptides or an anti αvβ integrin antibody; the treatment occurred in an appropriate adhesion buffer containing divalent cations essentials for receptor binding [ ] . then, cells were seeded onto vitronectin. as shown in figure a , rgdechi d greatly reduces the adhesion of hepg plated onto vitronectin of %; this result is even better than that obtained from the monoclonal anti-αvβ antibody ( %), used as positive control. incubation of cells with the scrambled peptide (negative control) does not decrease cell adhesion. considering the already reported rgdechi d binding specificity towards αvβ with respect to αvβ [ ] , this result confirms the specificity of action of rgdechi d towards αvβ on the hepg cell surface and indicates the ability of the peptide to compete with vitronectin and to prevent hepg cell adhesion. furthermore, it is worth noting that the inhibition occurred in a concentration-dependent manner with an ic of . µm ( figure b ). with the aim to evaluate the capability of the peptide to discriminate between αvβ and α β , cell adhesion assays on k cells, displaying α β at high levels and αvβ and αvβ at very low levels [ ] , were carried out. the cells were seeded both onto fibronectin, the main ecm protein that binds α β receptor, and onto α β antibody coated plates. the results obtained indicate that rgdechi d is not able to inhibit k adhesion neither on fibronectin nor that of the α β specific antibody; in this experiment the rgdechi peptide [ ] was used as a control to recognize αvβ integrin [ ] thus proving the validity of experimental data ( figure a ,b). the competition experiments here reported are all in the direction of corroborating rgdechi d specificity towards αvβ with respect to αvβ or α β integrins. with an ic of . µm ( figure b ). with the aim to evaluate the capability of the peptide to discriminate between αvβ and α β , cell adhesion assays on k cells, displaying α β at high levels and αvβ and αvβ at very low levels [ ] , were carried out. the cells were seeded both onto fibronectin, the main ecm protein that binds α β receptor, and onto α β antibody coated plates. the results obtained indicate that rgdechi d is not able to inhibit k adhesion neither on fibronectin nor that of the α β specific antibody; in this experiment the rgdechi peptide [ ] was used as a control to recognize αvβ integrin [ ] thus proving the validity of experimental data ( figure a ,b). the competition experiments here reported are all in the direction of corroborating rgdechi d specificity towards αvβ with respect to αvβ or α β integrins. the major life-threatening event in cancer patients is the metastasis formation which involves different events, such as cell migration and invasion into blood or lymphatic vessels in distal organs. firstly, to investigate whether rgdechi d could interfere with these mechanisms, an in vitro scratch assay was performed on hepg cells. after h of seeding, the cell monolayers were scratched linearly and incubated with the peptide rgdechi d ( µm). then, images were taken at , , and h after wounding ( figure a ). the results show that in the presence of the peptide, the wound healing was delayed compared to scrambled peptides and untreated cells. remarkably, rgdechi d k were pre-incubated with peptides ( µm) for min at • c then seeded on anti-α β coated plates. the results are presented as the percentage of adherent cells respect to the control (untreated cells) and are expressed as means ± se of at least three independent experiments performed in triplicate. statistical significance was analyzed using student's t test, unpaired, two-sided (* p < . ). the major life-threatening event in cancer patients is the metastasis formation which involves different events, such as cell migration and invasion into blood or lymphatic vessels in distal organs. firstly, to investigate whether rgdechi d could interfere with these mechanisms, an in vitro scratch assay was performed on hepg cells. after h of seeding, the cell monolayers were scratched linearly and incubated with the peptide rgdechi d ( µm). then, images were taken at , , and h after wounding ( figure a ). the results show that in the presence of the peptide, the wound healing was delayed compared to scrambled peptides and untreated cells. remarkably, rgdechi d inhibits closure of the gap both at and h after the scratch ( figure b ). afterwards, to evaluate the cell invasion inhibition, hepg cells were incubated with µm peptide for h and seeded on trans-well chambers coated with ecl cell attachment, an efficient model system that better mimics in vivo cell invasion. as shown in figure a , significant decrease in tumor cell invasiveness is observed when cells are treated with the rgdechi d peptide with respect to untreated cells (control) or scrambled peptides. the decrease in invasiveness was quantified and resulted in %, as indicated in the graph ( figure b ). the effect on cell adhesion, migration, and invasion, events closely correlated with the metastatic cascade, highlight promising features of this peptide in inhibiting critical steps of cancer progression. molecules , , x of inhibits closure of the gap both at and h after the scratch ( figure b ). afterwards, to evaluate the cell invasion inhibition, hepg cells were incubated with µm peptide for h and seeded on trans-well chambers coated with ecl cell attachment, an efficient model system that better mimics in vivo cell invasion. as shown in figure a , significant decrease in tumor cell invasiveness is observed when cells are treated with the rgdechi d peptide with respect to untreated cells (control) or scrambled peptides. the decrease in invasiveness was quantified and resulted in %, as indicated in the graph ( figure b ). the effect on cell adhesion, migration, and invasion, events closely correlated with the metastatic cascade, highlight promising features of this peptide in inhibiting critical steps of cancer progression. angiogenesis is required for invasive tumor growth and metastasis and constitutes a key process in the control of cancer progression, thus elucidating its tight correlation with tumor cell invasion and migration. the involvement of αvβ integrin in all these events together with its high expression in hcc tissues are well documented. since this tumor is highly aggressive and often culminates in extensive metastasis [ ] [ ] [ ] , ] , the targeting of integrin αvβ is a promising approach to improve the survival rate. the ability to inhibit the generation of new capillary blood vessels by the rgdechi d peptide was examined by an angiogenesis in vitro assay. human umbilical vein endothelial cells (huvec) angiogenesis is required for invasive tumor growth and metastasis and constitutes a key process in the control of cancer progression, thus elucidating its tight correlation with tumor cell invasion and migration. the involvement of αvβ integrin in all these events together with its high expression in hcc tissues are well documented. since this tumor is highly aggressive and often culminates in extensive metastasis [ ] [ ] [ ] , ] , the targeting of integrin αvβ is a promising approach to improve the survival rate. the ability to inhibit the generation of new capillary blood vessels by the rgdechi d peptide was examined by an angiogenesis in vitro assay. human umbilical vein endothelial cells (huvec) were used as specific system to evaluate tube assembly. the cells were incubated on a gel containing various matrix proteins such as laminin, collagen type iv, heparan sulfate. cellular network structures were already developed by h. in figure a the formation of new capillary tubes is evident and, interestingly, in the sample treated with rgdechi d results to be significantly reduced. the branch points were counted to better evaluate the percentage of inhibition. the results indicate that the rgdechi d peptide is able to inhibit the branch point formation of about % ( figure b ). were used as specific system to evaluate tube assembly. the cells were incubated on a gel containing various matrix proteins such as laminin, collagen type iv, heparan sulfate. cellular network structures were already developed by h. in figure a the formation of new capillary tubes is evident and, interestingly, in the sample treated with rgdechi d results to be significantly reduced. the branch points were counted to better evaluate the percentage of inhibition. the results indicate that the rgdechi d peptide is able to inhibit the branch point formation of about % ( figure b ). to assess if rgdechi d has cytotoxic activity besides ability to interfere with the initial stages of tumor dissemination such as adhesion, invasion and angiogenesis, we evaluated the peptide effect on cell proliferation and on related pathways. to this aim, hepg were thus incubated with the peptide at different concentrations ( - µm) for h; furthermore, h after the first incubation, one cell aliquot was incubated with a second addition of peptide ( µm). as indicated in figure , rgdechi d inhibits cell proliferation in a dose-dependent manner; in particular, at the higher concentration used, rgdechi d induces inhibition of proliferation ( %) with respect to the untreated cells. the scrambled peptide has no effect on cell proliferation. an interesting result was to assess if rgdechi d has cytotoxic activity besides ability to interfere with the initial stages of tumor dissemination such as adhesion, invasion and angiogenesis, we evaluated the peptide effect on cell proliferation and on related pathways. to this aim, hepg were thus incubated with the peptide at different concentrations ( - µm) for h; furthermore, h after the first incubation, one cell aliquot was incubated with a second addition of peptide ( µm). as indicated in figure , rgdechi d inhibits cell proliferation in a dose-dependent manner; in particular, at the higher concentration used, rgdechi d induces inhibition of proliferation ( %) with respect to the untreated cells. the scrambled peptide has no effect on cell proliferation. an interesting result was obtained by the double addition of the peptide, which results in a greater decrease in cell proliferation ( %) suggesting a probably poor serum stability of rgdechi d. akt is an important effector of cell survival whose phosphorylation determines the activation of pi k pathway triggered by integrin signaling [ ] . the effect of rgdechi d binding on the activation of the integrin receptor was analyzed in light of the results obtained with the anti-proliferative assay. for this purpose, the phosphorylation of akt was evaluated by western blotting analysis performed on hepg cell lysates after treatment with µm rgdechi d for h. in figure , a decreased signal level of pakt with rgdechi d treatment compared to not treated cell lysates can be observed. no effect on the signal is induced by scrambled peptide treatment. this result clearly suggests that rgdechi d can reduce akt phosphorylation in hepg cell line in good agreement with its capability to reduce the cell proliferation as well. akt is an important effector of cell survival whose phosphorylation determines the activation of pi k pathway triggered by integrin signaling [ ] . the effect of rgdechi d binding on the activation of the integrin receptor was analyzed in light of the results obtained with the anti-proliferative assay. for this purpose, the phosphorylation of akt was evaluated by western blotting analysis performed on hepg cell lysates after treatment with µm rgdechi d for h. in figure , a decreased signal level of pakt with rgdechi d treatment compared to not treated cell lysates can be observed. no effect on the signal is induced by scrambled peptide treatment. this result clearly suggests that rgdechi d can reduce akt phosphorylation in hepg cell line in good agreement with its capability to reduce the cell proliferation as well. molecules , , x of obtained by the double addition of the peptide, which results in a greater decrease in cell proliferation ( %) suggesting a probably poor serum stability of rgdechi d. akt is an important effector of cell survival whose phosphorylation determines the activation of pi k pathway triggered by integrin signaling [ ] . the effect of rgdechi d binding on the activation of the integrin receptor was analyzed in light of the results obtained with the anti-proliferative assay. for this purpose, the phosphorylation of akt was evaluated by western blotting analysis performed on hepg cell lysates after treatment with µm rgdechi d for h. in figure , a decreased signal level of pakt with rgdechi d treatment compared to not treated cell lysates can be observed. no effect on the signal is induced by scrambled peptide treatment. this result clearly suggests that rgdechi d can reduce akt phosphorylation in hepg cell line in good agreement with its capability to reduce the cell proliferation as well. finally, to investigate if cell death induced by the rgdechi d effect is due to the apoptosis process, a caspase- activity assay was carried out. hepg cells were treated with µm peptide for h and, as shown in the figure , the examined peptide induces a remarkable increase in caspase activity, the differently scrambled peptide does not induce apoptosis, thus suggesting that rgdechi d has a direct effect on cell viability mediated by an apoptotic pathway. these results are in good agreement with several reports demonstrating that cell detachment caused by the treatment with rgd-based peptides determines cell death by apoptosis, a process known as anoikis [ ] . treatment was carried out on hepg . cells were treated with the rgdechi d peptide for h. (a) total cellular extracts ( µg) were resolved by sds page and analyzed by western blotting with anti-pakt (ser ) and anti-akt antibodies. the western blotting is representative of three independent experiments. (b) bar graph illustrates the percentage of band quantification respect to the control. = control, = rgdechi d, = scrambled peptide. the results are expressed as mean ± se of percentage of pakt normalized to the density of akt. finally, to investigate if cell death induced by the rgdechi d effect is due to the apoptosis process, a caspase- activity assay was carried out. hepg cells were treated with µm peptide for h and, as shown in the figure , the examined peptide induces a remarkable increase in caspase activity, the differently scrambled peptide does not induce apoptosis, thus suggesting that rgdechi d has a direct effect on cell viability mediated by an apoptotic pathway. these results are in good agreement with several reports demonstrating that cell detachment caused by the treatment with rgd-based peptides determines cell death by apoptosis, a process known as anoikis [ ] . figure . apoptosis assay. caspase- activity on hepg was determined after incubation with peptides ( µm) for h. caspase- activity (calculated as nmol -amino- -trifluoromethylcoumarin (afc)/min/µg protein) was expressed as the percentage of caspase- activity with respect to control (untreated cells). the results are expressed as means ± se of at least three independent experiments performed in triplicate. statistical significance was analyzed using student's t test, unpaired, twosided (* p < . ). here, we demonstrate that in hepg cell line the rgdechi d peptide shows an interesting effect in some steps of the metastatic pathway. in addition, the peptide displays cytotoxic activity, ascribable to the interference with the pi k pathway, mediated by an apoptotic process, in accordance with other rgd based peptides that, through cell detachment, send cells into apoptosis. collectively, the obtained results give indications about a potential role of rgdechi d as a selective ligand for hcc targeting. moreover, considering that αvβ is an interesting player in different pathological events, ranging from tumor to viral diseases, selective antagonists could be considered appealing candidates for the treatment of a wide spectrum of disorders. scrambled peptide rgdechi d caspase- activity (% vs control) * figure . apoptosis assay. caspase- activity on hepg was determined after incubation with peptides ( µm) for h. caspase- activity (calculated as nmol -amino- -trifluoromethylcoumarin (afc)/min/µg protein) was expressed as the percentage of caspase- activity with respect to control (untreated cells). the results are expressed as means ± se of at least three independent experiments performed in triplicate. statistical significance was analyzed using student's t test, unpaired, two-sided (* p < . ). here, we demonstrate that in hepg cell line the rgdechi d peptide shows an interesting effect in some steps of the metastatic pathway. in addition, the peptide displays cytotoxic activity, ascribable to the interference with the pi k pathway, mediated by an apoptotic process, in accordance with other rgd based peptides that, through cell detachment, send cells into apoptosis. collectively, the obtained results give indications about a potential role of rgdechi d as a selective ligand for hcc targeting. moreover, considering that αvβ is an interesting player in different pathological events, ranging from tumor to viral diseases, selective antagonists could be considered appealing candidates for the treatment of a wide spectrum of disorders. human hepatocarcinoma cell line (hepg ) (atcc u.s.) were grown in dmem supplemented with % fetal bovin serum (fbs), % glutamine, u/ml penicillin and µg/ml streptomycin (euroclone, milan, italy). human umbilical vein endothelial cells (huvec) were purchased from lonza. cells were grown in endothelial cell growth medium (egm- ) from lonza, (basel, switzerland). all experiments were performed using low passage cell cultures [ ] . chronic myelogenous leukemia cells line (k ) (atcc, manassas, va, usa.) were grown in rpmi with added heat-inactivated % fbs (fetal bovine serum), mm glutamine, u/ml penicillin and µg/ml streptomycin (euroclone, milan, italy). the cells were maintained in humidified air containing % co at • c. for facs analysis adherent cells at about % confluence were detached using . mm edta in pbs (sigma aldrich, darmstads, germany), centrifuged and re-suspended in pbs containing . % bsa. cell aliquots ( . × cells) were treated with primary monoclonal antibody pe conjugate, (millipore, burlington, ma, usa) or isotype control (santa cruz biotechnology, dallas, tx, usa), at the same concentration ( µg/ml), whole a reaction volume of µl for min at • c. after washing, the cells were analyzed by using a flow cytometer equipped with a nm argon laser (facscan, becton dickinson, franklin lakes, nj, usa). a total of , events per sample were collected. integrin quantification with quantibrite pe beads (bectondickinson biosciences) was evaluated as previously described [ ] . values of fluorescence intensity were obtained from the histogram statistic of cellquest software. the effect of peptides on hepg or k adhesion was tested on nunc maxisorp well plates (dasit sciences, milan, italy) coated with µg/ml vitronectin (for hepg ), fibronectin or anti-α β antibody (for k ) (millipore, burlington, ma, usa). , cells/well were incubated in the presence of peptides ( µm) or antibodies ( µg/ml) at • c for min as previously reported [ ] and then cells were seeded on coated plates for h. non adherent cells were gently removed by repeated washing and adherent cell number was evaluated by crystal violet (sigma aldrich) assay, which correlates optical density with cell number. for the ic calculation, the peptides were added at different concentration starting from µm to µm. the mean value ± standard error (se) of adherent cells for each treatment was expressed as relative percentage of cell number vs. untreated cells (control). statistical differences were determined by student's t test, paired, two-sided. all experiments were performed in triplicate and repeated at least times; a p value < . was considered to be significant. the ic value was calculated by graphpad prism software. hepg cells were cultured in well plates until they reached the confluence, and were linearly scratched with a plastic pipette tip to create a wound [ ] . after washing with pbs, necessary to remove loose cells, the medium containing µm of peptides was then added, and cells were incubated at • c in a humidified incubator in % co . each scratch area was photographed at , , and h. the wound size for the different peptides was calculated as width at each end-points with respect to their value at h as follows: wound closure (%) = − (wound width t = x/wound width t = ) × . cell invasion was assayed using transwell chambers coated with ecl cell attachment matrix (millipore corporation) used according to manufacturer's instructions. two duplicates were set for the assay. cells at the logarithmic phase were detached using mm edta in pbs and washed twice with serum-free dmem. a total of × cells/ µl were seeded in the upper chamber. in the lower chamber, µl of dmem medium containing % fetal bovine serum was added for incubation at • c in a % co incubator for h. after removal of the upper chamber, they were washed twice with pbs and migrated cells were fixed with % formalin for min. next, the cells on the bottom surface of the membrane were stained with crystal violet for min. the cells which did not migrate were removed with cotton swabs [ ] . cell images were obtained under a phase contrast microscope (zeiss, oberkochen, germany) and the cells were counted by axiovert software (zeiss). transwell migration assays were repeated three times and performed in duplicate. all measurement data are presented as the mean ± se. statistical analysis was performed using a t-test where p < . was considered to indicate a statistically significant difference. ecmatrix tm solution ( µl) was plated in a pre-cooled well plate and incubated for h at • c. huvec cells were harvested, incubated ( cells) with µm peptide and seeded onto solidified matrix. after h of incubation the tube formation was inspected under an inverted light microscope at × magnification [ ] . imagines were acquired by axiovert zeiss microscopy fluorescence. the experiments were performed two times in duplicate. the effect of peptides on hepg proliferation was analyzed with an rtca icelligence™ instrument (acea, san diego, ca, usa) with a method that uses label-free, real time, non-invasive analysis [ ] . the instrument records a signal of electrical impedance due to the cell covering of gold electrodes located on the surface of a special microplate. the variation of the electrical impedance is named cell index (ci). ci values are proportional to the area of cells adherent and, as a consequence, to their numbers [ ] . in detail, , cells/well were seeded and incubated in the presence of peptides for h. the ci was calculated by measuring the slope of the proliferation curve between two selected time points for each curve, considering that the maximum ci values observed is the end-point of the experiments. statistical differences were determined by student's t test, paired, two-sided. all experiments were performed in triplicate and repeated at least three times; a p value less than . was considered to be significant. hepg cells were incubated with peptides ( µm) for h at • c. whole cell lysates were obtained by using lysis buffer ( mm hepes, ph . , mm nacl, % triton) supplemented with phosphatase (sigma aldrich, milan, italy) and protease inhibitor cocktails (roche, milan, italy). cell lysates were incubated on ice for min and then centrifuged at . rpm for min to remove cell debris. protein concentrations were determined by the bradford method using bio-rad reagent (bio-rad laboratories, hercules, ca, usa). proteins ( µg) were resolved by sds-polyacrylamide gel electrophoresis (sds-page) and transferred to a pvdf membrane (millipore). the membrane was probed with the primary antibodies (anti pakt (ser ) or anti akt (cell signaling technologies, boston, ma, usa)) over night at • c. proteins were visualized with an enhanced chemiluminescence detection system (euroclone, milan, italy) and images were acquired with chemidoc xrs system (bio-rad laboratories, italy) and analyzed with the quantityone software. reblot was performed using a harsh stripping buffer containing % sds, mm tris hcl, ph . , . % β-mercaptoethanol, incubating the membrane at • c for min with some agitation. determination of caspase- activity was performed by a fluorometric assay based on the proteolytic cleavage of the carbobenzoxy-asp-glu-val-asp- -amino- -methylcoumarin (acetyl-devd-afc alexis biochemicals, san diego, ca, usa) as described elsewhere [ ] . in detail, hepg were treated with the peptides ( µm) for h at • c. after h, cells were processed with reaction buffer ( mm hepes, ph . , . mm edta, . % nonidet p- , . % chaps and mm dtt) and µg of lysates were incubated with µm ac-devd-afc at • c for h. samples were analyzed using a microplate reader (biotek, winooski, vt, usa) (excitation wavelength nm; emission wavelength nm). an afc standard curve was determined, and caspase-specific activity was calculated as nmol of afc produced per min per µg proteins at • c at saturating substrate concentration ( µm). the diverse roles of integrins and their ligands in angiogenesis purification and functional characterization of integrin alpha v beta . an adhesion receptor for vitronectin ligand binding to integrins beyond rgd: virus interactions with integrins beta integrin is the major contributor to the alphavintegrin-mediated blockade of hiv- replication integrin alphavbeta internalizes zika virus during neural stem cells infection and provides a promising target for antiviral therapy a multi-targeting approach to fight sars-cov- attachment definition of two angiogenic pathways by distinct alpha v integrins integrin alphavbeta regulates lung vascular permeability and pulmonary endothelial barrier function src-mediated coupling of focal adhesion kinase to integrin alpha(v)beta in vascular endothelial growth factor signaling elevated expression of integrin alphav and beta subunit in laryngeal squamous-cell carcinoma associated with lymphatic metastasis and angiogenesis alphavbeta -integrins mediate early steps of metastasis formation significance of integrin alphavbeta and erbb in enhanced cell migration and liver metastasis of colon carcinomas stimulated by hepatocyte-derived heregulin mesenchymal to epithelial transition in development and disease inflammation and emt: an alliance towards organ fibrosis and cancer progression role of beta -integrin in epithelial-mesenchymal transition in response to tgf-beta p -activated kinase interacts with integrin alpha v beta and regulates alpha v beta -mediated cell migration increased expression of the coxsackie and adenovirus receptor downregulates alphavbeta and alphavbeta integrin expression and reduces cell adhesion and migration integrin beta contributes to the tumorigenic potential of breast cancer cells through the src-fak and mek-erk signaling pathways integrin targeted drug and gene delivery practice guidelines committee, management of hepatocellular carcinoma early and late recurrence after liver resection for hepatocellular carcinoma: prognostic and therapeutic implications tumour exosome integrins determine organotropic metastasis integrin-beta , a mir- -targeted gene, promotes hepatocellular carcinoma tumorigenesis by regulating beta-catenin stabilit elevated serum plasma fibrinogen is associated with advanced tumor stage and poor survival in hepatocellular carcinoma patients. medicine (baltimore) , , e hepatic stellate cells and the reversal of fibrosis fibrinogen induces cytokine and collagen production in pancreatic stellate cells expression, regulation, and function of alpha v integrins in hepatocellular carcinoma: an in vivo and in vitro study activation of hepatic stellate cells during liver carcinogenesis requires fibrinogen/integrin alphavbeta in zebrafish human integrin alphavbeta : homology modeling and ligand binding deciphering rgdechi peptide-α β integrin interaction mode in isolated cell membranes novel and selective alpha(v)beta receptor peptide antagonist: design, synthesis, and biological behavior conformational studies of rgdechi peptide by natural-abundance nmr spectroscopy imaging of alpha(v)beta( ) expression by a bifunctional chimeric rgd peptide not cross-reacting with alpha(v)beta( ) a combined nmr and computational approach to determine the rgdechi-hcit-alphav beta integrin recognition mode in isolated cell membranes anticancer effects of echinacoside in hepatocellular carcinoma mouse model and hepg cells affinity of integrins for damaged extracellular matrix: alpha v beta binds to denatured collagen type i through rgd sites matrix metalloproteinase and alphavbeta integrin-dependent vascular smooth muscle cell invasion through a type i collagen lattice dioscin inhibits the invasion and migration of hepatocellular carcinoma hepg cells by reversing tgf-beta -induced epithelial-mesenchymal transition cross-talk mechanism between endothelial cells and hepatocellular carcinoma cells via growth factors and integrin pathway promotes tumor angiogenesis and cell migration specific beta integrin site selectively regulates akt/protein kinase b signaling via local activation of protein phosphatase a rgdechi-hcit: alphavbeta selective pro-apoptotic peptide as potential carrier for drug delivery into melanoma metastatic cells unveiling a vegf-mimetic peptide sequence in the iqgap protein functional binding surface of a beta-hairpin vegf receptor targeting peptide determined by nmr spectroscopy in living cell chemical modification for proteolytic stabilization of the selective alphavbeta integrin rgdechi peptide: in vitro and in vivo activities on malignant melanoma cells therapeutic potential of a novel alphavbeta( ) antagonist to hamper the aggressiveness of mesenchymal triple negative breast cancer sub-type a selective αvβ integrin antagonist hidden into the anophelin family protein ce from the malaria vector anopheles gambiae application of real-time cell electronic sensing (rt-ces) technology to cell-based assays this article is an open access article distributed under the terms and conditions of the creative commons attribution we are grateful to maurizio amendola (institute of biostructures and bioimaging-cnr, naples) for technical assistance and luca de luca (institute of biostructures and bioimaging-cnr, naples) for computer assistance. the authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper. key: cord- -uysf sh authors: jia, meng-qi; xiong, ye-juan; xue, yun; wang, yan; yan, chao title: using uplc-ms/ms for characterization of active components in extracts of yupingfeng and application to a comparative pharmacokinetic study in rat plasma after oral administration date: - - journal: molecules doi: . /molecules sha: doc_id: cord_uid: uysf sh yupingfeng (ypf), a famous traditional chinese medicine, which contains a large array of compounds, has been effectually used in health protection. a two-dimensional liquid chromatography (( )d-lc) combined with quadrupole time-of-flight mass spectrometry (qtof-ms) method was firstly established to separate and identify chemical components in ypf. a total of compounds were identified, including constituents (flavonoids and saponins) in astragali radix; seven constituents (sesquiterpenoids and polysaccharide) in atractylodis rhizoma; and constituents (chromone and coumarins) in saposhnikoviae radix. the corresponding fragmentation pathway of typical substances was investigated. then, seven active constituents (astragaloside, calycosin, formononetin, cimicifugoside, -o-beta-d-glucosyl- -o-methylvisamminol, sec-o-glucosylhamaudol, and atractylenolide ii) derived from three medicinal plants were chosen to further investigate the pharmacokinetic behavior of ypf formula using ultrahigh-performance liquid chromatography with triple quadrupole mass spectrometry system. the method was sensitive, accurate and reliable. we also used the area under the plasma concentration–time curve from zero to infinity (auc( −∞)) as weighting factor to make an integrated pharmacokinetic curve. results show that the constituents of saposhnikoviae radix have the best absorption and pharmacokinetic behavior and may play important role in leading to the changes of overall therapeutic effects of ypf. further study is needed to confirm the association between them. currently, traditional chinese medicine (tcm) has drawn more and more attention worldwide. tcm is usually prepared as a formula by unique methods with a specific combination which contains multiple herbs [ ] [ ] [ ] [ ] . the multiple components in a formula work together to achieve satisfactory therapeutic efficacy and minimize adverse reactions [ ] . however, their synergistic mechanisms are poorly understood and must be addressed using a more holistic approach [ ] . pharmacokinetic studies might further provide physiologically relevant clues to the target identification and mechanistic study of tcm formula [ ] . yupingfeng (ypf), a famous tcm formula, has been efficaciously used in health protection, immunity enhancement and inflammatory disease control for thousands of years [ ] [ ] [ ] , including the herbs were processed as previously described [ ] : dried plants of astragali radix, atractylodis rhizoma and saposhnikoviae radix ( : : g) by weight were mixed and double extracted by refluxing with boiling water ( : and then : , w/v) for min; thereafter the water decoction was concentrated to . g/ml (crude drugs); and then the decoction was centrifuged for min at , rpm ( • c) after -min standing. the supernatant was centrifuged for min at , rpm ( • c), filtrated by the microvoid filter film ( . µm) before directly injected into the lc system for analysis. the primary standard stock solutions of astragaloside ( . mg/ml), calycosin ( . mg/ml), formononetin ( . mg/ml), atractylenolide ii ( . mg/ml), cimicifugoside ( . mg/ml), -o-beta-d-glucosyl- -o-methylvisamminol ( . mg/ml), and sec-o-glucosylhamaudol ( . mg/ml) were prepared by dissolving in methanol. these stock solutions were further appropriately diluted to give a serial of working standard solutions by spiking aliquots of the stock solutions into methanol. quality control (qc) samples (with low, medium, and high concentration, respectively) were independently prepared. all solutions were stored at − • c and brought to room temperature before use. for calibration standards, to an aliquot of µl blank plasma sample, µl of above working solutions were added to obtain final concentrations of . for the incurred samples, µl of methanol (volume of the corresponding standard solution for calibration curve) and µl of samples with µl acetonitrile were mixed, the mixture was then vortex-extracted for min, and centrifuged for min at , rpm ( • c). the supernatant was transferred to a new tube and evaporated under a gentle stream of nitrogen to dryness. the residue was reconstituted with µl of initial mobile phase, vortex-mixed for min and centrifuged at , rpm for min. two dimension-normal/reversed phase liquid chromatography combine with quadrupole time-of-flight mass spectrometry ( d-nplc/rplc-qtof-ms) was used in this study. in the first dimension, tsk gel amide- column ( . mm × mm, µm) was used. mobile phase water-acetonitrile (a:b) was run at . ml/min, the initial solvent was % b and kept for min, and then the gradient was - min, - % b; - min, - % b; - min, - % b; - min, - % b; - min, - % b. an automatic switching valve was used to collect the effluent of the amide column every min, for five fractions totally, and each fraction was collected times and dried by nitrogen. afterward, the residues were redissolved in µl methanol-water solution ( : , v/v) and vortex-mixed for min and centrifuged at , rpm for min, then µl supernatant was injected into the second dimension. in the second dimension, a waters uplc-beh c column ( mm × . mm, . µm) was used. the mobile phase were water (containing . % formic acid)-methanol (a:b) with a flow rate of . ml/min. the gradient was - min, - % b; - . min, - % b; . - min, - % b; . - min, - % b; - . min, - % b; . - . min, % b. the detection and identification were carried out on ultimate rslcnano liquid chromatography/maxis impact uhr-qtof-ms (bruker daltonics, bremen, germany; thermofisher, waltham, ma, usa), equipped with esi+ source, the conditions were as follow: capillary, . kv; sampling cone, . v; extraction cone, . v; cone gas flow, . l/hr; source temperature, • c; desolvation gas flow, . l/hr; desolvation temperature, • c; collision energy, . ; wavelength range, to nm; lock mass, . . the mass range was set at - . all the acquisition and analysis of data were controlled by masslynx v . software (waters corporation, milford, ma, usa) for peak detection and peak alignment. the chromatographic uplc-qqq-ms procedure of pharmacokinetics study was performed using acquity uplc (waters, milford, ma, usa) and sciex selexion triple quad™ system (ab sciex, framingham, ma, usa) equipped with a heated electrospray ionization source (esi) interface, separation was achieved using a waters beh c column ( . mm × mm, . µm) which was protected by an online filter, the mobile phase was at a flow rate of . ml/min, consisted of aqueous solvent a: water-acetic acid ( : , v/v) and organic phase solvent b: acetonitrile. a -min binary gradient elution was performed for the separation, and the consecutive program was as follows: an isocratic elution of % solvent b to equilibrate the column, then followed by a linear gradient elution: - . min, - % b; . - . min, - % b; . - . min, - % b; . - . min, - % b; . - . min, - % b; . - min, - % b; - min, - % b; and the re-equilibration time of gradient elution was min. the column and auto-sampler tray temperature were maintained constantly at • c and • c, respectively. the injection volume was µl. the electrospray ionization source was operated in positive mode. quantitation was performed by schedule multiple reactions monitoring (smrm). in the positive mode, the tandem mass spectrometry (ms/ms) typical instrumental conditions were: ion spray voltage . kv, ion source gas psi, ion source gas psi, temperature • c, and curtain gas psi. data acquisition and processing were performed using analyst software (ab sciex, corp., framingham, ma, usa). the method was fully validated in accordance with the us-fda document and other related guidelines with respect to specificity, linearity, precision and accuracy, recovery, matrix effect and stability. selectivity was assessed by comparing the chromatograms of a blank plasma sample, a blank plasma sample spiked with the working solutions and a rat plasma sample obtained from min after an oral administration of the mix standard solution of seven analytes. linearity and sensitivity: the linearity of assay for the test in vivo compounds with a total of six calibration standards were prepared in blank rat plasma at least six concentrations, respectively, obtained by plotting the peak area (y) of each analyte versus the theoretical standard concentrations (x) and assessed by weighed least-squares linear regression using concentrations (x) as the weighting factor in spss-based software. accuracy and precision: the accuracy and precision of intra-day and inter-day determination were carried out in six replicates samples at three qc levels (low, medium and high) within the same day and on three consecutive days. matrix effect and recovery: the matrix effect of the biological matrix (rat plasma) and extraction recoveries were evaluated at three qc levels for analytes with six replicates, matrix effect was assessed via comparison of the peak responses of the analytes in the extracted blank rat plasma matrix to those obtained from neat standard solutions at equivalent concentrations. while extraction recoveries were calculated by comparing the peak areas of samples added before extraction with those of samples added after extraction. stability the short-term and long-term stability of seven analytes in rat plasma in auto-sampler vials for h, at − • c for days and after three freeze-thaw cycles was assessed, and also carried out in six replicates samples at three qc levels (low, medium and high). the developed uplc-qqq-ms method was applied to a pharmacokinetic study of seven analytes in rat plasma. male healthy adult sprague-dawley rats (n = , ± g body weight) were supplied by shanghai jiao tong university laboratory animal centre (shanghai, china). animals were acclimatized in an environmental controlled breeding room with standard laboratory food and water for one week prior to the experiments. all the rats were fasted for h, with free access to water prior to the experiments, animal welfare and experimental procedures conformed to guidance for the care and use of laboratory animals, similarly the ethical regulations of sjtu. after oral administration of a single dose of mg/kg ( ml/kg) astragaloside, calycosin, formononetin, atractylenolide ii, cimicifugoside, -o-beta-d-glucosyl- -o-methylvisamminol, and sec-o-glucosylhamaudol mix standard solution prepared with . % carboxymethyl cellulose sodium (cmc-na), µl of blood samples were planned to be collected manually using heparinized tubes at scheduled time points ( , , , and min, and , . , , , , , and h). the total blood volume collected during each session did not exceed . % of the male rat total body weight. then immediately centrifuged at rpm for minutes, plasma was transferred into another via and stored at − • c until assay. the plasma concentrations of seven analytes at different time points are described as the mean ± s.d. the concentration versus time curve was plotted. compartmental model was chosen to calculate all the related pharmacokinetic parameters including c max (ng/ml), t max (h), auc −∞ (ng·h/ml), cl/f (l/h.kg), t / α (h) and t / β (h) were calculated by kinetica . software (thermo fisher scientific™, corp., waltham, ma, usa), and performed with graphpad (graphpad software, inc., la jolla, ca, usa). we also use the area under the plasma concentration-time curve from zero to infinity (auc −∞ ) as the weighting factor to make an integrated pharmacokinetic curve. by plugging various auc −∞ values into the following equations, integrated concentration of total analytes was calculated: where j and ω j , respectively, represent each analytes and the ratio of auc −∞ of single analyte to the total analytes; and c t was self-defined integrated concentration of total analytes after weight factor correction. the first step of this work was to develop an efficient method for enrichment and analysis of chemical constituents in the extracts of ypf, since the constituents are in wide range of polarity and molecular weight, and often co-eluted with analogs in low concentration, great efforts are needed to solve the encountering difficulties during the process of isolation and detection. firstly, d-nplc/rplc system based on two dimensions with orthogonal separation mechanisms was developed aiming at enhancing the peak capacity of system. given the complexity of chemical constituents of ypf, we needed to obtain the best peak capacity and maximize the meaningful difference between variable contents of chemicals in two dimensional system. the primary consideration is complete orthogonal separation mechanism, which is currently hard to achieve in reality; the most common used system consists of normal phase chromatographic columns combine with c (reverse phase) column. in addition, the major components of ypf decoction were all water-soluble, thus we evaluate the separation efficiency of cn ( figure a ), halo hilic (hydrophilic interaction liquid chromatography) ( figure b) , and tskgel amide ( figure c ) columns (normal phase chromatographic columns), which are hydrophilic as the first dimension because all separate substances based on differences in hydrophilicity. the d-nplc chromatograms of the crude decocted extract of ypf were shown on figure c . almost peaks could be seen clearly. it is obvious that amide column has the best peak resolution and sensitivity compared with the other two. the packing material of amide column is carbamoyl instead of aminopropyl groups bonded to silica gel, the bonded phase was significantly efficient in analysis of polysaccharides and glycosides, which exist in the herbs astragali radix [ , ] , atractylodis rhizoma [ ] and saposhnikoviae radix [ ] , as well as the flavonoids, lactones, volatile oil, etc. [ ] . astragali radix [ , ] , atractylodis rhizoma [ ] and saposhnikoviae radix [ ] , as well as the flavonoids, lactones, volatile oil, etc. [ ] . the fractions of ypf water decoction were collected and concentrated after separation in the first nplc system respectively, which would increase the concentration of the sample. samples diluted by mobile phase of first dimension would reduce the detection limit of the second dimension. in order to overcome this shortcoming as well as eliminate the influence of solvent incompatibility, this experiment adopted the method of concentrating samples manually, each fraction eluted from amide column in the same period of time were collected after times sample injection, and concentrated to dry under nitrogen, then dissolved in μl methanol-water ( : , v/v) for analysis by second dimension chromatography. to further analyze and characterize each fraction, the high resolution tandem mass spectrometry, particularly qtof-ms method which can offer great mass accuracy and selectivity with full-scan mode, should be considered. hence, uplc-qtof-ms using c chromatographic columns (as the second dimension) was established to isolate the five fractions from d-nplc. a representative d-nplc/uplc-qtof-ms chromatogram ( figure b ) of second fraction ( - min) from d-nplc can be seen in figure . for comparison, a d-uplc-qtof-ms chromatogram of the crude decocted extract of ypf is also shown (figure a ). it is obvious that the d method has successfully enriched the peak capacity and improved the resolution as well. there are molecular features presented in total from all five fractions. d-nplc/uplc-qtof-ms method was proven to be efficient in analysis of chemical constituents from the extract of ypf. the fractions of ypf water decoction were collected and concentrated after separation in the first nplc system respectively, which would increase the concentration of the sample. samples diluted by mobile phase of first dimension would reduce the detection limit of the second dimension. in order to overcome this shortcoming as well as eliminate the influence of solvent incompatibility, this experiment adopted the method of concentrating samples manually, each fraction eluted from amide column in the same period of time were collected after times sample injection, and concentrated to dry under nitrogen, then dissolved in µl methanol-water ( : , v/v) for analysis by second dimension chromatography. to further analyze and characterize each fraction, the high resolution tandem mass spectrometry, particularly qtof-ms method which can offer great mass accuracy and selectivity with full-scan mode, should be considered. hence, uplc-qtof-ms using c chromatographic columns (as the second dimension) was established to isolate the five fractions from d-nplc. a representative can be seen in figure . for comparison, a d-uplc-qtof-ms chromatogram of the crude decocted extract of ypf is also shown (figure a ). it is obvious that the d method has successfully enriched the peak capacity and improved the resolution as well. there are molecular features presented in total from all five fractions. d-nplc/uplc-qtof-ms method was proven to be efficient in analysis of chemical constituents from the extract of ypf. a large number of chemical constituents were effectively separated from the crude extracts of ypf through the d-nplc/uplc-qtof-ms system and characterized using uplc-qtof-ms, and all of their accurate masses, different retention times and abundances were extracted. however, some compounds were detected overlapped in the five fractions. all compound structures were characterized based on their chromatographic data and multiple fragmentations for structural information, comparing to their standards or referring to previous literature. some ion mass spectra and the corresponding fragmentation pathway of typical substances are shown in figure . ultimately, constituents (tables - ) were identified or tentatively characterized, including constituents (isoflavones, flavonoids, saponins and glycosides) in astragali radix; seven constituents (atractylodes lactone class, polysaccharide) in atractylodis rhizoma; and constituents (chromone and glycoside) in saposhnikoviae radix. a large number of chemical constituents were effectively separated from the crude extracts of ypf through the d-nplc/uplc-qtof-ms system and characterized using uplc-qtof-ms, and all of their accurate masses, different retention times and abundances were extracted. however, some compounds were detected overlapped in the five fractions. all compound structures were characterized based on their chromatographic data and multiple fragmentations for structural information, comparing to their standards or referring to previous literature. some ion mass spectra and the corresponding fragmentation pathway of typical substances are shown in figure . ultimately, constituents (tables - ) were identified or tentatively characterized, including constituents (isoflavones, flavonoids, saponins and glycosides) in astragali radix; seven constituents (atractylodes lactone class, polysaccharide) in atractylodis rhizoma; and constituents (chromone and glycoside) in saposhnikoviae radix. after chemical constituents were identified in the extract of ypf, seven major constituents were chosen to further investigate the pharmacokinetic behavior of ypf formula: astragaloside, calycosin and formononetin in astragali radix; atractylenolide ii in atractylodes rhizoma; and cimicifugoside, -o-beta-d-glucosyl- -o-methylvisamminol, and sec-o-glucosylhamaudol in saposhnikoviae radix. in early literature, some pharmacological studies [ ] [ ] [ ] [ ] [ ] [ ] have proven that these seven constituents are the main active compounds in the corresponding individual herbs, leading to changes of overall therapeutic effects, and they show remarkable anti-inflammatory, antiviral and immunosuppressive activities. therefore, we assume that these constituents could explain the effectiveness of ypf formula. however, it is challenging to simultaneously detect these seven compounds in biological samples because of the complicated metabolism in vivo and wide difference in the level of concentration. a proposed uplc-qqq-ms could offer much lower limit of detection, making it possible to analyze. thus, we optimized the most abundant ions and appropriate mass spectrum parameters for analysis after directly injecting a standard solution of each compound using the uplc-qqq-ms system. the list of selected srm parameters, detect mode, cone voltage (cv), collision energy (ce) and retention time (rt) for each analyte is presented at table figure s . in order to make a shorter analysis time and good chromatographic behavior and appropriate ionization, we used several volatile additives, such as formic acid, acetic acid, and ammonium acetate, in our experiment. it was finally found that the addition of . % acetic acid to aqueous mobile phase provides a better sensitivity and peak symmetry, and efficiently improves the ionization of all the analytes. meanwhile, acetonitrile has been proven to provide more stable and practical column back-pressure at the flowrate of . ml/min than methanol. when considering the initial percentage of organic mobile phase, we found that % acetonitrile can significantly change the peak shape of the analytes than other percentage. subsequently, sample pre-treatment procedures were also investigated. protein precipitation by methanol, acetonitrile, ethanol, dimethyl sulfoxide and mixed organic reagent were employed, and after chemical constituents were identified in the extract of ypf, seven major constituents were chosen to further investigate the pharmacokinetic behavior of ypf formula: astragaloside, calycosin and formononetin in astragali radix; atractylenolide ii in atractylodes rhizoma; and cimicifugoside, -o-beta-d-glucosyl- -o-methylvisamminol, and sec-o-glucosylhamaudol in saposhnikoviae radix. in early literature, some pharmacological studies [ ] [ ] [ ] [ ] [ ] [ ] have proven that these seven constituents are the main active compounds in the corresponding individual herbs, leading to changes of overall therapeutic effects, and they show remarkable anti-inflammatory, antiviral and immunosuppressive activities. therefore, we assume that these constituents could explain the effectiveness of ypf formula. however, it is challenging to simultaneously detect these seven compounds in biological samples because of the complicated metabolism in vivo and wide difference in the level of concentration. a proposed uplc-qqq-ms could offer much lower limit of detection, making it possible to analyze. thus, we optimized the most abundant ions and appropriate mass spectrum parameters for analysis after directly injecting a standard solution of each compound using the uplc-qqq-ms system. the list of selected srm parameters, detect mode, cone voltage (cv), collision energy (ce) and retention time (rt) for each analyte is presented at table figure s . in order to make a shorter analysis time and good chromatographic behavior and appropriate ionization, we used several volatile additives, such as formic acid, acetic acid, and ammonium acetate, in our experiment. it was finally found that the addition of . % acetic acid to aqueous mobile phase provides a better sensitivity and peak symmetry, and efficiently improves the ionization of all the analytes. meanwhile, acetonitrile has been proven to provide more stable and practical column back-pressure at the flowrate of . ml/min than methanol. when considering the initial percentage of organic mobile phase, we found that % acetonitrile can significantly change the peak shape of the analytes than other percentage. subsequently, sample pre-treatment procedures were also investigated. protein precipitation by methanol, acetonitrile, ethanol, dimethyl sulfoxide and mixed organic reagent were employed, and methanol and acetonitrile showed satisfied extraction recoveries and matrix effect for all analytes within a minimum number of steps. better yet, acetonitrile permitted a uniform extraction recoveries and a lower background noise. the method was fully validated in accordance with the us-fda document and other related guidelines with respect to specificity, linearity, precision and accuracy, recovery, matrix effect and stability. selectivity: six blank samples of rat plasma from different sources were analyzed. representative smrm chromatograms of astragaloside (figure b ), and plasma sample from a normal rat min after oral administration of mix-std ( figure c ). the background noise was very low, and the absence of endogenous interference at or close to the retention time of the analytes clearly proves the high selectivity of the method. the method was fully validated in accordance with the us-fda document and other related guidelines with respect to specificity, linearity, precision and accuracy, recovery, matrix effect and stability. selectivity: six blank samples of rat plasma from different sources were analyzed. representative smrm chromatograms of astragaloside figure , including blank plasma ( figure a ), blank plasma spiked with the analytes (figure b ), and plasma sample from a normal rat min after oral administration of mix-std ( figure c ). the background noise was very low, and the absence of endogenous interference at or close to the retention time of the analytes clearly proves the high selectivity of the method. linearity and sensitivity: lowest limit of quantification (lloq) is calculated as the lowest level of the analytes that can be reliably detected with a signal-to-noise (s/n) ratio above , and reproduced with an accuracy of - %. summary of regression equations, coefficient and lloqs of the seven analytes in blank rat plasma are presented in table . all calibration curves exhibited good linearity with coefficient of determination (r ) within the range of . - . . the lloqs were sensitive enough for quantitative detection of analytes in the pharmacokinetic studies. accuracy and precision: intra-day and inter-day precision was expressed as the relative standard deviation (rsd) should not exceed % (or ± % for lloq), and the accuracy was denoted by the proximity to the theoretical concentration relative error (re) ((true value of concentration − observed value of concentration)/(true value of concentration) × %) should be within ± % (or ± % for lloq). table summarizes the accuracy, precision, recovery and matrix effect data of the seven analytes with different concentrations of low, medium and high in rat plasma (n = ), intra-day and inter-day precision was less than % in terms of rsd%, and the accuracy (re%) was below %, with only one exception of cimicifugoside ( %) at its lowest concentration, which is still be acceptable (≤ %). these values indicate that the overall reproducibility of the method is within the bioanalytical method validation acceptance criteria as per fda and ema guidelines. table . summary of accuracy, precision, recovery and matrix effect of the seven analytes in rat plasma (n = ). matrix effect and recovery: the results of matrix effect and recovery are also summarized in table , from which we can see that the extraction recoveries of analytes and matrix effect are all in the range of - %, with only the recovery of calycosin at %, a little bit over the criteria. the other results show this method was consistent, precise and reproducible. stability: the analytes would be considered stable when the accuracy bias was within ± % of the nominal concentrations (or ± % for lloq). table shows the results of stability studies under different conditions: the measured concentrations for seven analytes at each qc level were within ± . % of nominal values, indicating that they were stable under all conditions tested. because these seven analytes are all difficult to dissolve in water, . % carboxymethyl cellulose sodium was used to make a suspension for oral administration. the dose of mg/kg mix-standard was determined by our previous pharmacokinetic experiments of ypf decoction. mean plasma concentration-time curves of astragaloside, calycosin, formononetin, atractylenolide ii, cimicifugoside, -o-beta-d-glucosyl- -o-methylvisamminol, and sec-o-glucosylhamaudol are presented in figure s . moreover, the integrated pharmacokinetics profile is showed in figure , while the results of auc −∞ and corresponding weighting coefficients (ωj) of seven analytes after oral administration and the main pharmacokinetic parameters are listed in tables and , respectively. stability: the analytes would be considered stable when the accuracy bias was within ± % of the nominal concentrations (or ± % for lloq). table shows the results of stability studies under different conditions: the measured concentrations for seven analytes at each qc level were within ± . % of nominal values, indicating that they were stable under all conditions tested. because these seven analytes are all difficult to dissolve in water, . % carboxymethyl cellulose sodium was used to make a suspension for oral administration. the dose of mg/kg mix-standard was determined by our previous pharmacokinetic experiments of ypf decoction. mean plasma concentration-time curves of astragaloside, calycosin, formononetin, atractylenolide ii, cimicifugoside, -o-beta-d-glucosyl- -o-methylvisamminol, and sec-o-glucosylhamaudol are presented in figure s . moreover, the integrated pharmacokinetics profile is showed in figure , while the results of auc −∞ and corresponding weighting coefficients (ωj) of seven analytes after oral administration and the main pharmacokinetic parameters are listed in tables and , respectively. table . pharmacokinetic parameters of analytes and integrated pk after oral administration ( mg/kg) to rats (n = , mean ± s.d.). the value of c max and auc -∞ of cimicifugoside, -o-beta-d-glucosyl- -o-methylvisamminol and sec-o-glucosylhamaudol were found higher than those of other compounds, one of the main reasons is that the glycoside counterparts can be absorbed through intestinal epithelium more efficiently than the aglycones, thus the aglycones show biological activities after being metabolized from their glycoside counterparts in vivo. they were all better fitted to a two-compartment model [ ] . in contrast, the relatively low plasma concentrations of atractylenolide ii was consistent with previous report, which showed that atracylenolide ii has poor intestinal absorption and rapid biliary excretion [ , ] . because of astragaloside's low bioavailability characteristics, it could be difficult to detect in rat plasma with such low concentration. with t max at . h and t / α at . h, formononetin was absorbed and eliminated more slowly than other isoflavones such as calycosin. taken together, the results suggests the constituents of saposhnikoviae radix have demonstrated the best absorption and pharmacokinetic behavior among all the major active components in the three medicinal herbs, thus they may play significant role in leading to the main changes of overall therapeutic effects of ypf. however, as discussed above, astragali radix constitutes the most important part of ypf prescription, and we chose only seven main ingredients that have clear anti-inflammatory activity in ypf formula in this study. therefore, it has its own limitations. we believed that plasma drug concentration of certain points can be regarded as the combination of all quantitative components in vivo, but, considering the huge differences of drug distribution and rate of elimination, the combination will be more complicated than simple multiplication, since the parameter of area under the plasma concentration-time curve from zero to infinity (auc −∞ ) is very important for drug efficacy and pharmacology, it can reflects the drug exposure directly. meanwhile, to measure the intracorporal drug exposure contribution of each component, we choose the ratio of single to overall components auc −∞ as the weighting factor. ct is self-defined integrated plasma drug concentration of total components after weight factor correction. the integrated pharmacokinetic curve shows that the integrated pharmacokinetic characteristics are different from that of any of the single ingredients, which indicates that the single ingredient pharmacokinetic characteristics cannot fully reflect the drug metabolism of the overall medicine formula in vivo. further study is needed to confirm the association between them. we hope to provide a new valuable method for the drug metabolism research of traditional chinese medicine. in the present study, we develop a d-nplc-uplc-qtof-ms platform to separate and identify chemical compounds in ypf. a total of compounds were identified or tentatively characterized, including constituents (isoflavones, flavonoids, saponins and glycosides) from astragali radix; seven constituents (atractylodes lactone class, polysaccharide) from atractylodis rhizoma; and constituents (chromone and glycoside) from saposhnikoviae radix. then, a rapid, simple and reproducible uplc-qqq-ms method was developed for simultaneous quantification of seven major constituents, astragaloside, calycosin, formononetin, atractylenolide ii, cimicifugoside, -o-beta-d-glucosyl- -o-methylvisamminol, and sec-o-glucosylhamaudol, in rat plasma with relatively low concentrations. this method was successfully validated and applied to a preclinical pharmacokinetic study. in addition, we also used the area under the plasma concentration-time curve from zero to infinity (auc −∞ ) as the weighting factor to make an integrated pharmacokinetic curve, which may offer a broader perspective for discovering and elucidating the pharmacokinetic behavior and clinical application of the whole ypf formula. metabolomics and traditional chinese medicine a pre-classification strategy for identification of compounds in traditional chinese medicine analogous formulas by high-performance liquid chromatography-mass spectrometry traditional chinese medicine in the prevention and treatment of cancer and cancer metastasis recent advances in traditional chinese medicine for kidney disease potentiating therapeutic effects by enhancing synergism based on active constituents from traditional medicine advancing drug discovery and development from active constituents of yinchenhao tang, a famous traditional chinese medicine formula insights into drug discovery from natural medicines using reverse pharmacokinetics anti-inflammatory and immunoregulatory effects of yupingfeng powder on chronic bronchitis rats jiawei-yupingfeng-tang, a chinese herbal formula, inhibits respiratory viral infections in vitro and in vivo effect of yupingfeng granules on ha and foxp (+) treg expression in patients with nasopharyngeal carcinoma. asian pac chinese medicinal herb radix astragali suppresses cardiac contractile dysfunction and inflammation in a rat model of autoimmune myocarditis studies on purification process of total saponins in radix astragali with resin and structural identification of compounds astragalus): latest advancements and trends in chemistry, analysis, pharmacology and pharmacokinetics the effects of the water-extraction of astragali radix and lycopi herba on the pathway of tgf-smads-upp in a rat model of diabetic nephropathy pharmacokinetic evidence on the contribution of intestinal bacterial conversion to beneficial effects of astragaloside iv, a marker compound of astragali radix, in traditional oral use of the herb anti-inflammatory constituents from the roots of saposhnikovia divaricata pharmacological mechanism responsible for the atractylodes japonica-induced distal colonic contraction in rats screening for the anti-inflammatory activity of fractions and compounds from atractylodes macrocephala koidz houshiheisan compound prescription protects neurovascular units after cerebral ischemia characterization and quantification of prim-o-glucosylcimifugin in the roots of saposhnikovia divaricata and its medicinal preparations by liquid chromatography-ion trap mass spectrometry preparative separation of chromones in plant extract of saposhnikovia divaricata by reverse-phase medium-pressure liquid chromatography and high performance counter-current chromatography development of an spe-hplc-ms method for simultaneous determination and pharmacokinetic study of bioactive constituents of yu ping feng san in rat plasma after oral administration identification and quantification of atractylenolide i and atractylenolide iii in rhizoma atractylodes macrocephala by liquid chromatographyion trap mass spectrometry determination of atractylenolide ii in rat plasma by reversed-phase high-performance liquid chromatography simultaneous determination and pharmacokinetic study of atractylenolide i, ii and iii in rat plasma after intragastric administration of baizhufuling extract and atractylodis extract by uplc-ms/ms the antioxidant effects of radix astragali (astragalus membranaceus and related species) in protecting tissues from injury and disease biological active ingredients of traditional chinese herb astragalus membranaceus on treatment of diabetes: a systematic review antiviral activities of atractylon from atractylodis rhizoma atractylenolide i inhibits lipopolysaccharide-induced inflammatory responses via mitogen-activated protein kinase pathways in raw . cells comparative pharmacokinetics of prim-o-glucosylcimifugin and cimifugin by liquid chromatography-mass spectrometry after oral administration of saposhnikoviae radix extract, cimifugin monomer solution and prim-o-glucosylcimifugin monomer solution to rats simultaneous determination of calycosin- -o-beta-d-glucoside, ononin, calycosin, formononetin, astragaloside iv, and astragaloside ii in rat plasma after oral administration of radix astragali extraction for their pharmacokinetic studies by ultra-pressure liquid chromatography with tandem mass spectrometry quantitative analysis of atractylenolide i in rat plasma by lc-ms/ms method and its application to pharmacokinetic study simultaneous determination of atractylenolide ii and atractylenolide iii by liquid chromatography-tandem mass spectrometry in rat plasma and its application in a pharmacokinetic study after oral administration of atractylodes macrocephala rhizoma extract this article is an open access article distributed under the terms and conditions of the creative commons attribution the authors declare no conflict of interest. key: cord- - vynwk authors: galdiero, stefania; falanga, annarita; vitiello, mariateresa; cantisani, marco; marra, veronica; galdiero, massimiliano title: silver nanoparticles as potential antiviral agents date: - - journal: molecules doi: . /molecules sha: doc_id: cord_uid: vynwk virus infections pose significant global health challenges, especially in view of the fact that the emergence of resistant viral strains and the adverse side effects associated with prolonged use continue to slow down the application of effective antiviral therapies. this makes imperative the need for the development of safe and potent alternatives to conventional antiviral drugs. in the present scenario, nanoscale materials have emerged as novel antiviral agents for the possibilities offered by their unique chemical and physical properties. silver nanoparticles have mainly been studied for their antimicrobial potential against bacteria, but have also proven to be active against several types of viruses including human imunodeficiency virus, hepatitis b virus, herpes simplex virus, respiratory syncytial virus, and monkey pox virus. the use of metal nanoparticles provides an interesting opportunity for novel antiviral therapies. since metals may attack a broad range of targets in the virus there is a lower possibility to develop resistance as compared to conventional antivirals. the present review focuses on the development of methods for the production of silver nanoparticles and on their use as antiviral therapeutics against pathogenic viruses. viruses represent one of the leading causes of disease and death worldwide. thanks to vaccination programmes, some of the numerous diseases that used to kill many and permanently disable others have been eradicated, such as smallpox in [ ] or have greatly reduced the burden of the disease, such as in the case of the paralytic disease poliomyelitis [ ] . however, for some of today's most pressing viral pathogens, there is still no vaccine available. to realize the huge economic impact that several viral diseases cause to the global community, we need only to think to common colds, influenza, various problems due to herpesviruses (from shingles, genital herpes, chickenpox, infectious mononucleosis, up to herpes keratitis, neonatal disseminated infections, or viral encephalitis). other viruses are also able to cause considerable distress and sometimes persistent infections that may lead to cancer or to acquired immunodeficiencies, such as hepatitis viruses (mainly hbv and hcv) or human immunodeficiency virus (hiv). much effort has been expended in attempts to develop vaccines for these diseases, without appreciable success, at least for some of these viruses, namely, hcv, hiv and some herpesviruses. presently, the development of new vaccines for such viruses seems likely to continue to be elusive. together with the risk of emerging or re-emerging viral agents, the field of antiviral compound discovery is very promising. emerging and re-emerging viruses are to be considered a continuing threat to human health because of their amazing ability to adapt to their current host, to switch to a new host and to evolve strategies to escape antiviral measures [ ] . viruses can emerge because of changes in the host, the environment, or the vector, and new pathogenic viruses can arise in humans from existing human viruses or from animal viruses. several viral diseases that emerged in the last few decades have now become entrenched in human populations worldwide. the best known examples are: sars coronavirus, west nile virus, monkey pox virus, hantavirus, nipah virus, hendravirus, chikungunya virus, and last but not least, the threat of pandemic influenza viruses, most recently of avian or swine origin. unfortunately the methodological advances that led to their detection have not been matched by equal advances in the ability to prevent or control these diseases. there have been improvements in antiviral therapy, but with a wide margin of ineffectiveness, therefore new antiviral agents are urgently needed to continue the battle between invading viruses and host responses. technological advances have led to the discovery and characterization of molecules required for viral replication and to the development of antiviral agents to inhibit them. most viruses are, indeed, provided by an extraordinary genetic adaptability, which has enabled them to escape antiviral inhibition and in certain cases to regain advantage over the host by mutagenesis that create new viral strains with acquired resistance to most of the antiviral compounds available [ ] . the course of viral infections is governed by complex interactions between the virus and the host cellular system. all viruses depend upon a host cell for their protein synthesis. thus, all viruses replicate via a broadly similar sequence of events ( figure ). the virus must first bind to the cell, and then the virus or its genome enters in the cytoplasm. the genome is liberated from the protective capsid and, either in the nucleus or in the cytoplasm, it is transcribed and viral mrna directs protein synthesis, in a generally well regulated fashion. finally, the virus undergoes genome replication and together with viral structural proteins assembles new virions which are then released from the cell. each of the single described phases represents a possible target for inhibition. drugs that target viral attachment or entrance have proved to be very difficult to be discovered. in fact, to date, only one entry inhibitor has been approved by the us food and drug administration (fda). this is enfuvirtide (t- ), a synthetic peptide that targets the hiv gp envelope protein to prevent fusion. targeting the early steps of virus entry is a very attractive strategy for therapeutic intervention since the site of action of the inhibitor is likely to be extracellular and therefore relatively accessible; this could be paired by a concomitant action of the same drug on multiple targets to obtain a more effective therapeutic compound. moreover one could expect, in the future, antiviral agents with a broad-spectrum of action against viruses of different families, to be used as first aid compounds against unforeseen viral epidemics or pandemics. due to the outbreak of the emerging infectious diseases caused by different pathogenic viruses and the development of antiviral resistance to classical antiviral drugs, pharmaceutical companies and numerous researchers are seeking new antiviral agents. in the present scenario, nanoscale materials have emerged as novel "antimicrobial agents" due to their high surface area to volume ratio and their unique chemical and physical properties [ , ] . nanotechnology is an emerging field of applied science and cutting edge technology that utilizes the physico-chemical properties of nanomaterials as a means to control their size, surface area, and shape in order to generate different nanoscale-sized materials. among such materials, metal-based ones seem the most interesting and promising, and represent the subject of the present review. nanotechnology is directly linked with physics, chemistry, biology, material science and medicine. in fact, it finds application in multiple aspects of research and in everyday life such as electronics and new material design. however, its use in medical research is probably one of the fastest growing areas in which the functional mechanisms of nanoparticles and especially metal-based nanoparticles are just beginning to be exploited. nanotechnologies have been used to develop nanoparticle-based targeted drug carriers [ , ] , rapid pathogen detection [ , ] , and biomolecular sensing [ ] , as well as nanoparticle-based cancer therapies [ , ] . the use of nanoparticles can be extended to the development of antivirals that act by interfering with viral infection, particularly during attachment and entry. nanoparticles are properly defined as particles with at least one dimension less than nm, and have attracted much attention because of their unique and interesting properties. their singular physical (e.g., plasmonic resonance, fluorescent enhancement) and chemical (e.g., catalytic activity enhancement) properties derive from the high quantity of surface atoms and the high area/volume relation, in fact, as their diameter decreases, the available surface area of the particle itself increases dramatically and as a consequence there is an increase over the original properties of their bulk materials. considering that biological interactions are generally multivalent, the interplay between microbes and host cells often involves multiple copies of receptors and ligands that bind in a coordinated manner, resulting in enhanced specificities, efficiencies, and strengths of such interactions that allow the microbial agent to take possess of the cell under attack. the attachment and entry of viruses into host cells represent a terrific example of such multivalent interactions between viral surface components and cell membrane receptors [ ] . interfering with these recognition events, and thereby blocking viral entry into the cells, is one of the most promising strategies being pursued in the development of new antiviral drugs and preventive topical microbicides [ ] [ ] [ ] . in recent years, the use of metal nanoparticles, that may or not have been functionalized on their surface for optimising interactions, is seeing increasing success. the idea of exploiting metals against microorganisms can be considered ancient; in fact, the use of silver was a common expedient for cooking procedures and for preserving water from contamination. the importance of silver for its curative properties has been known for centuries, in fact, silver has been the most extensively studied metal for purpose of fighting infections and preventing food spoilage, and notwithstanding the decline of its use as a consequence of the development of antibiotics, prophylaxis against gonococcal ophthalmia neonatorum with silver ions was considered the standard of care in many countries until the end of the th century [ ] . silver's mode of action is presumed to be dependent on ag + ions, which strongly inhibit bacterial growth through suppression of respiratory enzymes and electron transport components and through interference with dna functions [ ] . therefore, the antibacterial, antifungal and antiviral properties of silver ions and silver compounds have been extensively studied. silver has also been found to be non-toxic to humans at very small concentrations. the microorganisms are unlikely to develop resistance against silver as compared to antibiotics as silver attacks a broad range of targets in the microbes. considering the broad literature that describes silver, as a bulk material, effective against a wide range of pathogens, silver nanoparticles have been analysed and found to be extremely appealing. the silver nanoparticles have also found diverse applications in the form of wound dressings, coatings for medical devices, silver nanoparticles impregnated textile fabrics [ ] . the advantage of using silver nanoparticles for impregnation is that there is continuous release of silver ions enhancing its antimicrobial efficacy. the burn wounds treated with silver nanoparticles show better cosmetic appearance and scarless healing [ ] . silver nanoparticles have received considerable attention as antimicrobial agents and have been shown to be effective mainly as antibacterial. antimicrobial effectiveness was shown for both gram-positive and gram-negative bacteria [ , ] . the antibacterial activity of silver nanoparticles was mainly demonstrated by in vitro experiments. activity against methicillin-resistant staphylococcus aureus (mrsa) [ ] , escherichia coli [ , , , ] , pseudomonas aeruginosa [ ] , vibrio cholera [ ] and bacillus subtilis [ ] has been documented. low concentrations of silver nanoparticles were able to consistently inhibit e. coli [ ] while the growth-inhibitory effect on s. aureus was minor. synergistic antimicrobial activity of silver or zinc nanoparticles with ampicillin, penicillin g, amoxicillin, kanamycin, erythromycin, clindamycin, chloramphenicol and vancomycin against s. aureus, e. coli, salmonella typhi and micrococcus luteus was observed [ ] [ ] [ ] . also gold nanoparticles have been exploited as antimicrobial agents, mainly as a tool to deliver other antimicrobials or in order to enhance the photodynamic killing of bacteria [ ] . many studies have shown the antimicrobial effects of metal nanoparticles, but the effects of silver nanoparticles against fungal pathogens are mostly unknown; silver nanoparticles, indeed, showed significant antifungal activity against penicillium citrinum [ ] , aspergillus niger [ ] , trichophyton mentagrophytes [ ] and candida albicans [ ] . different types of nanomaterials like copper, zinc, titanium [ ] , magnesium, gold [ ] , alginate [ ] and silver have come up in recent years and most of them have proven to be effective against diverse microorganisms. the present review aims at a description of the reported antiviral activities of metal nanoparticles and their production methods, with particular regard to silver nanoparticles. metal nanoparticles have been studied for their antimicrobial potential and have proven to be antibacterial agents against both gram-negative and gram-positive bacteria [ , , , , ] . theoretically, any metal could be analysed for antiviral activity, however, little effort has been done to determine the interactions of metal nanoparticles with viruses, and only recently some studies have emerged showing that metal nanoparticles can be effective antiviral agents against hiv- [ ] [ ] [ ] [ ] , hepatitis b virus [ ] , respiratory syncytial virus [ ] , herpes simplex virus type [ , ] , monkeypox virus [ ] , influenza virus [ ] and tacaribe virus [ ] . seen the paucity of viruses that have been investigated and the fact that most of the nanoparticles used were made of silver, this section will be instrumental to analyse the inhibitory effect for each single virus (table ) . acquired immunodeficiency syndrome (aids), the disease caused by hiv, is responsible for over two million deaths per year, among more than million people that are infected. highly active anti-retroviral therapy (haart), a treatment regimen that employs a cocktail of drugs to suppress hiv infection, has significantly improved the quality of life and life expectancy of millions of hiv-infected individuals. numerous hiv-infected individuals are currently treated with haart, and these individuals harbor chronic long-term infection; as a result, hiv eventually develops resistance to these drugs, resulting in a need to change medication regimens and a subsequent increase in the cost of treatment [ ] . the replication cycle of hiv- is a complex multistep process that depends on both viral and host cell factors. entry into target cells is achieved through fusion of the viral lipid envelope and the cellular plasma membrane [ ] . the viral component that acts as a fulcrum for mediating fusion is the trimeric envelope glycoprotein composed of two subunits: gp , which binds to the cellular receptor, and gp , which is the subunit bearing the transmembrane segment, and that executes fusion [ ] . following gp binding to the cellular receptor, cd , and a subsequent interaction with ccr or cxcr co-receptors, a conformational change of gp leads to membrane fusion and delivery of the capsid to the cytoplasm. soon after entry, the rna is reverse-transcribed into a complementary dna which is converted to a double-stranded dna, and integrated into the cellular genome. the integrated proviral dna is transcribed to generate full-length progeny viral rna and a number of spliced mrna transcripts. transcription and translation, performed by the cellular machinery, result in the synthesis of viral proteins that together with the progeny viral rna are transported to the site of virus particle assembly at the plasma membrane, where the virus gains access to the extracellular milieu upon budding events [ ] . elechiguerra et al. [ ] were the first to describe the antiviral activity of metal nanoparticles, in fact, they found that silver nanoparticles undergo size-dependent interactions with hiv- . in their investigations, they explored the possibility that physicochemical properties of nanoparticles may depend on the nanoparticle interactions with a capping agent molecule. for this reason they tested silver nanoparticles with three different surface chemistries: foamy carbon, poly(n-vinyl- -pyrrolidone) (pvp), and bovine serum albumin (bsa). foamy carbon-coated silver nanoparticles were embedded in a foamy carbon matrix needed to preclude coalescence during their synthesis. pvp-coated nanoparticles were synthesized using glycerine as both reducing agent and solvent. in this method, a metal precursor is dissolved in a liquid polyol in the presence of a capping agent such as pvp [ ] . synthesis in aqueous solution was performed for bsa-conjugate silver nanoparticles. interactions of silver nanoparticles with hiv- were probed with the aid of high angle annular dark field (haadf) scanning transmission electron microscopy technology. it was possible to obtain sufficient data to determine that the interaction between hiv particles and silver nanoparticles is clearly due to the size of the silver nanoparticle since only nanoparticles within the range of - nm were able to bind to the virus. in particular, nanoparticles were not randomly attached to the virus, but all the three species of nanoparticles established regular spatial relationships with the viral envelope. the most probable sites for interaction were found to be the sulfur-bearing residues of the gp glycoprotein knobs, which being limited in number, may also explain the inability of larger nanoparticles to bind the virus. the capacity of silver nanoparticles to inhibit infectivity of a laboratory-adapted hiv- strain at non-cytotoxic concentrations was determined by in vitro assays, and a dose-dependant inhibition of viral infectivity was reported. in particular, bsa-and pvp-coated nanoparticles showed to possess a slightly lower inhibition efficacy, probably because the surface of the nanoparticle is directly bound to and encapsulated by the capping agent. in contrast, the silver nanoparticles released from the carbon matrix have a greater inhibitory effect due to their essentially free surface area. these findings, however, only provide indirect evidence for their proposed mode of interaction through the binding to gp , therefore, a panel of different in vitro assays was used to elucidate the silver nanoparticles mode of antiviral action against hiv- [ ] . a luciferase-based assay showed that silver nanoparticles coated with pvp were an effective virucidal agent against cell-free virus (including laboratory strains, clinical isolates, t and m tropic strains, and resistant strains) and cell-associated virus. the concentration of silver nanoparticles at which infectivity was inhibited by % (ic ) ranged from . to . mg/ml. the observed antiviral effect of silver nanoparticles was due to the nanoparticles, rather than just to the silver ions present in the solution. in fact silver salts exerting antibacterial effect through silver ions, inhibited hiv- with a therapeutic index times lower than the one of silver nanoparticles. silver nanoparticles inhibit the initial stages of the hiv- infection cycle by blocking adsorption and infectivity in a cell-fusion assay. the inhibitory activity of silver nanoparticles against the gp -cd interaction was also investigated in a competitive gp -capture elisa, which together with the cell-based fusion assay, showed that silver nanoparticles inhibit hiv- infection by blocking viral entry, particularly the gp -cd interaction. besides, silver nanoparticles inhibit post-entry stages of the hiv- life cycle, in fact, the antiviral activity was maintained also when the metal nanoparticles were added h after the cell had been infected with hiv. since silver ions can form complexes with electron donor groups containing sulfur, oxygen, or nitrogen that are normally present as thiols or phosphates on amino acids and nucleic acids they might inhibit post-entry stages of infection by blocking hiv- proteins other than gp , or reducing reverse transcription or proviral transcription rates by directly binding to the rna or dna molecules. silver nanoparticles proved to be virucidal to cell-free and cell-associated hiv- as judged by viral infectivity assays. hiv infectivity was effectively eliminated following short exposure of isolated virus to silver nanoparticles. these properties make silver nanoparticles a potential broad-spectrum agent not prone to inducing resistance that could be used preventively against a wide variety of circulating hiv- strains. pvp-coated silver nanoparticles, being an interesting virucidal candidate drug, have been further investigated as a potential topical vaginal microbicide to prevent transmission of hiv- infection [ ] using an in vitro human cervical tissue-based organ culture that simulates in vivo conditions [ , ] . when formulated into a non-spermicidal gel (replens) at a concentration of . mg/ml, pvp-coated silver nanoparticles were not toxic to the explant, even when the cervical tissues were exposed continuously to the metal for hours, but one minute of pre-treatment of the cervical explant with . to . mg/ml of pvp-coated silver nanoparticles prevented the transmission of cell-associated hiv- and cell-free hiv- isolates. when pre-treatment was carried on for minutes followed by extensive washing the drug conferred almost total protection against hiv- transmission for hours, indicating a long-lasting protective effect by the pvp-coated silver nanoparticles in the cervical explants. a different group [ ] also reported about the antiviral activity of silver nanoparticles that had been fabricated using hepes buffer. they showed that silver nanoparticles exhibited potent cytoprotective and post-infected anti-hiv- activities (at mm a % reduction was achieved) toward hut/ccr cells in a dose-dependent fashion. similar inhibitory activities were reported for the silver nanoparticles when a citrate solution with nabh as the reducing agent was used, while lower activity was observed for gold nanoparticles ( nm, fabricated in hepes buffer). the herpesvirus family consists of more than double-stranded dna viruses divided into , β and  subgroups. only eight herpesviruses are known to commonly infect humans and the remainder are animal herpesviruses infecting a wide variety of animal species. all members of the herpesvirus family cause life-long latent infections and, structurally, all have a linear, double-stranded dna genome packaged into an icosahedral capsid and covered by a lipid envelope with embedded proteins and glycoproteins [ ] . symptomatic diseases caused by hsv- (prototypic -herpesvirus) are generally limited to cold sores of the mouth and keratitis in the eyes, but hsv- is capable of causing life-threatening diseases in immunocompromised individuals, including newborns, patients with hiv or patients undergoing immunosuppressive treatment. transmission among humans requires physical contact and after the initial infection, the virus remains latent in neurons, a key feature of -herpesviruses [ ] . hsv entry into host cells marks the first and possibly most critical step in viral pathogenesis [ ] . five viral glycoproteins have been implicated in the viral entry process: gb, gc, gd, gh and gl. all but gc are essential for entry. the initial interaction, or binding to cells, is mediated via interactions of gc and/or gb with heparan sulfate (hs) [ ] . the significant reduction of hsv- infection in the absence of either viral gc or cell-surface hs [ ] point to a key role of gc high-affinity binding to heparan sulfate (hs) on the cell surface. following binding, hsv entry is achieved through fusion of the lipid bilayer of the viral envelope with a host cell membrane. the core fusion machinery is composed by gb and gh/gl [ , ] , in fact, the complete fusion is only achieved when the three proteins act together. glycoprotein b may act as the premier fusogen [ ] [ ] [ ] , but it seems to need the cooperation of several membranotropic sequences harboured in gh [ ] [ ] [ ] [ ] [ ] . transcription, replication of viral dna and assembly of progeny capsids take place within the host nucleus, and then there is a complex mechanism for the exit of the newly assembled viruses from the cell [ ] . baram-pinto et al. have described in two consecutive works [ , ] the potential of exploiting metal nanoparticles for viral inhibition. their strategy was proved valid against hsv- , but was probably intended and may prove useful against other viruses, such as papillomaviruses (hpv) and hiv. in fact, their anti-hsv- agents are based on the principle that they mimic hs and may compete for the binding of the virus to the cell. also hiv or hpv use hs as a docking site during infection, therefore the nanoparticles described by baram-pinto et al. may be useful as a broad topical microbicide for sexually-transmitted viral infections. another point of particular interest from their work is the analysis of the significance of the carrier core material, in fact they designed two different metal particles, one made of silver, and the other made with gold, but both with the same coating of mercaptoethane sulfonate (mes) intended to mimic the polysulfonated hs and therefore expected to create a high local concentration of binding molecules for improved inhibitory effect. the silver-(ag-mes) and gold-(au-mes) mes nanoparticles were tested in antiviral assays using the wild-type hsv- mcintyre strain. for the inhibition experiments, vero cells and/or virus solutions were treated with ag-mes and au-mes nanoparticles at different time points to analyse the different stages of the viral infection that may be blocked. taken together, their results indicate that sulfonate-capped silver and gold nanoparticles inhibit hsv- infections by blocking the attachment and thereby the entrance of the virus into the cells and/or by preventing the cell-to-cell spread of the virus. the inability of soluble mes and unmodified metal nanoparticles to control viral infectivity stressed the importance of spatially oriented functional groups anchored on a nanoparticle core for viral inhibition. at the same time, antiviral activity shown by both ag-mes and au-mes nanoparticles suggest the possibility of using alternative carrier core materials as well. while these results suggest the versatility of the idea of effective viral inhibitions using functionalized nanoparticles, it also indicates that other core materials could also be efficient as long as they are not toxic to the host cells. respiratory syncytial virus (rsv) belongs to the family paramyxoviridae and infects the epithelium of the lungs and the respiratory tract causing serious respiratory disease, especially in children and older people. no vaccine or adequate pharmaceutical compounds are available, underlining the need for the development of future rsv treatments. the rsv genome consists of a single rna molecule of negative-sense rna, which encodes, among others, for two surface glycoproteins, which are exposed on the viral envelope. these glycoproteins are the (g) protein, which serve as a receptor binding protein, and the (f) protein, which is responsible for the fusion between the cell membrane and the viral envelope. as within the name itself of the virus, following infection of cells, the f protein is expressed on the surface of cells and fuse adjacent cells, giving rise to syncytia formation, a well characterised cytopathic effect [ ] . sun et al. [ ] have utilized silver nanoparticles conjugated to various proteins to study the inhibition of rsv infection in hep- cell culture. in their study, the capping agents used for the silver nanoparticles were: ( ) poly(n-vinyl- -pyrrolidone) (pvp); ( ) bovine serum albumin (bsa); and ( ) a recombinant f protein from rsv (rf ). the preliminary analysis by transmission electron microscopy yielded interesting results on the interaction between silver nanoparticles with rsv virion particles. bsa-conjugated silver nanoparticles seemed to interact with rsv but without a specific association or spatial arrangement, while rf -conjugated silver nanoparticles appeared to be floating freely with no proof of regular attachment. on the other hand, pvp-coated silver nanoparticles were able to bind to the viral surface with a regular spatial arrangement, suggesting a possible interaction with g proteins that are evenly distributed on the envelope of the rsv virion. the hypothesised interpretation for the interaction of pvp-nanoparticles with g proteins is that their small size and uniformity ( - nm), compared to the other (bsa and rf ) coated nanoparticles ( - nm) may contribute to the effectiveness of the binding. since toxicity is an imperative issue regarding pharmaceutical compounds, the cytotoxicity of each of the nanoparticle conjugates was established using the trypan blue exclusion assay, and revealed that all of them (bsa-, pvp-and rf -silver nanoparticles) showed less than % cytotoxicity up to a concentration of μg/ml. silver nanoparticles have to be regarded as potentially harmful especially when intended for treating a respiratory disease such as rsv infections. sun et al. [ ] have, therefore considered that a saturated surface capping composed of a natural biomolecule (bsa) and a biocompatible chemical (pvp) could be able to mask the pure nano-silver surface and thus would reduce toxicity without hampering efficacy. nevertheless, further studies are needed to validate these in vitro data for the use into the clinical setting, and investigation of the toxic effects and fate of nanoparticles after their deposition in the respiratory tract is mandatory for the future development of anti-rsv silver nanoparticles-based therapeutic compounds. hep- cells were infected with rsv mixed with bsa-, pvp-and rf -coated silver nanoparticles and infectivity inhibition was evaluated by microscopic examination for syncytia formation and by immunofluorescence microscopy. neither bsa-nor rf -coated nanoparticles showed any significant inhibition of rsv infection, while pvp-coated silver nanoparticles inhibited rsv infection by %. these results led the authors to conclude that when the silver nanoparticles are conjugated to the pvp protein and mixed with rsv, they bind to the g protein on the viral surface and interfere with viral attachment to the hep- cells resulting in the inhibition of viral infection. hepatitis b virus (hbv) is a partially double-stranded dna virus provided with a lipidic envelope coat. hbv has a strong tropism for hepatocytes, and once it has entered the cell, viral particles migrate to the nucleus where the viral genome is completed to form a covalently closed circular dna (cccdna) that serves as the template for the subsequent steps of viral mrna transcription and formation of the pre-genomic rna (pgrna). the pgrna forms the template for the reserve transcription by the viral-encoded reverse-transcriptase that produce new viral genomes [ ] . nucleotide (adefovir) and nucleoside (lamivudine, entecavir, and telbivudine) analogue inhibitors, which represent approved pharmaceuticals with direct antiviral activity against hbv, target primarily the viral polymerase reverse-transcriptase. although their effectiveness has been proven, raising drug-resistant hbv strains is fast, therefore limiting the use of these antivirals. lu et al. [ ] have analysed monodisperse silver nanoparticles for their ability to inhibit hbv replication. the nanoparticles used in their study were prepared from agno in hepes and measured dimensions of ~ nm (ag ns), ~ nm (ag ns) and ~ nm (ag ns). silver nanoparticles with particle diameters of nm were too toxic for being evaluated as antiviral compound, but nm and nm particles showed only a minor toxicity at the concentration able to inhibit hbv replication. in fact, nanoparticles of both sizes showed potent anti-hbv activities. the ag ns reached % inhibition at µm and % at µm, while the ag ns were slightly more active with % and % at concentration of respectively µm and µm. in the same paper by lu et al. [ ] , the activity of silver nanoparticles was also compared to nm gold nanoparticles (au ns) and other silver compounds with silver in different oxidation states, and the overall results showed that the anti-hbv effects of silver nanoparticles are undoubtedly much more pronounced. in conclusion, silver nanoparticles were able to inhibit the production of hbv rna and extracellular virions probably via a specific interaction between the nanoparticles and the double-stranded dna of hbv and/or direct binding with viral particles. the influenza virus is a highly contagious pathogen that causes annual epidemics in the human population, and is much feared for its potential to generate new viruses able to jump to humans from different animal species and causing pandemics. recently, papp et al. [ ] have described their studies in which functionalized gold nanoparticles were used to inhibit the influenza virus. this is an orthomyxovirus containing a helical capsid with a genome of eight rna segments. the capsid is covered by a lipid envelope containing mainly two virally-encoded glycoproteins, namely hemoagglutinin (ha) and neuraminidase (na), that forms spiky projections on the surface. the virus binds to the cell plasma membrane through an interaction between ha and sialic acid (sa) residues present on glycoproteins and lipids on the surface of the host cell. this is soon followed by a mechanism of receptor-mediated endocytosis that brings the enveloped virus particle inside the cytoplasm but surrounded by a second lipid bilayer besides the envelope, the endosomal one. inside the late endosome, environment acidification triggers a conformational change of ha, which sets in motion a mechanism of protein (ha) mediated fusion of the endosomal membrane with the viral envelope ending with the release of the nucleoproteins and genome fragments into the cytoplasm [ ] . papp et al. [ ] strategy was to functionalize gold nanoparticles with sialic acid (sa)-terminated glycerol dendron with the objective to inhibit influenza virus binding to the plasma membrane. gold nanoparticles of different size were produced: one of nm and a second of nm. they found that nm had no inhibitory effect on the hemagglutination, used to test the ability of the influenza virus to bind to a target membrane. on the contrary, the nm gold nanoparticles inhibited hemagglutination at concentrations in the nanomolar range, demonstrating that the activity clearly depends on the particle dimension and the spatial distribution of the interacting ligand/receptor molecules. the same trend, with a more pronounced activity was observed in influenza virus inhibition assays where the sialylated particles of nm size were found to be effective for influenza virus inhibition, whereas the nm analogues did not show significant impact. therefore, they proved that sialic-acid-functionalized gold nanoparticles are able to effectively inhibit viral infection. monkeypox virus (mpv), an orthopoxvirus similar to variola virus, is the causative agent of monkeypox in many species of non-human primates, but it is also a human pathogen with a clinical presentation similar to that of smallpox. mpv is considered a big threat to human life and therefore research is being carried out to develop drugs and therapeutic agents against this virus [ ] . different size nanoparticles were produced by plasma gas synthesis and used by rogers et al. [ ] in a plaque reduction assay of mpv. the silver nanoparticles used in this work were (ag-np- ), (ag-np- ) and (ag-np- ) nm, and some nanoparticles were also coated with polysaccharide, (ag-ps- ), (ag-ps- ) and (ag-ps- ) nm nanoparticles. these nanoparticles, at concentrations ranging from . to g/ml, were evaluated for mpv inhibitory efficacy using a plaque reduction assay. the main results showed that the ag-ps- (polysaccharide-coated, nm) and ag-np- (non-coated, nm) exerted a significant dose-dependent inhibition of mpv plaque formation, but the mechanism by which this inhibition occurs has not been further investigated. poxviruses enter cells by endocytosis or direct fusion at the plasma membrane, and at least or envelope proteins are involved in the entry mechanism, this is followed by a regulated sequence of events that allow virus replication. many steps in the virus life cycle are still unknown, and this report on the activity of silver nanoparticles is too preliminary to attempt to give a satisfactory explanation of their mechanism of action. probably the silver nanoparticles may intervene in the early steps of binding and penetration by blocking virus-host cell binding by physical obstruction or, if internalised, they can disrupt intracellular pathways important for virus replication. rogers et al. [ ] also described that agno was active as a mpv inhibitor, but at the concentration of µg/ml its toxic effect on vero cells impeded the evaluation of the antiviral activity. interestingly, some of the nanoparticles analysed in the study promoted an increase in the mean number of mpv plaques/well at the highest concentrations used. a potential explanation for these contrasting results could lie in the fact that nanoparticles may tend to aggregate and consequently create on cells areas available for increased contacts between viral particles and the cell membrane, therefore augmenting internalization and plaque formation. however, these data are preliminary and need a more in-depth analysis to draw more significant conclusions. the family arenaviridae is composed of different species of viruses divided into two antigenic groups, the old world and new world (tacaribe complex) groups. the tacaribe complex, in addition to the tacaribe virus (tcrv), includes the viral hemorrhagic fever-inducing viruses junin, machupo, guanarito, and sabia. considering the high transmissibility by person-to-person via the respiratory route, the lack of diagnostic testing, and therapeutic options limited to ribavirin (not a satisfactory efficacy and easily emergence or resistant strains), the arenaviruses are included in the category a list of potential bio-weapons [ ] . tcrv is not a human pathogen, but exhibits a close antigenic relationship with junin and guanarito viruses [ ] , therefore could serve well as a model virus for arenavirus derived diseases without human pathogenic potential and adequate safety for laboratory manipulation. speshock et al. [ ] have recently analysed the activity of two types of silver nanoparticles against tcrv: uncoated (ag-np) and polysaccharide coated silver nanoparticles (ps-ag). they found that when tcrv was treated with μg/ml, μg/ml and even μg/ml of the nm ag-nps significant reduction in the progeny virus titer or no detectable progeny virus was produced. ps-ag particles showed a similar trend but were not as effective, but toxicity was reduced. therefore the polysaccharide coating may indeed protect the cell from the toxic effects of the ag-nps, but it also appears to interfere with the ag-np interaction with tcrv. silver nanoparticles seem to interact with tcrv prior to cellular exposure resulting in a decrease in viral infectivity with and nm ag-nps, therefore, the authors suggested that the silver nanoparticles may bind to virally-encoded membrane glycoproteins. in fact, tcrv glycoproteins are rich in cysteine residues [ ] and silver nanoparticles have been shown to bind easily to the thiol groups [ ] , which are found in cysteine residues. this interaction can either prevent the internalization of the viral particle by interfering with cellular receptor binding, or may favour the internalization of the silver nanoparticle together with the virus and produce an inhibitory effect on viral replication interfering with the tcrv rna-dependent rna polymerase (l protein). other possible mechanism of action could be related to the fact that the silver nanoparticles bound to the viral glycoproteins may prevent the virus uncoating in the endosome. finally, speshock et al. [ ] proved that pre-treatment of the cells with silver nanoparticles had no effect on viral replication, therefore they concluded that the ag-nps could be inactivating the virus prior to entry into the cell. the removal of viruses from water (and the environment in general) is of paramount important for health safety maintenance of our modern society that profoundly relies on water safety for drinking and leisure activities. pathogenic viruses such as adenovirus, norovirus, rotavirus, and hepatitis a commonly occur in both surface and groundwater sources [ ] [ ] [ ] and must be effectively inactivated to provide safe water. titanium dioxide has attracted much attention as a photocatalyst for water treatment, being resistant to corrosion and non-toxic when ingested. the antibacterial properties of tio have been well documented [ ] [ ] [ ] [ ] and are attributed to the generation of ros, especially hydroxyl free radicals and hydrogen peroxide [ , ] . while few studies have investigated the antiviral properties of tio , its potential for inactivating viruses has been demonstrated [ , , ] . however, the inactivation rates obtained in most of these studies were extremely low. for example, cho et al. [ ] demonstrated only minor removal of bacteriophage ms after h of irradiation using p tio suspended at g/l. the inactivation kinetics needs to be greatly improved in order to provide efficient drinking water disinfection. liga et al. [ ] have hypothesized a possible synergic mechanism occurring between silver and tio when silver doped titanium dioxide is used for inactivating microorganisms under uv radiation, therefore they demonstrated that silver doping tio greatly enhanced the photocatalytic inactivation of viruses primarily by increasing hydroxyl free radicals production in addition to slightly increasing virus adsorption. silver doping significantly enhanced ms inactivation by p tio and the inactivation rate increased with silver content. with silver doped tio nanoparticles a considerable removal of ms could be obtained in seconds, rendering feasible the goal of achieving virus removal from drinking water using photoreactors exploiting metal nanoparticles. although the continuous evolutions in the field of metal-based nanoparticles for drug delivery, medical imaging, diagnostics, therapeutics and engineering technology, there is a serious lack of information about the impact of metal nanoparticles on human health and environment, probably due to the intrinsic complex nature of nanoparticles that have led to different attitudes on their safety. therefore, an important issue in the use of metal nanoparticles is their potential toxicity. for metal nanoparticles to be effective as antiviral pharmaceuticals, it is imperative to gain a better understanding of their biodistribution/accumulation in living systems. the principal characteristic of metal nanoparticles is their size, which falls in between individual atoms or molecules and the corresponding bulk materials. particle size and surface area can modify the physicochemical properties of the metal material, but can also influence the reactivity of nanoparticles with themselves or with the cellular environment, leading to different modes of cellular uptake and further processing, leading to adverse biological effects in living cells that would not otherwise be possible with the same material in larger forms. in fact, as particle size decreases, some metal nanoparticles show increased toxicity, even if the same material is relatively inert in its bulk form (e.g., ag, au, and cu). apart from size, the biological consequences of metal nanoparticles also depend on chemical composition, surface structure, solubility, shape, and aggregation. all of these parameters can modify cellular uptake, protein binding, translocation to the target site, and most of the biological interactions with the possibility of causing tissue injury. therefore, in terms of safety, the effect of silver nanoparticles is a major consideration: even if they inhibit viral infections, it would not be beneficial if there are adverse effects to humans or animals. a commonly used strategy to reduce a possible toxicity is to use various capping agents to prevent the direct contact of the metal with the cells. potential routes of human exposure to metal nanoparticles used as therapeutic compounds include the gastrointestinal tract, the skin, the lungs, and systemic administration. considering the use of metal nanoparticles from the point of view of a potential antiviral therapy, it is straightforward that the safest and easiest results can be obtained with topical use of nanoparticles as microbicide for direct viral particles inactivation and/or inhibition of the early steps of the viral life cycle, attachment and entry. therefore, the dermal route seems the one of major concern. dermal exposure to metal nanoparticles often takes place when using sunscreen lotions, for example, tio and zno nanoparticles. in healthy skin, the epidermis provides excellent protection against particle spread to the dermis. however, in presence of damaged skin micrometer-size particles gain access to the dermis and regional lymph nodes. a further concern should be the potential of nanoparticles translocation to the brain via the olfactory nerve as a consequence of the vicinity of the nasal mucosa to the olfactory bulb. whether nanoparticles in such tissues have any pathological or clinical significance is uncertain, therefore, more data is needed to properly address the safety concern on the use of metal nanoparticles as pharmaceuticals. several studies have demonstrated the cytotoxic effects of metal nanoparticles [ ] [ ] [ ] [ ] , in fact, silver nanoparticles were found to be highly cytotoxic to mammalian cells based on the assessment of mitochondrial function, membrane leakage of lactate dehydrogenase, and abnormal cell morphologies [ ] [ ] [ ] [ ] . at a cellular level, metal nanoparticles interact with biological molecules within mammalian cells and can interfere with the antioxidant defence mechanism leading to the generation of reactive oxygen species (ros). such species, in excess, can cause damage to biological components through oxidation of lipids, proteins, and dna. oxidative stress may have a role in the induction or the enhancement of inflammation through upregulation of redox sensitive transcription factors (e.g., nf-κb), activator protein- , and kinases involved in inflammation [ ] [ ] [ ] [ ] . the generation of reactive oxygen species by cells exposed to silver nanoparticles [ ] has been showed in human lung fibroblast and human glioblastoma cells, and as a consequence dna damage and cell cycle abnormalities have been observed. accumulation of silver nanoparticles in various organs (lungs, kidneys, brain, liver, and testes) has been evidenced in animal studies [ ] . most of the in vitro studies show dose dependence, in fact, higher doses of silver induce a strongher cellular toxicity. nevertheless, should be considered that in vitro concentrations of nanoparticles are often much higher than the ones used in in vivo experiments, therefore such exposures do not represent a replica of the conditions expected for in vivo exposure. a recent study [ ] showed that mice exposed to silver nanoparticles showed minimal pulmonary inflammation or cytotoxicity following subacute exposures, but longer term exposures with higher lung burdens of nanosilver are not investigated, therefore eventual cronic effects may be underscored. this review presents only a brief description of the toxicity derived from the use of metal nanoparticles. a more detailed coverage of the topic is available in recently published reviews [ ] [ ] [ ] [ ] . although significant progress has been made to elucidate the mechanism of silver nanomaterial toxicity, a proved consensus on the immediate impact or long term effect on human health is still missing. further research is required to provide the necessary warranties to allow a safely exploitation of the interesting in vitro antiviral properties of silver nanoparticles and their transfer to the clinical setting. nanoparticles are nanoscale clusters of metallic atoms, engineered for some practical purpose, most typically antimicrobial and sterile applications. different wet chemical methods have been used for the synthesis of metallic nanoparticles dispersions. the early methods to produce nanoparticles of noble-metals are still used today and continue to be the standard by which other synthesis methods are compared. the most common methods involve the use of an excess of reducing agents such as sodium citrate [ ] or nabh [ ] . ayyanppan et al. [ ] obtained ag, au, pd and cu nanoparticles by reducing metallic salts in dry ethanol. longenberger et al. [ ] produced au, ag and pd metal colloids from air-saturated aqueous solutions of poly(ethylene glycol) (peg). reduction methods can also be used for the production of pt, pd, cu, mi etc., although specific protocols depend on the reduction potential of the source ion [ ] . cu and ni are not very stable as the metal particles are easily oxidized requiring strong capping ligands to prevent the oxidation. initially, the reduction of various complexes with metallic ions leads to the formation of atoms, which is followed by agglomeration into oligomeric clusters. controlled synthesis is usually based on a two-step reduction process: in the first step a strong reducing agent is used to produce small particles; in the second step these small particles are enlarged by further reduction with a weaker reducing agent [ ] . strong reductants lead to small monodisperse particles, while the generation of larger size particles can be difficult to control. weaker reductants produce slower reduction reactions, but the nanoparticles obtained tend to be more polydisperse in size. different studies reported the enlargement of particles in the secondary step from about - nm to - nm [ ] . another general method for the production of different metal nanoparticles (au, ag, pt, pd) uses commonly available sugars, e.g., glucose, fructose and sucrose as reducing agents [ ] . this approach has several important features: ( ) sugars (glucose, fructose, and sucrose) are easily available and are used as reducing agents; ( ) upon their exploitation no other stabilizing agent or capping agent is required to stabilize the nanoparticles; ( ) sugars are very cheap and biofriendly ( ) the nanoparticles can be safely preserved in a essiccator for months and redispersed in aqueous solution whenever required instead of being kept in aqueous solution. an array of other physical and chemical methods have been used to produce nanomaterials. in order to synthesize noble metal nanoparticles of particular shape and size specific methodologies have been formulated, such as ultraviolet irradiation, aerosol technologies, lithography, laser ablation, ultrasonic fields, and photochemical reduction techniques, although they remain expensive and involve the use of hazardous chemicals. therefore, there is a growing concern to develop environment-friendly and sustainable methods. biosynthesis of gold, silver, gold-silver alloy, selenium, tellurium, platinum, palladium, silica, titania, zirconia, quantum dots, magnetite and uraninite nanoparticles by bacteria, actinomycetes, fungi, yeasts and viruses have been reported. however, despite the stability, biological nanoparticles are not monodispersed and the rate of synthesis is slow. to overcome these problems, several factors such as microbial cultivation methods and extraction techniques have to be optimized and factors such as shape, size and nature can be controlled through just modifying ph, temperature and nutrient media composition. owing to the rich biodiversity of microbes, their potential as biological materials for nanoparticle synthesis is yet to be fully explored. the production of metal nanoparticles involves three main steps, including ( ) selection of solvent medium; ( ) selection of environmentally benign reducing agent; ( ) selection of nontoxic substances for the nanoparticles stability [ ] . biomineralization is also an attractive technique, being the best nature friendly method of nanoparticle synthesis. in one of the biomimetic approaches towards generation of nanocrystals of silver, reduction of silver ions has been carried out using bacteria and unicellular organisms. the reduction is mediated by means of an enzyme and the presence of the enzyme in the organism has been found to be responsible of the synthesis [ , ] . therefore in search of a methodology that could provide safer and easier synthesis of metal nanoparticles, it seems that the biogenic synthesis using the filtrated supernatant of different bacterial and fungal cultures is having a considerable impact, where the reduction of metal ions occurs through the release of reductase enzymes into the solution [ , [ ] [ ] [ ] . for an extensive coverage of the biological synthesis of metal nanoparticles by microbes, refer to the recent review by narayanan and sakthivel [ ] . in the crusade toward the development of drugs for the therapy of viral diseases, the emergence of resistant viral strains and adverse side effects associated with a prolonged use represent huge obstacles that are difficult to circumvent. therefore, multidisciplinary research efforts, integrated with classical epidemiology and clinical approaches, are crucial for the development of improved antivirals through alternative strategies. nanotechnology has emerged giving the opportunity to re-explore biological properties of known antimicrobial compounds, such as metals, by the manipulation of their sizes. metal nanoparticles, especially the ones produced with silver or gold, have proven to exhibit virucidal activity against a broad-spectrum of viruses, and surely to reduce viral infectivity of cultured cells. in most cases, a direct interaction between the nanoparticle and the virus surface proteins could be demonstrated or hypothesized. the intriguing problem to be solved is to understand the exact site of interaction and how to modify the nanoparticle surface characteristics for a broader and more effective use. besides the direct interaction with viral surface glycoproteins, metal nanoparticles may gain access into the cell and exert their antiviral activity through interactions with the viral genome (dna or rna). furthermore, the intracellular compartment of an infected cell is overcrowded by virally encoded and host cellular factors that are needed to allow viral replication and a proper production of progeny virions. the interaction of metal nanoparticles with these factors, which are the key to an efficient viral replication, may also represent a further mechanism of action ( figure ). most of the published literature describes the antiviral activity of silver or gold nanoparticles against enveloped viruses, with both a dna or an rna genome. considering that one of the main arguments toward the efficacy of the analysed nanoparticles is the fact that they in virtue of their shape and size, can interact with virus particles with a well-defined spatial arrangement, the possibility of metal nanoparticles being active against naked viruses seems appealing. moreover, it has been already proven that both silver and gold nanoparticles may be used as a core material. however, no reports are yet available for the use of other metals, but the future holds many surprises, especially considering that the capping molecules that could be investigated are virtually unlimited. nonetheless, for metal nanoparticles to be used in therapeutic or prophylactic treatment regimens, it is critical to understand the in vivo toxicity and potential for long-term sequelae associated with the exposure to these compounds. additional research is needed to determine how to safely design, use, and dispose products containing metal nanomaterials without creating new risk to humans or the environment. the authors declare no conflict of interest. principles and lessons from the smallpox eradication programme paralytic poliomyelitis: seasoned strategies, disappearing disease mechanisms of viral emergence the bactericidal effect of silver nanoparticles antimicrobial effects of silver nanoparticles a peptide derived from herpes simplex virus type glycoprotein h: membrane translocation and applications to the delivery of quantum dots delivery of nanoparticulate drug delivery systems via the intravenous route for cancer gene therapy dual enlargement of gold nanoparticles: from mechanism to scanometric detection of pathogenic bacteria high-throughput detection and sizing of individual low-index nanoparticles and viruses for pathogen identification array-based sensing with nanoparticles: chemical noses' for sensing biomolecules and cell surfaces a new era for cancer treatment: gold-nanoparticle-mediated thermal therapies nano-oncology: drug delivery, imaging, and sensing virology" fifth edition: virus entry and uncoating virus entry: molecular mechanisms and biomedical applications inhibition of viral-induced membrane fusion by peptides inhibitors of viral entry clinical significance of credé's prophylaxis in germany at present antimicrobial effect of surgical masks coated with nanoparticles application of anisotropic silver nanoparticles: multifunctionalization of wool fabric nanosilver as a new generation of nanoproduct in biomedical applications silver nanoparticles as antimicrobial agent: a case study on e. coli as a model for gram-negative bacteria the synthesis of chitosan-based silver nanoparticles and their antibacterial activity silver colloid nanoparticles: synthesis, characterization, and their antibacterial activity does the antibacterial activity of silver nanoparticles depend on the shape of the nanoparticle? a study of the gram-negative bacterium escherichia coli susceptibility constants of escherichia coli and bacillus subtilis to silver and copper nanoparticles synthesis and effect of silver nanoparticles on the antibacterial activity of different antibiotics against staphylococcus aureus and escherichia coli zno nanoparticles enhanced antibacterial activity of ciprofloxacin against staphylococcus aureus and escherichia coli biogenic synthesis of silver nanoparticles and their synergistic effect with antibiotics: a study against gram-positive and gram-negative bacteria destruction and control of toxoplasma gondii tachyzoites using gold nanosphere/antibody conjugates facile preparation and characterization of highly antimicrobial colloid ag or au nanoparticles antifungal effect of silver nanoparticles on dermatophytes antifungal activity and mode of action of silver nano-particles on candida albicans biosynthesis and characterization of ti/ni bimetallic nanoparticles small molecule-capped gold nanoparticles as potent antibacterial agents that target gram-negative bacteria inhalable alginate nanoparticles as antitubercular drug carriers against experimental tuberculosis silver nanoparticles as a new generation of antimicrobials interaction of silver nanoparticles with hiv- silver nanoparticles fabricated in hepes buffer exhibit cytoprotective activities toward hiv- infected cells mode of antiviral action of silver nanoparticles against hiv- pvp-coated silver nanoparticles block the transmission of cell-free and cell-associated hiv- in human cervical culture silver nanoparticles inhibit hepatitis b virus replication silver nanoparticles inhibit replication of respiratory sincitial virus inhibition of herpes simplex virus type infection by silver nanoparticles capped with mercaptoethane sulfonate inhibition of hsv- attachment, entry, and cell-to-cell spread by functionalized multivalent gold nanoparticles a preliminary assessment of silver nanoparticles inhibition of monkeypox virus plaque formation inhibition of influenza virus infection by multivalent sialic-acid-functionalized gold nanoparticles interaction of silver nanoparticles with tacaribe virus genotypic resistance profiles in antiretroviral-naive hiv- infections before and after initiation of first-line haart: impact of polymorphism on resistance to therapy hiv- membrane fusion: targets of opportunity aids virus envelope spike structure the retroviruses and their replication electrochemical reduction of noble metal compounds in ethylene glycol development of an in vitro organ culture model to study transmission of hiv- in the female genital tract blocking of cell-free and cell-associated hiv- transmission through human cervix organ culture with uc the family herpesviridae: a brief introduction fields herpes simplex viruses fusing structure and function: a structural view of the herpesvirus entry machinery herpes simplex virus: receptors and ligands for cell entry herpesviruses and heparin sulfate: an intimate relationship in aid of viral entry glycoproteins gb, gd, and ghgl of herpes simplex virus type are necessary and sufficient to mediate membrane fusion in a cos cell transfection system entry of herpesviruses into mammalian cells crystal structure of glycoprotein b from herpes simplex virus mutational evidence of internal fusion loops in herpes simplex virus glycoprotein b structural basis of local, ph-dependent conformational changes in glycoprotein b from herpes simplex virus type fusogenic domains in herpes simplex virus type glycoprotein h analysis of synthetic peptides from heptad-repeat domains of herpes simplex virus type glycoproteins h and b evidence for a role of the membrane-proximal region of herpes simplex virus type glycoprotein h in membrane fusion and virus inhibition analysis of a membrane interacting region of herpes simplex virus type glycoprotein h the presence of a single n-terminal histidine residue enhances the fusogenic properties of a membranotropic peptide derived from herpes simplex virus type glycoprotein h herpesviruses remodel host membranes for virus egress respiratory syncytial virus and metapneumovirus virology the viruses and their replication. in virology human monkeypox: an emerging zoonotic disease the viruses and their replication properties and characterization of monoclonal antibodies to tacaribe virus the ' end termini of the tacaribe arenavirus subgenomic rnas antimicrobial effect of silver-impregnated cellulose: potential for antimicrobial therapy occurrence of viruses in us groundwaters detection and quantification of human bocavirus in river water evaluation of public health risks at recreational beaches in lake michigan via detection of enteric viruses and a human-specific bacteriological marker bactericidal activity of tio photocatalyst in aqueous media: toward a solar-assisted water disinfection system photocatalitic bactericidal effect of tio thin films: dynamic view of the active oxygen species responsible for the effect different inactivation behaviors of ms- phage and escherichia coli in tio photocatalytic disinfection photocatalytic inactivation of escherischia coli. effect of concentration of tio and microorganism, nature, and intensity of uv irradiation environmental applications of semiconductor photocatalysis inactivation of microorganisms in a flow-through photoreactor with an immobilized tio layer kinetic evaluation of biocidal activity of titanium dioxide against phage ms considering interaction between the phage and photocatalyst particles virus inactivation by silver doped titanium dioxide nanoparticles for drinking water treatment in vitro cytotoxicity of nanoparticles in mammalian germline stem cells cytotoxicity and genotoxicity of silver nanoparticles in human cells in vitro toxicity of silver nanoparticles at noncytotoxic doses to hepg human hepatoma cells in vitro toxicity of nanoparticles in brl a rat liver cells regulation of nuclear factor-κb, activator protein- , and glutathione levels by tumor necrosis factor-α and dexamethasone in alveolar epithelial cells glutathione, stress responses, and redox signaling in lung inflammation biomedical applications and potential health risks of nanomaterials: molecular mechanisms effects of nanomaterial physicochemical properties on in vivo toxicity twenty-eight-day oral toxicity, genotoxicity, and genderrelated tissue distribution of silver nanoparticles in sprague-dawley rats nanosilver induces minimal lung toxicity or inflammation in a subacute murine inhalation model silver or silver nanoparticles: a hazardous threat to the environment and human health? nanosilver: a nanoproduct in medical application a review of the antibacterial effects of silver nanomaterials and potential implications for human health and the environment metal-based nanoparticles and their toxicity assessment adsorption and surface-enhanced raman of dyes on silver and gold sols plasma resonance enhancement of raman scattering by pyridine adsorbed on silver or gold sol particles of size comparable to the excitation wavelength nanoparticles of ag, au, pd, and cu produced by alcohol reduction of the salts formation of metal particles in aqueous solutions by reactions of metal complexes with polymers synthesis, characterization, and stability of dendrimer-encapsulated palladium nanoparticles reproducible preparation of silver sols with small particle size using borohydride reduction: for use as nuclei for preparation of larger particles general method of synthesis for metal nanoparticles completely "green" synthesis and stabilization of metal nanoparticles biosynthesis of silver nanocrystals by bacillus licheniformis biosynthesis of silver and gold nanoparticles using brevibacterium casei fungus-mediated synthesis of silver nanoparticles and their activity against pathogenic fungi in combination with fluconazole biosynthesis of silver nanoparticles from staphylococcus aureus and its antimicrobial activity against mrsa and mrse extracellular synthesis of silver bionanoparticles from aspergillus clavatus and its antimicrobial activity against mrsa and mrse biological synthesis of metal nanoparticles by microbes key: cord- - rzibpbl authors: eng, yi shin; lee, chien hsing; lee, wei chang; huang, ching chun; chang, jung san title: unraveling the molecular mechanism of traditional chinese medicine: formulas against acute airway viral infections as examples date: - - journal: molecules doi: . /molecules sha: doc_id: cord_uid: rzibpbl herbal medicine, including traditional chinese medicine (tcm), is widely used worldwide. herbs and tcm formulas contain numerous active molecules. basically, they are a kind of cocktail therapy. herb-drug, herb-food, herb-herb, herb-microbiome, and herb-disease interactions are complex. there is potential for both benefit and harm, so only after understanding more of their mechanisms and clinical effects can herbal medicine and tcm be helpful to users. many pharmacologic studies have been performed to unravel the molecular mechanisms; however, basic and clinical studies of good validity are still not enough to translate experimental results into clinical understanding and to provide tough evidence for better use of herbal medicines. there are still issues regarding the conflicting pharmacologic effects, pharmacokinetics, drug interactions, adverse and clinical effects of herbal medicine and tcm. understanding study validation, pharmacologic effects, drug interactions, indications and clinical effects, adverse effects and limitations, can all help clinicians in providing adequate suggestions to patients. at present, it would be better to use herbs and tcm formulas according to their traditional indications matching the disease pathophysiology and their molecular mechanisms. to unravel the molecular mechanisms and understand the benefits and harms of herbal medicine and tcm, there is still much work to be done. it is common to initiate the therapy of orthodox western medicine by fitting the pharmacologic characteristics of a drug, including pharmacokinetic and pharmacodynamic effects, to the disease pathophysiology. physicians further validate the clinical application according to evidence-based medicine (ebm); however, this is not the case for tcm. tcm developed in ancient china, and at that time, physicians managed diseases with herbs only by clinical experience, without any knowledge of disease pathophysiology, nor the pharmacologic activities of herbs, to say nothing of the molecular mechanisms of herbs. to unravel the molecular mechanism of tcm formulas, it would be better to understand how physicians prescribe them first. tcm includes herbal therapy, acupuncture, massage, and dietary therapy. in the current work, tcm will be simply defined as herbal and dietary therapies. tcm is widely popular in east asia and forms the kampo medicine in japan, as well as traditional korean medicine; importantly, traditional medicines form the mainstream of healthcare in these countries. in ancient china, several famous tcm textbooks summarized the clinical experiences of using tcm formulas against various diseases, including endemic diseases, and each formula has its indication, including specific symptoms of patients. this is quite different from using tcm formulas based on the yin-yang theory (two opposite, but complementary forces) and five elements theories (everything in the world can be classified into the natural five elements. these elements promote as well as feedback each other to keep everything in balance). kampo medicine in japan classified tcm theories into ancient formula sect and recent formula sect for prescribing tcm formulas. the physicians of the ancient formula sect (a-physicians) use the indications of formula formed before formula can have active molecules that are pharmacologically different from that ingredient or the tcm formula. obversely, the pharmacological activities of a tcm formula may differ from those of their active ingredient or active molecules of ingredients. as a consequence, unraveling the molecular mechanism of a tcm formula needs comparison of the pharmacological activities between the formula, its ingredients, and the active molecules in the ingredients. the amount of most bioactive compounds in the herbs is very low. combination of herbs to form a tcm formula can further decrease their concentrations. is it possible that herbs and tcm formulas can be effective in this low concentration of bioactive compounds? is it possible that little amount of bioactive molecules can cause interactions? in orthodox western medicine, vitamins of little amount can show their clinical effects. the molecular mechanisms are the key, instead of their amount. in the real world practice, herbs and tcm formulas are bioactive. several side effects of tcm formulas have been reported [ ] [ ] [ ] [ ] [ ] [ ] [ ] that raise the safety issue of tcm formula. natural products and tcm formulas might not be safe; however, some a-physicians suppose that the side effects may come from the misuse of tcm formulas without fulfilling their indications, while r-physicians consider the side effects developing from the misinterpretation of the disharmony. most tcm physicians do not agree that these side effects come from tcm formulas themselves, although with the same indications or disharmony, it is common to find that some patients respond well while others do not-while some patients develop side effects, some show responses opposite to the in vitro pharmacological effects. for example, panax quinquefolius prolongs thromboplastin time, prothrombin time (pt) and thrombin time in vitro [ ] . however, in combination therapy with warfarin, panax quinquefolius actually decreases seral concentration of warfarin and has shortened inr in a clinical trial [ ] . with therapy using panax ginseng, one can develop thrombosis [ ] , bleed [ , ] , or remain without any particular response [ ] . several factors may affect the molecular mechanisms and subsequent clinical effects of tcm formulas, including individual gene-based response, composition and amount of active molecules in tcm formulas, complex interactions, and appropriateness of use of tcm formulas. individual genetic basis is unique to metabolize tcm formulas and produces different responses. from the results of pharmacokinetic study of gan-lu-siao-du-yin, a tcm formula (submitted data), the blood concentrations of structurally-related index molecules, baicalin and baicalein, wogonin, and wogonoside, are highly variable between participants. the different patterns of blood concentrations support the unique pharmacokinetic profile based on individual genes. different concentrations of active molecules may affect the pharmacologic activities. therefore, individual gene-based metabolism could be one of the major factors affecting the molecular mechanisms and subsequent clinical effects of tcm formulas. to provide insights into action mechanisms of tcm formulas, metabolomic technologies might be helpful. a metabolomics integrative approach accepts a 'top-down' strategy to express the function of organisms through terminal symptoms of metabolic network and will gain a revolution in understanding of the holistic concept of tcm [ , ] . such technologies have been used to investigate the biological mechanisms of different syndromes of patients by studying the functional activities of the human body from a system-wide perspective. for example, the overall biological characterization of the urine of psoriasis patients with tcm blood stasis syndrome was performed to investigate the therapeutic metabolomic mechanism of the optimized yinxieling formula [ ] . in addition, metabolomics have been considered a powerful tool in diagnosis and treatment of primary dysmenorrhea by supporting information on changes of metabolites and changes in endocrinal, neural, and immune pathways [ ] . the xiang-fu-si-wu formula has been demonstrated to affect some significant perturbations in sphingolipid and glycerophospholipid metabolism as well as steroid hormone biosynthesis to make the metabolic discrepancy return to the normal level [ ] . tcm formulas are complex with numerous active chemical molecules in variable amounts. among these molecules with different pharmacologic activities, it is unclear which one mainly accounts for the clinical effect of a tcm formula, as the most abundant molecule might not be the most important one for a specific activity. the amount of an active molecule can be easily changed in different batches of product, or by different agriculture and collection of medicinal plants, therefore, understanding the molecular mechanisms of a tcm formula requires analysis not only of the mechanisms of the tcm formula as a whole, but those of individual active molecules and ingredient respectively as well. understanding the molecular mechanisms of an active molecule can facilitate its development into an investigational new drug (ind). meanwhile, unraveling the molecular mechanisms of a tcm formula can help to validate its traditional use and avoid its misuse and side effects. to keep a relatively constant amount of active molecules and pharmacologic activities, several things should be paid attention to, including use of right specie, use of right part of a plant, and use of a plant harvested in the right season. both use of closely related but wrong species and use of wrong part of herbs might lead to different active molecules with different pharmacologic activities, and various clinical effects and side effects. plants harvested in different seasons might contain variable amounts of active molecules thereby affecting their activities. some active molecules are secondary metabolites of plants against physical, chemical, or biological stimulants. active molecules of some plants can vary from year to year and place to place. therefore, confirmation of its authenticity is the cornerstone. in addition to this, fingerprints of the active molecules are needed to confirm the authenticity of a plant or a formula and to confirm the amount of active molecules via high-performance liquid chromatography (hplc) or liquid chromatography coupled with mass spectrometry (lcms). this is highly necessary for quality control and efficacy assessment. to have quality control of the products of tcm formulas, good manufacturing practice (gmp) procedure should be followed to avoid (a) inadequate processing that might lead to different chemical compositions of the final product; (b) inadequate storage conditions or prolonged storage that might lead to microbial contamination and early decay of the active molecules; and (c) adulteration of formulas and accidental contamination creating serious uncertainty in quality. adulteration is a plant or formula containing active pharmaceuticals or other bioactive agents for the purpose of claiming better efficacy or broader indications. accidental contamination is the plant raw materials containing heavy metals or other toxic substances from the manufacturing process due to ecological collapse. lead, mercury, and arsenic contamination in traditional chinese herbs has been reported [ ] [ ] [ ] [ ] . . complex interplays between herb-drug, herb-food, herb-herb, herb-microbiome, and herb-disease in the clinical practice of orthodox medicine, the more drugs used, the more adverse drug reaction (adr) occurred [ ] . this is commonly caused by drug-drug interactions. tcm formulas are mixtures of several ingredients. each ingredient has several bioactive compounds, so a tcm formula has numerous bioactive compounds. thinking of dozens or even hundreds of active molecules in a tcm formula been taken at once implies that the probability of drug interaction could be high. such interactions may be found between herb and drug, herb and food, herb and herb, herb and microbiome, and even between herb and disease. for example, in herb-drug interaction, scutellariae baicalensis is a common ingredient in tcm formulas. s. baicalensis contains baicalin, a flavonoid, as one of its major molecules. interactions between s. baicalensis and drugs are found due to baicalin affecting metabolic enzymes of drugs, displacing plasma protein binding, and regulating various transporters involved in the pharmacokinetics [ ] . baicalin may inhibit the expression and hydroxylation activity of cyp a in the liver to change the pharmacokinetics of drugs [ ] . co-administration of extract of s. baicalensis, and mefenamic acid, a kind of nsaid, can potentiate its anti-inflammatory effect [ ] . co-administration of baicalin and rosuvastatin, a hmg-coa reductase inhibitor commonly used to reduce serum cholesterol level, might find reduced plasma concentration of rosuvastatin in certain patients with certain genomes [ ] . as for herb-food and herb-food-drug interactions, baicalin can potentiate the antioxidant activity of β-carotene, which is a terpenoid of red-orange color, abundant in plants and fruits [ ] . the intakes of flavonoid-rich foods and beverages, containing baicalin and rutin, might compete with the binding site of calcium channel blockers on human serum albumin to affect their clinical effects [ , ] . by contrast, baicalin and rutin will increase the binding affinity of curcumin on human serum albumin to change its bioavailability [ ] . herbs may also interact with each other. for example, baicalin and berberine are important coexisting molecules of the combination of s. baicalensis and coptidis chinensis. berberine, but not baicalin, can increase glucose consumption. co-administration of berberine and baicalin had a synergetic effect on glucose utilization [ ] . additionally, tcm herbs are commonly used as food supplements and dietary therapy. foods have been reported to modify the intestinal microbiome [ ] . commensal microbiota have been thought to be involved in the development of the innate and adaptive immunity, nutrient metabolism of humans, and protection from the overgrowth of intestinal pathogens [ ] . herbs can change pharmacokinetics of drugs by intestinal microbiota [ ] . the intestinal microbiome is metabolically active to play an important role in the absorption of certain active molecules and change their bio-availabilities, particularly in those containing glycosidic linkages [ , ] . therefore, change of intestinal microbiome by herbs-containing foods may affect human health care and drug therapy. as for interactions between disease and herb, more absorption of active molecules of maxing shigan decoction (mxgst), including liquiritin, glycyrrhizin, amygdalin, prunasin, ephedrine, pseudoephedrine, and methylephedrine, can be found in rsv pneumonia-infected rats vis-à-vis normal rats by reducing the clearance rates of these active molecules [ ] . there are highly complex interactions between herbs and drugs, foods, herbs, microbiome, and diseases, and most of these complex interactions are not completely discovered or remain unseen, just like the submerged part of an iceberg. therefore, there are still insufficient data to completely understand the molecular mechanisms, pharmacokinetics, pharmacodynamics, and interactions of tcm formulas. acute airway infections, including acute bronchitis, viral pneumonia, and acute exacerbation of chronic obstructive pulmonary disease (copd), are commonly caused by viruses of different families, including rhinovirus, influenza, and parainfluenza virus, enteroviruses, coronavirus, adenovirus, respiratory syncytial virus, etc. [ , ] . these viruses infect epithelia, produce inflammation, induce immune response, and cause symptoms. from the viewpoint of pathophysiology, tcm formulas used to manage airway viral infections need to have antiviral activity against such viruses listed above, and/or to induce antiviral cytokines, and/or anti-inflammatory effect, and/or to relieve symptoms commonly presented in airway infections ( figure ). to simplify the molecular mechanisms and to correlate the pharmacologic activities with their clinical effects, five formulas of a-physicians will be used as examples against airway infections: ge-gen-tang (ggt; table ) [ ] has been reported to be effective in the treatment of common colds, chronic sinusitis, allergic rhinitis, and pneumonia. the indication to use ggt is patients with symptoms of headache, fever without sweating, and particularly stiffness of neck and shoulders. ggt can successfully reduce various symptoms of dogs infected with common cold viruses [ ] . ggt has antiviral activities against respiratory syncytial virus (rsv) [ ] and influenza virus [ ] . ggt can reduce the mortality of influenza virus-infected mice [ ] . among its active molecules, uralsaponins from glycyrrhiza uralensis [ ] and procyanidin from cinnamomum cassia [ ] may effectively inhibit the replication of the influenza virus. allicin in ginger (zingiber officinale) and coumarin in cinnamomum cassia might have the activity to inhibit influenza neuraminidase [ ] . paeonol and , , , , -penta-o-galloyl-β-d-glucopyranose from paeonia lactiflora show antiviral activity against rhinovirus [ ] . of its anti-inflammatory effects, ggt can suppress the interleukin- α (il- α) production induced by interferons (ifn) in influenza [ ] . ggt was found to decrease cigarette smoking-(cs-) and lipopolysaccharide (lps)-induced elevated counts of inflammatory cells, and expression of inflammatory cytokines and proteins (il- , tnf-α, inos, and cox- ) [ ] . ggt can stimulate il- and ifn-β to counteract viral infection [ ] , and can also enhance the phagocytic activity of macrophages [ ] . among its active molecules, paeoniflorin, a major constituent of paeonia lactiflora, exerts anti-inflammatory and immunomodulatory effects by balancing the function of th /th [ ] . glycyrrhizin, a major constituent of glycyrrhiza uralensis, ge-gen-tang (ggt; table ) [ ] has been reported to be effective in the treatment of common colds, chronic sinusitis, allergic rhinitis, and pneumonia. the indication to use ggt is patients with symptoms of headache, fever without sweating, and particularly stiffness of neck and shoulders. ggt can successfully reduce various symptoms of dogs infected with common cold viruses [ ] . ggt has antiviral activities against respiratory syncytial virus (rsv) [ ] and influenza virus [ ] . ggt can reduce the mortality of influenza virus-infected mice [ ] . among its active molecules, uralsaponins from glycyrrhiza uralensis [ ] and procyanidin from cinnamomum cassia [ ] may effectively inhibit the replication of the influenza virus. allicin in ginger (zingiber officinale) and coumarin in cinnamomum cassia might have the activity to inhibit influenza neuraminidase [ ] . paeonol and , , , , -penta-o-galloyl-β-d-glucopyranose from paeonia lactiflora show antiviral activity against rhinovirus [ ] . of its anti-inflammatory effects, ggt can suppress the interleukin- α (il- α) production induced by interferons (ifn) in influenza [ ] . ggt was found to decrease cigarette smoking-(cs-) and lipopolysaccharide (lps)-induced elevated counts of inflammatory cells, and expression of inflammatory cytokines and proteins (il- , tnf-α, inos, and cox- ) [ ] . ggt can stimulate il- and ifn-β to counteract viral infection [ ] , and can also enhance the phagocytic activity of macrophages [ ] . among its active molecules, paeoniflorin, a major constituent of paeonia lactiflora, exerts anti-inflammatory and immunomodulatory effects by balancing the function of th /th [ ] . glycyrrhizin, a major constituent of glycyrrhiza uralensis, suppresses nuclear factor-kappa b (nf-κb) via the phosphoinositide -kinase (pi k) pathway, inhibits the production of nitric oxides (no), prostaglandin e (pge ), and reactive oxygen species (ros), and reduces the protein and mrna levels of inducible no synthase (inos) and cyclooxygenase- (cox- ) [ ]( figure ). meanwhile, glycyrrhiza polysaccharide, isolated from glycyrrhiza uralensis, significantly induces no production and inos transcription in peritoneal macrophages [ ] . however, this study design uses intraperitoneal injection to show the induction of no [ ] , instead of the traditional oral route. polysaccharides will be normally digested in the gastrointestinal tract into monosaccharide, so that polysaccharides can hardly reach intraperitoneal macrophages in the regular oral route, so this pharmacologic activity has been questioned. isoliquiritigenin, a flavonoid from glycyrrhiza uralensis, inhibits nf-κb activation to suppress inflammatory response [ ] . cinnamaldehyde, from cinnamomum cassia, inhibits the secretion of pge , il- β and tumor necrosis factor-α (tnf-α), and the activation of nf-κb to show the anti-inflammatory effect [ ] [ ] [ ] . particularly, e-cinnamaldehyde and o-methoxy-cinnamaldehyde down-regulate no and tnf-α production to show anti-inflammatory activity [ ] . although it can mediate antiviral activity, tnf-α plays only a minor role in clearance of various airway viruses; rather, it is the major contributor of t-cell-mediated lung injury [ ] . for enhancement of antiviral immunity, puerarin, an isoflavonoid from pueraria lobate, increased ifn-γ [ ] . -gingerol ( -g), the main bioactive component of ginger (zingiber officinale), increases ifn-γ and il- , but decreases il- and transforming growth factor-β (tgf-β ) levels [ ] . on the contrary, gingerol was also reported to suppress t cell response and inhibit ifn-γ synthesis [ ] (figure ). these conflicting data may come from different study designs and raise questions about the actual pharmacological activity in humans. (ros), and reduces the protein and mrna levels of inducible no synthase (inos) and cyclooxygenase- (cox- ) [ ] (figure ). meanwhile, glycyrrhiza polysaccharide, isolated from glycyrrhiza uralensis, significantly induces no production and inos transcription in peritoneal macrophages [ ] . however, this study design uses intraperitoneal injection to show the induction of no [ ] , instead of the traditional oral route. polysaccharides will be normally digested in the gastrointestinal tract into monosaccharide, so that polysaccharides can hardly reach intraperitoneal macrophages in the regular oral route, so this pharmacologic activity has been questioned. isoliquiritigenin, a flavonoid from glycyrrhiza uralensis, inhibits nf-κb activation to suppress inflammatory response [ ] . cinnamaldehyde, from cinnamomum cassia, inhibits the secretion of pge , il- β and tumor necrosis factor-α (tnf-α), and the activation of nf-κb to show the antiinflammatory effect [ ] [ ] [ ] . particularly, e-cinnamaldehyde and o-methoxy-cinnamaldehyde downregulate no and tnf-α production to show anti-inflammatory activity [ ] . although it can mediate antiviral activity, tnf-α plays only a minor role in clearance of various airway viruses; rather, it is the major contributor of t-cell-mediated lung injury [ ] . for enhancement of antiviral immunity, puerarin, an isoflavonoid from pueraria lobate, increased ifn-γ [ ] . -gingerol ( -g), the main bioactive component of ginger (zingiber officinale), increases ifn-γ and il- , but decreases il- and transforming growth factor-β (tgf-β ) levels [ ] . on the contrary, gingerol was also reported to suppress t cell response and inhibit ifn-γ synthesis [ ] (figure ). these conflicting data may come from different study designs and raise questions about the actual pharmacological activity in humans. ma-huang- tang (mht; table ) [ ] have been reported to be effective in the treatment of influenza, upper respiratory tract infection, acute and chronic bronchitis, and asthma [ , ] . the indication to use mht is patients with chills, fever without sweating, headache, shortness of breath, and joint pain. clinically, mht can effectively reduce fever and flu symptoms, including myalgia, headache, arthralgia, fatigue and cough, in patients with seasonal influenza type a [ ] . among its active molecules, l-ephedrine from ephedra sinica and amygdalin from prunus armeniacae, possess the antitussive effect [ ] , so co-administration of ephedra sinica and prunus armeniacae shows a better antitussive effect than use singly [ ] . mht clearly shows anti-inflammatory activity via suppressing the no/pge pathway [ ] , reducing inflammatory cells infiltration and reducing pro-inflammatory cytokine, including tnf-α, il- β, and il- , in lung experiments [ ] . in an acute bronchial asthma mice model, mht can also mitigate the pathological changes of acute asthma-like syndrome through inhibition of the toll-like receptor (tlr ) pathway [ ] . mht can modulate th /th cytokines via decreasing il- & il- and increasing ifn-γ levels. mht can inhibit th cells [ ] , and decreases il- , il- , tnf-α, cd +, cd + t cell levels (th response), but increases il- , ifn-γ, and cd + t cell levels (th response) to increase cd +/cd + ratio [ ] . among its active ingredients, ephedra sinica inhibits pge biosynthesis, reduces ige-mediated histamine release, reduces the mrna or protein levels of il- β, il- , tnf-α, cox , and nf-κb [ ] and inhibits complement activation of both classical and alternative pathways [ ] . additionally, ephedra sinica can directly activate both alpha-and beta-adrenergic receptors to reduce bronchial mucosal edema and to dilate the bronchus respectively [ , ] . to understand the molecular mechanism, several active molecules have been identified. ephedrannin a and b, from ephedra sinica, effectively suppressed the transcription of tnf-α, il- β, and nf-κb, and the phosphorylation of p mitogen-activated protein (map) kinase to exert their anti-inflammatory actions on lps-stimulated macrophages [ ] . glycyrrhiza uralensis and cinnamomum cassia contain active molecules mediating anti-inflammatory and immunomodulatory effects mentioned in the ggt section. for its antiviral activity, mht was initially thought to inhibit airway viruses through inducing antiviral ifn. however, herbacetin from ephedrine alkaloid-free extract of ephedra sinica might have anti-influenza activity similar to its extract containing ephedrine and pseudoephedrine [ ] . the study of ephedra sinica is relative rare owing to it containing ephedrine and pseudoephedrine, which are illegal in several countries. glycyrrhiza uralensis and cinnamomum cassia also have active antiviral constituents mentioned in the ggt section ( figure ). ma-xing-gan-shi- tang (mxgst; table ) [ ] , a similar formula to ma-huang-tang, is effective against influenza virus infection [ ] . mxgst has only one ingredient different from that of mht, i.e., using gypsum instead of cinnamomum cassia, so their pharmacologic effects and active molecules are similar, except that gypsum possesses a more powerful anti-pyretic effect by decreasing the pge level in the hypothalamus [ ] . co-treatment with ephedra sinica and gypsum can have synergistic effects to manage fever and asthma than single use [ ] . mxgst add-on therapy may improve pulmonary function indicies, such as forced expiratory volume in one second (fev ), forced vital capacity (fvc), and fev /fvc in patients with acute exacerbation of copd [ ] . additionally, at higher cumulative doses, mxgst might reduce the incidence of pneumonia and protect against admission [ ] . the indication to use mxgst is patients with fever, cough with yellow and sticky sputum, chest pain, and shortness of breath. mxgst has been found to have antitussive and anti-pyretic effects in an lps-induced hyperthermia rat model [ ] . mxgst has bronchodilation effect mediated by stimulation of β -adrenoceptors in pigs [ ] and can block acetyl-cholinergic and histaminergic receptor-induced bronchial contraction in rats [ ] , reduce neutrophilic inflammation [ ] , and in a copd rat model, can decrease il- , il- , and tnf-α, but increase ifn-γ [ ] . these effects may be beneficial to manage airway viral infections with cough. the molecular mechanism of mxgst is summarized (figure ). molecules , , x; doi: www.mdpi.com/journal/molecules ma-xing-gan-shi- tang (mxgst; table ) [ ] , a similar formula to ma-huang-tang, is effective against influenza virus infection [ ] . mxgst has only one ingredient different from that of mht, i.e., using gypsum instead of cinnamomum cassia, so their pharmacologic effects and active molecules are similar, except that gypsum possesses a more powerful anti-pyretic effect by decreasing the pge level in the hypothalamus [ ] . co-treatment with ephedra sinica and gypsum can have synergistic effects to manage fever and asthma than single use [ ] . mxgst add-on therapy may improve pulmonary function indicies, such as forced expiratory volume in one second (fev ), forced vital capacity (fvc), and fev /fvc in patients with acute exacerbation of copd [ ] . additionally, at higher cumulative doses, mxgst might reduce the incidence of pneumonia and protect against admission [ ] . the indication to use mxgst is patients with fever, cough with yellow and sticky sputum, chest pain, and shortness of breath. mxgst has been found to have antitussive and antipyretic effects in an lps-induced hyperthermia rat model [ ] . mxgst has bronchodilation effect mediated by stimulation of β -adrenoceptors in pigs [ ] and can block acetyl-cholinergic and histaminergic receptor-induced bronchial contraction in rats [ ] , reduce neutrophilic inflammation [ ] , and in a copd rat model, can decrease il- , il- , and tnf-α, but increase ifn-γ [ ] . these effects may be beneficial to manage airway viral infections with cough. the molecular mechanism of mxgst is summarized (figure ). xiao-qing-long- tang (xqlt; table ) [ ] is one of the most common prescriptions used against allergic rhinitis [ ] . xqlt, at higher cumulative doses, might reduce the incidence of pneumonia and protect against admission [ ] . xqlt with/without ma-xing-gan-shi-tang (mxgst) is the most frequently prescribed tcm formula for copd [ ] , and is also commonly used in the treatment of patients with respiratory diseases, such as common cold, flu, bronchitis, asthma, bronchiectasis, and emphysema. the indication to use xqlt is patients with cough, watery rhinorrhea or watery sputum, but without thirst. to manage airway viral infection, xqlt is effective against human respiratory syncytial virus (rsv) infection by preventing viral attachment, internalization, syncytial formation, and by stimulating ifn-β secretion [ ] , and has been proven beneficial against influenza virus in vivo through the augmentation of antiviral iga antibody [ , ] . xqlt can reduce the airway inflammation with the decrease of eosinophils count, the ovalbumin (ova)-specific immunoglobulin e (ige) antibody, and histamine release [ ] [ ] [ ] , can also modulate th /th balance thereby reducing il- and restoring ifn-γ levels [ , ] . among its active molecules, schisandrin a, a bioactive lignin of schisandra sphenanthera, inhibits the il β-induced inflammation via suppression of mitogen-activated protein kinase (mapk) and nf-κb signal pathways [ ] , and can also inhibit the nf-κb, mapk and pi k/akt pathways, partially mediated by the activation of the nuclear factor erythroid -related factor /heme oxygenase- (nrf /ho- ) pathway to manage inflammatory and oxidative disorders caused by over-activation of macrophages [ ] . schisandrin b, another bioactive lignin of schisandra sphenanthera, increases the expression of nrf and ho- and blocks the activation of nf-κb induced by lps to suppress the production of vascular cell adhesion molecule (vcam- ), intercellular adhesion molecule (icam- ), tnf-α, and il- expressions in human umbilical vein endothelial cells (huvecs) [ ] . α-cubebenoate, isolated from schisandra chinensis, can block the increase of il- β and il- during inflammation [ ] , and inhibit lps-induced expression of inos and cox- [ ] . oral polysaccharide from schisandra chinensis showed antitussive effect in a guinea pig model [ ] . the active molecules of common ingredients, including ephedra sinica, cinnamomum cassia, paeonia lactiflora, glycyrrhiza uralensis, and zingiber officinale, have several pharmacologic activities mentioned in the above sections. most of these activities aim at inhibiting inflammation induced by airway infection, however, lectin from pinellia ternate may activate macrophages, induce neutrophil migration, cytokine release, ros overproduction and the activation of the nf-κb signaling pathway to produce pro-inflammatory activity [ ] (figure ). therefore, interactions, including both synergistic and antagonistic between active molecules in a tcm formula could be so complex as to affect the final effects. from more than two thousand years ago in china and japan, ye-gan-ma-huang- tang (ygmht; table ) [ ] has been used to manage flu-like symptoms. a meta-analysis shows that ygmht can improve the total effective rate, fev , and asthma control test (act) score of refractory asthmatic patients [ ] . when combined with salbutamol aerosol, ygmht can obviously improve their pulmonary functions, including act score, pef, fev , and fev % predicted value. the indication to use ygmht is patients with chill and fever, hyperinflation of lung, cough with stridor and rales associated with frothy or whitish sputum [ ] . ygmht has been reported effective against enterovirus infection, including coxsackievirus [ ] and ev [ ] . it can regulate the serum levels of tnf-α, il- , and il- to show clinical effect in management of cough and variant asthmatic symptoms in children [ ] . additionally, a modified ygmht (also named san-long-ping-chuan-decoction; slpcd) can significantly inhibit airway inflammation, reduce inflammatory cells in bronchoalveolar lavage fluid (balf), and decrease the serum total ige levels [ ] . slpcd can significantly down-regulate the mrna expression levels of th cytokines (il- , il- , il- , and il- ) and up-regulate those of th cytokines (il- and ifn-γ) in lungs of asthmatic mice [ ] . for this anti-inflammatory activity, aster tataricus can protect from lps-induced acute lung injury mainly through inhibiting the release of inflammatory cells (wbc, macrophage, neutrophil, lymphocyte), regulating the pro-inflammatory cytokines (il- β, il- , tnf-α), and attenuating the pulmonary edema [ ] . among its active molecules, irigenin, a major active constituent of belamcanda chinensis, can reduce no and pge production by decreasing the mrna and protein expression of inos and cox- , respectively, as well as by suppressing nf-κb activation [ ] (figure ). molecules , , x; doi: www.mdpi.com/journal/molecules from more than two thousand years ago in china and japan, ye-gan-ma-huang- tang (ygmht; table ) [ ] has been used to manage flu-like symptoms. a meta-analysis shows that ygmht can improve the total effective rate, fev , and asthma control test (act) score of refractory asthmatic patients [ ] . when combined with salbutamol aerosol, ygmht can obviously improve their pulmonary functions, including act score, pef, fev , and fev % predicted value. the indication to use ygmht is patients with chill and fever, hyperinflation of lung, cough with stridor and rales associated with frothy or whitish sputum [ ] . ygmht has been reported effective against enterovirus infection, including coxsackievirus [ ] and ev [ ] . it can regulate the serum levels of tnf-α, il- , and il- to show clinical effect in management of cough and variant asthmatic symptoms in children [ ] . additionally, a modified ygmht (also named san-long-ping-chuan-decoction; slpcd) can significantly inhibit airway inflammation, reduce inflammatory cells in bronchoalveolar lavage fluid (balf), and decrease the serum total ige levels [ ] . slpcd can significantly down-regulate the mrna expression levels of th cytokines (il- , il- , il- , and il- ) and up-regulate those of th cytokines (il- and ifn-) in lungs of asthmatic mice [ ] . for this anti-inflammatory activity, aster tataricus can protect from lps-induced acute lung injury mainly through inhibiting the release of inflammatory cells (wbc, macrophage, neutrophil, lymphocyte), regulating the pro-inflammatory cytokines (il- β, il- , tnf-α), and attenuating the pulmonary edema [ ] . among its active molecules, irigenin, a major active constituent of belamcanda chinensis, can reduce no and pge production by decreasing the mrna and protein expression of inos and cox- , respectively, as well as by suppressing nf-κb activation [ ] (figure ). to unravel the molecular mechanism of tcm formulas, several issues need to be solved. many pharmacologic activities are obtained from in vitro and animal studies. could these activities be extrapolated into humans? there are so many active molecules with various pharmacologic activities in a tcm formula or herbs. are these molecules specific for that specific activity of a tcm formula or herbs? could they reach a minimal serum level to exert that particular pharmacologic activity in humans? active molecules may have the same activity with different potency. the most abundant one may not be the most important one for a particular activity. is it possible that there might be unidentified active molecules that actually account for a particular pharmacologic activity? several pharmacologic activities of a tcm formula cannot find a corresponding active molecule. is it possible that interactions between active molecules, e.g., synergism, but not active molecules themselves, account for a specific activity? is it possible that new active molecules are generated during preparation of the tcm formula? the genetic basis will affect the drug pharmacokinetics. is there a specific gene or single nucleotide polymorphism (snp) largely affecting the pharmacokinetic profile? does publication bias exist so that undesired pharmacologic effects are not published? is it possible that a particular gene is prone to a specific adr of a tcm formula or herbs? could a specific adr come from interactions between specific active molecules? several tcm formulas clearly show that some ingredients are not active in managing their traditional applications. could these inactive ingredients be omitted? all these questions need many studies to determine valid answers. at this moment, it would be better to use herbs and tcm formulas according to their traditional indications and the disease pathophysiology by matching their molecular mechanisms. as the world population has continued to age, the elderly have become associated with chronic diseases requiring multiple medications. increasing dissatisfaction with orthodox medicine and/or preference for alternative therapists and/or naturopathy has meant people continue to seek alternatives to maintain or improve their health, and this has spurred the growth of complementary and alternative medicine (cam) therapies. the validity of pharmacologic studies, safety issues, and validation of clinical effects of herbal medicine and tcm are limitations that should be highly considered; however, most clinicians are not familiar with herbal medicine and tcm so they do not recommend herbal therapies. more effort should be placed on unraveling the molecular mechanisms in order to solve the above issues. the reasons that limit clinicians from becoming more familiar with herbal medicine and for not recommending herbal therapies are explored below. herbal medicine and tcm form a part of complementary and alternative medicine (cam). however, evidence-based research in the field of cam therapies is still limited. there is a wrong perception that a naturally derived product is relatively safe. it is highly important to identify both usefulness and safety of cam and integrate these health approaches with orthodox medicine through rigorous scientific investigation to improve health care. it is also important for medical educators and providers to recognize the trend, the evidence, the benefits, and the risks of herbal medicine and tcm to educate clinicians for appropriate patient management and education. tcm studies published in chinese are usually not translated into english. the terminology of tcm is also difficult to translate, particularly those used by r-physicians, so medical education of herbal medicine and tcm has been neglected worldwide, except in germany and china. with their increasing use since the s, understanding the molecular mechanisms, benefits, and limitations by physicians has become increasingly important to monitor their benefits and harmfulness. most of the evidence supporting clinical claims of herbs and natural products come from studies of inadequate design that do not provide tough evidence of the effects. relatively few well-designed studies support their clinical claims. sometimes, adequately powered, double-blinded, placebo-controlled clinical trials come to conclusions against previous reports, such as echinacea for upper respiratory infection [ ] , or ginkgo for dementia or mild cognitive impairment [ ] . additionally, the amounts of most bioactive compounds in herbs and tcm formulas are very low. is it possible that the clinical effects of herbs and tcm formulas can be effective with such a small amount of bioactive compounds? one study discussed the clinical effect of ma-huang-tang (mht) against seasonal influenza [ ] . mht showed an equivalent clinical effect as neuraminidase inhibitors. however, not every tcm formula can provide such evidence. several tcm formulas, such as ma-xing-gan-shi-tang (mxgst), ge-gen-tang (ggt), and xiao-qing-long-tang (xqlt), are among the top ten most common tcm prescriptions for patients with upper respiratory tract infections (urtis) in taiwan [ ] . they are commonly used for urtis, but more research is required to validate their clinical effects and mechanisms. without tough clinical evidence and clear molecular mechanisms, physicians tend to avoid herbal therapies. most clinical claims of herbal therapies are based on bench studies that do not possess external validity to support their conclusions; for example, using animal-derived cancer cell lines to study antiviral effects in humans; using cancer cell lines to study physical changes; using intraperitoneal injection to study oral medications; and using high-dose pharmacological designs to study physiological responses, etc. therefore, there is still much space for improvement of our understanding of the mechanisms of herbal medicine. herbs are pharmacologically active and can positively or negatively affect patient's health. with increasing use of herbs as dietary supplements and alternative therapy, there is an increased risk of negative impacts, such as adverse effects and interactions. herbal medicine is among the most common causes of drug-induced liver injury (dili) [ ] . additionally, several adverse effects of commonly used herbs have been reviewed, including hypoglycemic or hyperglycemic effect, hypolipidemic or hyperlipidemic effect, hormonal activities, hypertensive, cardioactive [ ] , and hepatotoxic effects [ ] . some of them are serious even in recommended dosage, such as ephedra alkaloids (derived from ephedra sinica or ma huang) [ ] [ ] [ ] . however, most of the molecular mechanisms causing side effects are unknown. plants containing pyrrolizidine alkaloids can lead to hepatotoxicity and venoocclusive disease, possibly by the accumulation of toxic metabolites produced via the cytochrome system [ ] . some herbal treatments containing aristolochic acid (aa), including aristolochia fangchi, aristolochia debilis sieb. et zucc., aristolochia manshuriensis, aristolochia debilis, can cause aa nephropathy requiring renal replacement therapy [ , ] ; additionally, aa is associated with urothelial cancers [ ] . although most of the above issues have been identified, the unrecognized issues are just the tip of the iceberg. tcm formulas have numerous bioactive compounds to form a kind of cocktail therapy. however, the amounts of each bioactive compound in herbs and tcm formulas are very low. this may raise questions about the likelihood of their interactions with others during administration in a decoction. ginseng, a common natural product used among adults [ ] , has about ginsenosides as its major bioactive compounds [ ] , although the actual amount of each ginsenoside is small. after oral administration, ginsenosides are metabolized and transformed by intestinal microbiota. diet can markedly influence the transformation of ginsenosides. they exert pharmacologic effects in animals, and also show various clinical effects in randomized controlled trials, including several side effects and drug interactions [ ] , so small amounts of bioactive compounds do have clinical effects and side effects, which might come from the individual pharmacological activity of bioactive compounds or their synergism. the side effects may also come from individual unfavorable bioactivity or from interactions. additionally, herbal medicine and tcm therapy are commonly used in combination with orthodox medicine by patients, sometimes unknown to their doctors. the complex and unknown interactions, including herb-drug, herb-herb, herb-food, herb-microbiome, and herb-disease interactions, make use of herbal medicine more complicated and requiring frequent monitoring. the mechanisms of these interactions can be divided into molecular mimicry and pharmacologic interactions, such as pharmacodynamic and pharmacokinetic interactions. for example, with molecular mimicry, several herbs naturally has coumarin or salicylate analogue that may potentiate the bleeding risk of warfarin and salicylate, respectively. for pharmacodynamic interactions, ephedra sinica (ma huang) should not be used with sedative or anti-hypertensive agents. for pharmacokinetic interactions, st john's wort and several tcm formulas have been noted to affect the cyp system of liver, which may increase or decrease the effects of other drugs [ ] . additionally, the herb-drug interaction may be individualized, e.g., in combination with warfarin, panax ginseng may cause thrombotic event [ ] , bleeding [ , ] , or neither [ ] . currently, most of the molecular mechanisms of these identified interactions are not fully understood, to say nothing of the unrecognized interactions. quality uncertainty impacts the reproducibility of clinical efficacy and safety of commercially available natural products. variability of the quality of natural products may come from a lack of standardization of manufacturing, including misidentification of authenticity, inadequate processing, inadequate storage, adulteration of formulas, and accidental contamination [ ] . most of those quality problems can be gradually solved by following the who guidelines on good agriculture and collection practices for medicinal plants [ ] and botanical drug development guidance for industry of fda [ ] . several health benefits of herbal medicine and tcm are claimed; for example, herbs and tcm formulas, including those discussed above, are believed to have anti-oxidative activities helpful against several diseases. this idea is based on reactive oxygen and nitrosative species (ros/rns) as metabolic byproducts that can cause damage to cellular macromolecules; thus, many diseases can be triggered by oxidative stress under high levels of ros/rns. these diseases include cancer, inflammation, and degenerative diseases. oxidative stress causes damage either with an overwhelming production of ros/rns or under insufficient levels of antioxidants or repair mechanisms, so blocking the generation of ros/rns might prevent and/or manage these diseases [ ] . however, ros/rns are also signaling molecules for several physiological functions, including regulation of vascular tone, control of ventilation, and erythropoietin production, etc. actually, ros-mediated responses may protect against oxidative stress [ ] . additionally, ros/rns may play a dual role in different diseases, i.e., ros/rns might contribute to, or counteract, the disease progression. it remains unclear that more dosage of antioxidants is not better and may even worsen a medical condition [ ] . there are insufficient data to establish the ability of tcm to decrease ros/rns levels and establish its effects on the disease, and this affects the interpretation of any claims of benefit. to validate such claims of the benefits of herbal medicine and tcm, much work remains to be done. only when these limitations can be minimized, can the molecular mechanisms of herbs and tcm formulas be understood. consequently, clinicians can help patients by giving adequate prescriptions and suggestions to minimize the harmfulness and maximize the benefits to healthcare. herbal medicine, including tcm, is commonly used worldwide. herbal remedies contain numerous active molecules to form a kind of cocktail therapy. these active molecules may interact with each other to affect the therapeutic effects and produce side effects. understanding their pharmacological effects, interactions, side effects, clinical effects, and the underlying molecular mechanisms is very important to provide benefits, but avoid harm, to the patient. to unravel the molecular mechanisms, much work remains to be done. the abc clinical guide to herbs trends in alternative medicine use in the united states, - : results of a follow-up national survey trends in the use of complementary health approaches among adults: united states complementary and alternative medicine use among adults and children: united states current state of phytotherapy in germany glycyrrhizic acid inhibits virus growth and inactivates virus particles antiviral effect of cimicifugin from cimicifuga foetida against human respiratory syncytial virus pterodontic acid isolated from laggera pterodonta inhibits viral replication and inflammation induced by influenza a virus a possible mechanism of interstitial pneumonia during interferon therapy with sho-saiko-to a case of acute eosinophilic pneumonia due to sho-saiko-to autoimmune hepatitis with drug-induced pneumonia due to sho-saiko-to two cases of pneumonia caused by sho-saiko-to acute lymphoblastic leukemia complicated by type c hepatitis during treatment and further by acute interstitial pneumonia due to sho-saiko-to in -year-old an autopsy case of interstitial pneumonia probably induced by sho-saiko-to a case of interstitial pneumonia with chronic hepatitis c following interferon-alfa and sho-saiko-to therapy a comparative study on anticoagulant activities of three chinese herbal medicines from the genus panax and anticoagulant activities of ginsenosides rg and rg brief communication: american ginseng reduces warfarin's effect in healthy patients: a randomized thrombosis of a prosthetic aortic valve disclosing a hazardous interaction between warfarin and a commercial ginseng product ginseng and vaginal bleeding ginseng face cream and unexplained vaginal bleeding interaction between warfarin and panax ginseng in ischemic stroke patients systems biology: metabonomics potential role of metabolomics apporoaches in the area of traditional chinese medicine: as pillars of the bridge between chinese and western medicine application of metabolomics on diagnosis and treatment of patients with psoriasis in traditional chinese medicine biomarkers of primary dysmenorrhea and herbal formula intervention: an exploratory metabonomics study of blood plasma and urine plasma metabolic profiling of normal and dysmenorrhea syndrome rats and the effects of xiang-fu-si-wu decoction intervention adulterants in asian patent medicines heavy metal content of ayurvedic herbal medicine products arsenic and mercury intoxication due to indian ethnic remedies lead, mercury, and arsenic in us-and indian-manufactured ayurvedic medicines sold via the internet risk factors for adverse drug events among nursing home residents role of intestinal microbiota in baicalin-induced drug interaction and its pharmacokinetics inhibitory effects of baicalin on the expression and activity of cyp a induce the pharmacokinetic changes of midazolam in rats herb-drug interactions between scutellariae radix and mefenamic acid: simultaneous investigation of pharmacokinetics, anti-inflammatory effect and gastric damage in rats the effect of herbal medicine baicalin on pharmacokinetics of rosuvastatin, substrate of organic anion-transporting polypeptide b baicalin in radical scavenging and its synergistic effect with beta-carotene in antilipoxidation studies on the competitive binding of cleviprex and flavonoids to plasma protein by multi-spectroscopic methods: a prediction of food-drug interaction spectroscopic investigation on the food components-drug interaction: the influence of flavonoids on the affinity of nifedipine to human serum albumin the increased binding affinity of curcumin with human serum albumin in the presence of rutin and baicalin: a potential for drug delivery system interaction of baicalin with berberine for glucose uptake in t -l adipocytes and hepg hepatocytes could the gut microbiota reconcile the oral bioavailability conundrum of traditional herbs? role of metabolism by intestinal microbiota in pharmacokinetics of oral baicalin pharmacokinetics of maxing shigan decoction in normal rats and rsv pneumonia model rats by hplc-ms/ms upper respiratory infections common viral respiratory infections. in harrison's principles of ministry of health and welfare. taiwan herbal pharmacopeia a pharmacologic study on the mechanism of action of kakkon-to: body temperature elevation and phagocytic activation of macrophages in dogs ge-gen-tang has anti-viral activity against human respiratory syncytial virus in human respiratory tract cell lines mechanism of action of the suppression of influenza virus replication by ko-ken tang through inhibition of the phosphatidylinositol -kinase/akt signaling pathway and viral rnp nuclear export effect of interleukin- level augmented by kakkon-to, a herbal medicine, on the early stage of influenza infection in mice uralsaponins m-y, antiviral triterpenoid saponins from the roots of glycyrrhiza uralensis high-throughput screening for anti-influenza a virus drugs and study of the mechanism of procyanidin on influenza a virus-induced autophagy identification of suitable natural inhibitor against influenza a (h n ) neuraminidase protein by molecular docking antiviral activity and possible mechanism of action of constituents identified in paeonia lactiflora root toward human rhinoviruses kakkon-to suppressed interleukin-la production responsive galgeun-tang attenuates cigarette smoke and lipopolysaccharide induced pulmonary inflammation via ikappabalpha/nf-kappab signaling anti-inflammatory and immunomodulatory effects of paeonia lactiflora pall. a traditional chinese herbal medicine the pharmacological activities of licorice nitrite oxide and inducible nitric oxide synthase were regulated by polysaccharides isolated from glycyrrhiza uralensis fisch isoliquiritigenin attenuates adipose tissue inflammation in vitro and adipose tissue fibrosis through inhibition of innate immune responses in mice cinnamaldehyde reduces il- beta-induced cyclooxygenase- activity in rat cerebral microvascular endothelial cells cinnamaldehyde inhibits pro-inflammatory cytokines secretion from monocytes/macrophages through suppression of intracellular signaling cinnamaldehyde and -methoxycinnamaldehyde as nf-kappab inhibitors from cinnamomum cassia anti-inflammatory activity of cinnamon (c. zeylanicum and c. cassia) extracts-identification of e-cinnamaldehyde and o-methoxy cinnamaldehyde as the most potent bioactive compounds cellular immunity and lung injury in respiratory virus infection puerarin attenuates airway inflammation by regulation of eotaxin- -gingerol as an arginase inhibitor prevents urethane-induced lung carcinogenesis by reprogramming tumor supporting m macrophages to m phenotype differential inhibition of t lymphocyte proliferation and cytokine synthesis by [ ]-gingerol antipyretic effect of mao-to, a japanese herbal medicine, for treatment of type a influenza infection in children prescription pattern of chinese herbal products for adult-onset asthma in taiwan: a population-based study the efficacy of ma-huang-tang (maoto) against influenza antitussive effects of l-ephedrine, amygdalin, and makyokansekito (chinese traditional medicine) using a cough model induced by sulfur dioxide gas in mice effect of ephedra with semen armeniacae amarum by compatibility of different ratio in acute toxicity test and antiasthmatic simultaneous determination and anti-inflammatory effects of traditional herbal medicine, mahwang-tang post-treatment with ma-huang-tang ameliorates cold-warm-cycles induced rat lung injury huang tang ameliorates bronchial asthma symptoms through the tlr pathway huang tang ameliorates asthma though modulation of th /th cytokines and inhibition of th cells in ovalbumin-sensitized mice antiviral effects of ma huang tang against h n influenza virus infection in vitro and in an icr pneumonia mouse model phytochemistry and pharmacology of genus ephedra. chin a component of the medicinal herb ephedra blocks activation in the classical and alternative pathways of complement ephedra in perspective-a current review ephedrannin a and b from roots of ephedra sinica inhibit lipopolysaccharide-induced inflammatory mediators by suppressing nuclear factor-kappab activation in raw . macrophages the pharmacological actions of ephedrine alkaloids-free ephreda herb extract and preparation for clinical application anti-influenza agents from plants and traditional chinese medicine an study on gypsum compounds and their antipyretic function and anti-inflammatory mechanisms antipyretic and anti-asthmatic activities of traditional chinese herb-pairs, ephedra and gypsum maxingshigan decoction for treating aecopd in cases and nursing measures. china pharm traditional chinese medicine therapy decreases the pneumonia risk in patients with dementia antitussive, anti-pyretic and toxicological evaluation of ma-xing-gan-shi-tang in rodents the effects of ma-xing-gan-shi-tang on respiratory resistance and airway leukocyte infiltration in asthmatic guinea pigs changes in the level of cytokine in rats with chronic obstructive pulmonary disease of phlegm heat cumber lung type after treatment of maxing shigan decoction. chin the prescriptions frequencies and patterns of chinese herbal medicine for allergic rhinitis in taiwan the use of chinese herbal medicine in the treatment of chronic obstructive pulmonary disease (copd) xiao-qing-long-tang (sho-seiryu-to) inhibited cytopathic effect of human respiratory syncytial virus in cell lines of human respiratory tract in vivo anti-influenza virus activity of kampo (japanese herbal) medicine "sho-seiryu-to" to indicate a break in thought or interpretation and its mode of action in vivo anti-influenza virus activity of kampo (japanese herbal) medicine "sho-seiryu-to"-effects on aged mice, against subtypes of a viruses and b virus, and therapeutic effect proteomic analysis of anti-inflammatory effects of a kampo (japanese herbal) medicine "shoseiryuto (xiao-qing-long-tang)" on airway inflammation in a mouse model xiao-qing-long-tang attenuates allergic airway inflammation and remodeling in repetitive dermatogoides pteronyssinus challenged chronic asthmatic mice model effects of xiaoqiongtang decoction on airway inflammation and airway remodeling in patients with copd anti-allergic activity of a kampo (japanese herbal) medicine "sho-seiryu-to (xiao-qing-long-tang)" on airway inflammation in a mouse model schisandrin a inhibits the il- beta-induced inflammation and cartilage degradation via suppression of mapk and nf-kappab signal pathways in rat chondrocytes schisandrin a suppresses lipopolysaccharide-induced inflammation and oxidative stress in raw . macrophages by suppressing the nf-kappab, mapks and pi k/akt pathways and activating nrf /ho- signaling schisandrin b inhibits lps-induced inflammatory response in human umbilical vein endothelial cells by activating nrf anti-septic activity of alpha-cubebenoate isolated from schisandra chinensis identification of a novel anti-inflammatory compound, alpha-cubebenoate from schisandra chinensis antitussive activity of the schisandra chinensis fruit polysaccharide (scfp- ) in guinea pigs models pinellia ternata lectin exerts a pro-inflammatory effect on macrophages by inducing the release of pro-inflammatory cytokines, the activation of the nuclear factor-kappab signaling pathway and the overproduction of reactive oxygen species meta-analysis on shegan mahuang tang for refractory asthma clinical observation on therapeutic effect of traditional chinese medicine granules made by formula of shegan mahuang decoction for patients with asthma yakammaoto inhibited human coxsackievirus b (cvb )-induced airway and renal tubular injuries by preventing viral attachment, internalization, and replication yakammaoto inhibits enterovirus infection by reducing viral attachment, internalization, replication, and translation effect of modified shegan mahuang decoction on cytokines in children patients with cough and variant asthma antiasthmatic effects of sanglong pingchuan decoction through inducing a balanced th /th immune response network pharmacology-based investigation of protective mechanism of aster tataricus on lipopolysaccharide-induced acute lung injury inhibitory effects of irigenin from the rhizomes of belamcanda chinensis on nitric oxide and prostaglandin e( ) production in murine macrophage raw . cells echinacea for preventing and treating the common cold ginkgo biloba for preventing cognitive decline in older adults: a randomized trial a randomized, controlled trial comparing traditional herbal medicine and neuraminidase inhibitors in the treatment of seasonal influenza traditional chinese medicine treatments for upper respiratory tract infections/common colds in taiwan acg clinical guideline: the diagnosis and management of idiosyncratic drug-induced liver injury herbal medicines: a guide for health-care professionals hepatotoxicity of drug used in the treatment of gastrointestinal disorders adverse cardiovascular and central nervous system events associated with dietary supplements containing ephedra alkaloids is there an association between ephedra and heart failure? a case series efficacy and safety of ephedra and ephedrine for weight loss and athletic performance: a meta-analysis hepatotoxicity of herbal remedies rapidly progressive interstitial renal fibrosis in young women: association with slimming regimen including chinese herbs chinese herbs nephropathy: a clue to balkan endemic nephropathy? kidney int aristolochic acid as a probable human cancer hazard in herbal remedies: a review panax ginseng and panax quinquefolius: from pharmacology to toxicology progress in cytochrome p enzyme in toxicity of traditional chinese medicines. chin the state pharmacopoeia commission of the people's republic of china. pharmacopoeia of the people's republic of china who guidelines on good agricultural and collection practices (gacp) for medicinal plants drug development guidance for industry positive or negative actors? biomolecules free radicals in the physiological control of cell function the authors would like to thank teachers of the center for languages and culture of kaohsiung medical university for the technical support. the authors declare no conflict of interest. key: cord- - hgrs authors: miazga-karska, malgorzata; michalak, katarzyna; ginalska, grazyna title: anti-acne action of peptides isolated from burdock root—preliminary studies and pilot testing date: - - journal: molecules doi: . /molecules sha: doc_id: cord_uid: hgrs this work aimed to study the anti-bacterial, anti-biofilm and anti-oxidant potential effects of low molecular weight (lmw) peptides (br-p) isolated from burdock (arctium lappa l.) roots. we conducted a preliminary study to exclude or confirm the antibiotic activity of the lmw peptides fraction of this plant. br-p were isolated using gel filtration and a kda cut-off membrane. the obtained peptides were identified by maldi tof/tof. antibacterial activity was tested against acne strains using diffusion tests, mic and mbc. the fibroblast cytotoxicity of br-p was tested, and the selectivity index (si) value was determined. the fraction of br-p peptides isolated from burdock root with a molecular weight below da and theoretic pi (isoelectric point) of . – . showed a narrow spectrum of activity against gram-positive acne bacterial strains. one of the br-p peptides assessed on maldi rapiddenovo was lrcdygrffaskslydplkkrr cationic peptide. it was analogous to that contained in a. lappa protein, and theoretically it was matched as a peptide with antibiotic nature. br-p did not show toxicity to fibroblasts in the tested concentration up to mg/ml, obtaining cc( ) mg/ml. the si value for the tested propionibacterium strains ranged from to . finally, an active dressing based on chitosan/alginate/genipin was prepared using freeze-drying. the formed dressing was evaluated for its anti-acne activity. to sum up: preliminary biological studies confirmed the anti-acne properties of the isolated peptide fraction from burdock root and pointed to the possibility of using it to create an active dressing on the skin. the world health organization (who) warns against epidemiological threats. today it is coronavirus, tomorrow drug-resistant bacteria can cause a large number of complications and high mortality. irrational antibiotic therapy leads to microbial resistance, which is the ability of microorganisms (like bacteria, viruses, and some parasites) to stop antimicrobial drugs from resisting against them. as a result, standard treatments become ineffective, and infections persist and may spread to others. who reports that the misuse of antibiotics is putting us all at risk, and the world is heading towards a pre-penicillin era. however, if effective anti-infection medication runs out, most invasive medical procedures, from tooth extraction to complicated surgery, will again be at high risk of fatal infection [ ] . propionibacterium acnes is the most important skin inflammatory factor. an acne skin treatment lasting several years is a difficult and complex problem not only for dermatologists. it is also noticeable by surgeons; for patients with acne around the shoulders and the back, chest or thighs, it can cause postoperative infections and prolong healing due to the presence of propionibacterium acnes. in such cases, bactericidal treatment before and after surgery is recommended. in most european the burdock roots samples, isolated using ultrasound and salting out procedure, were fractionated using a sephadex g- gel filtration (materials and methods, section . .) the obtained fractions were evaluated for the amount of protein and antibacterial properties (the diffusion test in solid medium). the active fractions were combined and resulting in obtaining a peptide fraction named br-f (the amount of protein for the br-f fraction was µg/ml). br-f was initially tested for antibacterial activity (figure , table ), and the remaining br-f part was further ultrafiltrated in amicon ( kda cut-off), followed by freeze-drying to obtain final br-p peptide samples (the protein content was µg/ml). it turns out that the antimicrobial activity increased with purification steps, during which the protein levels decreased. the data in table and figure indicate that both peptide samples of burdock root (br-f and br-p) did not inhibit the growth of gram-negative bacteria. in contrast, there was a noticeable inhibitory effect on gram-positive bacteria by both peptide samples: br-f and the purified peptide fraction of br-p inhibited the actions of bacterial strains, both aerobic and anaerobic. however, the purified br-p peptide fraction worked almost twice as strong compared to br-f. these br-f created zones of gram-positive bacteria growth inhibition in the range of . - mm, while br-p peptides showed growth inhibition of the tested strains in the range of . - . mm (figure ). the above results were confirmed with a minimum inhibitory concentration (mic) evaluation. data in table indicate the most favorable, low mic concentration values were obtained by br-p peptide samples primarily against the acne strains p. acnes pcm (mic . µg/ml) and p. acnes pcm (mic . µg/ml). higher mic values were determined against other gram-positive staphylococcus aureus and s. epidermidis bacteria (mic and µg/ml, respectively). however, a twice as high br-f concentration was needed to achieve such mic values for s. aureus and s. epidermidis and up to eight times higher to reach mics for both p. acnes species. in the mic assay, both peptide burdock samples were not active against gram-negative strains. another mbc/mic test confirmed that the bactericidal or bacteriostatic nature of burdock samples depends on the step of their purification (table ). the br-p peptide fraction was bactericidal against all tested gram-positive strains, both aerobic and anaerobic. however, br-f fractionated using a sephadexg- gel filtration had only a bacteriostatic character to these gram-positive strains. due to the low br-p and br-f activity against gram-negative strains, the mbc/mic value could not be determined. in subsequent experiments, the br-p peptide sample was used because of its good antibacterial activity. in the next stage of the study, the ms spectrum of the peptide mixture was assessed by maldi-tof. the obtained spectrum was smoothed, and the list of peaks for the signal-to-noise ratio was digitally generated (figure , figure s ). obtained data confirmed that the br-p sample consisted of peptides with molecular masses lower than da. the major peaks (corresponding to peptides) with a signal-to-noise ratio greater than were loaded and subjected to biotools rapidenovo sequencing. the signal-to-noise parameter response to intensity of the peak corresponds to the number of certain peptides. these peaks were selected for analysis due to the supposition that they had the greatest effect on antibacterial action. the peptide sequences obtained from the br-p burdock root sample were further analyzed using the basic local alignment search tool (blast) homology base. this base allowed the peptides present in the br-p sample to be matched (in the homology field) with analogous peptides and proteins found in the asteraceae family. the selected peptides of a given amino acid sequence were included in the enzymatic (oxygenases, dehydrogenases, transferases) or plant immune proteins from the asteraceae family. they are specified in table . in the case of nwfkpg sequence fragment, there are suggestions of proteins from which they may originate. additionally, for a m/z two equally likely sequence fragments were selected (fsrerd, nkfsre). in the case of antimicrobial peptides, it was important to determine the pi value. in our mixture of peptides from burdock, ten occurring in the largest amount were distinguished. based on the data contained in table , it can be concluded that the br-p peptide mixture contained peptides with a pi in the range of . - . . among them there was a cationic peptide lrcdygrffaskslydplkkrr with theoretically inhibitory activity on bacterial permease and with a pi of . . thus, due to the type of activity and cationic nature, the peptide may be responsible for antibiotic activity. * signal-to-noise ratio on mass spectrum. ** protein sequences matched to sequence in databases in basic local alignment search tool (blast). *** activities determined in biopep-uwm database of proteins and biopep-uwm database of bioactive peptides. **** theoretical pi determined in peptidemass expasy. the next step was to determine the strength of scavenging the free radicals of br-p in the dpph assay. such an activity of the peptide sample would be desirable when preparing an anti-acne formulation. in the experiment, radical scavenging assays were used to evaluate the activity of br-p and glutathione (gsh) as a positive control ( figure ). the dpph free radical inhibition assay of the new peptide fraction br-p revealed the potent antioxidant activity as observed through the plot of effective concentration ( - µg/ml) ( figure ). the ic value of br-p for dpph inhibition was found to be . µg/ml. the results were comparable to that of the reference peptide gsh (tested at - µg/ml) and with ic of . µg/ml. in analyzing the sequence of peptides present in the sample, it can be seen that they contained fragments of peptides considered to be antioxidant, hence high antioxidant properties may result. additionally, during the isolation process there is a possibility of nonspecific breaking of amino acid chains, resulting in the occurrence of small di-or tripeptides that are not observed on the maldi spectrum. it may mean that there are many more peptide fragments with antioxidant activity in the br-p sample. the investigated br-p peptides sample was evaluated for its cytotoxic activity towards the human skin fibroblast cell line (bj) after hours of incubation. based on mtt assay results, the half maximal cytotoxic concentration (cc ) was determined and summarized in figure . data show that br-p peptides exhibited no cytotoxicity against bj cells in the entire tested concentration range ( . - mg/ml). the fibroblast viability was maintained with a tested br-p concentration range at a high level of over %. this result means that the cc values of br-p peptides were higher than mg/ml. thus, for biocompatibility and selectivity determination of extracts, the cc value of br-p was assigned as mg/ml (table ) . to estimate the in vitro therapeutic safety and efficacy of the br-p peptide sample, the selectivity index (si) was determined. si index is defined as the ratio of cytotoxic activity (cc ) to antibacterial activity (mic). high values of the selectivity index indicate that the tested sample may be more effective against bacterial strains and safer for eukaryotic cells. thus, si values below indicate lack of therapeutic safety, and si values higher than allow to perform in vivo evaluation [ ] . it is extremely therapeutically important that the br-p exhibited a favourable and highly safe therapeutic potential against acne strains p. acnes pcm and p. acnes pcm , with safety si values of and , respectively. however, towards the tested gram-negative bacteria (e. coli and p. aeruginosa) br-p did not meet the safety requirements. the last stage of research was the production of an exemplary functional anti-acne dressing modified with the br-p peptide fraction from burdock root. the dressing material was created with genipine cross-linked chitosan and alginate. obtained after freeze-drying, the hydrogel sheet composed of polysaccharides was a uniform, thin foam resistant to chipping or cracking. the control unmodified and modified with br-p peptide dressings were immersed in a medium with a plankton form of acne strains. it was compared whether such planktonic forms of bacteria could create colonies resulting in biofilm formation to the same extent on control and modified materials. the conducted test imaged in a confocal laser-scanning microscope showed the beneficial effects of modification with br-p ( figure ). namely, the confocal laser-scanning microscope (clsm) images showed p. acnes or s. aureus biofilms on the surface of the dressing with viable and dead colonies (green and yellow-red fluorescence, respectively). the control dressings group demonstrated viable p. acnes or s. aureus bacteria, which formed green fluorescent biofilm architecture indicating that bacteria were viable during the evaluation period. whereas, only red colonies of dead bacteria were seen on the tested dressings. images show weaker or no adherence of tested bacteria to the br-p-modified dressing in comparison to the unmodified control ( figure ). acne vulgaris is a chronic disease of the hair sebaceous gland accompanied by seborrhoea. it is not an infectious disease, but patients have a multiplication of propionibacterium acnes [ ] . achermann's team presented relative abundances of propionibacterium species in different skin areas, proving that these bacteria are not only present in the face area, resulting in not only cosmetic trouble, but make it difficult to carry out surgical procedures in a larger area [ ] . the roots of burdock (arctium lappa l.) are commonly used as herbal medicine based on its valuable phytochemical content. pereira et al. [ ] , gentil et al. [ ] and pirvu et al. [ ] showed antibacterial activity against gram-positive and gram-negative bacteria of crude extracts from burdock, and knott et al. and jingvi et al. [ , ] proved skin improvement when using burdock extracts. one of the molecules in burdock roots responsible for antibacterial properties can be peptides because every organism (from microorganisms to mammalia) has an innate defense system, which is determined by the presence of, among others, antimicrobial peptides (amps) [ ] . therefore, it was desirable in our work to purify the crude extract of burdock root to assess the biological activity of the peptide solution. the presence of fractions of peptides with molecular masses lower than in da br-p was determined by maldi tof. the major peaks were subjected to biotools rapiddenovo sequencing ( figure ). the determined theoretical pi values confirmed the presence of some cationic peptides with a preference for antibacterial activity with pi values of . , . , . , and . . based on table , we can theoretically assume that the peptide lrcdygrffaskslydplkkrr with pi of . and kk amino acid fragment, with activity indicated as a bacterial permease ligand, may be responsible for the antibiotic activity. in the mechanism of energy transduction in bacterial membranes, a bulk-phase, transmembrane electrochemical ion gradient is an important problem and concerns, e.g., secondary active transport, oxidative phosphorylation, and rotation of the bacterial flagellar motor. transport involves substrate-specific membrane proteins that catalyze equilibration or uphill translocation of solute across a membrane. these carrier proteins are named transporters, cotransporters, symporters, or permeases. thus, permeases are involved in active transport of energy sources, e.g., glucose, fructose, various sugars, and sugar alcohols. thus, permeases, by providing the bacterial cell with a source of energy, may be the target of molecular inhibition of bacterial growth [ ] . both burdock root samples (br-f, br-p) obtained in subsequent purification stages were evaluated for antimicrobial character. the data of bacterial growth inhibition show that burdock root polypeptide samples had the ability to inhibit the growth of acne bacteria (figure ) . the results indicate that the antibacterial nature of the samples increased with the number of purification steps, as the br-p-purified sample was at least twice as strong as br-f. the mic value confirmed that br-p had stronger antibacterial properties than the mixture of crude br-f polypeptides (table ) . br-p possessed bactericidal nature against all gram-positive strains, whereas br-f was only active against anaerobic gram-positive strains. the tested preparations from burdock roots did not inhibit the activity of gram-negative strains. the narrow spectrum demonstrated by burdock root samples, limited only to gram-positive bacteria, may be beneficial. it suggests that chemotherapy with a single drug may be used in the fight against acne, which is preferred for the identified pathogen, minimizes the impact on the body's physiological flora, and reduces side effects and toxicity. our tests also showed that a br-p sample in an acid environment ph (bhi broth and bhi agar ph . ) had antibacterial activity. this is a promising result and therapeutically useful for cosmetic formulations requiring acidic ph. besides, antibacterial peptides active under acidic conditions have a protective effect against bacterial infections (the skin, oral cavity, urinary tract, and vagina possess acidic ph). a korean team [ ] noted that the high antibacterial properties of nod and nod peptides isolated from nordotis discus in an acidic environment might be useful when these peptides are applied as skin therapy in ph of about . . the clinically important measurement results of the selectivity index si value are interesting. the high values of the selectivity index indicated that the tested sample may be more effective against bacterial strains and safer for eukaryotic cells. thus, si values below determine a lack of therapeutic safety, and si values higher than allow to perform in vivo evaluation [ ] . si values of br-p for gram-positive strains were at least and, even compared to acne strains, had an extremely favorable selectivity of up to . low toxicity and good antibacterial activity result in a safe value on therapeutic index, suggesting that the obtained purified burdock root peptides could be promising as an anti-acne agent. other authors also report favorable, high si values of tested antimicrobial peptides (amps) from plants and the possibilities of their application in the cosmetics, medical, food, or agriculture industries [ ] [ ] [ ] [ ] [ ] . on the other hand, kumar et al. [ ] showed that, although many amps have reached clinical trials, not many of them have been approved by the us food and drug administration (fda) due to issues with toxicity. authors have pointed out strategies to improve the activity and biocompatibility of amps, such as chemical modifications and the use of delivery systems [ ] . another georgian-american team of scientists working on de novo amp synthesis pointed out that the design of amps with high therapeutic indexes, low cost of synthesis, high resistance to proteases, and high bioavailability remains a challenge. authors have developed a digital tool to predict amp algorithms for the design of de novo amps, and short peptides with high therapeutic indexes against gram-negative bacteria have been designed [ ] . due to the nature of acne skin diseases, anti-acne drugs should also have an antioxidative nature. free radicals are not only dangerous for normal and acne skin, but they also can lead to a variety of various human diseases (cardiovascular diseases, rheumatoid arthritis, alzheimer's disease, and cancer). according to some of the authors, regardless of the solvent used, the crude burdock root extract possessed significant antioxidant activity, as determined with dpph; thus, crude extract seems to be a promising antioxidant [ , , [ ] [ ] [ ] . in our work the dpph free radical scavenging assay was performed to assess the antioxidant capacity of the br-p peptide samples against the free dpph radical. the experiment showed potent antioxidant activity (range - µg/ml) (figure ). the ic value of br-p for dpph inhibition was found to be . µg/ml. the possibility of scavenging free radicals through burdock peptides is promising when applied to dermatological cosmetic formulations. it is significant that also proteins, polypeptides and peptides are proper antioxidants because they can inhibit biomolecule oxidation though some pathways (including inactivation of reactive oxygen species, scavenging free radicals, chelation of pro-oxidative transition metals, and reduction of hydroperoxides). changes to the tertiary protein structure increase the accessibility of amino acid residues that can scavenge free radicals and chelate pro-oxidative metals. peptides, not proteins, show the most promise as proteinaceous antioxidants, and a number of studies have shown that they have a substantially higher activity than proteins with a whole, intact structure [ , ] . for diseases with keratinolytic dysfunction, it is beneficial to combat both free radicals and the bacteria that cause these diseases, with a lack of cytotoxicity. low-weight molecular fractions of burdock root peptides exhibit such combined characteristics. in the last stage of the experiments, we made an antibacterial and antioxidant active dressing for acne skin. the dressing base created de novo with polysaccharides (chitosan and sodium alginate) was crosslinked by genipin and modified with br-p peptides. the hydrogel thus obtained, after freeze-drying, had a dry foam structure that was tested for bacterial biofilm adhesion. the br-p-modified dressing was found to have beneficial antibacterial properties; it showed no adhesion or poor adhesion of the tested bacteria to the surface compared to the unmodified control material, as evidenced by clsm images (figure ) . to use the developed modification of active br-p peptide from burdock roots to create commercial dressings, in the future, research should be carried out with clinical bacterial strains isolated from dermatological patients. it would be advisable to carry out in vivo tests that would confirm or exclude the effectiveness of antibacterial dressings in the prevention and treatment of peri-operative acne skin infections. burdock roots (arctium lappa l, bardanae radix dried, kawon, gostyń, poland) ( g) were grated in a mortar, and proteins were extracted with buffer ( mm na hpo , mm nah po , mm kcl, pepsin . mg/g defatted powder, mm edta, ph . ) by sonication at • c in a series of × min. ammonium sulphate was added to get % saturation, and precipitate obtained after h at room temperature was removed after centrifugation ( min at × g). collected supernatant was adjusted to % ammonium sulphate saturation, and precipitated proteins (after centrifugation at × g for min at • c) were resuspended in distilled water and dialyzed against distilled water using da cut-off dialysis tubing (sigma-aldrich, saint louis, mo, usa). the peptide fraction after dialyse was next purified by size exclusion chromatography on a sephadex g- (sigma-aldrich) on a column ( × cm) using the same buffer as that used in extraction. each eluted fraction ( ml) was collected and monitored for peptides at λ = nm. additionally, fractions were collected and analyzed using a screening antibacterial test on solid medium (br-f fractions). the samples active against bacteria were combined together and ultrafiltrated through a kda cut-off membrane (amicon). the antibacterial activity was again controlled after freeze-drying and treated as an active peptide fraction from burdock roots (br-p fractions). the freeze-dried extract br-p sample was weighed, the overall process yield was calculated, and then mg/ml stock solutions were prepared. maldi technique was used to monitor the peptide purification. for measuring molecular masses of the peptides, the br-p sample was analyzed on a matrix-assisted laser desorption/ionization time of flight mass spectrometer ultraflextreme (maldi tof/tof; bruker, bremen, germany). maldi analysis was performed according to a previously proposed procedure [ ] . briefly, . µl of peptide sample was mixed with the same volume of matrix saturated solution containing α-cyano- -hydroxycinnamic acid (hcca, bruker, bremen, germany) and , -dihydroxy benzoic acid (dhb, bruker, bremen, germany). next, the obtained mixture was spotted on an anchorchip maldi plate (bruker, bremen, germany) with prespotted hcca in acetone. analyzed br-p peptides were subjected to mild ionization using maldi-tof in the linear mode and recorded in active positive reflector mode within the - m/z range with laser frequency hz and shot counts. the collected spectrum was smoothed using the savitzky-golay method, baseline corrected (top hat baseline algorithm), and the list of peaks for the signal-to-noise ratio of > was generated using flex analysis . software (bruker, bremen, germany). the major peaks with signal-to-noise ratios greater than were assigned to rapiddenovo sequencing using fragmentation spectra obtained in ms/ms tandem mode. in this aim, the ms/ms data were loaded and recognized automatically, b-and y-ions were detected, and the software predicted the sequence or its fragment based on the peaks which were presented. automatically predicted sequences were given with their probability of occurrence. the most probable sequence (or sequence fragment) was next analyzed by the blast algorithm for finding regions of amino acid similarity in order to assign them to proteins from which they originated. we selected "protein-protein blast" as a search algorithm which simply compares a peptide fragment to a protein database. we chose only proteins from asteraceae family to compare and designated peptide fragments were % identical to the matched proteins. knowing a sequence of a given protein enabled the determination of the peptide sequence with a given mass (m/z) using peptidemass expasy [ ] . the peptide sequence was then analyzed for active fragments in the biopep-uwm database, while theoretical biological activity and isoelectric point were indicated [ ] . antibacterial assays were done using the micro-aerobic strains propionibacterium acnes pcm and propionibacterium acnes pcm as well as the aerobic strains staphylococcus aureus atcc , staphylococcus epidermidis atcc , pseudomonas aeruginosa atcc , and escherichia coli atcc as a microbial cause of acne appearance. before setting up the experiments, micro-aerobic bacteria were grown separately on bhi agar (biomaxima s.a., lublin, poland), ph . the preliminary antibacterial activity of the burdock roots samples against pathogenic gram-positive micro-aerobic and aerobic bacterial acne was evaluated by measuring the zones of inhibition in the disk diffusion method [ ] . antibacterial disc diffusion assays were carried out on petri plates with solid medium (bhi agar (ph . [ ] , or m-h agar). appropriate strain cultures were separately spread over the agar surface with a sterile cotton swab. next, each sample ( µl) was placed on petri plates with an agar medium. after h of incubation at • c (for aerobic strains) or h at • c (for micro-aerobic strains), zones of microbial growth created around the tested burdock samples were measured and recorded as the diameters of inhibition (mm). a broth microdilution method was used to evaluate the minimum inhibitory concentration (mic) according to the clsi document (clsi performance standards for antimicrobial susceptibility testing, eighteenth international supplement, clsi document m -mic, clinical laboratory standards institute, wayne) with some modifications. the lowest concentration of the tested compound (µg/ml) which did not result in any visible growth of bacteria was considered as the mic value. a serial doubling dilution of the burdock samples was prepared in -well plates ( µl per well). a suitable medium (m-h broth, bhi broth) was used as a diluent. the final concentrations of burdock samples were - . µg/ml. finally, µl of inoculum of the tested bacterial strain ( . × cfu/ml) was added to each well. the tests were performed either at • c for h (aerobic strains) or at h (micro-aerobic strains). after incubation, the panel was digitally analyzed at nm using the microplate reader bio tech synergy (usa) with a dedicated software system. the growth intensity in each well was compared to the negative (clear broth) and positive (broth with inoculum) controls. additionally, minimal bactericidal concentration (mbc) was obtained by spreading on agar µl of medium from the clear well, which did not show any visible growth after incubation during the mic test. the plates were incubated at • c for h, and the mbc was defined as the lowest concentration of sample without bacterial growth. each experiment was repeated in triplicate. the cell culture experiment was performed using a human skin fibroblast cell line (bj cells from american type culture collection atcc, uk). bj cells were cultured in emem supplemented with % fbs, u/ml penicillin, and µg/ml streptomycin and maintained at • c in a humidified atmosphere of % co and % air. the cells were seeded in -well plates in µl of a complete growth medium (supplemented with % fbs) at a concentration of . × cells/well and incubated for h at • c in a humidified atmosphere of % co . after this time, the br-p sample was firstly diluted in dmso to obtain the stock solution at mg/ml, and the solution was further diluted in culture medium supplemented with % fbs. subsequently, the culture medium was replaced with µl of the serial dilutions of the br-p peptide sample. untreated cells were used as a control of cytotoxicity, and different concentrations of dmso were used as a solvent control. the cell cultures were incubated at • c for h. cytotoxic effects were estimated using the mtt test. the experiment was repeated in three separate measurements. the half-maximal cytotoxic concentration (cc ) was defined as the sample concentration required to reduce cell viability to %. the cc values were calculated via four-parameter nonlinear regression analysis (graphpad prism , version . ) and were presented as mean values ± standard deviation (sd). the present study evaluated the antioxidant activity of br-p samples by employing a diphenylpicrylhydrazyl (dpph • ) (sigma-aldrich, usa) radical scavenging assay based on the abdel moneim method [ ] . this assay was conducted spectrophotometrically in -wells plates using digital reader bio tech synergy (usa). color disappearance of dpph ( µl . mm in % ethanol) after min was monitored at nm. this dpph decolouration, or lack thereof, was caused by the presence of µl tested samples in the concentration range - µg/ml. due to the precipitate formed (in some samples) during incubation (as a result of the ethanol of dpph with the sample combination), after min of incubation the mixture had to be centrifuged, the sediment rejected, and the clear solution read in a clean -well plate. ascorbic acid and glutathione (gsh) were used as standards for the , -diphenyl- -picrylhydrazyl scavenging assay. the following equation was used to calculate the percentage of dpph decolorization of the sample: % dpph • radical inhibition = [(abs control − abs sample)/abs control] × ( ) where abs is absorbance at nm. radical scavenging activity was expressed as the inhibition concentration (ic ), i.e., concentration of sample required to inhibit % of dpph free radicals. ic was determined graphically from the curve plot between the percentages of dpph scavenging activity and the sample's concentration. dressing material for acne skin disease treatment was created using natural polysaccharides crosslinked with genipin (sigma-aldrich, usa). briefly, . % chitosan ( - % deacetylation degree, - kda, sigma-aldrich, usa) and . % sodium alginate (sigma-aldrich, usa) were separately dissolved in ml of . % acetic acid and water, respectively. then, alginate was poured in portions into the chitosan standing on a magnetic stirrer. after the two polysaccharides were completely mixed, calcium β-glycerophosphate ( mg/ml) used as inorganic filler was added and mixed for min at • c. the obtained hydrogel was divided into two parts: one as an unmodified control and another modified with br-p ( . mg/ml) as the active substance. finally, % genipin ( . µg/ml) was added as the bifunctional cross-linker, with further mixing until a homogeneous mixture was obtained. the resulting modified and unmodified gels were transferred quantitatively into appropriate forms and left for h at room temperature for crosslinking. the final stage of dressing formation was deep frozen (− • c) and freeze-dried (lyo gt basic srk systemtechnik, gmbh, germany), resulting in discs with a diameter of about . mm and thickness of about . mm. modified and unmodified dressings were sterilized using a plastic/paper peel pouch in ethylene oxide prior to further testing. dry dressing samples (unmodified dressing as a control; dressing modified by br-p) were washed in µl of pbs and then transferred to the bottoms of -well polystyrene plates (cytoone, usa). each well with a disc sample inside was filled up with µl of appropriate broth (bhi or m-h). finally, µl of inoculum ( . × cfu/ml) was added to each well. for mono-species biofilm assay it was p. acnes pcm and s. aureus atcc . sterility controls (only m-h or bhi broth) were included in all experiments. to allow biofilm formation, i.e., the adhesion of planktonic forms of bacteria in the colonies attached to the biomaterial, the plates were incubated twice as long. namely, plates with aerobic bacteria were incubated for h, and with micro-aerobic bacteria h, both at • c. the tests were performed using three replicates. the viability of bacteria and their adhesion to the material surface were determined using double fluorescent staining for both live and dead bacteria using viability/cytotoxicity assay kit for bacteria live/dead cells (biotium, hayward, ca, usa). dressing samples with bacterial suspensions were prepared for confocal microscopy assay after the incubation. first, the medium from wells was removed and gently washed twice with µl of . % nacl, to remove the loosely adherent planktonic bacteria and to leave only the biofilm attached to the material. next, samples were transferred into fresh wells and filled with µl of . % nacl and live/dead dye. the solution of this dye was prepared by mixing µl of dmao with µl of ethd-iii in µl of . % nacl. three microliters of such obtained live/dead dye solution was added to disc-containing wells with µl of pbs. the samples were incubated for min at room temperature in the darkness, and bacterial colonies attached to dressing samples were visualized using a confocal microscope (clsm) with dedicated software. the results are expressed as mean ± rsd. statistical analysis was performed by analysis of variance (anova) tests, and statistical significance was set accordingly at the p = . level. our research allowed us to obtain promising results regarding the antibacterial and antioxidant activities of the tested burdock peptides. the obtained solution containing br-p peptides with a value below da from burdock roots had anti-acne properties and were not toxic to fibroblast cultures. these properties of active peptides have allowed them to be used to produce therapeutic dressing material (br-p/chitosan/sodium alginate) for use in infections against gram-positive bacteria in acne skin disease. one of the peptides is lrcdygrffasksldplkkrr, whose theoretical pi and bacterial permease ligand activity may possess antibiotic, anti-oxidative and anti-seborrheic properties. however, in-depth research is necessary on the biochemical analysis of the isolated br-p peptide fraction. supplementary materials: the following are available online at http://www.mdpi.com/ - / / / /s , figure s : title: br-p sample analyse in maldi/tof. ms/ms fragmentation spectra for rapiddenovo sequencing. antibiotic-resistant acne: lessons from europe magainins, a class of antimicrobial peptides from xenopus skin: isolation, characterization of two active forms, and partial cdna sequence of a precursor cell membrane lipid composition and distribution: implications for cell function and lessons learned from photoreceptors and platelets peptide antimicrobial agents new type of antimicrobial protein produced by the plant pathogen clavibacter michiganensis subsp. michiganensis antimicrobial peptides. ups phytamp: a database dedicated to antimicrobial plant peptides a hevein-like protein and a class i chitinase with antifungal activity from leaves of the paper mulberry purification and antipathogenic activity of lipid transfer proteins (ltps) from the leaves of arabidopsis and spinach de la canal, l. misctletoe viscotoxins induce membrane permeabilization and spore death in phytopathogenic fungi defensins-components of the innate immune system in plants, flowers and stems analysis of two novel classes of plant antifungal proteins from radish (raphanus sativus l.) seeds novel antifungal defensins from nigella sativa l. seeds expression of a novel small antimicrobial protein from the seeds of motherwort (leonurus japonicus) confers disease resistance in tobacco purification biochemical characterization and antifungal activity of a new lipid transfer protein (ltp) from coffea canephora seeds with α-amylase inhibitor properties an antimicrobial peptide ar-amp from amaranth (amaranthus retroflexus l.) seeds committee on herbal medicinal products (hmpc). assessment report on arctium lappa l. radix, european medicines agency antimicrobial assays of tyree native british plants used in anglo-saxon medicine for wound healing formulations in th century england antibiofilm effect of burdock (arctium lappa l.) leaf fraction and its efficiency in meat preservation antioxidant antifungal and anti-inflammatory effects of wild-growning romaniam native arctium lappa l. (asteraceae) and veronica perlica poiret (plantaginaceae) polysaccharides from arctium lappa l.: chemical structure and biological activity arctium lappa contributes to the management of type diabetes mellitus by regulating glucose homeostasis and improving oxidative stress: a critical review of in vitro and in vivo animal-based studies effects of aqueous extract of arctium lappa l. roots on serum lipid metabolism synthesis and biological activities of novel indole- -carbinol and (benzimidazolyl-methyl)triazole- . -diamine derivatives propionibacterium acnes: from commensal to opportunistic biofilm-associated implant pathogen antimicrobial activity of arctium lappa constituents against microorganisms commonly found in endodontic infections in vitro evaluation of the antibacterial activity of arctium lappa as a phytotherapeutic agent used in intracanal dressings arctium lappa) leaf extracts increase the in vitro antimicrobial efficacy of common antibiotics on gram-positive and gram-negative bacteria natural arctium lappa fruit extract improves the clinical signs of aging skin comparative analysis of caffeoylquinic acids and lignans in roots and seeds among various burdock (arctium lappa) genotypes with high antioxidant activity cell-penetrating antimicrobial peptides-prospectives for targeting intracellular infections transpoter classification database antibacterial action of new antibacterial peptides, nod and nod , isolated from nordotis discus discus cell-penetrating peptides as a novel transdermal drug delivery system phaseococcin, an antifungal protein with antiproliferative and anti-hiv- reverse transcriptase activities from small scarlet runner beans plant antimicrobial peptides plant defensins: defense. development and application antimicrobial activity, improved cell selectivity and mode of action of short pmap- -derived peptides against bacteria and candida antimicrobial peptides: diversity. mechanism of action and strategies to improve the activity and de novo design and in vitro testing of antimicrobial peptides against gram-negative bacteria antioxidative and in vitro antiproliferative activity of arctium lappa root extracts hydrophilic antioxidant capacities of vegetables and fruits commonly consumed in japan and estimated average daily intake of hydrophilic antioxidants from these foods caffeoylquinic acid derivatives from the roots of arctium lappa l. (burdock) and their structure-activity relationships (sars) of free radical scavenging activities antioxidant activity of proteins and peptides purification and identification of novel antioxidant peptides from enzymatic hydrolysate of ginkgo biloba seed proteins matrix-assisted laser desorption/ionization time of flight (maldi-tof) mass spectrometric analysis of intact proteins larger than kda biopep-uwm database of bioactive peptides current opportunities manual of clinical microbiology the neuroprotective effects of purslane (portulaca oleracea) on rotenone-induced biochemical changes and apoptosis in brain of rat this article is an open access article distributed under the terms and conditions of the creative commons attribution key: cord- -b yuh y authors: shi, yanhong; zheng, cheng; li, jinhang; yang, li; wang, zhengtao; wang, rui title: separation and quantification of four main chiral glucosinolates in radix isatidis and its granules using high-performance liquid chromatography/diode array detector coupled with circular dichroism detection date: - - journal: molecules doi: . /molecules sha: doc_id: cord_uid: b yuh y as chemical drugs, separation and quantification of the specific enantiomer from the chiral compounds in herbal medicines are becoming more important. to clarify the chemical characterization of chiral glucosinolates—the antiviral active ingredients of radix isatidis, an optimized efficient method of hplc-uv-cd was developed to simultaneously separate and quantify the four main chiral glucosinolates: progoitrin, epiprogoitrin, and r,s-goitrin. the first step was to determine progoitrin, epiprogoitrin, and r,s-goitrin using hplc-uv, and then determine the r-goitrin and s-goitrin by coupling with cd detection. subsequently, through the linear relations between anisotropy factor (g factor) and the percent optical purity of r-goitrin, the contents of r-goitrin and s-goitrin from the r,s-goitrin mixture were calculated separately. furthermore, the chemical composition features of the four chiral glucosinolates in samples from crude drugs, decoction pieces, and granules of r. isatidis were conducted. the total content of the four glucosinolates was obviously higher in crude drugs, and the variance character of each glucosinolate contents was different. in summary, the accurate measurement method reported here allows for better control of the internal quality of r. isatidis and its granules and provides a powerful approach for the analysis of other chiral components in traditional chinese medicines. natural products from traditional chinese medicines (tcms) represent a major part of today's pharmaceutical market as they possess a number of biological activities, such as antimicrobial, antitumor, anti-inflammatory, antiviral, cardiovascular activities, and so on [ ] . since chirality is a fundamental characteristic of nature, a large number of well-known therapeutic ingredients from elution were compared. comparison with different compositions of acetonitrile/methanol-water, methanol-water with different modifiers including formic acid and ammonium acetate showed the combination of methanol and mm ammonium acetate (adjusted ph for . with formic acid) gave an optimal mobile phase system for glucosinolates separation. as shown in figure s , in order to acquire better peak intensity, five uv wavelengths- , , , , nm-were detected and nm gave the best results. in addition, the maximum absorption cd wavelength of rand s-goitrin was focused on and nm, respectively, whereas the detection range of the present cd- detector was - nm. therefore, the cd wavelength was set at nm. as shown in table , all calibration curves of the glucosinolates showed good linearity (r > . ) within a relatively wide range of concentration ( . - . mg/ml). the limit of detection (lod) and the limit of quantification (loq) were lower than . and . µg/ml, respectively. table . regression equations, linear range, limit of detection (lod) and limit of quantification (loq) for three analytes. the known purities of r-goitrin (%) and s-goitrin (%) were mixed and simultaneously determined by uv and cd to calculate the g factors ( table ). the relative purities of r-goitrin (%) and g factors made calibration curves. the calibration curve between g factors (x ) and the different relative purities of r-goitrin (y ) was y = − . x + . (r = . ), which showed a good linearity with a wide range of r-goitrin purities ( ~ %). the precision, repeatability, and stability of the developed method were also validated for each analyte, and the rsd values were less than . %. the recoveries of five glucosinolates were measured from . to . %, and the rsd values were less than . % (table ) . compared to previous published methods [ ] [ ] [ ] [ ] , glucosinolates profiles of crude drug, decoction pieces, and granules of r. isatidis were performed using hplc-uv-cd, which improved the limitation of high-cost chiral columns, complex operation of sample preparation, and simultaneous quantification of the characteristic glucosinolates. in the typical uv and cd chromatograms of r. isatidis samples (figure ), r-goitrin and s-goitrin showed the same retention time, uv absorption feature, and overlapping peak. they could not be separated by uv detection without using the specific chiral columns ( figure a ,b; upper). in contrast to that, r-goitrin and s-goitrin had completely opposite chromatographic profiles at the same retention time as shown in cd chromatograms ( figure a ,b; lower). notably, in the typical cd chromatograms, only r-goitrin could be detected in the mixed r,s-goitrin, crude drugs, decoction pieces, and granules of r. isatidis. therefore, the r-goitrin content could be quantified using cd detector, while the s-goitrin content was calculated using the mixed r,s-goitrin and the determined r-goitrin content. combining the cd chromatographic properties with total content of the mixed r,s-goitrin, it provided an efficient method to determine the content of r-goitrin in the mixed r,s-goitrin without chiral separation. compared to previous published methods [ ] [ ] [ ] [ ] , glucosinolates profiles of crude drug, decoction pieces, and granules of r. isatidis were performed using hplc-uv-cd, which improved the limitation of high-cost chiral columns, complex operation of sample preparation, and simultaneous quantification of the characteristic glucosinolates. in the typical uv and cd chromatograms of r. isatidis samples (figure ), r-goitrin and s-goitrin showed the same retention time, uv absorption feature, and overlapping peak. they could not be separated by uv detection without using the specific chiral columns ( figure a ,b; upper). in contrast to that, r-goitrin and s-goitrin had completely opposite chromatographic profiles at the same retention time as shown in cd chromatograms ( figure a ,b; lower). notably, in the typical cd chromatograms, only r-goitrin could be detected in the mixed r,s-goitrin, crude drugs, decoction pieces, and granules of r. isatidis. therefore, the r-goitrin content could be quantified using cd detector, while the s-goitrin content was calculated using the mixed r,s-goitrin and the determined r-goitrin content. combining the cd chromatographic properties with total content of the mixed r,s-goitrin, it provided an efficient method to determine the content of r-goitrin in the mixed r,s-goitrin without chiral separation. as shown in figure , the chemical composition of progoitrin, epiprogoitrin, and r,s-goitrin in crude drugs, decoction pieces, and granules of r. isatidis showed that the chromatographic patterns were found to be consistent between all samples but their contents obviously varied. the three analytes could be easily detected in the uv chromatograms, while only r-goitrin was determined in cd chromatograms. we think the different absorption intensity between uv and cd detection might be due to the differences in chemical structures. therefore, the content variations of these characteristic glucosinolates should be conducted. the contents of four glucosinolates-progoitrin, epiprogoitrin, r-goitrin, and s-goitrin-were simultaneously quantified in samples using hplc-uv coupled with cd detection. as shown in figure and table s , the total contents of four glucosinolates in three types of r. isatidis were significantly different. it was obviously higher in crude drugs ( . - . mg/g, mean ± sd: . ± . ) and decoction pieces ( . - . mg/g, mean ± sd: . ± . ), while the glucosinolates occupied relatively lower contents in its granules ( . - . mg/g, mean ± sd: . ± . ). as shown in figure , the chemical composition of progoitrin, epiprogoitrin, and r,s-goitrin in crude drugs, decoction pieces, and granules of r. isatidis showed that the chromatographic patterns were found to be consistent between all samples but their contents obviously varied. the three analytes could be easily detected in the uv chromatograms, while only r-goitrin was determined in cd chromatograms. we think the different absorption intensity between uv and cd detection might be due to the differences in chemical structures. therefore, the content variations of these characteristic glucosinolates should be conducted. the contents of four glucosinolates-progoitrin, epiprogoitrin, r-goitrin, and s-goitrin-were simultaneously quantified in samples using hplc-uv coupled with cd detection. as shown in figure and table s , the total contents of four glucosinolates in three types of r. isatidis were significantly different. it was obviously higher in crude drugs ( . - . mg/g, mean ± sd: . ± . ) and decoction pieces ( . - . mg/g, mean ± sd: . ± . ), while the glucosinolates occupied relatively lower contents in its granules ( . - . mg/g, mean ± sd: . ± . ). most previous studies have focused only on the content of r,s-goitrin mixture in r. isatidis and its related products. in the present study, r-goitrin accounted for a majority of the r,s-goitrin component in crude drugs, decoction pieces, and granules of r. isatidis; the r-goitrin content was twice as much as that of s-goitrin. the results might provide positive evidence that r-goitrin is responsible for the pharmacological properties of r. isatidis. it was noticed that progoitrin and epiprogoitrin contents showed an obvious declining trend in crude drugs and decoction pieces of r. isatidis. in particular, they could not be detected in most of the granule samples. compared with crude drugs, r-goitrin and s-goitrin contents in decoction most previous studies have focused only on the content of r,s-goitrin mixture in r. isatidis and its related products. in the present study, r-goitrin accounted for a majority of the r,s-goitrin component in crude drugs, decoction pieces, and granules of r. isatidis; the r-goitrin content was twice as much as that of s-goitrin. the results might provide positive evidence that r-goitrin is responsible for the pharmacological properties of r. isatidis. it was noticed that progoitrin and epiprogoitrin contents showed an obvious declining trend in crude drugs and decoction pieces of r. isatidis. in particular, they could not be detected in most of the granule samples. compared with crude drugs, r-goitrin and s-goitrin contents in decoction pieces increased slightly. the traditional processing and extraction methods could improve the biotransformation of progoitrin and epiprogoitrin, and then increase the content of the degradation products (r-and s-goitrin). this is consistent with our previous report [ , ] . however, the content dynamic change features of the glucosinolates in crude drugs, decoction pieces, and granules as well as the main influencing parameters for biotransformation among these glucosinolates need to be further investigated. fifteen crude drugs (b -b ), nine decoction pieces (b -b ), and thirteen granules (b -b ) of r. isatidis were collected from the main commercial herbal markets and different herbal manufactories in china (table s ). the vouchers have been deposited in shanghai r&d centre for standardization of chinese medicines, shanghai university of traditional chinese medicine. the four glucosinolates-epiprogoitrin, progoitrin, and r,s-goitrin-were isolated and purified from the root of i. indigotica fort. in our laboratory. r-goitrin and s-goitrin were prepared from r,s-goitrin using the shiseido ® cd-ph chiral column ( µm, × . mm) with acetonitrile-water ( : , v/v). their chemical structures ( figure ) were elucidated by a series of spectroscopic and chemical analyses [ ] . the purities of five glucosinolates were determined to be higher than . % through hplc-dad analysis. hplc-grade acetonitrile, methanol, formic acid (≥ . %, hplc grade), and ammonium acetate (≥ . %, hplc grade) for hplc analysis were purchased from thermo fisher scientific (swedesboro, nj, usa). water was prepared by a milli-q ® system (millipore, ma, usa). all other reagents of analytical grade for extraction were purchased from tianjin damao chemical reagent factory (tianjin, china). molecules , , x for peer review of pieces increased slightly. the traditional processing and extraction methods could improve the biotransformation of progoitrin and epiprogoitrin, and then increase the content of the degradation products (r-and s-goitrin). this is consistent with our previous report [ , ] . however, the content dynamic change features of the glucosinolates in crude drugs, decoction pieces, and granules as well as the main influencing parameters for biotransformation among these glucosinolates need to be further investigated. fifteen crude drugs (b -b ), nine decoction pieces (b -b ), and thirteen granules (b -b ) of r. isatidis were collected from the main commercial herbal markets and different herbal manufactories in china (table s ). the vouchers have been deposited in shanghai r&d centre for standardization of chinese medicines, shanghai university of traditional chinese medicine. the four glucosinolates-epiprogoitrin, progoitrin, and r,s-goitrin-were isolated and purified from the root of i. indigotica fort. in our laboratory. r-goitrin and s-goitrin were prepared from r,s-goitrin using the shiseido ® cd-ph chiral column ( μm, × . mm) with acetonitrile-water ( : , v/v). their chemical structures ( figure ) were elucidated by a series of spectroscopic and chemical analyses [ ] . the purities of five glucosinolates were determined to be higher than . % through hplc-dad analysis. hplc-grade acetonitrile, methanol, formic acid (≥ . %, hplc grade), and ammonium acetate (≥ . %, hplc grade) for hplc analysis were purchased from thermo fisher scientific (swedesboro, nj, usa). water was prepared by a milli-q ® system (millipore, ma, usa). all other reagents of analytical grade for extraction were purchased from tianjin damao chemical reagent factory (tianjin, china). hplc system (agilent , santa clara, ca, usa) was equipped with a g dad, a quaternary pump and an online degasser. a cd detector (cd- , jasco, tokyo, japan) was epiprogoitrin ( ) progoitrin ( ) s-goitrin ( b) r-goitrin ( a) detector for the chiral analysis. the mobile phase consisted of methanol (a) and mm ammonium acetate (adjusted ph for . with formic acid b). the sample solutions were analyzed on a ultimate aq-c column ( µm, × . mm) with a gradient elution system as follows: - min, % a; - min, - % a; - min, % a. the flow rate was . ml/min, and the column temperature was set at • c. uv and cd detective wavelengths were set at nm and nm, respectively. each of the five reference compounds was accurately weighed and then dissolved in water to prepare the standard solutions of . mg/ml for progoitrin, . mg/ml for epiprogoitrin, . mg/ml for r,s-goitrin, . mg/ml for r-goitrin, and . mg/ml for s-goitrin. for uv detection, a series of standard solutions were prepared by appropriate dilution of the stock solutions to make calibration curves. the calibration curve was obtained by plotting peak area (y ) of each reference glucosinolate against the concentration of the corresponding compound (x ). furthermore, the known purities of r-goitrin (%) were determined by uv and cd to calculate the anisotropy factor (g = ∆a/a). the calibration curve depended on g factors (y ) of each reference r-goitrin against the relative purities of r-goitrin (x ). crude drugs and decoction pieces: the respective sample was pulverized to obtain homogeneous fine powder. . g of the fine powder was accurately weighed and extracted with ml water by refluxing in a water bath for h, then cooled and filtered. ml of the filtrate was dissolved with ml water containing % formic acid (v/v). the mixed solution was packed into a spe column (waters oasis wax cc cartridge/ mg, waters, usa), which was activated with ml methanol and then washed with ml water before adding sample solution. the spe column was orderly eluted with ml water containing % formic acid, ml methanol (a) and ml methanol containing % ammonium hydroxide (v/v, b). subsequently, the eluted solutions of a and b was dried by flushing with nitrogen (n ). the residue was dissolved with . ml methanol for hplc analysis. granules: the granules were pulverized and screened through a µm sieve to obtain homogeneous fine powder. . g (containing sugar) and . g (no sugar) of the fine powder was accurately weighed and extracted with ml water by ultrasonication at room temperature for min, then cooled and filtered. ml of the filtrate was dissolved with ml water containing % formic acid (v/v). the mixed solution was packed into a spe column. subsequently, the sample solution was prepared through the same operation procedure with sample solutions of crude drugs and decoction pieces. linearity was assessed by generating six-point calibration curves for each reference compound. the precision was evaluated by replicate injections of a mixture solution containing four glucosinolates and a sample solution of r. isatidis (b ). six injections of the same preparation solutions per day were investigated for three days. six preparation solutions of a sample (b ) were analyzed for repeatability. to determine the recovery rate of extraction, the four reference components (approximately %, % and % of the original amount) were added to the weighed powder of r. isatidis that was extracted and analyzed. as the cd signal depends only on the enantiomeric composition of the chiral molecule whereas uv absorbance is related to the analyte concentration, the measurement method of the enantiomeric content from the chiral sample was referred to the reported literatures [ , ] . firstly, the cd detector recorded both dichroic (∆ε or ∆a) and uv (ε or a) signals at the optimized wavelength and calculated the anisotropy factor (g = ∆ε/ε or ∆a/a), which is dependent of the enantiomeric purity. secondly, screening for the bioactive constituents of traditional chinese medicine-progress and challenges the market of chiral drugs: chiral switches versus de novo enantiomerically pure compounds putting chirality to work: the strategy of chiral switches fda drug approvals recent progress in the studies of chemical consituents, pharmacological effects and quality control methods on the roots of isatis indigotica english), part i; china medical science and technology press progress of studies on chemical constituents and pharmacological effects of radix isatidis. chin. wild plant reour efficacy of treatment if influenza a (h n ) with oseltamivir phosphate and isatis root granules antiviral activity of isatis indigotica extract and its derived indirubin against japanese encephalitis virus. evid. based complement. altern in vitro inhibition of influenza virus infection by a crude extract from isatis indigotica root resulting in the prevention of viral attachment progress of studies on antiviral effective constituents from radix isatidis antivirus constituents of radix of isatis indigotica the in vitro anti-virus effects and dose-effect relationship of epigoitrin and fructopyrano-( → )-glucopyranose based on "deletion/increment" strategy determination of r,s-goitrin of banlangen preparation by rp-hplc biotransformation of glucosinolates epiprogoitrin and progoitrin to (r)-and (s)-goitrin in radix isatidis quantitative and chemical fingerprint analysis for the quality evaluation of isatis indigotica based on ultra-performace liquid chromatography with photodiode array detector combined with chemometric methods - -vinyl- -thiooxazolidone, an antithyroid compound from yellow turip and from brassica seeds determination of epigoitrin and goitrin in isatidis radix by chiral high performance liquid chromatography. chin stereospecific assay of (r)-and (s)-goitrin in commercial formulation of radix isatidis by reversed phase high-performance liqiud chromatography qualitative and quantitative analyses of goitrin-epigoitrin in isatis indigotica using supercritical fluid chromatography-photodiode array detector-mass spectrometry improved chiral separation of (r,s)-goitrin by sfc: an application in traditional chinese medicine an efficient method for separation and purification of glucosinolate stereoisomers from radix isatidis chiral analysis of milnacipran by a nonchrial hplc-circular dichroism: improvement of the linearity of dichroic response by temperature control reliable assay of extreme enantiomeric purity values by a new circular dichroism based on hplc detection system author contributions: l.y., z.w., and r.w. designed the research; y.s., c.z., and j.l. performed the experiments and analyzed the data; y.s. and c.z. wrote the paper. all of the authors have read and approved the content of the manuscript. the authors declare no conflict of interest. of through the linear relations between g factor and the percent optical purity of the enantiomeric, the relative proportion of the enantiomeric in the chiral molecule were calculated. finally, the absolute content of enantiomeric in the chiral molecule was measured by its relative purity and uv absorbance. the present study proposed an efficient method of hplc-uv-cd for simultaneous separation and quantification of the main chiral glucosinolates in r. isatidis. progoitrin, epiprogoitrin and r,s-goitrin were easily determined by uv detection, whereas r-goitrin and s-goitrin were separated by coupling with cd detection. through the linear relations between g factor and the percent optical purities of r-goitrin, the contents of r-goitrin and s-goitrin were successfully calculated. subsequently, glucosinolate profiles in crude drugs, decoction pieces and granules of r. isatidis were conducted and found that the variance character of each of the glucosinolate contents was different. in summary, the new method allows better control of the internal quality of r. isatidis and its granules and provides a powerful approach and foundation for further investigation of dynamic change features of the contents and the main influencing parameters for biotransformation of glucosinolates in r. isatidis. the following are available online. figure s : the cd absorbance features of rand s-goitrin (a), maximum uv absorbance spectrum of the four glucosinolates (b), and the chemical profiling of the four glucosinolates in five different wavelength (c); table s : crude drugs, decoction pieces and granules of radix isatidis used in this study; table s : the contents of four glucosinolates in samples from crude drugs, decoction pieces and granules of radix isatidis (n = ). key: cord- - q zg authors: illesca, paola; valenzuela, rodrigo; espinosa, alejandra; echeverría, francisca; soto-alarcon, sandra; campos, cristian; rodriguez, alicia; vargas, romina; magrone, thea; videla, luis a. title: protective effects of eicosapentaenoic acid plus hydroxytyrosol supplementation against white adipose tissue abnormalities in mice fed a high-fat diet date: - - journal: molecules doi: . /molecules sha: doc_id: cord_uid: q zg objective: obesity induced by high-fat diet (hfd) elicits white adipose tissue dysfunction. in this study, we have hypothesized that the metabolic modulator eicosapentaenoic acid (epa) combined with the antioxidant hydroxytyrosol (ht) attenuates hfd-induced white adipose tissue (wat) alterations. methods: c bl/ j mice were administered with a hfd ( % fat, % protein, % carbohydrates) or control diet (cd; % fat, % protein, % carbohydrates), with or without epa ( mg/kg/day), ht ( mg/kg/day), or both for weeks. determinations in wat include morphological parameters, epa and docosahexaenoic acid content in phospholipids (gas chromatography), lipogenesis, oxidative stress (os) and inflammation markers, and gene expression and activities of transcription factors, such as sterol regulatory element-binding protein- c (srebp- c), peroxisome proliferator-activated receptor-gamma (ppar-γ), nuclear factor kappa-light-chain-enhancer of activated b cells (nf-κb) (p subunit) and nuclear factor erythroid -related factor (nrf ) (quantitative polymerase chain reaction and enzyme linked immunosorbent assay). results: hfd led to wat hypertrophy in relation to ppar-γ downregulation. wat metabolic dysfunction was characterized by upregulation of lipogenic srebp- c system, mitochondrial energy metabolism depression, loss of the antioxidant nrf signaling with os enhancement, n- long-chain polyunsaturated fatty acids depletion and activation of the pro-inflammatory nf-κb system. epa and ht co-supplementation diminished hfd-dependent effects additively, reaching values close or similar to controls. conclusion: data presented strengthen the importance of combined protocols such as epa plus ht to attenuate metabolic-inflammatory states triggered by obesity. assay (cayman chemical company, ann arbor, mi, usa) after adjusting the total protein concentration to . mg/ml per sample. reduced glutathione (gsh) and glutathione disulfide (gssg) contents in ewat were assessed with an enzymatic recycling method described by rahman et al. [ ] . samples of ewat were homogenized in three volumes of phosphate buffer ( mm), ph . containing edta ( mm) and sucrose ( mm) for determination of antioxidant enzyme activities. the homogenate was subjected to a first centrifugation ( × g, min, • c) and one supernatant aliquot was used to evaluate superoxide dismutase (sod) (cayman chemical co., ann arbor, mi, usa) and catalase (cat) activities [ ] . subsequently, the remaining supernatants were centrifuged at , × g for min at • c to perform glutathione peroxidase (gpx) and glutathione reductase (gr) assays [ ] . on the other hand, ewat samples were homogenized in three volumes of buffer, ph , containing kh po ( mm), k hpo ( mm), dithiothreitol (dtt) ( mm) and khco ( mm) and centrifuged ( × g, min, • c). the fat of the upper layers were discarded, the supernatants were again centrifuged at , × g for min at • c and the aqueous fractions were used to study acetyl-coa carboxylase (acc), fatty acid synthase (fas) and the glucose- -phosphate dehydrogenase (g pd) activities as previously described [ , ] . to assay malic enzyme (me) activity, ewat homogenates were centrifuged for min at , × g at • c, then the me activity was evaluated in the resulting supernatant following the method performed by wise and ball [ ] . in addition, the activity of lipoprotein lipase (lpl) (enzyme associated with the tissue incorporation of fatty acids) was measured in an epididymal fat pad according to llobera et al. [ ] . the protein content in enzyme extracts was determined by the bradford assay (bio-rad reagent, california, usa). nuclear extracts from ewat were obtained using a commercial extraction kit (cayman chemical company number - - , ann arbor, mi, usa). srebp- c, ppar-γ, nuclear factor-kb (nf-κb) (p subunit) and nuclear factor e -related factor (nrf ) dna-binding activities were assessed with a commercial elisa kit (cayman chemical company, number . srebp- c, number by ppar-γ, number by nf-κb (p ) and number by nrf , ann arbor, mi, usa) and according to the manufacturer's instructions. values were expressed as percentage of srebp- c, ppar-γ, nf-κb (p subunit) and nrf dna-binding with respect to a positive control provided by the respective elisa kit. total rna was isolated from ewat samples using trizol (invitrogen, carlsbad, ca, usa), according to the supplier's protocols. purified rna ( µg) was then treated with dnaase i (dna free kit; ambion, austin, tx, usa) and used to generate first-strand cdna with moloney murine leukemia virus reverse transcriptase (invitrogen, carlsbad, ca, usa), utilizing random hexamers (invitrogen, carlsbad, ca, usa) and deoxynucleotide triphosphate (dntp) mix (bioline, london, united kingdom), according to the manufacturer's protocol. the resultant cdna was amplified with specific primers for mice in a total volume of µl. gene-specific primer sequences used are shown in table . primers were optimized to yield %- % of reaction efficiency with pcr products by development in agarose gel to verify the correct amplification length. the quality of the dna extracted was checked by agarose gel ( %) electrophoresis according to lee et al. [ ] . briefly, samples were mixed with gel loading dye ( . % bromophenol blue, . % xylene cyanol and % glycerol). electrophoresis was run at volt for min. dna was stained using gelred ® nucleic acid gel stain (biotum, fremont, ca, usa). real-time pcr was performed in a stratagen mx p system using brilliant ii sybr ® green quantitative real time pcr master mix (agilent technologies, la jolla, ca, usa) following the manufacturer's recommendations (applied biosystems, foster city, ca, usa). all the expression levels of target genes under study were normalized by the expression of β-actin as an internal control (applied biosystems, foster city, ca, usa). fold changes between groups were calculated by the -(∆∆ct) method, as established by pfaffl [ ] and livak and schmittgen [ ] . sequences are listed in the → direction. nuclear factor erythroid -related factor (nrf ), glutathione s-transferase (gst), glutamate-cysteine ligase (gcl), sterol regulatory element-binding protein- c (srebp- c), fatty acid synthase (fas), acetyl-coa carboxylase (acc), peroxisome proliferator-activated receptor-gamma (ppar-γ), fatty acid transport protein (fatp ), lipoprotein lipase (lpl), nuclear factor kappa-light-chain-enhancer of activated b cells (nf-κb), tumor necrosis factor alpha (tnf-α), interleukin (il- ), beta actin (β-actin). molecules , , of statistical analysis was performed with graphpad prism . software (graphpad prism, inc., san diego, usa). the values shown represent the mean ± standard deviation (sd) for - animals per each experimental group. evaluation of normality data distribution was performed using the shapiro-wilk test. assessment of the statistical significance of differences between mean values was performed by two-way analysis of variance (anova) and the tukey's test. a p < . was considered significant. the body weight (bw) of mice at the beginning of the study was similar. after weeks, all groups fed with cd, with or without supplementation, exhibited comparable final bw. hfd led to an increase in bw gain of % (p < . ), compared with cd groups, which is related with to augmented adiposity (table a , group e). in fact, all fat pads evaluated, namely, subcutaneous, perirenal and epididymal, in mice fed the hfd showed significant weight increases (table b) , therefore the total mass of wat increased considerably ( %; p < . ) over the cd group. dietary intake was comparable in all experimental groups, but energy consumption was higher in mice subjected to the hfd with or without supplementation over cd groups (data not shown). epa and epa + ht supplementation diminished bw gain produced by the hfd by % and % respectively (p < . ), accompanied with a decrease in fat pads and total wat of % and % (p < . ), respectively (table b ). ht administration was not able to prevent the increased bw observed in hdf-fed mice; however, it reduced the localized and total adiposity (total wat) by % (table b) . specific and morphological parameters were evaluated in the ewat (table c ). the expansion of epididymal fat pad of hfd-fed mice exhibited a lower number of cells per gram of wat, adipocytes of increased size with augmented tag content compared with the control group, indicating hypertrophy. the supplementation with epa, ht or both significantly decreased the hypertrophy observed in wat of the hfd-fed mice, improving all the parameters evaluated without observing differences between treatments and without reaching control values (table c) . levels of serum adipokines are shown in the table d . serum adiponectin and leptin were not modified by supplementation when mice were fed the cd. however, the hfd significantly decreased serum adiponectin levels ( %; p < . ) while increasing those of leptin ( %; p < . ). supplementation with epa, ht or epa + ht improved the profile of serum adipokines in hfd-fed mice, showing a similar effect independently of the treatment, although the values did not reach those observed in cd-fed mice. comparable content of total saturated fatty acids (sfa), monounsaturated fatty acids (mufa) and polyunsaturated fatty acids (pufa) were found in ewat phospholipids of all groups receiving cd. hfd-fed mice exhibit an increase of total sfa and decrease of total pufa (table ) . regarding the ewat palmitate levels, comparable values were observed in all groups fed with cd, a significant increment in hfd saline group, and a decrease caused by the administration of epa or ht alone, with the total recovery towards cd values when supplemented with epa+ht (table ) . moreover, hfd led to a severe reduction in epa content ( %; p < . ) with consequent decrease in dha content ( %; p < . ) in ewat phospholipids compared to cd-fed animals ( table ). the high content of epa in the adipose tissue of the animals that received the supplementation evidence the effectiveness of the treatment, increasing by . -fold and -fold epa levels in cd groups supplemented with epa and epa+ht, respectively. moreover, the supplementation with epa to hfd-mice (epa and epa+ht groups) restored the depleted levels of epa in ewat phospholipids reaching control levels. the incorporation of epa into ewat also improved the content of dha in this tissue, increasing dha levels in cd animals, with normalization in hfd mice achieving levels observed in cd group (table ) . it should be noted that in the hfd-fed group that received ht exclusively, the levels of epa and dha were also increased in ewat phospholipids, confirming previous results [ , ] . surprisingly, the effect of the supplements given together, improved the tissue content of epa and dha even more than when each administered individually in animals fed hfd. furthermore, epa reduced the n- /n- ratio ewat of cd animals, and ameliorate this ratio in both groups of mice fed hfd that received epa supplementation. also, ht given alone diminished the ratio n- /n- in ewat of hfd-mice, an effect that was more pronounce after epa and ht co-supplementation (table ) . table . general and metabolic parameters in control diet (cd) and high-fat diet (hfd)-fed mice subjected to eicosapentaenoic acid (epa), hydroxytyrosol (ht) or epa + ht supplementation. values are expressed as mean ± sd; eight to ten mice were included in each experimental group. significant differences between the groups are indicated by the letter identifying each values are expressed as mean ± standard deviation (sd). eight to ten mice were included in each experimental group. significant differences between the groups are indicated by the letter identifying each group, namely, (a monounsaturated fatty acids (mufas) correspond to c : n- , c : n- and c : n- . polyunsaturated fatty acids (pufas) correspond to c : n- , c : n- , c : n- , c : n- , c : n- and c : n- . n- lcpufas are c : n- and c : n- , n- lcpufas are c : n- , c : n- and c : n- , n- /n- lcpufas ratio: c : n- /(c : n- + c : n- + c : n- ). the treatments showed no effect on any of the parameters associated with oxidative stress evaluated in ewat of cd-fed mice ( figure ). hfd feeding induced oxidative stress in ewat, as evidenced by the increase in markers of oxidative damage tbars ( figure a ), f -isoprostanes ( figure b ) and protein carbonyls ( figure c ) accompanied by alteration of the glutathione status ( figure d -g). supplementation with epa alone only reduced levels of protein carbonyls ( figure c ), without improving the rest of the parameters evaluated in ewat of hfd animals ( figure a ,b,d-g). when ht was given alone, tbars and f -isoprostanes levels were reduced ( figure a ,b), and the content of protein carbonyls ( figure c ) and glutathione levels ( figure d -g) were normalized, reaching control values. notably, the administration of both supplements, epa + ht, prevented oxidative damage in all parameters evaluated and deterioration of glutathione status in ewat of mice receiving the hfd ( figure a -d). molecules , , x for peer review of the treatments showed no effect on any of the parameters associated with oxidative stress evaluated in ewat of cd-fed mice (figure ). hfd feeding induced oxidative stress in ewat, as evidenced by the increase in markers of oxidative damage tbars ( figure a ), f -isoprostanes ( figure b ) and protein carbonyls ( figure c ) accompanied by alteration of the glutathione status ( figure d -g). supplementation with epa alone only reduced levels of protein carbonyls ( figure c ), without improving the rest of the parameters evaluated in ewat of hfd animals ( figure a ,b,d-g). when ht was given alone, tbars and f -isoprostanes levels were reduced ( figure a and b), and the content of protein carbonyls ( figure c ) and glutathione levels ( figure d -g) were normalized, reaching control values. notably, the administration of both supplements, epa + ht, prevented oxidative damage in all parameters evaluated and deterioration of glutathione status in ewat of mice receiving the hfd ( figure a-d) . the fall in the mrna levels and the dna binding capacity impaired of the cytoprotective transcription factor nrf in ewat of hfd-fed mice were partially recovered when animals received epa or ht supplementation (figure a,b) . likewise, the hfd-induced diminution in the expression levels of its downstream genes glutamate-cysteine ligase (gcl) ( figure c ) and glutathione-s-transferase (gst) ( figure d ) involved in glutathione metabolism were equally improved by epa or ht. the supplementation with epa plus ht in hfd mice showed an even greater effect, restoring the expression levels of nrf , gcl and gst to control values, and normalizing the dna binding activity of nrf (figure a-d) . a similar behavior was observed in the activity of antioxidant enzymes cat ( figure e ), sod ( figure f ), gr ( figure g ) and gpx ( figure h ) in ewat of hfd-mice that received epa, ht or both supplements, which were partially enhanced by epa or ht after reduction by hfd and totally recovered by combined epa and ht ( figure d-h) . molecules , , x for peer review of the fall in the mrna levels and the dna binding capacity impaired of the cytoprotective transcription factor nrf in ewat of hfd-fed mice were partially recovered when animals received epa or ht supplementation (figure a,b) . likewise, the hfd-induced diminution in the expression levels of its downstream genes glutamate-cysteine ligase (gcl) ( figure c ) and glutathione-s-transferase (gst) ( figure d ) involved in glutathione metabolism were equally improved by epa or ht. the supplementation with epa plus ht in hfd mice showed an even greater effect, restoring the expression levels of nrf , gcl and gst to control values, and normalizing the dna binding activity of nrf (figure a-d) . a similar behavior was observed in the activity of antioxidant enzymes cat ( figure e ), sod ( figure f ), gr ( figure g ) and gpx ( figure h ) in ewat of hfd-mice that received epa, ht or both supplements, which were partially enhanced by epa or ht after reduction by hfd and totally recovered by combined epa and ht ( figure d-h) . the ewat of mice subjected to hfd exhibit a significant increase in dna-binding activity ( figure a ) and mrna levels of srebp- c ( figure b ). the activation of srebp- c was accompanied by higher gene expression and activities of lipogenic enzymes acc ( figure e ,f) and fas ( figure c ,d) and higher activities of g pd ( figure g ) and me ( figure h ) compared to cd values. in addition, mrna levels of enzyme lpl were decreased, whereas their activity increased in ewat of hfd-fed mice ( figure d,e) . the administration of epa or ht supplied alone to hfd mice led to a partial recovery of all the lipogenic parameters evaluated in ewat (figure ) including the lpl enzyme ( figure d,e) , showing comparable results with both supplements, except for the fas activity ( figure g ). the effect of epa and ht given together led to a significantly greater recovery of srebp- c ( figure a ,b) and its target genes fas, acc, g pd, me ( figure c -h) and lpl ( figure d,e) , although without achieving values observed in ewat of cd mice. the ewat of mice subjected to hfd exhibit a significant increase in dna-binding activity ( figure a ) and mrna levels of srebp- c ( figure b ). the activation of srebp- c was accompanied by higher gene expression and activities of lipogenic enzymes acc ( figure e ,f) and fas ( figure c ,d) and higher activities of g pd ( figure g ) and me ( figure h ) compared to cd values. in addition, mrna levels of enzyme lpl were decreased, whereas their activity increased in ewat of hfd-fed mice ( figure d,e) . the administration of epa or ht supplied alone to hfd mice led to a partial recovery of all the lipogenic parameters evaluated in ewat (figure ) including the lpl enzyme ( figure d,e) , showing comparable results with both supplements, except for the fas activity ( figure g ). the effect of epa and ht given together led to a significantly greater recovery of srebp- c ( figure a ,b) and its target genes fas, acc, g pd, me ( figure transport protein (fatp ) ( figure c ) were decreased in ewat of hfd-fed mice compared to the control group. supplementation whit epa or ht alone similarly improved the activity and mrna levels of ppar-γ and gene expression of fatp . moreover, epa plus ht supplementation led to a significant increase in the binding activity and gene expression of ppar-γ and in the gene expression of the fatp transporter compared to groups that received epa or ht, without reaching cd values ( figure a-c) . mice subjected to cd with or without supplementation showed comparable results in all parameters evaluated in ewat mentioned in figures and . the dna-binding activity and the gene expression of the transcription factor nf-κΒ were significantly increased in ewat of mice fed with the hfd compared to those that received the cd ( figure a,b) . in parallel, the mrna levels of the pro-inflammatory cytokine tumor necrosis factor alpha (tnf-α) and interleukin (il- ) were increased in ewat of hfd-fed mice ( figure c,d) . in the hfd-fed mice that received epa or ht, the dna-binding activity of nf-κΒ and the mrna levels of nf-κΒ, tnf-α and il- were moderately improved, with comparable results between both groups, respectively. the group that received epa plus ht showed a significantly greater decrease in these values in comparison to the groups supplemented with epa or ht alone, without reaching those of the control group ( figure ). the dna-binding activity ( figure a ) and mrna levels of ppar-γ ( figure b ) and fatty acid transport protein (fatp ) ( figure c ) were decreased in ewat of hfd-fed mice compared to the control group. supplementation whit epa or ht alone similarly improved the activity and mrna levels of ppar-γ and gene expression of fatp . moreover, epa plus ht supplementation led to a significant increase in the binding activity and gene expression of ppar-γ and in the gene expression of the fatp transporter compared to groups that received epa or ht, without reaching cd values ( figure a-c) . mice subjected to cd with or without supplementation showed comparable results in all parameters evaluated in ewat mentioned in figures and . the dna-binding activity and the gene expression of the transcription factor nf-κb were significantly increased in ewat of mice fed with the hfd compared to those that received the cd ( figure a,b) . in parallel, the mrna levels of the pro-inflammatory cytokine tumor necrosis factor alpha (tnf-α) and interleukin (il- ) were increased in ewat of hfd-fed mice ( figure c,d) . in the hfd-fed mice that received epa or ht, the dna-binding activity of nf-κb and the mrna levels of nf-κb, tnf-α and il- were moderately improved, with comparable results between both groups, respectively. the group that received epa plus ht showed a significantly greater decrease in these values in comparison to the groups supplemented with epa or ht alone, without reaching those of the control group ( figure ). diet-induced obesity (dio) in mice represents a model of choice for preclinical nutritional studies [ ] [ ] [ ] . hfd feeding comprising % of the total calories as fat for weeks achieved significant bw gain with concomitant liver and wat dysfunction. in the case of the liver, hfd induced steatosis in association with n- lcpufas depletion, oxidative stress with loss of antioxidant defenses [ ] , a pro-inflammatory state without histological inflammation [ ] and a decline in mitochondrial energy metabolism [ ] . the drastic metabolic alterations induced by hfd are evidenced by the deranged levels of (i) serum-free fatty acids, the adipokines adiponectin and leptin, and the cytokines tnf-α and il- , which are associated with ir development [ ] , (ii) the development of adipocyte hypertrophy and enhancement in total tissue mass, with higher cell size and tag content and lower cell number per g of tissue over control values and (iii) the derangement of metabolic parameters in wat favoring oxidative stress, lipogenesis, mitochondrial dysfunction and inflammation, features that agree with a previous report [ ] and resemble those elicited in the liver [ , , ] . metabolic alterations can be prevented or attenuated by the combination of active substances, including n- lcpufas, antioxidants and insulin sensitizers, as reported for dha, epa, ht, rosiglitazone and astaxantin [ , , , ] . in the present study, epa and ht co-administration diminished bw gain in hfd-fed mice over cd animals, an effect that was accompanied by a partial diet-induced obesity (dio) in mice represents a model of choice for preclinical nutritional studies [ ] [ ] [ ] . hfd feeding comprising % of the total calories as fat for weeks achieved significant bw gain with concomitant liver and wat dysfunction. in the case of the liver, hfd induced steatosis in association with n- lcpufas depletion, oxidative stress with loss of antioxidant defenses [ ] , a pro-inflammatory state without histological inflammation [ ] and a decline in mitochondrial energy metabolism [ ] . the drastic metabolic alterations induced by hfd are evidenced by the deranged levels of (i) serum-free fatty acids, the adipokines adiponectin and leptin, and the cytokines tnf-α and il- , which are associated with ir development [ ] , (ii) the development of adipocyte hypertrophy and enhancement in total tissue mass, with higher cell size and tag content and lower cell number per g of tissue over control values and (iii) the derangement of metabolic parameters in wat favoring oxidative stress, lipogenesis, mitochondrial dysfunction and inflammation, features that agree with a previous report [ ] and resemble those elicited in the liver [ , , ] . metabolic alterations can be prevented or attenuated by the combination of active substances, including n- lcpufas, antioxidants and insulin sensitizers, as reported for dha, epa, ht, rosiglitazone and astaxantin [ , , , ] . in the present study, epa and ht co-administration diminished bw gain in hfd-fed mice over cd animals, an effect that was accompanied by a partial wat reduction and ewat cellularity preservation similar to those observed in the separate supplementations. in agreement with these findings, replacement of dietary lipids in a hfd by % to % (wt/wt) of epa plus dha (dha > epa) limited the bw gain and ewat accumulation in c bl/ j hfd-fed mice, with similar results being found by replacing a hfd rich in ala or linoleic acid (c : n- , la) by mixtures of epa and dha at different ratios [ ] . the effect of epa on obesity may be partly mediated by binding to g protein-coupled receptor (gpr ) present in adipocytes and adipocyte tissue macrophages (atms) [ ] , thus regulating adipogenesis, glucose uptake, inflammation and insulin sensitivity [ ] . epa binding and activation of gpr promotes several effects, including (i) the interaction with ppar-γ that favors the differentiation of pre-adipocytes into white adipocytes [ ] , (ii) the binding to β-arrestin and their interaction with transforming growth factor-β-activated kinase binding protein (tab ) that blocks nf-κb and c jun n-terminal kinase (jnk) functioning, with inhibition of the low-grade inflammation due to tnf-α and il- production by atms triggered by obesity [ ] and (iii) the release of fibroblast growth factor (fgf ), resulting in the promotion of brown adipose tissue (bat) activity and wat browning [ ] , with stimulation of mitochondrial energy metabolism depressed by hfd. it is important to note that epa supplementation in hfd-fed mice upregulates the expression of ppar-α and the micro-rnas mir- and mir- - p in bat [ ] . these mediators enhance the expression of thermogenic markers that stimulate mitochondrial fao and energy expenditure, pointing to bat as a crucial anti-obesity target [ ] . this contention is further supported by data showing that ht prevents visceral adipogenesis and decreased metabolic activity in bat and wat, induced by the exposure to fine particulate matter in mice [ ] . the contribution of epa to the induction of a healthy adipocyte phenotype may also involve its direct binding to ppar-γ, whose expression and activity are diminished by hfd and partially rescued by epa. activation of ppar-γ induces adipogenesis as an important mechanism of wat remodeling, which is accomplished through downregulation of srebp- c functioning [ , ] . the ht component of the combined protocol elicited diminution of wat lipid accumulation by regulating genes related to lipogenesis and fao, but mainly due to the improvement of the cellular antioxidant status that preserves n- lcpufas loss by hfd [ , ] . the latter mechanism of ht action is exerted directly as a free-radical scavenger or by recovering the nrf system that induces antioxidant enzymes (sod, cat, gpx) and those associated with glutathione repletion (gcl, gr) or utilization (gst). at the doses used in our study, the combined epa and ht administration did not exhibit more potentiated effects on wat morphology parameters and lipid content than those of the individual compounds. however, the parameters related to wat metabolic dysfunction are diminished in an additive manner, considering that the specific effects exerted by epa or ht exhibit convergence in the combined protocol. in agreement with previous data, wat expansion in hfd-fed mice led to a significant decrease of serum levels of adiponectin and an increase in those of leptin [ ] , which are correlated with ir [ ] and are partially restored by epa and ht single or combined supplementations. in this context, the administration of epa is known to enhance the gene expression and release of adiponectin from adipocytes, either in ewat from hfd-fed mice [ ] or in lean and overweight rats [ ] , thus evidencing the insulin-sensitizing properties of epa [ , , ] . interestingly, the administration of epa to obese subjects elevated adiponectin levels without altering bw, suggesting direct involvement of epa on adiponectin secretion [ ] . this conclusion is also supported by epa supplementation to hfd-fed mice showing maintenance of normal adiponectin levels without diminishing adiposity, pointing to adiponectin as a major factor in epa-mediated prevention and reversal of ir [ ] . regarding the molecular mechanisms underlying these effects, epa increase the expression of adiponectin through binding to ppar-γ, promoting the expression of fgf and the activation of amp-activated protein kinase (ampk), the energy sensor that favors fao over lipogenesis [ , , , ] . the epa-induced ampk activation by phosphorylation in t -l adipocytes [ ] is in agreement with in vivo studies showing that n- lcpufas increase the content of wat ampk α subunit and its phosphorylation status, in association with an enhancement in adiponectin levels [ , ] . in the case of leptin, the enhancement in its serum levels by hfd feeding in mice and their diminution by epa (table d ) agree with studies in humans and rodents [ ] , suggesting that improved leptin sensitivity concomitantly with decreased production would be secondary to the anti-inflammatory effect of n- lcpufas [ ] . regarding ht, several preclinical studies reported higher circulating levels of adiponectin with lower leptin values than controls achieved by this polyphenol [ , , , ] . improved expression and activity of the ppar-γ gene by co-administration of epa and ht also leads to improved expression of the target genes fatp and lpl, an effect that is additive over the individual supplementations after hfd feeding. in particular, upregulation of wat fatp may contribute to the smaller healthy phenotype of adipocytes due to its fa transport function across cell membranes and its acyl-coa synthase activity to activate fas for further utilization [ ] . lpl, however, exhibited opposite changes regarding its mrna expression and activity, which may be related to different mechanisms regulating lpl at transcriptional and post-translational levels [ ] . the small healthy adipocyte phenotype is also associated with downregulation of key lipogenic genes in epa-ht-hfd-treated mice. these include genes coding for transcription factor srebp- c that controls the expression of the lipogenic enzymes acc and fas and the reduced nicotinamide adenine dinucleotide phosphate (nadph) generating systems g pd and me that are required for fa synthesis. the anti-lipogenic effect of the epa plus ht protocol is related to the repletion of n- lcpufas due to lower oxidative deterioration by the normoxic state achieved, which downregulates lipogenic srebp- c functioning [ ] . this contention is supported by previous studies showing (i) diminution in the lipogenic enzyme activities in wat of sucrose-rich diet-fed rats subjected to fish oil administration [ ] , (ii) the decrease in the expression of the lipogenic enzyme glycerol- -phosphate dehydrogenase in epa-treated t -l adipocytes [ ] and (iii) the diminished srebp- c functioning with prevention of dio in adipocytes from hfd-fed mice supplemented with epa [ ] . additionally, hfd-induced prolipogenic state in ewat with n- lcpufas depletion are attenuated by ht supplementation, which may preserve the activity of delta- and delta- desaturases that contribute to n- lcpufas synthesis, as previously reported in the liver [ ] , downregulate srebp- c or reduce oxidative stress [ ] . a major hallmark in the expansion of wat in obesity is the enhancement in the adipocyte oxidative stress status, which underlies significant mitochondrial dysfunction. this is evidenced by the decreases in the activities of the carboxylic acid cycle regulatory enzyme citrate synthase and the respiratory chain complexes i and ii [ ] , a condition that favors energy deprivation and mitochondrial reactive oxygen species (ros) production, thus contributing to the attainment of a hypertrophic adipocyte phenotype [ , ] . data presented indicate that hfd coupled to epa plus ht supplementation totally avoided oxidative damage to biomolecules and the depletion of antioxidant systems in wat. this finding mainly relies on the ht component of the combined protocol and may effectively contribute to the hypertrophic to the small healthy adipocyte phenotype transition observed. the reduction of the enlarged wat in obesity achieved by epa plus ht is in agreement with studies on the antioxidant behavior exerted by epa, dha and the carotenoid astaxantin, alone or in combinations in hepg -c cells [ ] , epa and dha in t -l adipocytes and schwann cells [ , ] or in human macrophages [ ] , with responses exhibiting synergism. the epa plus ht protocol also improved the pro-inflammatory status of wat in hfd-fed mice in an additive manner. possible mechanisms involved include: (i) disruption of the sfa inflammatory signal in toll-like receptor that activate nf-κb, (ii) ppar-γ activation that interferes with the translocation of nf-κb to the nucleus, (iii) binding to gpr and prevention of nf-κb activation, (iv) decreased pro-inflammatory adipokine expression and macrophage accumulation, promoting repolarization towards the m anti-inflammatory phenotype by spms [ , , ] and (v) preservation of wat n- lcpufas and downregulation of nf-κb by ht [ , ] . hfd feeding in mice drastically impacts wat functioning through the induction of hypertrophy and metabolic dysfunction. wat hypertrophy involves higher cell size with tag accumulation and lower cell number, which is related to epa depletion that blunts ppar-γ operation. metabolic dysfunction includes (i) upregulation of the lipogenic srebp- c system, (ii) loss of mitochondrial energy metabolism and (iii) downregulation of the antioxidant nrf system, leading to oxidative stress and the secondary n- lcpufas depletion and activation of the redox-sensitive pro-inflammatory nf-κb system. these effects of hfd were significantly diminished by the co-supplementation with epa and ht additively, with resulting values being close or similar to those observed for the cd-fed animals. these observations strengthen the contention that epa and ht co-administration is an important strategy to attenuate obesity-driven metabolic inflammation states. the data reported concerning ppar-γ upregulation by epa and ht co-administration may also be of importance as a possible adjuvant therapy to diminish the severity of sars-cov- infection [ ] , considering the anti-inflammatory response involving ppar-γ binding to gpr and/or spms production from epa and its product dha, and the anti-fibrotic effect of ppar-γ [ ] . author contributions: l.a.v. and r.v. designed the study. p.i., l.a.v. and r.v. analyzed and interpreted the data. a.e., f.e., s.s.-a., c.c., a.r., and r.v. performed the experiments. p.i., l.a.v., r.v. and t.m. structured and wrote the manuscript. all authors have read and agreed to the published version of the manuscript. funding: this research was funded by initiation fondecyt to rodrigo valenzuela grant number [ ] and regular fondecyt to alejandra espinosa grant number [ ] . health effects of overweight and obesity in countries over years the epidemiology of obesity obesity: global epidemiology and pathogenesis obesity and nonalcoholic fatty liver disease: biochemical, metabolic and clinical implications the cell biology of fat expansion the importance of the long-chain polyunsaturated fatty acid n- /n- ratio in development of non-alcoholic fatty liver associated with obesity targeting n- polyunsaturated fatty acids in non-alcoholic fatty liver disease omega- fatty acids and adipose tissue biology olive leaf extracts act as modulators of the human immune response hydroxytyrosol and cytoprotection: a projection for clinical interventions molecular adaptations underlying the beneficial effects of hydroxytyrosol in the pathogenic alterations induced by a high-fat diet in mouse liver: ppar-α and nrf activation, and nf-κb down-regulation hydroxytyrosol supplementation ameliorates the metabolic disturbances in white adipose tissue from mice fed a high-fat diet through recovery of transcription factors nrf , srebp- c, ppar-γ and nf-κb crosstalk mechanisms in hepatoprotection: thyroid hormone-docosahexaenoic acid (dha) and dha-extra virgin olive oil combined protocols impact of the co-administration of n- fatty acids and olive oil components in preclinical nonalcoholic fatty liver disease models: a mechanistic view long-term vitamin d and high-dose n- fatty acids' supplementation improve markers of cardiometabolic risk in type diabetic patients with chd reduction of high-fat diet-induced liver proinflammatory state by eicosapentaenoic acid plus hydroxytyrosol supplementation: involvement of resolvins rve / and rvd / high-fat diet induces mouse liver steatosis with a concomitant decline in energy metabolism: attenuation by eicosapentaenoic acid (epa) or hydroxytyrosol (ht) supplementation and the additive effects upon epa and ht co-administration reduction in the desaturation capacity of the liver in mice subjected to high fat diet: relation to lcpufa depletion in liver and extrahepatic tissues attenuation of high-fat diet-induced rat liver oxidative stress and steatosis by combined hydroxytyrosol-(ht-) eicosapentaenoic acid supplementation mainly relies on ht metabolism of isolated fat cells:i. effects on hormones on glucose metabolism and lipolysis a simple method to determine fat cell size and number in four mammalian species a simple method for the isolation and purification of total lipides from animal tissuers hydroxytyrosol prevents reduction in liver activity of ∆- and ∆- desaturases, oxidative stress, and depletion in long chain polyunsaturated fatty acid content in different tissues of high-fat diet fed mice a rapid method of total lipid extraction and purification assay for quantitative determination of glutathione and glutathione disulfide levels using enzymatic recycling method supplementation with docosahexaenoic acid and extra virgin olive oil prevents liver steatosis induced by a high-fat diet in mice through ppar-α and nrf upregulation with concomitant srebp- c and nf-kb downregulation malic enzyme and lipogenesis lipoprotein lipase activity activity in liver of the rat fetus agarose gel electrophoresis for the separation of dna fragments a new mathematical model for relative quantification in real-time rt-pcr analysis of relative gene expression data using real-time quantitative pcr and the -∆∆ct method laboratory animals as surrogate models of human obesity animal models of metabolic syndrome: a review c bl/ j mice as a polygenic developmental model of diet-induced obesity n- fatty acids and rosiglitazone improve insulin sensitivity through additive stimulatory effects on muscle glycogen synthesis in mice fed a high-fat diet astaxanthin and omega- fatty acids individually and in combination protect against oxidative stress via the nrf -are pathway omega- pufa of marine origin limit diet-induced obesity in mice by reducing cellularity of adipose tissue gpr ) as a fatty acid sensor involved in appetite control, insulin sensitivity and inflammation regulation gpr : a critical role in adipogenesis, inflammation, and energy metabolism in adipose tissue gpr is an omega- fatty acid receptor mediating potent anti-inflammatory and insulin sensitizing effects the lipid sensor gpr promotes brown fat activation and fgf release from adipocytes moustaid-moussa, n. transcriptic and microrna analyses of gene meteorks regulated by eicosapentaenoic acid in brown adipose tissue of diet-induced obese mice hydroxytyrosol prevents pm . -indiced adiposity and onsulin resistance by restraining oxidative stress related ti nf-κb pathway and modulation of gut microbiota in a murine model. free radic hydroxytyrosol prevents diet-induced metabolic syndrome and attenuates mitochondrial abnormalities in obese mice. free radic leptin, and fatty acids in the maintenance of metabolic homeostasis through adipose tissue crosstalk polyunsaturated fatty acids of marine origin induce adiponectin in mice fed a high-fat diet eicosapentaenoic acid actions on adiposity and insulin resistance in control and high-fat-fed rats: role of apoptosis, adiponectin and tumour necrosis factor-α increased adiponectin secretion by highly purified eicosapentaenoic acid in rodent models of obesity and human obese subjects eicosapentaenoic acid prevents and reverses insulin resistance in high-fat diet-induced obese mice via modulation of adipose tissue inflammation eicosapentaenoic acid stimulates amp-activated protein kinase and increases visfatin secretion in cultured murine adipocytes obesity-induced insulin resistance and hepatic steatosis are alleviated by ω- fatty acids: a role for resolvins and protectins pufa: bioavailability and modulation of adipose tissue function regulation of adipokine secretion by n- fatty acids omega- fatty acids: a review of the effects on adiponectin and leptin and potential implications for obesity management chronic hydroxytyrosol feeding modulates glutathione-mediated oxido-reduction pathways in adipose tissue: a nutrigenomic study slc fatty acid transport proteins lipogenic enzyme activities and glucose uptake in fat tissue of dyslipemic, insulin-resistant rats: effects of fish oil eicosapentaenoic acid reduces adipocyte hypertrophy and inflammation in diet-induced obese mice in an adiposity-independent manner adipose mitochondrial biogenesis is suppressed in db/db and high-fat diet-fed mice and improved by rosiglitazone the perfect storm: obesity, adipocyte dysfunction, and metabolic consequences the impact of oxidative stress on adipose tissue energy balance omega- polyunsaturated fatty acid has an anti-oxidant effect via the nrf- /ho- pathway in t -l adipocytes omega- polyunsaturated fatty acids exert anti-oxidant effects through the nuclear factor (erythroidderived )-related factor pathway in immortalized mouse schwann cells omega- fatty acids decrease oxidative stress and inflammation in macrophages from patients with small abdominal aortic aneurysm omega- fatty acids and inflammatory processes: from molecules to man eicosapentaenoic acid shows anti-inflammatory effect via gpr in t -l adipocytes and attenuates adipose tissue inflammation in diet-induced obese mice the role of adipocytes and adipocyte-like cells in the severity of covid- infections ppar-gama is a gatekeeper for extracellular matrix and vascular cell homeostasis: beneficial role is n pulmonary hypertension and renal/cardiac/pulmonary fibrosis this article is an open access article distributed under the terms and conditions of the creative commons attribution the authors gratefully acknowledge yolanda b. lombardo, school of biochemistry, university of litoral, santa fe, argentina, for the technical support received in her research group. the authors declare no conflict to interest. key: cord- -gj yei authors: lebedeva, natalya sh.; gubarev, yury a.; koifman, mikhail o.; koifman, oskar i. title: the application of porphyrins and their analogues for inactivation of viruses date: - - journal: molecules doi: . /molecules sha: doc_id: cord_uid: gj yei the problem of treating viral infections is extremely relevant due to both the emergence of new viral diseases and to the low effectiveness of existing approaches to the treatment of known viral infections. this review focuses on the application of porphyrin, chlorin, and phthalocyanine series for combating viral infections by chemical and photochemical inactivation methods. the purpose of this review paper is to summarize the main approaches developed to date in the chemical and photodynamic inactivation of human and animal viruses using porphyrins and their analogues and to analyze and discuss the information on viral targets and antiviral activity of porphyrins, chlorins, of their conjugates with organic/inorganic compounds obtained in the last – years in order to identify the most promising areas. over the past decades, the onset of epidemics and pandemics caused by sars viruses (sars-cov, ( ) ( ) , and the sars-cov- coronavirus conditioned the need to consider a healthcare system not only as a public sector organizing and protecting the health of the population of a particular country but also as a global international task. change of priorities for threat evaluation takes place throughout a civilized society. the reason for this is the emergence of new viral infections that cause epidemics and pandemics, as well as the existence of drug-resistant infections, which are increasing each year [ ] [ ] [ ] [ ] . therefore, the search and development of new alternative approaches to the treatment of viral and bacterial infections are necessary. the proposed review will consider the main achievements over the past few years in the search for alternative therapeutic approaches and of the drugs based on porphyrins and their analogues (p&a) for the treatment of viral infections. for an objective and comparative analysis of existing approaches and for revealing the most promising areas, we briefly consider the structure of viral particles. this will allow us to establish potential vulnerabilities, the so-called molecular targets. viruses are microscopic pathogens, translated from latin word virus, which means "poison and poisonous source". mammalian viruses have various shapes, most often spherical or ellipsoidal with sizes ranging from to nm [ ] . viral particles contain genetic material in the form of dna or rna, often a protein shell (capsid), and may also include additional lipid shells (supercapsid or peplos). as noted above, despite the comparative simplicity, all types of viruses differ in their genetic material, replication mechanism, and in chemical structure of structural and nonstructural proteins. therefore, an understanding of the biomechanics and biochemistry of the functioning of structural proteins of viral particles is necessary for a scientifically based search for virucidal compounds. now, we will consider the methods of inactivating viral particles, using porphyrins and their analogues at the first stage of the life cycle, taking as the example human immunodeficiency virus (hiv). the vast majority of studies focus on hiv inactivation, partly because this virus appeared relatively long ago (in ) and has been well studied, but to a greater extent, owing to the slowly progressive disease caused by it, which often leads to death. according to who, there are about million people diagnosed with hiv in the world. the human immunodeficiency virus belongs to the genus lentiviruses (lentivirus) of the family of retroviruses (retroviridae). hiv virus is relatively simple (figure ). in addition to structural proteins, it contains two rna helices and three enzymes: reverse transcriptase (revertase), integrase, and protease. the first stage of attachment of the virion to the surface of the cell and fusion with the cell membrane is determined by the spikes on the surface of the virion. the spikes on the viral particle are formed by env glycoprotein (envelope protein), which consists of two subunits: the variable protein gp performing the function of binding to the cd receptor and of the more conservative transmembrane gp , which holds the gp molecule in the membrane (figure ). the gp viral glycoprotein strongly links the cd receptor ( figure ). the first stage of attachment of the virion to the surface of the cell and fusion with the cell membrane is determined by the spikes on the surface of the virion. the spikes on the viral particle are formed by env glycoprotein (envelope protein), which consists of two subunits: the variable protein gp performing the function of binding to the cd receptor and of the more conservative transmembrane gp , which holds the gp molecule in the membrane (figure ). the gp viral glycoprotein strongly links the cd receptor ( figure ). to glycoprotein (gp) exposes a coreceptor binding site in gp ; (c and d) coreceptor binding causes the exposure of the gp fusion peptide and its insertion into the membrane of the target cell in a triple-stranded coiled-coil; and (e) formation of a helical hairpin structure in which gp folds back on itself is coincident with membrane fusion [ ] . as a result of this interaction, gp undergoes conformational changes that also provide binding the coreceptor molecule cxcr or ccr ( figure ). this is followed by the stage of internalization of the virus into the host cell; the latter ensures the fusion of the cell membranes of the virus and the host due to the gp viral transmembrane protein. thus, the proteins of the gp or gp virus are the most important and critical targets, since if they are damaged or linked, further propagation of the hiv virus becomes impossible. among the porphyrins and their analogues, several very promising compounds have been identified that are capable of forming strong complexes with the gp protein glycoprotein [ ] , and the complex formed in this way does not allow the viral gp of protein to bind to the host cell cd receptor (figure ) [ ] . the first stage of attachment of the virion to the surface of the cell and fusion with the cell membrane is determined by the spikes on the surface of the virion. the spikes on the viral particle are formed by env glycoprotein (envelope protein), which consists of two subunits: the variable protein gp performing the function of binding to the cd receptor and of the more conservative transmembrane gp , which holds the gp molecule in the membrane ( figure ). the gp viral glycoprotein strongly links the cd receptor ( figure ). to glycoprotein (gp) exposes a coreceptor binding site in gp ; (c and d) coreceptor binding causes the exposure of the gp fusion peptide and its insertion into the membrane of the target cell in a triple-stranded coiled-coil; and (e) formation of a helical hairpin structure in which gp folds back on itself is coincident with membrane fusion [ ] . as a result of this interaction, gp undergoes conformational changes that also provide binding the coreceptor molecule cxcr or ccr ( figure ). this is followed by the stage of internalization of the virus into the host cell; the latter ensures the fusion of the cell membranes of the virus and the host due to the gp viral transmembrane protein. thus, the proteins of the gp or gp virus are the most important and critical targets, since if they are damaged or linked, further propagation of the hiv virus becomes impossible. among the porphyrins and their analogues, several very promising compounds have been identified that are capable of forming strong complexes with the gp protein glycoprotein [ ] , and the complex formed in this way does not allow the viral gp of protein to bind to the host cell cd receptor ( figure ) [ ] . to glycoprotein (gp) exposes a coreceptor binding site in gp ; (c,d) coreceptor binding causes the exposure of the gp fusion peptide and its insertion into the membrane of the target cell in a triple-stranded coiled-coil; and (e) formation of a helical hairpin structure in which gp folds back on itself is coincident with membrane fusion [ ] . as a result of this interaction, gp undergoes conformational changes that also provide binding the coreceptor molecule cxcr or ccr ( figure ). this is followed by the stage of internalization of the virus into the host cell; the latter ensures the fusion of the cell membranes of the virus and the host due to the gp viral transmembrane protein. thus, the proteins of the gp or gp virus are the most important and critical targets, since if they are damaged or linked, further propagation of the hiv virus becomes impossible. among the porphyrins and their analogues, several very promising compounds have been identified that are capable of forming strong complexes with the gp protein glycoprotein [ ] , and the complex formed in this way does not allow the viral gp of protein to bind to the host cell cd receptor ( figure ) [ ] . the structure of the porphyrins and their analogues plays a key role in linking gp protein with the glycoprotein as illustrated by the example of the compounds shown in figure . it was found that the porphyrin-zinc complex containing three -nitrophenyl groups and one methylpyridinium group in the mesoposition ( figure , compound ) most effectively blocks the penetration of virions ( μm, > %). a cationic substituent is necessary to increase the solubility of porphyrins and their analogues in aqueous media, and three nitro groups provide linking of the gp protein with the glycoprotein (figure ) and thereby inhibit fusion between the virus and the cell membrane. a wider range of porphyrins and their analogues, including deutero-, proto-, hemato-, and meso-porphyrins, derivatives of metal-tetraphenylporphyrin-tetrasulfonate and derivatives of sulfonated tetraarylporphyrins was tested for anti-hiv activity ( figure ) [ ] . the structure of the porphyrins and their analogues plays a key role in linking gp protein with the glycoprotein as illustrated by the example of the compounds shown in figure . it was found that the porphyrin-zinc complex containing three -nitrophenyl groups and one -methylpyridinium group in the mesoposition ( figure , compound ) most effectively blocks the penetration of virions ( µm, > %). a cationic substituent is necessary to increase the solubility of porphyrins and their analogues in aqueous media, and three nitro groups provide linking of the gp protein with the glycoprotein ( figure ) and thereby inhibit fusion between the virus and the cell membrane. a wider range of porphyrins and their analogues, including deutero-, proto-, hemato-, and meso-porphyrins, derivatives of metal-tetraphenylporphyrin-tetrasulfonate and derivatives of sulfonated tetraarylporphyrins was tested for anti-hiv activity ( figure ) [ ] . screening showed that none of the deutero-, proto-, hemato-, and meso-porphyrins reduced infection by more than % at a concentration of µg/ml. the most active compounds were sulfonated tetranaphthyl-porphyrin, sulfonated tetraanthracenyl-porphyrin and sulfonated , -difluoromesotetraphenylporphine and its copper complex. their antiviral activity was %, %, %, and %, respectively. such a high virucidal activity, according to the authors of [ ] , is due to the linking of hiv- gp with porphyrins and their analogues, and this reaction completely inhibits the ability of env proteins to induce cell fusion with the receptors of cd host cells. a comparison of the results of studies [ , ] allows us to conclude that the charge of porphyrins and their analogues is not determining in linking with viral gp , since both cationic [ ] and anionic [ ] porphyrins showed a similar high virucidal activity. it can be assumed that the electrostatic interactions in the mgc-gp system are not significant, and it is logical, since the outer part of gp consists of sites that are very variable and carry multiple sugars (the basis for the virus to evade the immune response to this protein in the body). detailed studies of the structure of the gp target showed that this protein contains five variable regions (v -v ) with five conserved regions (c -c ). the region involved in coreceptor linking has been identified as a polycation, which includes the v loop of the gp protein and conservative elements adjacent to this loop [ ] . to target the v loop, porphyrins and their analogues must be negatively charged. screening showed that none of the deutero-, proto-, hemato-, and meso-porphyrins reduced infection by more than % at a concentration of μg/ml. the most active compounds were sulfonated tetranaphthyl-porphyrin, sulfonated tetraanthracenyl-porphyrin and sulfonated , difluoro-mesotetraphenylporphine and its copper complex. their antiviral activity was %, %, %, and %, respectively. such a high virucidal activity, according to the authors of [ ] , is due to the linking of hiv- gp with porphyrins and their analogues, and this reaction completely inhibits the ability of env proteins to induce cell fusion with the receptors of cd host cells. a comparison of the results of studies [ , ] allows us to conclude that the charge of porphyrins and their analogues is not determining in linking with viral gp , since both cationic [ ] and anionic [ ] porphyrins showed a similar high virucidal activity. it can be assumed that the electrostatic interactions in the mgc-gp system are not significant, and it is logical, since the outer part of gp consists of sites that are very variable and carry multiple sugars (the basis for the virus to evade the immune response to this protein in the body). detailed studies of the structure of the gp target showed that this protein contains five variable regions (v -v ) with five conserved regions (c -c ). the region involved in coreceptor linking has been identified as a polycation, which includes the v loop of the gp protein and conservative elements adjacent to this loop [ ] . to target the v loop, porphyrins and their analogues must be negatively charged. it should be noted that targeted modelling of the porphyrins and their analogues structure for linking with a specific region of the target has not yet been carried out. at this stage of development of this research direction, the porphyrins and their analogues are mainly screened, both using computer modelling [ ] and experimental methods [ ] . for example, the antiviral activity of methyl and metal-free porphyrin and chlorin compounds against a wide range of viruses with and without envelope was evaluated in [ ] . chlorophyllide was most effective against hepatitis b virus (hbv). only μm chlorophyllide reduced hbv dna signal by %. to establish the mechanism of it should be noted that targeted modelling of the porphyrins and their analogues structure for linking with a specific region of the target has not yet been carried out. at this stage of development of this research direction, the porphyrins and their analogues are mainly screened, both using computer modelling [ ] and experimental methods [ ] . for example, the antiviral activity of methyl and metal-free porphyrin and chlorin compounds against a wide range of viruses with and without envelope was evaluated in [ ] . chlorophyllide was most effective against hepatitis b virus (hbv). only µm chlorophyllide reduced hbv dna signal by %. to establish the mechanism of the virucidal activity of chlorophyllide, the authors conducted in vitro biochemical studies in which chlorophyllide was introduced on each life cycle of the virus. it turned out that chlorophyllide changed the structure of the capsid of hbv virions and directly destroyed the hbv virions in the culture medium of producer cells, and it resulted in the loss of dna virion. chlorophyllide does not have a noticeable effect on the viability of the host cells and intracellular products of viral genes; it is safe for humans at a dose of mg/d for months [ ] . chlorin e has demonstrated the strongest antiviral activity against hepatitis b virus as well as a deep antiviral effect on other enveloped viruses, such as hepatitis c virus (hcv), human immunodeficiency virus (hiv), dengue virus (denv), marburg virus (marv), tacaribe virus (tcrv), and junin viruses (junv). it is noteworthy that chlorin e inactivated denv at the subnanomolar level. however, the compound did not exert an antiviral effect against the enveloped virus of encephalomyocarditis virus (the genome of the virus is single-stranded plus-rna) and adenovirus (dna containing) [ ] . chemical inhibition at the stage of attachment of the virion to the cell surface is possible not only by the action of the porphyrins and their analogues on the viral protein but also by an alternative linking of the host cell receptor itself. for example, oxovanadium mesoporphyrin ix forms very stable complexes with the cd receptor, which in its stability compete with the best peptidomimetics (cyclometallic complexes of platinum (ii)) [ ] . all the studies reviewed above are based on the method of chemical inactivation of viruses. without any doubt, this approach is promising, but in the formation of complexes between the target and the porphyrins and their analogues due to π-π-interaction, axial coordination, h-bonding, and electrostatic interaction, there always remains the option of destruction (collapse) of the complexes. in addition, it should be borne in mind that for a positive antiviral effect, the required amount of p&a can be significant. first, this follows from the basics of thermodynamics and is associated with the need for an excess of p&a to increase the number of the combined surface protein structures. second, the number of spines themselves on the virion of viruses is very different e.g., on the surface of the hiv- virion there are about - spines (env) [ ] , influenza virus has - spines, vesicular stomatitis virus has about , and the more the spines present, the greater the number of porphyrins and their analogues required. third, the problem of target linking selectivity arises, i.e., part of the porphyrins and their analogues can interact with other biosubstrates. in addition, the determination of the required dose of porphyrins and their analogues should be correlated with the degree of damage to the body. finally, another equally important factor-like resistance to bacterial antibiotics-is the resistance to antiviral drugs that also remains an urgent issue [ ] , because with chemical inhibition, the virus still exists, it is only inactivated. in this sense, the use of photoinactivation of viral particles seems more promising, since photoinactivation involves the complete destruction of the viral infection. it is well known that porphyrin, chlorin, and phthalocyanine series is capable of absorbing light energy and generating reactive oxygen species. this process can proceed according to two mechanisms-type i and type ii [ ] , the essence of which is as follows: in the ground state, the porphyrins and their analogues are a singlet, the absorption of a photon of light leads to the excitation of one electron and its transition to an orbit with a higher energy ( figure ). the singlet state is short-lived (nanosecond lifetime), and excess energy can be emitted by the p&a in the form of light (fluorescence) or dissipated into the energy of disordered processes and, ultimately, emitted into heat. the third option, "intersystem crossing," is also possible, in which the p&a goes into a more stable excited triplet state (microsecond lifetime). from the point of view of photochemistry, triplet state allows the p&a to transfer energy upon contact with molecular oxygen (o ); as a result, the p&a returns to its original singlet state, and oxygen passes into the excited triplet state ( o ). the described photochemical reaction is called the type ii photochemical process [ ] . a type i photochemical process can occur when a p&a photosensitizer in an excited state undergoes electron transfer reactions, which ultimately lead to the formation of reactive oxygen species (ros). their formation occurs as follows: at the first stage, electron transfer is the singlet state is short-lived (nanosecond lifetime), and excess energy can be emitted by the p&a in the form of light (fluorescence) or dissipated into the energy of disordered processes and, ultimately, emitted into heat. the third option, "intersystem crossing," is also possible, in which the p&a goes into a more stable excited triplet state (microsecond lifetime). from the point of view of photochemistry, triplet state allows the p&a to transfer energy upon contact with molecular oxygen molecules , , of (o ); as a result, the p&a returns to its original singlet state, and oxygen passes into the excited triplet state ( o ). the described photochemical reaction is called the type ii photochemical process [ ] . a type i photochemical process can occur when a p&a photosensitizer in an excited state undergoes electron transfer reactions, which ultimately lead to the formation of reactive oxygen species (ros). their formation occurs as follows: at the first stage, electron transfer is carried out with the formation of a cation/anion radical. a radical anion can react with oxygen to form a superoxide anion radical (o •− ). the dismutation or one-electron reduction of o •− produces hydrogen peroxide (h o ), which, in turn, can undergo another one-electron reduction with the formation of hydroxyl radicals (ho • ) [ ] . active oxygen species obtained during photoexposure to the p&a will react with the immediate environment of the p&a and lead to irreversible chemical changes in the target. according to a number of researchers [ , ] , the majority of p&as used in photoinduced oxidative processes of biomolecules "operate" via a type ii mechanism. this opinion is based on a comparison of the rate constants of energy transfer processes in the production of o (k ≈ - × dm ·mol − ·s − ) and electron transfer in the production of o •− (k ≤ × dm ·mol − ·s − ) [ ] . according to other researchers, both mechanisms proceed simultaneously, and the contribution to the photooxidation of each of them depends on the nature of the p&a, the excitation wavelength [ ] , the duration and intensity of photoirradiation [ ] , and on other external conditions. as noted above, all ros have a short lifetime and high reactivity that results in damage of the immediate environment of the p&a. for the unenveloped viruses, capsid proteins, including host cell recognition proteins, are direct targets. for the enveloped viruses, the main targets are enveloped glycoproteins, including host recognition proteins, as well as the lipid membrane [ ] . moreover, in both cases, structural proteins play a decisive role in the interaction of the host virus and in the internalization of the virus into the host cell. therefore, their irreversible photochemical damage can lead to the destruction of the virus. the principal possibility of virus inactivation in photoinduced capsid damage was demonstrated on the bacteriophage ms . the bacteriophage ms is often used as a model of the virus due to its nonpathogenicity to humans and ease of handling [ ] [ ] [ ] [ ] . it turned out that , , , -tetrakis-(n-methyl- -pyridyl)porphin (tmpyp ) with a concentration of . µm destroys the bacteriophage under -min illumination with a power of mw·sm − . the authors attribute such rapid inactivation [ ] to the oxidation of one of the sites of the viral protein responsible for the attachment of ms to the bacterial pilus and the delivery of its protein genome to the host genome. this site is enriched with amino acids residues of histidine and tryptophan known as amino acids of the increased vulnerability to o (by the rate of the chemical reaction between o and amino acids, the most vulnerable acids cysteine, methionine, tryptophan, tyrosine, and histidine are revealed (k = . × , . × , . × , × and . × М − ·с − , respectively) [ ] . the opposite conclusion was reached in [ , ] , where it was shown that the complete cleavage of the capsid of the bacteriophage ms occurred due to the oxidation of cys , which is the only susceptible and accessible o amino acid residue in this peptide. one cannot fail to note the research [ ] in which the photoinduced tmpyp inactivation of four x-shell-free rna viruses (bacteriophages ms and qβ, bovine enterovirus (bev- ), and mouse norovirus (mnv- )) were studied. viruses virions are of the similar size in - nm and contain icosahedral capsids. this work is noteworthy because the authors tried to establish a relationship between the rate of photoinactivation of the virus and the content of vulnerable amino acids in the capsid protein (histidine, tryptophan, methionine, and cysteine), and they determined the possibility of the effect of photooxidation on the inner surface of the capsid protein and on the viral genome. for icosahedral viruses, the phenomenon of capsid respiration is known [ ] , so the question if photoactivation can affect the viral genome has some sense. it turned out that rna extracted from virion susceptible to photooxidation and transfected into raw . cells exhibited titers compared to viral rna not subjected to photooxidation. the obtained results confirm that in this case, two targets were hit during tmpyp photoactivation: capsid protein and rna. cationic tetrakis(n-methyl- -pyridiniumyl) porphyrin, tetrakis(n-[n-butyl]- -pyridiniumyl) porphyrin, tetrakis(n-[n-octyl]- -pyridiniumyl) porphyrin, and anionic porphyrins (tetrakis ( -sulfonatophenyl) porphyrin) were evaluated as potential drugs for inactivation of nonenveloped viruses (hepatitis a virus and bacteriophage ms ). the authors set themselves the task of determining the effect of the charge and hydrophobicity of the p&a on its virucidal activity [ ] . unfortunately, the quantum yield of singlet oxygen and sor was not taken into account in this work; therefore, the conclusions drawn need to be revised. a similar error was made in [ ] , aimed at establishing the effect of the p&a charge on the photoinactivation of a t -like bacteriophage. enveloped viruses such as vesicular stomatitis virus (vsv) are photoinactivated by hydrophobic p&as (blood type porphyrins). the envelope of the vesicular stomatitis virus is a lipid bilayer obtained from the host cell; it contains trimers of one type of integral glycoprotein, called the g-protein, which allows the virus to enter the target cell through endocytosis, and it catalyzes the fusion of viral and endosomal membranes. dose-dependent inactivation of the virus was enhanced during photoactivation of porphyrins (protoporphyrin ix, zn (ii) protoporphin, mesoporphyrin ix) [ ] . it turned out that all three porphyrins intercalate into lipid vesicles and disrupt the structure of the viral membrane. in addition, these photoporphyrins caused viral glycoproteins vsv to crosslink. incubation of viruses with sodium azide and α-tocopherol partially protected vsv from inactivation by porphyrins, and it indicates that the process proceeded according to type ii. heme, cobalt protoporphyrin ix (coppix), and tin protoporphyrin ix (snppix) display antiviral activity against arboviruses, such as dengue virus, yellow fever virus, zika virus, mayaro virus, sindbis virus, and dental vesicular virus. porphyrin treatment results in the loss of viral enveloped protein, affects the morphology of the virus, adsorption, and also blocks penetration into host cells. photoirradiation enhanced snppix activity against all tested arboviruses [ ] . another example of photoinactivation of a virus with an envelope by porphyrin, the hiv virus, is presented in [ ] . the authors, taking into account the positively charged v loop of the gp protein, selected a series of anionic porphyrins containing two sulfonate and two carboxyl groups with different degrees of esterification as photosensitizers. in the dark phase, the esterified porphyrin compounds inhibited anti-v antibodies, but combined with the conserved region of c . after photoirradiation, inhibition of antibodies of the c region was exclusively observed. the results are rather unexpected, as it is reliably known that ros are very reactive and short-lived [ ] . according to the evaluations of [ ] , the lifetime of o in water is µs, while its maximum possible path does not exceed - nm in the absence of quenchers [ ] . in the system under consideration, judging by the data of [ ] , porphyrins are localized in region v , and they cause photodamage in region c . the authors explained this unusual phenomenon by the presence of a large number of vulnerable amino acids in the c protein fragment. speaking about the photodynamic inactivation of viruses with the envelope, one cannot fail to note the work [ ] devoted to the creation of influenza drugs. influenza virus (type a, b, and c) is one of the main human pathogens responsible for respiratory diseases, causing high morbidity and mortality from seasonal flu. the influenza virus is a (−)rna virus. a virion consists of a core, a shell, and matrix proteins. these proteins are hemagglutinin (ha), neuraminidase (na), matrix protein (m ), proton channel protein (m ), nucleoprotein (np), rna polymerase (pa, pb , and pb ), nonstructural protein (ns ), and nuclear export protein (nep, ns ). the rna genome of the influenza virus constantly mutates with the formation of new viral subtypes. although a vaccine is the most effective way to prevent influenza, vaccine formulations should be updated annually due to changes in circulating influenza viruses, and it takes several months to produce a flu vaccine. if the prognosis of incoming influenza strains is wrong, vaccines can offer only limited protective effectiveness [ ] . the stage of attachment of the virion to the cell surface (receptor binding) and fusion with the cell membrane is mediated by hemagglutinin (ha), which interacts with the host cell glycoproteins containing terminal sialic acid (n-acetylneuraminic acid, neu ac) [ , ] (figure ). thus, hemagglutinin is a target for inactivating the influenza virus. flu vaccine. if the prognosis of incoming influenza strains is wrong, vaccines can offer only limited protective effectiveness [ ] . the stage of attachment of the virion to the cell surface (receptor binding) and fusion with the cell membrane is mediated by hemagglutinin (ha), which interacts with the host cell glycoproteins containing terminal sialic acid (n-acetylneuraminic acid, neu ac) [ , ] (figure ). thus, hemagglutinin is a target for inactivating the influenza virus. a synthetic approach was developed to create a conjugate of porphyrin with zanamivir. the optimal length of the spacer was determined to ensure retaining of the affinity of zanamivir ( figure ) [ ] . zanamivir due to its high affinity for the hemagglutinin of the virus promoted targeted delivery of porphyrin, and porphyrin generated photocontact with singlet oxygen, which oxidized viral neuraminidase. the photochemical oxidation of this enzyme did not allow it to catalyze the hydrolytic reaction of cleavage of the terminal residue of neu ac from the sialo-receptor on the surface of the host cell (for the release of new virions). it should be noted that this is one of the few works in which the construction of the p&a photosensitizer for inactivation of the virus was carried out. a synthetic approach was developed to create a conjugate of porphyrin with zanamivir. the optimal length of the spacer was determined to ensure retaining of the affinity of zanamivir ( figure ) [ ] . zanamivir due to its high affinity for the hemagglutinin of the virus promoted targeted delivery of porphyrin, and porphyrin generated photocontact with singlet oxygen, which oxidized viral neuraminidase. the photochemical oxidation of this enzyme did not allow it to catalyze the hydrolytic reaction of cleavage of the terminal residue of neu ac from the sialo-receptor on the surface of the host cell (for the release of new virions). it should be noted that this is one of the few works in which the construction of the p&a photosensitizer for inactivation of the virus was carried out. some studies have reported that photoactivation of viruses without a membrane is faster and with a lower amount of p&a photosensitizer [ ] than viruses with a membrane. other works make the opposite conclusions [ ] , e.g., cationic tetraplatinized porphyrins do not exhibit any virucidal activity against enveloped viruses, but they effectively inactivate enveloped viruses (bvdv and bohv- ), which cause cattle diseases. it is probably not correct to compare the virucidal activity of p&as with respect to enveloped and unenveloped viruses, since: ( ) a priori, it should be different p&as, in the first case, they should be hydrophobic for localization in lipid structures, whereas in the second one, more hydrophilic; ( ) different p&as will have different ros quantum yield; ( ) capsid and supercapsid possess different local oxygen concentration; and ( ) photoinduced damages in the capsid and supercapsid are different [ ] (figure ). some studies have reported that photoactivation of viruses without a membrane is faster and with a lower amount of p&a photosensitizer [ ] than viruses with a membrane. other works make the opposite conclusions [ ] , e.g., cationic tetraplatinized porphyrins do not exhibit any virucidal activity against enveloped viruses, but they effectively inactivate enveloped viruses (bvdv and bohv- ), which cause cattle diseases. it is probably not correct to compare the virucidal activity of p&as with respect to enveloped and unenveloped viruses, since: ( ) a priori, it should be different p&as, in the first case, they should be hydrophobic for localization in lipid structures, whereas in the second one, more hydrophilic; ( ) different p&as will have different ros quantum yield; ( ) capsid and supercapsid possess different local oxygen concentration; and ( ) photoinduced damages in the capsid and supercapsid are different [ ] (figure ). some studies have reported that photoactivation of viruses without a membrane is faster and with a lower amount of p&a photosensitizer [ ] than viruses with a membrane. other works make the opposite conclusions [ ] , e.g., cationic tetraplatinized porphyrins do not exhibit any virucidal activity against enveloped viruses, but they effectively inactivate enveloped viruses (bvdv and bohv- ), which cause cattle diseases. it is probably not correct to compare the virucidal activity of p&as with respect to enveloped and unenveloped viruses, since: ( ) a priori, it should be different p&as, in the first case, they should be hydrophobic for localization in lipid structures, whereas in the second one, more hydrophilic; ( ) different p&as will have different ros quantum yield; ( ) capsid and supercapsid possess different local oxygen concentration; and ( ) photoinduced damages in the capsid and supercapsid are different [ ] (figure ). these damages are revealed to a greater extent on protein than in lipid structures [ ] . protein oxidation can lead to a change in protein conformation, oxidation of amino acid residues [ , ] , crosslinking, and destruction of the main chain of the polypeptide up to the capsid cleavage. there are several approaches to studying the state of viral proteins after photoinitiated reactions. these approaches are the traditional polyacrylamide gel electrophoresis (sds-page), ir spectroscopy [ ] , mass spectrometry with matrix laser desorption ionization (maldi) [ ] , and ionization by sputtering in an electric field (esi) [ , ] , and the method based on the use of antibodies [ ] , as well as the classical biochemical method for evaluating the virucidal effect of the oxidation process by means of cultivation procedure. the use of sds-page and ir spectroscopy provides general information about the state of viral proteins, their aggregation and cleavage, but it is impossible to obtain a detailed information on the affected domains, on oxidation of side chains, loops, and on conformation changes in the secondary and tertiary structure of the protein. the advent of mass spectrometric methods provided accurate mass measurements of whole viral particles and individual capsid proteins before and after oxidation. these methods make it possible to determine the degree of fragmentation of the main chain of the polypeptide and to identify areas of damage of the protein capsid, of photo-induced dimerization, and aggregation, i.e., those processes that lead to significant changes in the molecular weight of the protein. protein oxidation can also result in structural changes in the side chains of amino acid residues, to transformations that have only a moderate effect on the mass of the protein but can have dramatic consequences for its functioning. antibodies can help in detecting even small damaged areas [ ] . lipid oxidation causes less dramatic changes if the photoinduced process is carried out according to the type ii mechanism. then singlet oxygen reacts with unsaturated lipids with the formation of lipid hydroperoxides (looh) and it leads to a change in the thickness, area [ ] , and permeability of the lipid layer [ , ] . in the case of oxidation by mechanism i, i.e., when free radical mechanisms are involved in the first stage, the hydrogen atom is cleaved from unsaturated fatty acid (lh) and carbon-centered lipid radical (l • ) is formed, which, when interacting with an oxygen molecule, gives the radical loo • . the loo • radical is capable of reacting with other lh fatty acids and this reaction leads to the formation of lipid hydroperoxide (looh) and another lipid radical (l • ) [ ] . thus, numerous studies have shown that p&a compounds can act as virucidal chemotherapeutic and photoinactivating compounds at the stage of attachment of the virion to the cell surface (receptor binding) and fusion with the cell membrane, regardless of the presence or absence of supercapsid. it is likely that the degree of virulence of the p&a will depend significantly on the localization of the p&a and the selectivity of linking with a target. for targeted modelling of the mgc structure, reliable information on the structure of the target is needed. there is a definite correlation between virucidal activity and chemotherapeutic activity and photoinactivating ability. as a rule, p&as exhibiting both of the latter properties are virucidal [ , ] . as noted above, hiv virion contains three enzymes: reverse transcriptase (rna-dependent dna polymerase), which is involved in the synthesis of dna on an rna matrix; an integrase that catalyzes the incorporation of the generated viral dna into the chromosome of the host cell; and a protease that splits synthesized polyproteins into structural proteins. the stage of transcription and replication of viral rna/dna is impossible without the participation of these enzymes which are the target for chemotherapeutic agents used in the treatment of aids. in this context, a publication [ ] should be noted in which it was reported that a new site for the reverse reductase enzyme has been identified, and it was capable of binding compounds of the porphyrin class. to identify it, the authors compared the protein sequences of reverse reductase and heme-binding proteins and detected an identical conserved site ( - position in the hiv- reverse reductase polypeptide chain (wetwwteywq) [ ] . this site contained an excessive amount of aromatic amino acid residues which, according to the authors of [ ] , led to high stability of the complexes of this site with iron(iii) and zinc(ii) protoporphyrins (the binding constant is about ( . ÷ ) × dm ·mol − ). it should be noted that the binding of protoporphyrins to the indicated site by metal complexes was not highly selective, and the antiviral activity exhibited was relatively low ( µm, > %). the significance of the nature of the metal complexing agent in the processes of hiv inactivation is evidenced by the results of [ ] . according to them, reverse transcriptase is inhibited by porphyrins containing aminosulfonyl peripheral substituents. in this case, the metal-free porphyrin and its zinc complex were active at % and . % inhibition, respectively, while the vanadium complex of porphyrin showed much better results ( µm with an inhibition of more than %). however, as shown in [ ] , porphyrins demonstrated a low selectivity for reverse transferase binding. the selective binding of reverse transferase with iron-protoporphyrin dimethyl ether, protoporphyrin dimethyl ether, and its sodium salt has been established [ ] , but the virucidal activity of natural porphyrins has not been evaluated. unfortunately, it was not possible to find publications in which highly selective binding of p&a to reverse transferase was achieved due to the introduction of peripheral substituents containing an oligopeptide sequence complementary to the hiv fragment, although this approach is used to increase the tumorigenicity and selectivity of the p&a used in pdt. for example, porphyrin conjugates can be synthesized, the peripheral substituents of which contain the hiv peptide sequence [ ] , and the latter provides targeting of cancer cells. analysis of nonpeptide compounds with useful pharmacological properties synthesized for neutron capture therapy revealed several porphyrins containing carborane esters at positions and ( figure ); these can inhibit micromolar amounts of hiv- and hiv- protease [ ] . is used to increase the tumorigenicity and selectivity of the p&a used in pdt. for example, porphyrin conjugates can be synthesized, the peripheral substituents of which contain the hiv peptide sequence [ ] , and the latter provides targeting of cancer cells. analysis of nonpeptide compounds with useful pharmacological properties synthesized for neutron capture therapy revealed several porphyrins containing carborane esters at positions and ( figure ); these can inhibit micromolar amounts of hiv- and hiv- protease [ ] . porphyrin compounds containing carborane esters as peripheral substituents have a greater inhibitory effect on the protease compared with protoporphyrin ix and porphyrins not containing carboranes. the introduction of metals, both coordinatively saturated and unsaturated, negatively affects the ability of the p&a to inhibit the protease. important factors are not only the presence of carborane groups but also their isomerism. the introduction of a methyl group into carborane also significantly reduces the affinity of p&a linking with the protease. apparently, carborane substituents specifically interact with hiv protease, and it leads to a high affinity between the p&a and the enzyme. replacing carborane cells with similar in size, but less hydrophobic groups, such as benzoyl, adamantoyl, β-napthoyl, allows one to obtain inhibitors in the low micromolar range, and it indicates the importance of hydrophobic interactions in stabilizing the complex of the p&a with the enzyme. the best result was obtained with porphyrin-tetrakiscarborane carboxylate ester of , -bis-(α,β-dihydroxyethyl) deuteroporphyrin ix which is a submicromolar hiv protease inhibitor [ ] . moreover, for hiv- protease, the ic value is nm, whereas for hiv- , it is nm. the stage of transcription and replication of viral rna/dna is naturally impossible without the source of genetic material. viral genomes contain dna or rna, which can be either double- porphyrin compounds containing carborane esters as peripheral substituents have a greater inhibitory effect on the protease compared with protoporphyrin ix and porphyrins not containing carboranes. the introduction of metals, both coordinatively saturated and unsaturated, negatively affects the ability of the p&a to inhibit the protease. important factors are not only the presence of carborane groups but also their isomerism. the introduction of a methyl group into carborane also significantly reduces the affinity of p&a linking with the protease. apparently, carborane substituents specifically interact with hiv protease, and it leads to a high affinity between the p&a and the enzyme. replacing carborane cells with similar in size, but less hydrophobic groups, such as benzoyl, adamantoyl, β-napthoyl, allows one to obtain inhibitors in the low micromolar range, and it indicates the importance of hydrophobic interactions in stabilizing the complex of the p&a with the enzyme. the best result was obtained with porphyrin-tetrakiscarborane carboxylate ester of , -bis-(α,β-dihydroxyethyl) deuteroporphyrin ix which is a submicromolar hiv protease inhibitor [ ] . moreover, for hiv- protease, the ic value is nm, whereas for hiv- , it is nm. the stage of transcription and replication of viral rna/dna is naturally impossible without the source of genetic material. viral genomes contain dna or rna, which can be either double-stranded or single-stranded, linear, or circular in topology, and monocistronic or polycistronic. genomes can be divided into several segments [ ] . reflecting the diversity of genetic material and replication mechanisms, baltimore's classification divides viruses into seven groups: double-stranded (ds) dna genomes (group i), single-stranded (ss)dna genomes (group ii), dsrna genomes, (group iii), ss(+)rna genomes (group iv), ss(−)rna genomes in (group v), ssrna genomes with reverse transcription of the dsdna replication of intermediate (group vi), and dsdna genomes with the ssrna replication intermediate (group vii) [ ] . the genome of the vast majority of viruses has been decoded and can be found in specialized literature. obviously, this genetic material of the virus is a supertarget for the fight against viral infection. moreover, the linking of the p&a with the genetic material of the virus and its further photoinactivation are very promising. porphyrins and their analogues can form various types of complexes with dna: intercalates (internal complex) ( figure a ) [ ] , binding to a small groove of dna ( figure b ) [ ] , binding to a large groove of dna ( figure c ) [ ] , and external binding with self-stacking along the dna surface (external complex) ( figure d ) [ ] . linking of the p&a with the genetic material of the virus and its further photoinactivation are very promising. porphyrins and their analogues can form various types of complexes with dna: intercalates (internal complex) (figure a ) [ ] , binding to a small groove of dna (figure b ) [ ] , binding to a large groove of dna (figure c ) [ ] , and external binding with self-stacking along the dna surface (external complex) (figure d ) [ ] . the way of linking porphyrins with dna depends on the nature of the peripheral substituents of the p&a, on the metal of the complexing agent, the type of dna [ ] [ ] [ ] , external conditions, and even the concentration of the p&a [ ] . one of the first cationic porphyrins studied in terms of interaction with dna was tmpyp . the first publication dated [ ] reported on the spectral manifestation of the interaction of tmpyp with dna. subsequently, the data were supplemented, and it was found that intercalation of porphyrin in dna leads to a bathochromic shift of the soret band (> nm) in the uv-vis spectrum of porphyrin, to a decrease in its intensity by (~ %), while in the spectra of circular dichroism of the intercalate in the soret region, the negative induced signal is recorded. the formation of an external complex (figure d ) is spectrally discovered in the bathochromic shift of the soret band to nm and a slight decrease in intensity (~ - %) [ ] is observed. the spectrum of circular dichroism of the complex (figure d ) includes one positive band in the soret region [ ] . in a number of works, it is noted that a positive induced cd band in the soret region is indicative of external (minor) groove binding. in the case of the formation of an intercalate (figure a ), the interaction between porphyrin and a pair of nitrogenous bases guaninecytosine is most likely [ , ] . during the formation of the complex (figure d) , the π-π interaction is realized between the aromatic system of the porphyrin macrocycle and the adeninethymine pair. the spectral changes listed above are called "fingerprints", i.e., they allow one to identify what type of complex is being formed (figure ). the way of linking porphyrins with dna depends on the nature of the peripheral substituents of the p&a, on the metal of the complexing agent, the type of dna [ ] [ ] [ ] , external conditions, and even the concentration of the p&a [ ] . one of the first cationic porphyrins studied in terms of interaction with dna was tmpyp . the first publication dated [ ] reported on the spectral manifestation of the interaction of tmpyp with dna. subsequently, the data were supplemented, and it was found that intercalation of porphyrin in dna leads to a bathochromic shift of the soret band (> nm) in the uv-vis spectrum of porphyrin, to a decrease in its intensity by (~ %), while in the spectra of circular dichroism of the intercalate in the soret region, the negative induced signal is recorded. the formation of an external complex ( figure d ) is spectrally discovered in the bathochromic shift of the soret band to nm and a slight decrease in intensity (~ - %) [ ] is observed. the spectrum of circular dichroism of the complex ( figure d ) includes one positive band in the soret region [ ] . in a number of works, it is noted that a positive induced cd band in the soret region is indicative of external (minor) groove binding. in the case of the formation of an intercalate ( figure a ), the interaction between porphyrin and a pair of nitrogenous bases guanine-cytosine is most likely [ , ] . during the formation of the complex ( figure d ), the π-π interaction is realized between the aromatic system of the porphyrin macrocycle and the adenine-thymine pair. the spectral changes listed above are called "fingerprints", i.e., they allow one to identify what type of complex is being formed (figure ) . when changing the composition of the medium or the molar ratio r (r ratio is the ratio of the concentration of dna base to porphyrin, [dna]/[p] ranges), the internal complex can transform into the external [ , , ] . as a rule, the formation of intercalation complexes requires the planar structure of the p&a, small peripheral substituents, and the p&a:dna ratio (for nitrogen base pairs) of less than : . cationic p&as are preferred since phosphate groups of dna are negatively charged. the formation of intercalation of metal complexes of the p&a with dna containing unsaturated or multiply charged metals is difficult or does not occur at all, due to steric hindrances caused by the existing axial ligand or metal counterion [ ] . the thermodynamic stability of the complexes of p&a with dna depends not only on the nature of p&a but also on the type of complex formed [ ] . this condition is important when using p&a as a chemotherapeutic agent, as well, as it is shown below, when p&a is applied as a photoinactivator. as our own [ ] and literary studies [ ] [ ] [ ] have shown, depending on the type of photochemical process i or ii (figure ), different results of dna photooxidation can be obtained. the process according to mechanism i leads to oxidation of bases, oxidation of a phosphoric ester group, and, as a consequence, dna cleavage [ ] . the process according to mechanism ii causes deamination, release of free purine bases, and oxidation of a phosphoric ester group and leads to dna fragmentation [ , , ] . as our own [ ] and literary studies [ ] [ ] [ ] have shown, depending on the type of photochemical process i or ii (figure ), different results of dna photooxidation can be obtained. the process according to mechanism i leads to oxidation of bases, oxidation of a phosphoric ester group, and, as a consequence, dna cleavage [ ] . the process according to mechanism ii causes deamination, release of free purine bases, and oxidation of a phosphoric ester group and leads to dna fragmentation [ , , ] . in particular, the guanine fragments ( figure , compound ) are susceptible to oxidative damage, resulting in the formation of -oxo- , -dihydroguanine ( figure , compound ) , one of the main products of type i photosensitized oxidation. porphyrin * electron transfer generation oh • energy transfer generation o oxidation of bases. oxidation of a phosphoric ester group deamination release of free purine bases oxidation of a phosphoric ester group dna cleavage dna fragmentation figure . scheme of dna photoreactions of type i and type ii. * excided triplet state. in particular, the guanine fragments ( figure , compound ) are susceptible to oxidative damage, resulting in the formation of -oxo- , -dihydroguanine ( figure , compound ) , one of the main products of type i photosensitized oxidation. photochemical process i or ii (figure ), different results of dna photooxidation can be obtained. the process according to mechanism i leads to oxidation of bases, oxidation of a phosphoric ester group, and, as a consequence, dna cleavage [ ] . the process according to mechanism ii causes deamination, release of free purine bases, and oxidation of a phosphoric ester group and leads to dna fragmentation [ , , ] . in particular, the guanine fragments ( figure , compound ) are susceptible to oxidative damage, resulting in the formation of -oxo- , -dihydroguanine ( figure , compound ) , one of the main products of type i photosensitized oxidation. porphyrin * electron transfer generation oh the result of photoirradiation of complexes of p&a with dna substantially depends on the type of complex being formed. for example, in [ ] , it was shown that irradiation of porphyrin intercalates (tmpyp and tmpyp ) with dna leads to dna fragmentation, and in the case of tmpyp , dna fragments of different sizes are formed. irradiation of external complexes of porphyrins with dna results in the cleavage of dna , , , -tetrakis-(n-methyl- -pyridyl)porphin (tmpyp ) and tmpyp and is accompanied by photolysis of porphyrin (tmpyp ). according to the researchers [ ] , the binding of porphyrins tmpyp and meso-tri-( -nmethylpyridyl) monophenylporphyrin (tmpympp) to dna is not a prerequisite for photoinactivation. the authors of [ ] consider that free porphyrins are more effective in inactivating the virus than dna-related species and explain it by a lower quantum yield of singlet oxygen of the p&a in the complex as compared with free p&a [ ] . this conclusion contradicts the generally accepted view that the p&a should be in close proximity to the place of photodamage. the reason for the controversial conclusions may be that the authors did not take into account the very probable [ ] inversion of the intercalation complex into the external complex [ ] . having obtained intercalates at the initial stage, then increasing the concentration of porphyrins, they mistakenly believed that saturation of the intercalation complex was achieved and unbound porphyrin was present in the solution [ ] . it is possible to achieve the formation of only one type of complexes with dna due to a significant increase in the volume of peripheral porphyrin substituents, making intercalation sterically impossible. this was demonstrated in [ ] by the example of oligo-and polypeptide conjugates of cationic porphyrins, which form exclusively external complexes with dna. in addition to the type of complex formed, an important influence is exerted by the intensity and duration of irradiation-these conditions are described in detail in the review [ ] . prevention of viral replication and reduction of virulence after dna damage by active oxygen forms, generated by the p&a, was shown in [ , , , ] . as noted above, the genetic material of the virus can be represented by dna and rna. the most serious human diseases caused by rna viruses are ebola hemorrhagic fever, sars, covid- , rabies, influenza, hepatitis c and e, west nile fever, poliomyelitis, measles, etc. the viral genome can be represented as single-stranded (ssrna single-stranded) and double-stranded rna (dsrna double-stranded) [ ] . single-stranded rnas in accordance with their polarity are divided into single-stranded rna viruses with a negative strand ((−)rna) and single-stranded rna viruses with a positive strand ((+)rna). single-stranded (+)rna acts as a messenger rna and can be directly translated by an infected cell (it can individually cause an infectious disease). therefore (+)rna is also called semantic. antisemantic (−)rna of the virus before translation must be converted to (+)rna by the action of rna polymerase. in terms of targeting, rna has many attractive properties similar to those of dna and/or proteins. both forms of nucleic acids take a regular helix with large and small grooves. however, the usual helix of the a-form of rna is often disturbed by regions of inappropriate or unpaired bases; they lead to a change in the secondary structure of rna ( figure ). compared with the small and large grooves of dna, these structural motifs in rna are much smaller, since the pitch of the helix and the inclination of the bases are smaller. according to [ , ] , the width of the main groove of the a-shape helix is only Å and this circumstance excludes the binding of small organic compounds, including p&a. thus, the targets available for binding to the p&a are the duplex rna regions presented above ( figure ). during the interaction of p&a with duplex rna, as in the case of dna, the formation of types of complex is possible-external with self-stacking p&a and intercalation. the first publication on the interaction of cationic tmpyp porphyrin with rna duplexes dates back to [ ] . a similar approach based on the interaction of cationic porphyrins with mrna g-quadruplexes is used to evaluate the use of porphyrins in photodynamic therapy [ ] . the introduction of copper ion into the tmpyp reaction center leads to the binding of cutmpyp to ra•ru base pairs in duplex rnas. the introduction of metal contributes to the formation of compared with the small and large grooves of dna, these structural motifs in rna are much smaller, since the pitch of the helix and the inclination of the bases are smaller. according to [ , ] , the width of the main groove of the a-shape helix is only Å and this circumstance excludes the binding of small organic compounds, including p&a. thus, the targets available for binding to the p&a are the duplex rna regions presented above ( figure ). during the interaction of p&a with duplex rna, as in the case of dna, the formation of types of complex is possible-external with self-stacking p&a and intercalation. the first publication on the interaction of cationic tmpyp porphyrin with rna duplexes dates back to [ ] . a similar approach based on the interaction of cationic porphyrins with mrna g-quadruplexes is used to evaluate the use of porphyrins in photodynamic therapy [ ] . the introduction of copper ion into the tmpyp reaction center leads to the binding of cutmpyp to ra·ru base pairs in duplex rnas. the introduction of metal contributes to the formation of stronger complexes with rna duplexes compared with similar complexes of metal-free porphyrin [ ] . there are also known data on the interaction of rna g-quadruplexes with phthalocyanines [ ] . a series of works is devoted to md modelling of the binding of tmpyp to rna and oligonucleotides [ , ] , the result of which was the conclusion about the binding of tmpyp in large and small grooves and an unusual external binding in which porphyrin is positioned so that its plane is oriented parallel to the main chain of the biopolymer. similar schemes of complexes contradict the existing views [ , ] , whereas another work [ ] of the same authors reports on the intercalation binding of tmpyp to a synthetic polynucleotide poly(adenyl), poly(uridyl) acid (poly(a)-poly(u)). the effect of the modification of the cationic porphyrin substituent (figure ) on the binding to poly(ra) poly(ru) and poly(ri) poly(rc) homopolymer rna duplexes was studied in [ ] . tetohpyp and talpyp intercalate into duplexes poly (ra) poly (ru) and poly (ri) poly (rc), whereas tmetalpyp binds to these rna duplexes, forming an external complex with self-stacking [ ] . it is noteworthy that from the spectral data obtained in the temperature range of and °c, the authors determined the thermodynamic parameters of binding [ ] , while not taking into account that the van't hoff approach is not acceptable for thermosensitive biopolymers and their complexes with a large fraction of the electrostatic contribution. as a rule, p&a compounds form not very strong complexes with rna due to structural factors, therefore, chemotherapeutic agents for the inactivation of rna viruses have not yet been proposed. on the other hand, there remains the possibility of photoinduced inactivation of rna viruses at the stage of transcription and replication of the viral genome. as shown above, in the case of dna, all four bases are oxidized, but guanine is the most susceptible; it produces -oxo- , dihydro- '-deoxyguanosine under the influence of singlet oxygen. it is this compound that is often used as a marker of oxidative damage in dna [ ] . in the case of rna, there is no such marker. confirmation of rna photooxidation is carried out using microbiological and analytical methods. however, there are certain difficulties, as many rna viruses are uncultivated or difficult to cultivate (e.g., hepatitis a, human norovirus, etc.). analytical methods (pcr) in the case of (−) rna imply pretreatment with reverse transcriptase, which may be accompanied by a large number of errors [ ] . these factors limit the study of photoinactivation of viral rna. there is relatively scarce evidence that inactivation of rna viruses with photo-irradiation of the p&a is possible, and it can lead to mutations in the genome. oxidized rnas can lead to the synthesis of defective proteins that are not able to form a capsid or provide a binding to the host cell receptor [ ] . it is the violation of the biochemical functions of rna during photoirradiation of the external stacking complexes of meso-tetrakis( -n-methylpyridyl)porphin, meso-tetrakis(para-ntrimethylanilinium)porphin, and meso-tetrakis( -n-methylpyridyl) porphin and rna and complexes of porphyrins with elements of the secondary structure of rna [ ] that causes inactivation of the hiv- virus. from a chemical point of view, the photoinduced reaction of o with viral rna leads to the modification of nucleosides in the region of localization of porphyrins tetohpyp and talpyp intercalate into duplexes poly(ra) poly(ru) and poly(ri) poly(rc), whereas tmetalpyp binds to these rna duplexes, forming an external complex with self-stacking [ ] . it is noteworthy that from the spectral data obtained in the temperature range of and • c, the authors determined the thermodynamic parameters of binding [ ] , while not taking into account that the van't hoff approach is not acceptable for thermosensitive biopolymers and their complexes with a large fraction of the electrostatic contribution. as a rule, p&a compounds form not very strong complexes with rna due to structural factors, therefore, chemotherapeutic agents for the inactivation of rna viruses have not yet been proposed. on the other hand, there remains the possibility of photoinduced inactivation of rna viruses at the stage of transcription and replication of the viral genome. as shown above, in the case of dna, all four bases are oxidized, but guanine is the most susceptible; it produces -oxo- , -dihydro- -deoxyguanosine under the influence of singlet oxygen. it is this compound that is often used as a marker of oxidative damage in dna [ ] . in the case of rna, there is no such marker. confirmation of rna photooxidation is carried out using microbiological and analytical methods. however, there are certain difficulties, as many rna viruses are uncultivated or difficult to cultivate (e.g., hepatitis a, human norovirus, etc.). analytical methods (pcr) in the case of (−)rna imply pretreatment with reverse transcriptase, which may be accompanied by a large number of errors [ ] . these factors limit the study of photoinactivation of viral rna. there is relatively scarce evidence that inactivation of rna viruses with photo-irradiation of the p&a is possible, and it can lead to mutations in the genome. oxidized rnas can lead to the synthesis of defective proteins that are not able to form a capsid or provide a binding to the host cell receptor [ ] . it is the violation of the biochemical functions of rna during photoirradiation of the external stacking complexes of meso-tetrakis( -n-methylpyridyl)porphin, meso-tetrakis(para-n-trimethylanilinium) porphin, and meso-tetrakis( -n-methylpyridyl) porphin and rna and complexes of porphyrins with elements of the secondary structure of rna [ ] that causes inactivation of the hiv- virus. from a chemical point of view, the photoinduced reaction of o with viral rna leads to the modification of nucleosides in the region of localization of porphyrins (inner and stem loops and hairpins, figure ). chemical modification of these areas of the secondary structure of rna conditions a change in the tertiary structure, namely, a change in coaxially laid rna helices [ ] . thus, the formation of complexes between the p&a and dna/rna viruses can result in chemical and/or photochemical inactivation. currently, the possibility of inactivation of viruses in vitro and in vivo is mainly determined by the mechanism of chemical inactivation. in this case, there is a potential danger, since the chemical modification of the genome may not inactivate the virus, but lead to a mutation [ ] . the main difficulty in using p&a to inactivate viruses with a molecular target (the genetic material of viruses) lies in the low selectivity of p&a and the active binding of p&a to dna by host cells. perhaps, the solution to this problem is not far off. a chemical approach to dna recognition [ ] based on the use of polyamides has already been found. analogues of the n-methylpyrrole rings of these polyamides provide a set of five-membered heterocycles that can be combined (in the form of asymmetric pairs of rings) in a modular manner to recognize predetermined dna sequences with affinity and specificity comparable to dna-binding proteins. the presence of such a peripheral substituent in the p&a can solve the problem of precise targeting of viral dna. another possible solution to the problem of selectivity for targeting the p&a to the viral genome is to accurately target the sections of the genetic material of viruses, such as, e.g., g-quadruplexes. guanine-rich rna or dna sequences are capable of folding and accepting four strands, called g-quadruplexes or "g " ( figure e ). unique features of the g topology, very different from dna duplex or single-stranded rna, make it a potential therapeutic target. the orientation of the strands determines a parallel, antiparallel, or mixed topology of g , and it is directly related to the conformational state, the anti-or syn-glycosidic bond between the base g and sugar ( figure ) [ ] . another possible solution to the problem of selectivity for targeting the p&a to the viral genome is to accurately target the sections of the genetic material of viruses, such as, e.g., gquadruplexes. guanine-rich rna or dna sequences are capable of folding and accepting four strands, called g-quadruplexes or "g " (figure e ). unique features of the g topology, very different from dna duplex or single-stranded rna, make it a potential therapeutic target. the orientation of the strands determines a parallel, antiparallel, or mixed topology of g , and it is directly related to the conformational state, the anti-or syn-glycosidic bond between the base g and sugar ( figure ) [ ] . g structures are widely represented in the human genome, especially in telomeres and oncogenic promoters. in recent years, the presence of g s in viruses has attracted increasing interest. g-quadruplexes were found in several viruses, including those associated with recent epidemics, such as zika and ebola viruses, sars, and covid- [ ] . viral g s are usually located in regulatory regions of the genome and are involved in the control of key viral processes, such as virulence, virus replication, recombination, and structural changes to evade the immune response [ ] . comprehensive information on the number of g-quadruplexes and their functions in viruses is presented in reviews [ ] [ ] [ ] . inactivation of viruses is possible due to the formation of a strong complex between the p&a and g-quadruplexes of viruses. when designing porphyrins and their analogues, it is important to achieve high stability of their complexes with gquadruplexes, and it is possible with strict chemical and geometric compliance. cationic porphyrins tmpyp and ( , , , -tetrakis-(n-methyl- -pyridyl)porphin) (tmpyp ) were proposed as a g structures are widely represented in the human genome, especially in telomeres and oncogenic promoters. in recent years, the presence of g s in viruses has attracted increasing interest. g-quadruplexes were found in several viruses, including those associated with recent epidemics, such as zika and ebola viruses, sars, and covid- [ ] . viral g s are usually located in regulatory regions of the genome and are involved in the control of key viral processes, such as virulence, virus replication, recombination, and structural changes to evade the immune response [ ] . comprehensive information on the number of g-quadruplexes and their functions in viruses is presented in reviews [ ] [ ] [ ] . inactivation of viruses is possible due to the formation of a strong complex between the p&a and g-quadruplexes of viruses. when designing porphyrins and their analogues, it is important to achieve high stability of their complexes with g-quadruplexes, and it is possible with strict chemical and geometric compliance. cationic porphyrins tmpyp and ( , , , -tetrakis-(n-methyl- -pyridyl)porphin) (tmpyp ) were proposed as a complexing compound for g of a number of viruses [ ] . tmpyp compared with the tmpyp isomer showed the best results [ ] in inactivation of hiv- virus. after incubation with tmpyp , the expression of nef protein, the main necessary protein for virus replication, was reduced. the authors suppose that it occurred due to the formation of a stable complex between tmpyp and g-quadruplex structures in the nef hiv- coding region [ ] . it is interesting to note that the stability of the resulting complexes, on which the antiviral activity of porphyrin depends, was evaluated according to the melting points of free and complexed g-quadruplex. later, tetracationic porphyrins exhibiting antiviral activity against different strains of hiv were patented [ ] . it is noteworthy that exploratory studies are carried out in the direction of modifying cationic peripheral substituents [ ] , but it is overlooked that the complex of g-quadruplexes with p&a is stabilized not only due to anion-cationic interactions but also due to the extended π-π-interaction between the aromatic system of porphyrin and guanine bases. the cationic porphyrins tmpyp , tmpyp , and tmpyp were considered as potential binding agents for epstein-barr virus g-quadruplex rna. it turned out that tmpyp and tmpyp form a stable complex with g regions of the virus genome, and it does not allow to encode the ebna protein, which is crucial for replication and maintenance of the genome during latency in proliferating cells [ ] . in numerous studies, it was shown that cationic porphyrins containing peripheral charged substituents in the para position, as a rule, demonstrate greater efficiency compared to both the orthoand metaisomers. in this sense, the approach of the authors [ ] comparing the antiviral activity of the tmpyp mesoisomer and the chemotherapeutic agent of braco- (trisubstituted acridine, characterized by excellent g-quadruplex binding properties) against herpes simplex virus (hiv- ) is not clear as well as their conclusion about the absence of the p&a study prospects in general. tmpyp was also used to study the role of g s in ebola virus replication [ ] . for this, the genetic materials of the original virus and the mutant that does not contain g quadruplexes in the l gene were studied. it was established that tmpyp exhibited high stabilization of only the initial rna. more important is that after treatment with elevated concentrations of tmpyp , gene transcription gradually decreased. if we compare the affinity of tmpyp with respect to g -structures and duplex dna, then it will be lower [ ] , respectively, and the antiviral activity of tmpyp can have several mechanisms of action and they limit its biological and clinical use. it is quite remarkable that in the vast majority of works on the inactivation of viruses, cationic porphyrins are considered as a binding agent for g-quadruplex structures. although, as shown in [ ] , anionic p&as are also capable of binding to g structures, moreover, more selectively. and if the issue of the selectivity of virus inactivation in living organisms remains open, then one can definitely say that recently successful attempts have been made to use p&a to reduce the transmission of pathogenic microorganisms during blood transfusion. viral infections, the potential risk of their transmission, narrowly targeted viral tests, and the lack of adequate and systematic screening for the virus and bacteria in the blood and red blood cells make the problem of increasing blood products safety very urgent. impressive results were demonstrated by the authors of [ ] by the example of inactivation of the vesicular stomatitis virus in red blood cells for blood transfusion. to evaluate virucidal and erythrocyte-damaging activity, a number of cationic porphyrins were considered ( figure ) . the most effective photosensitizer from the tested series, which at the same time maintained the integrity of red blood cells, was tri-p ( ). it caused the least hemolysis under conditions that led to a -fold decrease in the extracellular virus of vesicular stomatitis. all positively charged tested porphyrins were able to inactivate the extracellular virus of vesicular stomatitis, but caused a violation of the integrity of red blood cells during storage. vesicular stomatitis virus is a single-stranded lipid rna virus. unfortunately, the authors did not advance in the analysis of the "structure (porphyrin)-property (virucidal ability)," but they only suggested that the cause is the interaction of porphyrins with rna. judging by the data presented [ ] , the virucidal activity decreases in the series: tri-p( ) < tetohpyp( ) < trans-p( ) < cis-p( ) < tmpyp < mono-p( ). moreover, the last three porphyrins caused the maximum destruction of red blood cells. obviously, in addition to the photochemical properties of the photosensitizer, penetration (through the lipid membrane) and binding to rna are significant. in conclusion, we would like to note that the chemo-and photoinactivation of p&a in viruses is very promising and can become an alternative and highly effective way to treat viral infections. to increase the selectivity of the action of p&a on viral targets, a targeted modification of the structure of p&a is necessary. the complexity and versatility of the objects of research emphasize the need to combine the efforts of chemists, microbiologists, and virologists. the authors declare no conflict of interest. is antimicrobial resistance a slowly emerging disaster? public health ethics world health organization releases global priority list of antibiotic-resistant bacteria to guide research, discovery, and development of new antibiotics attributable deaths and disability-adjusted life-years caused by infections with antibiotic-resistant bacteria in the eu and the european economic area in : a population-level modelling analysis the high prevalence of antibiotic heteroresistance in pathogenic bacteria is mainly caused by gene amplification international committee on taxonomy of viruses executive committee. the new scope of virus taxonomy: partitioning the virosphere into hierarchical ranks endocytosis of viruses and bacteria. cold spring harbor perspect progress in hiv- integrase inhibitors: a review of their chemical structure diversity phages and hiv- : from display to interplay lentiviruses and macrophages: molecular and cellular interactions structure and immune recognition of the hiv glycan shield dual activity of amphiphilic zn(ii) nitroporphyrin derivatives as hiv- entry inhibitors and in cancer photodynamic therapy inactivation of human immunodeficiency virus type by porphyrins difficult-to-neutralize global hiv- isolates are neutralized by antibodies targeting open envelope conformations prediction of anti-hiv- activity of a series of tetrapyrrole molecules synthesis and antiviral activity evaluation of nitroporphyrins and nitrocorroles as potential agents against human cytomegalovirus infection alkylated porphyrins have broad antiviral activity against hepadnaviruses, flaviviruses, filoviruses, and arenaviruses chemoprevention of hepatocellular carcinoma in aflatoxin endemic areas some uses of transition metal complexes as anti-cancer and anti-hiv agents review of hiv- reverse transcriptase three-dimensional structure: implications for drug design antiviral drug resistance as an adaptive process new photosensitizers for photodynamic therapy oxygen-independent antimicrobial photoinactivation: type iii photochemical mechanism? antibiotics mechanisms of photosensitized oxidation. there are several different types of photosensitized oxidation which may be important in biological systems photodynamic efficiency: from molecular photochemistry to cell death singlet oxygen-mediated damage to proteins and its consequences effect of irradiation spectral range on porphyrin-protein complexes sewage bacteriophage inactivation by cationic porphyrins: influence of light parameters photodynamic inactivation of bacteriophage ms : the a-protein is the target of virus inactivation tmpyp functionalised chitosan membrane for efficient sunlight driven water disinfection oxidation of virus proteins during uv and singlet oxygen mediated inactivation photodynamic inactivation of mammalian viruses and bacteriophages photo-oxidation of proteins and its role in cataractogenesis oxidative modification of cytochrome c by singlet oxygen. free radic photodynamic inactivation of non-enveloped rna viruses maturation of a tetravirus capsid alters the dynamic properties and creates a metastable complex photoinactivation of hepatitis a virus by synthetic porphyrins mechanisms of vesicular stomatitis virus inactivation by protoporphyrin ix, zinc-protoporphyrin ix, and mesoporphyrin ix co-protoporphyrin ix and sn-protoporphyrin ix inactivate zika, chikungunya and other arboviruses by targeting the viral envelope interaction of sulfonated anionic porphyrins with hiv glycoprotein gp : photodamages revealed by inhibition of antibody binding to v and c domains an introduction to reactive oxygen species: measurement of ros in cells lifetime of oxygen (o ( .delta.g)) in liquid water as determined by time-resolved infrared luminescence measurements spatially resolved cellular responses to singlet oxygen synergistic effect of zanamivir-porphyrin conjugates on inhibition of neuraminidase and inactivation of influenza virus universal vaccines and vaccine platforms to protect against influenza viruses in humans and agriculture development of effective anti-influenza drugs: congeners and conjugates-a review influenza virus assembly and budding susceptibility of non-enveloped dna-and rna-type viruses to photodynamic inactivation photodynamic inactivation of selected bovine viruses by isomeric cationic tetra-platinated porphyrins generation of reactive oxygen species by photosensitizers and their modes of action on proteins sds-page and ir spectroscopy to evaluate modifications in the viral protein profile induced by a cationic porphyrinic photosensitizer lipid oxidation induces structural changes in biomimetic membranes giant vesicles under oxidative stress induced by a membrane-anchored photosensitizer membrane changes under oxidative stress: the impact of oxidized lipids photodynamic effect of some phthalocyanines on enveloped and naked viruses photodynamic inactivation of bovine herpesvirus type (bohv- ) by porphyrins the connection domain is implicated in metalloporphyrin binding and inhibition of hiv reverse transcriptase in vitro inhibition of human immunodeficiency virus type- (hiv- ) reverse transcriptase by gold (iii) porphyrins physiologically stable vanadium (iv) porphyrins as a new class of anti-hiv agents inhibition of hepadnavirus reverse transcriptase-ε rna interaction by porphyrin compounds enhanced cellular uptake with a cobaltacarboraneporphyrin-hiv- tat - conjugate specific inhibition of hiv- protease by boronated porphyrins evaluation of the genomic diversity of viruses infecting bacteria, archaea and eukaryotes using a common bioinformatic platform: steps towards a unified taxonomy interactions of tetracationic porphyrins with dna and their effects on dna cleavage comparison of the effects of cationic porphyrins on dna properties: influence of gc content of native and synthetic polymers intercalative and nonintercalative binding of large cationic porphyrin ligands to calf thymus dna rotation of periphery methylpyridine of meso-tetrakis(n-n-methylpyridiniumyl)porphyrin (n = , , ) and its selective bindingto native and synthetic dnas interaction of dna with a porphyrin ligand: evidence for intercalation small-molecule interaction with a five-guanine-tract g-quadruplex structure from the human myc promoter structural basis for binding of porphyrin to human telomeres -tetra-(n-methyl- -pyridyl)porphyrin destabilizes the antiparallel telomeric quadruplex d(ttaggg) dna-photocleavage agents potential involvement of both type i and type ii mechanisms in m virus inactivation by methylene blue photosensitization involvement of type i and type ii mechanisms on the photoinactivation of non-enveloped dna and rna bacteriophages photosensitizers mediated photodynamic inactivation against virus particles trends and targets in antiviral phototherapy type i and type ii photosensitized oxidation reactions: guidelines and mechanistic pathways comparison of the efficiency and the specificity of dna-bound and free cationic porphyrin in photodynamic virus inactivation oligo-and polypeptide conjugates of cationic porphyrins: binding, cellular uptake, and cellular localization light-activated nanotube-porphyrin conjugates as effective antiviral agents origins and evolution of the global rna virome targeting rna with small molecules stitching together rna tertiary architectures rna bulges as architectural and recognition motifs thermal unwinding of polyadenylic·polyuridylic acid complex with tmpyp porphyrin in aqueous solutions photodynamic therapy for ras-driven cancers: targeting g-quadruplex rna structures with bifunctional alkyl-modified porphyrins copper insertion facilitates water-soluble porphyrin binding to ra·ru and ra·dt base pairs in duplex rna and rna·dna hybrids an anionic phthalocyanine decreases nras expression by breaking down its rna g-quadruplex surface complex of zntmpyp metalloporphyrin with double-stranded poly(a)-poly(u) viral rna targets and their small molecule ligands mutual influence between contiguous tmpyp ligands when bound to a synthetic double-stranded poly(a)-poly(u) thermodynamics of interactions of water-soluble porphyrins with rna duplexes positively charged porphyrins: a new series of photosensitizers for sterilization of rbcs mechanistic aspects of the oxidation of dna constituents mediated by singlet molecular oxygen direct and indirect photochemical reactions in viral rna measured with rt-qpcr and mass spectrometry oxidized messenger rna induces translation errors efficient modification of rna by porphyrin cation photochemistry: monitoring the folding of coaxially stacked rna helices in trnaphe and the human immunodeficiency virus type rev response element rna local treatment of viral disease using photodynamic therapy recognition of the dna minor groove by pyrrole-imidazole polyamides dna: sequence, topology and structure the g-quadruplex/helicase world as a potential antiviral approach against covid- targets and tools in antiviral therapy g-quadruplexes in pathogens: a common route to virulence control? g-quadruplexes in viruses: function and potential therapeutic applications formation of a unique cluster of g-quadruplex structures in the hiv- nef coding region: implications for antiviral activity a dynamic g-quadruplex region regulates the hiv- long terminal repeat promoter derivatives of porphyrins, their process of preparation and their use for treating viral infections the binding mode of porphyrins with cation side arms to (tg t) g-quadruplex: spectroscopic evidence role for g-quadruplex rna binding by epstein-barr virus nuclear antigen in dna replication and metaphase chromosome attachment the herpes simplex virus- genome contains multiple clusters of repeated g-quadruplex: implications for the antiviral activity of a g-quadruplex ligand chemical targeting of a g-quadruplex rna in the ebola virus l gene shedding light on the interaction between tmpyp and human telomeric quadruplexes specific binding of anionic porphyrin and phthalocyanine to the g-quadruplex with a variety of in vitro and in vivo applications key: cord- -jetmocvk authors: wang, denong; tang, jin; tang, jiulai; wang, lai-xi title: targeting n-glycan cryptic sugar moieties for broad-spectrum virus neutralization: progress in identifying conserved molecular targets in viruses of distinct phylogenetic origins date: - - journal: molecules doi: . /molecules sha: doc_id: cord_uid: jetmocvk identifying molecular targets for eliciting broadly virus-neutralizing antibodies is one of the key steps toward development of vaccines against emerging viral pathogens. owing to genomic and somatic diversities among viral species, identifying protein targets for broad-spectrum virus neutralization is highly challenging even for the same virus, such as hiv- . however, viruses rely on host glycosylation machineries to synthesize and express glycans and, thereby, may display common carbohydrate moieties. thus, exploring glycan-binding profiles of broad-spectrum virus-neutralizing agents may provide key information to uncover the carbohydrate-based virus-neutralizing epitopes. in this study, we characterized two broadly hiv-neutralizing agents, human monoclonal antibody g and galanthus nivalis lectin (gna), for their viral targeting activities. although these agents were known to be specific for oligomannosyl antigens, they differ strikingly in virus-binding activities. the former is hiv- specific; the latter is broadly reactive and is able to neutralize viruses of distinct phylogenetic origins, such as hiv- , severe acute respiratory syndrome coronavirus (sars-cov), and human cytomegalovirus (hcmv). in carbohydrate microarray analyses, we explored the molecular basis underlying the striking differences in the spectrum of anti-virus activities of the two probes. unlike g , which is strictly specific for the high-density man( )glcnac( )asn (man )-clusters, gna recognizes a number of n-glycan cryptic sugar moieties. these include not only the known oligomannosyl antigens but also previously unrecognized tri-antennary or multi-valent glcnac-terminating n-glycan epitopes (tri/m-gn). these findings highlight the potential of n-glycan cryptic sugar moieties as conserved targets for broad-spectrum virus neutralization and suggest the gna-model of glycan-binding warrants focused investigation. developing broad-range virus-neutralizing antibodies (bnabs) requires identifying the immunological targets that are conserved among the targeted viruses and are suitable for antibody targeting. discovery of oligomannosyl moieties as targets of bnabs against hiv- has stimulated substantial interest in carbohydrate moieties as vaccine candidates [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] . the heavy glycosylation of the hiv envelope with oligomannoses appears to be a defense mechanism for the virus to evade host immune recognition of protein-based neutralizing epitopes. however, isolation of a number of glycan-dependent hiv bnabs from hiv-infected individuals, such as g , pg /pg , and other potent bnabs [ , , , , , ] , strongly suggests that the "glycan shield" of hiv virions may also display important targets for immunological intervention against viral infections. like hiv- , virtually all human viruses decorate their virions with carbohydrate moieties. thus, the potential of viral carbohydrates as immunological targets for other viral pathogens warrants exploration. one intriguing question is whether human viruses of distinct phylogenetic origins, such as hiv- and sars-cov, may display conserved glycan targets that are suitable for broad virus neutralization. availability of bnabs targeting such conserved glycans is clearly important for improving a biodefensive public health response to unexpected viral epidemics. in response to the sars epidemic, our team introduced carbohydrate microarrays to study sars-cov-elicited anti-glycan antibodies [ ] . in essence, we characterized the carbohydrate-binding activity of the sars-cov-neutralizing antibodies elicited by immunizing horses with an inactivated sars-cov vaccine. in these horse antibodies, we detected unexpected auto-antibody reactivity specific for the carbohydrate moieties of an abundant human serum glycoprotein, asialo-orosomucoid (asor). the targeted carbohydrates are tri-antennary type ii (galβ → glcnac) or multi-valent type ii (tri/m-ii) sugar moieties. the n-glycosylation pathway has potential to generate numerous cryptic n-glycans of distinct structural characteristics. the spike glycoprotein of sars-cov has potential n-linked glycosylation sites [ ] [ ] [ ] [ ] . ritchie et al. determined the glycan profiles of the spike protein produced by monkey vero-e cells [ ] . its major glycans include high-mannose (man - glcnac ), hybrid, and bi-, tri-and tetra-antennary complex carbohydrates with and without bisecting glcnac and core fucose. interestingly, sialylation was negligible in the spike proteins produced by monkey vero-e cells, which led to exposure of terminal galactoses in tri/m-ii glyco-determinants. thus, induction of anti-asor auto-antibodies by inactivated sars-cov can be attributed to the fact that asor and the sars-cov spike glycoprotein commonly express the tri/m-ii cryptic glyco-determinants. of note, this structural glycomics study revealed that sars-cov also expresses the high-mannose series of carbohydrate structures as its major glycan moieties. thus, despite the fact that the two viruses differ in their general glycan profiles, they commonly overexpress oligomannosyl cores of n-glycans. a significant number of virus-neutralizing agents target oligomannosyl moieties. notably, these include mab g and lectin gna. however, the two probes represent distinct classes of virus-neutralizing agents. the former is "mono-specific" for hiv- ; the latter is "pauci reactive", being a potent neutralizer for several viruses, including at least hiv- , hcmv, and sars-cov [ ] [ ] [ ] [ ] [ ] . given the spectrum of anti-virus activities, gna may serve as a model for exploration of broad-spectrum virus-neutralizing epitopes. an essential step toward this goal is to identify the natural ligands of gna that are preserved among gna-targeted viral pathogens, which is the focus of this study. we reasoned that exploring glycan-binding profiles of broadly virus-neutralizing agents may provide key information to uncover the carbohydrate-based viral neutralization epitopes. in the first set of the experiments, we verified that the preparations of gna or g we utilized recognize corresponding epitopes presented by the native viral antigens. subsequently, we performed a comparative carbohydrate microarray analysis to characterize the glycan-binding profiles of g and gna and to pinpoint specific glyco-epitopes they recognize. in figure a ,b, we examined detection of gna-and g -epitopes in hiv- gp glycoproteins. to preserve the native glyco-epitopes, we produced two gp preparations in hek cells, i.e., bal-gp -man and bal-gp . bal-gp -man was expressed in hek cells in the presence of kifunensine ( µg/ml), a potent α-mannosidase i inhibitor, following the previously described procedures [ , ] . the use of the α-mannosidase i inhibitor allows the enrichment of high-mannose type (man ) glycoform through blocking further glycosylation processing to complex or hybrid carbohydrates. thus, bal-gp -man was produced to resemble the native envelope spikes of hiv, which are almost entirely coated with oligomannose antigens [ ] . these proteins were applied on elisa plates at . µg/ml, and the glycan staining was performed in comparison with lectins phaseolus vulgaris-l lectin (pha-l) and sambucus nigra i agglutinin (sna-i). with gna and g targeting oligomannosyl epitopes, pha-l for tri/m-ii cryptic epitopes, and sna-i recognizing α - -linked neu ac residues, this panel of probes monitors expression of layers of cryptic glyco-epitopes as schematically shown in figure a . this assay demonstrated that both bal-gp -man ( figure a ) and bal-gp ( figure b ) were strongly positive with gna and g . pha-l was selectively reactive with bal-gp but not with bal-gp -man . this pha-l-differential staining pattern reflects the effective blockage of biosynthesis of tri/m-ii complex moieties during bal-gp -man production as we initially planned. sna- was negative to both glycoproteins under the same staining condition. elisa results were presented without background subtraction in a-c or with background subtraction in d and e. lectins were applied at the concentrations (µg/ml) as specified. the elisa data shown here are representative results of multiple assays. we further examined whether sars-cov expresses the native gna-epitopes and pha-l-epitopes. in figure c , the elisa plates were coated with the native sars-cov antigens that were expressed by vero e cells. these plates were treated with a disinfecting fixing agent and gamma-irradiated to inactivate infectious virus particles while preserving antigenic structures of sars-cov. lectins were applied at the concentrations specified in the figures. this assay demonstrates that the pha-l-epitopes were readily detected in the sars-cov-coated elisa plates as we previously observed [ ] . moreover, it shows that gna is strongly positive with sars-cov. by contrast, other gal/galnac-reactive lectins, including helix pomatia (hpa) and griffonia (bandeiraea) simplicifolia-i (gs-i), were only marginally reactive with sars-cov antigens. hpa is specific for galnac-and o-glcnac-moieties [ ] ; gs-i recognizes the α-gal-epitope [ , ] . the latter is conserved in many microorganisms but is negligible in sars-cov as expected. in figure d , we examined whether hcmv expresses oligomannosyl epitopes using gna and concanavalin a (con a) in comparison with other lectins of different specificities. in this experiment, elisa was coated with purified virus (ad , creative diagnostics, new york, ny, usa) at protein concentration approximately . µg/ml for lectin-screening. the two anti-mannose agents differ in epitope-binding specificities. gna is specific for the manα , man and manα , man moieties of oligomannoses; con a recognizes terminal manα →moieties that are more broadly expressed by mannose-containing antigens. figure e illustrates lectin-binding of a synthetic glycoconjugate, man -klh, which bears both gna-and con-a-glyco-epitopes [ ] . inspection of figure d readily reveals that the relative binding activity of gna is markedly higher than the con-a activity in the hcmv-elisa assay although the two mannose-specific lectins are similarly reactive with the man -klh standard in the assay. other lectins of different glycan-binding specificities show certain reactivities with hcmv lysates, which may reflect the glycome complexity of this most intricate human virus [ , ] , which requires further study. in summary, we verified expression of the native gna-epitopes by three phylogenetically distinct viruses, hiv- , sars-cov, and hcmv. in the context of a panel of lectins of different specificities, gna was identified to have the highest relative binding activities with sars-cov and hcmv. the ability of gna to bind and neutralize viruses of distinct phylogenetic origins unavoidably raises questions about its glycan-binding specificity and potential cross-reactivity. one plausible explanation is that these targeted viruses commonly express the defined gna-epitopes of manα , man and manα , man moieties. however, the selective gna-positive staining of hcmv without parallel con a reactivity ( figure d ) suggests an alternative possibility, i.e., presence of other carbohydrate moieties that are negative or weakly reactive with con a but strongly reactive with gna. a carbohydrate antigen with such differential reactivity between gna and con a is a yeast-derived phosphomannan polysaccharide (p-man), which is nevertheless not present in the viral glycome [ , ] . to support exploration of the potential gna glyco-epitopes in this study, we produced a set of comprehensive antigen microarrays, which include a large-panel of carbohydrates, lipids/liposomes, and protein antigens (supplementary table s ). key compounds supporting glyco-epitope analysis include the following: ( ) orosomucoid (or) (neu ac), asor (tri/m-ii), and agalacto-or (agor) (tri/m-gn) (figure a ). these autoantigens display distinct glyco-epitopes with identical protein carriers. asor is an asialo-derivative of or, and agor is an agalacto-derivative of asor. they are crucial for defining binding-specificities for n-glycan cryptic epitopes, tri/m-ii, and tri/m-gn. ( ) phosphomannan (p-man), a yeast polysaccharide. both m - -rb and p-man are known to be positive with gna; the former but not the latter also binds to con a. using these microarrays, we characterized gna, pha-l, and g for their epitope-binding profiles. results are summarized in figures and . figure a is a schematic view of structural relationship among autoantigens, or, asor, and agor and the glyco-epitopes they display. figure b shows microarray images of pha-l, gna, or g staining against a panel of autoantigens, including or, asor, agor, man , (m ) , which stands for [(man glcnac asn) ]n-klh (m _ g ) conjugates, and m - -rb. the two control probes spotted are klh and e. coli k polysaccharide. microarray datasets corresponding to figure b are shown in figure c . as illustrated, each antigen was spotted in triplicate at given concentrations as specified. microarray detections are shown as the mean fluorescent intensities (mfis) of triplicate microspots for the arrays stained with pha-l, gna, and g , respectively. results were compared using overlay plots of the mfis of staining signal (blue bars) versus those of local backgrounds surrounding the antigen microarrays (red bars). figure b ,e show that pha-l and g are specific for asor (tri/m-ii) and (m ) , respectively, which is expected. however, gna highly and selectively binds to a number of n-glycan cryptic sugar moieties, including man , (m ) , m - , and agor. gna-binding of agor is a novel observation. given that gna had no binding to asor (tri/m-ii), which only differs from agor (tri/m-gn) by having the terminal galactoses. this result demonstrates, therefore, that gna is most likely specific for the tri/m-gn-glyco-epitopes of agor with exposed terminal glcnac moieties. in figure below, we illustrate the global antigen-binding profiles of gna and g against the full panel of antigen preparations. microarray results were plotted as the ratio of antigen-specific signals over the corresponding spots' background readings, i.e., gna ag/bg, or g ag/bg. to assist a comparative analysis of binding profiles between gna and g , datasets were plotted in the same scale in the y-axis. the positive detections labeled with numbers in the graphs are agor ( ), man ( ), (m ) ( ), m - -rb ( ) , and p-man in two dilutions ( and ) . the whole datasets for these graphs are listed in supplementary table s . in this microarray analysis, gna and g show minimal or no cross-relativities with irrelevant antigens but illustrate strikingly different glycan-binding profiles. g is "mono-specific" for man -clusters ( and ) as expected. by contrast, gna is "pauci reactive" with a number of glycan targets. its binding to the spotted oligomannose antigens ( , , , and ) and p-man ( and ) can be attributed to the known gna-specificity for recognition of the shared manα , man and/or manα , man moieties. however, gna-binding to agor, but not to asor or or, indicates that gna also specifically recognize the tri/m-gn-glyco-determinants displayed by agor. supporting to this prediction is the fact that agor, asor, and or are negative to other anti-mannose agents, including lectin con a, mabs g , tm , and polyclonal antisera that recognize oligomannosyl moieties of varies cluster configurations [ , ] . molecular mechanisms underlying gna-recognition of the tri/m-gn-determinants of agor are yet to be explored. a high-precision microarray robot (pixsys c, cartesian technologies, irvine, ca, usa) was used to spot antigen preparations onto glass slides pre-coated with nitrocellulose polymer (fast slides; schleicher & schuell, keene, nh, usa) as described [ ] . the antigen preparations applied include carbohydrates, proteins/peptides, and liposomes of various compositions as listed in supplementary table s . proteins and carbohydrates were dissolved in phosphate-buffered saline (pbs; ph . ) and saline ( . % nacl), respectively. liposome preparations were suspended in saline ( . % nacl) at the concentrations specified and were printed in triplicate with spot sizes of ~ µm and at -µm intervals, center to center. the printed microarrays were air-dried and stored at room temperature without desiccant before application. immediately before use, the printed microarrays were rinsed with pbs, ph . , with . % (v/v) tween and then blocked by incubating the slides in % (w/v) bovine serum albumin (bsa) in pbs containing . % (w/v) nan at room temperature (rt) for min. they were then incubated at rt with g (nih aids reagent program, germantown, md, usa), biotinylated pha-l (pha-l bi ), or gna bi (ey laboratories, inc., san mateo, ca, usa) at an indicated titration in % (w/v) bsa in pbs containing . % (w/v) nan and . % (v/v) tween . the secondary antibodies or streptavidin conjugates applied for microarray staining are specified in the figure legends. the stained slides were rinsed five times with pbs with . % (v/v) tween , air-dried at room temperature, and then scanned for fluorescent signals using a scanarray a microarray scanner (perkinelmer life science, boston, ma, usa) following the manufacturer's manual. fluorescence intensity values for each array spot and its background were calculated using scanarray express software (perkinelmer life science, boston, ma, usa). sas institute's jmp-genomics software package (cary, nc, usa) was used for further microarray data processing and statistical analysis. in figure , microarray detections are shown as the mean fluorescent intensities (mfis) of triplicate detections captured by scanarray a for the arrays stained with pha-l, gna, or g . analysis of such microarray data in association with visual inspection of the microarray image provided an evaluation of reproducibility and variation of this antigen microarray technology. in figure , microarray results were plotted as the ratio of antigen-specific signal over corresponding spots' background reading, i.e., gna ag/bg, or g ag/bg. the whole microarray datasets for pha-l, gna, and g were presented in supplementary table s . two hiv- gp preparations, bal-gp -man and bal-gp , were produced in hek cells as previously described [ , ] . bal-gp -man was expressed in the presence of the α-mannosidase i inhibitor, kifunensine ( µg/ml), to enrich high-mannose type glycoforms. a preparation of sucrose density-gradient-purified hcmv (ad ) was obtained from creative diagnostics (ny, ny, usa). an elisa protocol described previously [ ] for detection of anti-carbohydrate antibodies was followed with minor modifications. in figure a -e, antigen preparations were diluted in . m sodium bicarbonate buffer solution, ph . , for coating on elisa microplates (nunc, maxisorp, thermo fisher scientific inc., santa clara, ca, usa) followed by blocking using % bsa, pbst. in figure c , a sars-cov-specific elisa kit (euroimmun ag, lübeck, germany) was used to measure sars-cov-expression of lectin-specific glyco-epitopes. anti-glycan antibodies or biotinylated lectins were pre-titrated in % bsa, pbst for elisa. the bound antibodies were revealed by an alkaline phosphatase (ap)-conjugate of goat anti-human igg-fc-specific antibody, and the biotinylated lectins captured were quantified by ap-streptavidin-conjugate. in this pilot study, we examined whether human viruses of distinct phylogenetic origins may express common carbohydrate moieties. using carbohydrate microarrays and elisa-based viral glycan-profiling analysis, we characterized two broadly hiv-neutralizing agents, human monoclonal antibody g and lectin gna. although these agents were known to target oligomannosyl antigens, they differ strikingly in the spectrum of viruses they effectively neutralize. the former is solely hiv-specific; the latter is broadly reactive with human viruses, including hiv- , sars-cov, and hcmv, that are phylogenetically and pathogenically distinct. our carbohydrate microarray analyses demonstrate the two probes differ strikingly in glycan-targeting specificities and the spectrum of glyco-epitopes they recognize. although g is strictly specific for the high-density man clusters that decorate the hiv envelope spike, gna recognizes a number of n-glycan cryptic sugar moieties. these include oligomannoses and the previously unrecognized tri/m-gn-glyco-determinants. the latter appear to be the potent natural ligands of gna. molecular mechanisms underlying the gna-model of "pauci reactive" glycan-binding and broad virus-neutralization warrant further investigation. owing to the potential immunogenic activity as a plant-derived lectin, gna is unlikely suitable for anti-virus therapy in vivo. thus, effort must also be made to establish gna-like potent and broadly virus-neutralizing antibodies, especially humanized or fully human mabs that are readily applicable in the front-line biodefense against emerging viral pathogens. supplementary materials can be accessed at: http://www.mdpi.com/ - / / / /s . antibody domain exchange is an immunological solution to carbohydrate cluster recognition rational antibody-based hiv- vaccine design: current approaches and future directions the broadly neutralizing anti-human immunodeficiency virus type antibody g recognizes a cluster of alpha --> mannose residues on the outer face of gp the mannose-dependent epitope for neutralizing antibody g on human immunodeficiency virus type glycoprotein gp a limited number of antibody specificities mediate broad and potent serum neutralization in selected hiv- infected individuals structure and function of broadly reactive antibody pg reveal an h subdomain that mediates potent neutralization of hiv- pgv , an hiv- gp cd binding site antibody, is broad and potent in neutralization but does not induce conformational changes characteristic of cd rapid development of glycan-specific, broad, and potent anti-hiv- gp neutralizing antibodies in an r siv/hiv chimeric virus infected macaque a potent and broad neutralizing antibody recognizes and penetrates the hiv glycan shield broad neutralization coverage of hiv by multiple highly potent antibodies synthetic carbohydrate antigens for hiv vaccine design human monoclonal antibody g defines a distinctive neutralization epitope on the gp glycoprotein of human immunodeficiency virus type structure of hiv- gp v /v domain with broadly neutralizing antibody pg glycan arrays lead to the discovery of autoimmunogenic activity of sars-cov the genome sequence of the sars-associated coronavirus characterization of a novel coronavirus associated with severe acute respiratory syndrome the sars-cov s glycoprotein: expression and functional characterization identification of n-linked carbohydrates from severe acute respiratory syndrome (sars) spike glycoprotein alpha-( - )-and alpha-( - )-d-mannose-specific plant lectins are markedly inhibitory to human immunodeficiency virus and cytomegalovirus infections in vitro marked depletion of glycosylation sites in hiv- gp under selection pressure by the mannose-specific plant lectins of hippeastrum hybrid and galanthus nivalis profile of resistance of human immunodeficiency virus to mannose-specific plant lectins mannose-specific plant lectins from the amaryllidaceae family qualify as efficient microbicides for prevention of human immunodeficiency virus infection plant lectins are potent inhibitors of coronaviruses by interfering with two targets in the viral replication cycle expression, glycoform characterization, and antibody-binding of hiv- v glycopeptide domain fused with human igg -fc inhibition of mammalian glycan biosynthesis produces non-self antigens for a broadly neutralising, hiv- specific antibody envelope glycans of immunodeficiency virions are almost entirely oligomannose antigens the lectin helix pomatia agglutinin recognizes o-glcnac containing glycoproteins in human breast cancer the xenograft antigen bound to griffonia simplicifolia lectin -b . x-ray crystal structure of the complex and molecular dynamics characterization of the binding site evolutionary relationship between the natural anti-gal antibody and the gal alpha ---- gal epitope in primates uncovering cryptic glycan markers in multiple sclerosis (ms) and experimental autoimmune encephalomyelitis (eae) primate cytomegalovirus glycoproteins: lectin-binding properties and sensitivities to glycosidases quantitative temporal viromics: an approach to investigate host-pathogen interaction anti-oligomannose antibodies as potential serum biomarkers of aggressive prostate cancer n-glycan cryptic antigens as active immunological targets in prostate cancer patients carbohydrate antigen microarrays carbohydrate microarrays for the recognition of cross-reactive molecular markers of microbes and host cells this article is an open access article distributed under the terms and conditions of the creative commons attribution license the authors acknowledge the nih aids reagent program for mab g and the kabat collection of carbohydrate antigens at sri international for a number of carbohydrate antigens that were applied in this study. this work is supported in part by nih grant numbers r ai d.w. and j.l.t. conceived and designed the experiments; d.w. and j.t. performed the experiments; l.x.w. contributed key reagents/materials and edited the manuscript; d.w. analyzed the data and wrote the paper.abbreviations or (orosomucoid); asor (asialo-orosomucoid); agor (agalacto-orosomucoid); tri/m-ii (tri-antennary and multivalent type ii (galβ → glcnac) chain epitopes); tri/m-gn (tri-antennary or multi-valent glcnac-terminating epitopes); gna (galanthus nivalis agglutinin); pha-l (phaseolus vulgaris-l lectin); sna-i (sambucus nigra i agglutinin); hcmv (human cytomegalovirus); hiv- (human immunodeficiency virus- ); sars-cov (severe acute respiratory syndrome coronavirus). the authors declare no conflict of interest. key: cord- -vx cypf authors: li, shi-fang; gong, mei-jiao; sun, yue-feng; shao, jun-jun; zhang, yong-guang; chang, hui-yun title: in vitro and in vivo antiviral activity of mizoribine against foot-and-mouth disease virus date: - - journal: molecules doi: . /molecules sha: doc_id: cord_uid: vx cypf foot-and-mouth disease (fmd) is a highly contagious viral disease of cloven-hoofed animals, which has significant economic consequences in affected countries. as the currently available vaccines against fmd provide no protection until – days post-vaccination, the only alternative method to control the spread of fmd virus (fmdv) during outbreaks is the application of antiviral agents. hence, it is important to identify effective antiviral agents against fmdv infection. in this study, we found that mizoribine has potent antiviral activity against fmdv replication in ibrs- cells. a time-of-drug-addition assay demonstrated that mizoribine functions at the early stage of replication. moreover, mizoribine also showed antiviral effect on fmdv in vivo. in summary, these results revealed that mizoribine could be a potential antiviral drug against fmdv. foot-and-mouth disease (fmd) is one of the most economically and socially devastating diseases affecting cloven-hoofed animals [ ] . the infectious agent, foot-and-mouth disease virus (fmdv), is a member of the aphthovirus genus of the picornaviridae family, and contains single-stranded positive-sense rna genomes of about , nucleotides [ ] . as an antigenically variable virus, fmdv consists of seven serotypes (a, o, c, asia , and south african territories , , and ) and a large number of subtypes. in general, slaughtering fmdv-infected/exposed or fmdv-susceptible animals, restricting animal movement, and, in some cases, vaccinating against fmdv and then slaughtering these animals are used as control measures for potential outbreaks in disease-free areas [ ] . although inactivated fmd vaccines have been available since the early s and new novel vaccines are being continuously developed, they offer little or no cross-protection against various serotypes and subtypes of fmdv. in addition, these vaccines do not provide complete clinical protection until seven days post-vaccination. therefore, there is a need for developing effective and safe alternative antiviral strategies against fmdv [ ] [ ] [ ] . mizoribine, an imidazole nucleoside ( figure a ) [ ] , has been used as an immunosuppressive agent for the treatment of renal transplantation, autoimmune diseases, and steroid-resistant nephrotic syndrome in some countries owing to its antiproliferative activity against t and b lymphocytes [ ] . this drug could be phosphorylated by adenosine kinase and converted to mizoribine -monophosphate, replication of some dna and rna viruses, such as cytomegalovirus [ ] , respiratory syncytial virus [ ] , severe acute respiratory syndrome-associated coronavirus (sars-cov) [ ] , bovine viral diarrhea virus (bvdv) [ ] , vaccinia virus [ ] , influenza virus types a and b, and herpesviruses, in combination with acyclovir [ , ] . however, the antiviral activity of mizoribine against fmdv has not yet been investigated. hence, in this study, the antiviral effect of mizoribine against fmdv was evaluated in vitro using ibrs- cells and confirmed in vivo using suckling mice. figure b illustrates the results of the mts assay. as is shown in figure b , mizoribine presented little or no cytotoxicity to the cells. the cell viability was . %, . %, . %, . %, . %, and . % at mizoribine concentrations of , , , , , and μm, respectively, and the % cytotoxic concentration (cc ) of mizoribine was estimated to be more than μm on ibrs- cells. the inhibitory effect of mizoribine on fmdv infection in ibrs- cells was calculated by measuring cell viability using the results of mts assay. as indicated in figure a , the inhibition rates were approximately . %, . %, . %, and . % in cells treated with , , , and μm mizoribine, respectively, whereas other lower mizoribine concentrations demonstrated limited or no inhibitory effect on fmdv. the ic and si values were calculated to be . μm and . , respectively. interestingly, mizoribine also displayed activity against another fmdv strain, a/gd/mm/ , with an ic of . μm and si value of . ( figure c ). these data supported the potential broad-spectrum activity of mizoribine against rna viruses. figure b illustrates the results of the mts assay. as is shown in figure b , mizoribine presented little or no cytotoxicity to the cells. the cell viability was . %, . %, . %, . %, . %, and . % at mizoribine concentrations of , , , , , and µm, respectively, and the % cytotoxic concentration (cc ) of mizoribine was estimated to be more than µm on ibrs- cells. the inhibitory effect of mizoribine on fmdv infection in ibrs- cells was calculated by measuring cell viability using the results of mts assay. as indicated in figure a , the inhibition rates were approximately . %, . %, . %, and . % in cells treated with , , , and µm mizoribine, respectively, whereas other lower mizoribine concentrations demonstrated limited or no inhibitory effect on fmdv. the ic and si values were calculated to be . µm and . , respectively. interestingly, mizoribine also displayed activity against another fmdv strain, a/gd/mm/ , with an ic of . µm and si value of . ( figure c ). these data supported the potential broad-spectrum activity of mizoribine against rna viruses. the transcription of fmdv mrna was significantly reduced with , , , and µm mizoribine treatments relative to the dmso control group ( figure b ). consistent with mrna expression, immunofluorescence assay (ifa) revealed that mizoribine inhibited the expression of fmdv protein in a dose-dependent manner ( figure ). the concentration of µm, which showed more protection against cpe than other concentration, was chosen for the further study. the transcription of fmdv mrna was significantly reduced with , , , and μm mizoribine treatments relative to the dmso control group ( figure b ). consistent with mrna expression, immunofluorescence assay (ifa) revealed that mizoribine inhibited the expression of fmdv protein in a dose-dependent manner ( figure ). the concentration of μm, which showed more protection against cpe than other concentration, was chosen for the further study. subsequently, the antiviral efficacy of mizoribine was further evaluated at various intervals post-fmdv infection, we found that the viral b mrna and protein expressions were continuously inhibited at different time points ( , , , and h) after treatment with μm mizoribine; however, no significant differences were observed between h post-infection (hpi) and the control group ( figure ). taken together, these results suggested that mizoribine exhibited potent antiviral activity against fmdv in ibrs- cells at the early stages of viral infection. the supernatants were used for viral rna quantification using qpcr. the expression values relative to that of β-actin were calculated using the −∆∆ct method. cpe, cytopathic effect. statistically significant differences are indicated by asterisks (* p < . , *** p < . ). the transcription of fmdv mrna was significantly reduced with , , , and μm mizoribine treatments relative to the dmso control group ( figure b ). consistent with mrna expression, immunofluorescence assay (ifa) revealed that mizoribine inhibited the expression of fmdv protein in a dose-dependent manner ( figure ). the concentration of μm, which showed more protection against cpe than other concentration, was chosen for the further study. subsequently, the antiviral efficacy of mizoribine was further evaluated at various intervals post-fmdv infection, we found that the viral b mrna and protein expressions were continuously inhibited at different time points ( , , , and h) after treatment with μm mizoribine; however, no significant differences were observed between h post-infection (hpi) and the control group ( figure ). taken together, these results suggested that mizoribine exhibited potent antiviral activity against fmdv in ibrs- cells at the early stages of viral infection. subsequently, the antiviral efficacy of mizoribine was further evaluated at various intervals post-fmdv infection, we found that the viral b mrna and protein expressions were continuously inhibited at different time points ( , , , and h) after treatment with µm mizoribine; however, no significant differences were observed between h post-infection (hpi) and the control group ( figure ). taken together, these results suggested that mizoribine exhibited potent antiviral activity against fmdv in ibrs- cells at the early stages of viral infection. ''vc'' represents those cells treated with % dmso without mizoribine. values represent the mean ± standard deviation for three independent experiments. the asterisks indicate significant differences between mock-treated and drug-treated cells (** p < . ). inosine- ′-monophosphate dehydrogenase (impdh) is required for de novo purine nucleotide synthesis and its inhibition can lead to depletion of intracellular gtp pools. to investigate the effect of mizoribine on purine synthesis in fmdv, serially diluted guanosine was added to the infected cells treated with mizoribine. while guanosine had no effect on mizoribine, it significantly attenuated the anti-fmdv effect of mizoribine in a dose-dependent manner ( figure ). these data indicated that mizoribine activity against fmdv involved inhibition of impdh-dependent purine synthesis. inosine- -monophosphate dehydrogenase (impdh) is required for de novo purine nucleotide synthesis and its inhibition can lead to depletion of intracellular gtp pools. to investigate the effect of mizoribine on purine synthesis in fmdv, serially diluted guanosine was added to the infected cells treated with mizoribine. while guanosine had no effect on mizoribine, it significantly attenuated the anti-fmdv effect of mizoribine in a dose-dependent manner ( figure ). these data indicated that mizoribine activity against fmdv involved inhibition of impdh-dependent purine synthesis. ''vc'' represents those cells treated with % dmso without mizoribine. values represent the mean ± standard deviation for three independent experiments. the asterisks indicate significant differences between mock-treated and drug-treated cells (** p < . ). inosine- ′-monophosphate dehydrogenase (impdh) is required for de novo purine nucleotide synthesis and its inhibition can lead to depletion of intracellular gtp pools. to investigate the effect of mizoribine on purine synthesis in fmdv, serially diluted guanosine was added to the infected cells treated with mizoribine. while guanosine had no effect on mizoribine, it significantly attenuated the anti-fmdv effect of mizoribine in a dose-dependent manner ( figure ). these data indicated that mizoribine activity against fmdv involved inhibition of impdh-dependent purine synthesis. the suckling mice pretreated by subcutaneous injection of mizoribine or pbs in the neck were infected with fmdv to determine the antiviral activity of mizoribine in vivo. all the solvent-treated mice died within h after ld of o/my /by/ challenge. in contrast, a -h delay in death post-infection was noted in the mizoribine-treated group, and all the mice died within h after the viral challenge. the overall death time of mice treated with mizoribine was delayed by h, when compared with the control, and a significant difference in mouse survival was noted between the treatment group and control (p = . ) ( figure a ). the suckling mice pretreated by subcutaneous injection of mizoribine or pbs in the neck were infected with fmdv to determine the antiviral activity of mizoribine in vivo. all the solvent-treated mice died within h after ld of o/my /by/ challenge. in contrast, a -h delay in death post-infection was noted in the mizoribine-treated group, and all the mice died within h after the viral challenge. the overall death time of mice treated with mizoribine was delayed by h, when compared with the control, and a significant difference in mouse survival was noted between the treatment group and control (p = . ) ( figure a ). furthermore, significant histopathological damage was observed in the heart tissue of fmdv-infected mice, including considerable myocardial interstitial hemorrhage, myocardial fibronectin degeneration, and extensive inflammatory cell infiltration, as indicated by black arrows ( figure a ). in addition, myocardial fiber edema and incomplete fibrous structure were also observed. however, mice treated with mizoribine showed mild histopathological changes and only a small amount of inflammatory cell infiltration in the heart ( figure b ). intriguingly, although fmdv antigen was detected in the heart tissue of both mizoribine-treated and control mice, it was not statistically significant ( figure c,d) . these findings suggested that histopathological damage may be the main cause of death resulting from fmdv infection, and mizoribine can effectively alleviate these effects. furthermore, significant histopathological damage was observed in the heart tissue of fmdv-infected mice, including considerable myocardial interstitial hemorrhage, myocardial fibronectin degeneration, and extensive inflammatory cell infiltration, as indicated by black arrows ( figure a ). in addition, myocardial fiber edema and incomplete fibrous structure were also observed. however, mice treated with mizoribine showed mild histopathological changes and only a small amount of inflammatory cell infiltration in the heart ( figure b ). intriguingly, although fmdv antigen was detected in the heart tissue of both mizoribine-treated and control mice, it was not statistically significant ( figure c,d) . these findings suggested that histopathological damage may be the main cause of death resulting from fmdv infection, and mizoribine can effectively alleviate these effects. as fmdv exhibits high mutation rates and produces significant economic loss in affected countries, it is important to adopt effective measures to control this virus. it has been demonstrated that mizoribine does not produce tumorigenic and gonadal suppression effects, and exerts low bone marrow inhibition and hepatotoxic outcomes, and has been used for the treatment of renal diseases in humans [ , ] . thus, considering the safe, reliable, and acceptable efficacy of mizoribine in humans, it might also be employed for the treatment of animal diseases. as fmdv exhibits high mutation rates and produces significant economic loss in affected countries, it is important to adopt effective measures to control this virus. it has been demonstrated that mizoribine does not produce tumorigenic and gonadal suppression effects, and exerts low bone marrow inhibition and hepatotoxic outcomes, and has been used for the treatment of renal diseases in humans [ , ] . thus, considering the safe, reliable, and acceptable efficacy of mizoribine in humans, it might also be employed for the treatment of animal diseases. to the best of our knowledge, the present study is the first to report on the antiviral effect of mizoribine on fmdv both in vitro and in vivo. by using mts assay, the cytotoxicity of mizoribine was determined to be very weak, with a cc value higher than µm. furthermore, mizoribine showed significant anti-fmdv activity, not only against type o fmdv o/my /by/ strain, but also against type a fmdv a/gd/mm/ , with si values of . and . , respectively. these results indicated that mizoribine could be a better drug to prevent fmdv a/gd/mm/ infection. moreover, qpcr and ifa findings revealed that mizoribine can significantly inhibit the viral mrna and fmdv protein levels. to understand the preliminary antiviral mechanism of mizoribine, time-of-drug-addition assay was performed, and the results demonstrated that mizoribine mainly functions at the early stages of infection. it has been reported that the antiviral activity of mizoribine involves inhibition of impdh [ ] , an essential enzyme for the synthesis of guanosine monophosphate from inosine monophosphate through de novo pathway [ , ] . similarly, in the present study, the antiviral activity of mizoribine against fmdv was found to be attenuated by guanosine supplementation. the animal experiment demonstrated that mizoribine had an inhibitory effect on fmdv in vivo, and treatment with µg of mizoribine significantly prolonged the survival of fmdv-infected suckling mice. although the in vivo results did not show a significant increase in the survival rates of infected mice, the delay in death and alleviated histopathological changes suggested the inhibitory effect of mizoribine on viral replication. overall, the weak cytotoxicity and strong antiviral activities of mizoribine both in vitro and in vivo favored its further potential clinical applications in the treatment of viral infection. combination treatment strategies have been proposed to enhance the efficacy of antiviral agents, because of their advantages in overcoming viral mechanisms of resistance to antiviral treatments [ ] . it has been demonstrated that mizoribine enhances the anti-caprine-herpesvirus- activity of acyclovir, and the combination of mizoribine and acyclovir resulted in an almost complete inhibition of viral replication. thus, combined therapy of acyclovir and mizoribine could be exploited for the treatment of genital infection by herpesviruses [ ] . moreover, mizoribine has been reported to be active against the replication of bvdv in madin-darby bovine kidney (mdbk) cells, and a combination of interferons (ifns) and mizoribine had been noted to synergistically inhibit bvdv replication in bovine kidney cells [ ] . with regard to fmd, fmdv have been demonstrated to be very sensitive to ifns, and ifn-based strategies have been established to be an efficient biotherapeutic option against fmdv [ ] . similarly, the combination of antiviral agents, such as sirna, ribavirin, and ifns, has been determined to produce enhanced antiviral effect against fmdv [ ] . therefore, future studies must investigate whether a combination of ifns and mizoribine could produce increased inhibitory effect on fmdv replication both in vitro and in vivo. in conclusion, to the best of the authors' knowledge, the present study is the first to demonstrate that mizoribine can suppress fmdv replication in vitro as well as prolong the survival of suckling mice in vivo, suggesting the potential applications of this drug in antiviral regimens for fmd treatment. the findings of this study may warrant further investigations on the efficacy and safety of combined use of mizoribine and other antiviral agents in vitro and in vivo. thirty-two ( ) two-and three-day-old balb/c mice weighing - g were used to investigate the efficacy of mizoribine in vivo. all the animal trials were performed in a biosafety level- laboratory and approved by the animal ethics committee of lanzhou veterinary research institute, chinese academy of agricultural science (no. lvriaec - ). fmdv type o (o/mya /by/ ) strain was used for viral challenge. the cytotoxicity of mizoribine was evaluated using mts assay. briefly, × cells were seeded into each well of a -well plate containing µl of complete medium. on the following day, the cells were incubated with µl of mizoribine at various concentrations ( , , , , , and µm) for h. as a control, cells were treated with % dmso. after treatment, the supernatants were discarded, and the cells were washed three times. then, µl of dmem were added to each well along with µl of mts solution and incubated for an additional h at • c. the optical density of each well at nm was determined using a microplate reader (bio-rad, hercules, ca, usa). the cell viability was expressed as the percentage of absorbance of the treated cells to that of the control cells. the antiviral activity of mizoribine against fmdv was determined using an mts-based cytopathic effect (cpe) inhibition assay. briefly, the viral suspension ( tcid o/my /by/ ) was added to the ibrs- cell monolayers. after incubation for h, the cells were washed three times with dmem, and serially diluted mizoribine solution was added to the wells (eight wells for each concentration). then, the plates were incubated at • c in % co . after h, when maximum cpe was noted in the virus control group (vc), the cell viability was measured using mts assay as described earlier, and the percentage of inhibition associated with each mizoribine concentration was normalized with respect to the vc. the virus inhibition rate was calculated as follows: inhibition rate = (optical density of mizoribine group − optical density of vc)/(optical density of cell control group − optical density of vc) × %. the supernatant of each well was collected and the viral b mrna was analyzed using q-pcr. the % inhibitory concentration (ic ) values were calculated with graphpad software (version . , la jolla, ca, usa). the monolayers of cells were seeded in -well plates infected with tcid fmdv o/my /by/ at • c for h. then, a µm portion of mizoribine supplemented with serial dilutions of guanosine (from to µm) was added and incubated for h. after incubation, the viral protein and gene expressions were assessed by western blot analysis and q-pcr, respectively. the ibrs- cells were incubated with tcid o/my /by/ for h. subsequently, the viruses were removed and the medium was replaced. mizoribine ( µm) was added to the cells during infection (co) or post-infection ( , , and h). after h incubation, the fmdv b mrna and vp protein in the cells were determined by q-pcr and western blot analysis, respectively. the expression levels of fmdv b mrna and β-actin were determined by real-time pcr as previously reported [ ] . briefly, the total rna from the ibrs- cells was extracted using trizol reagent, and µg of rna was used in reverse transcription reaction using a primescript™ rt reagent kit containing gdna eraser, following the manufacturer's instructions. the reaction mixture for real-time pcr comprised diluted cdna ( µl), µm primers, and . µl of sybr green master mix to a final volume of µl. the amplification conditions were as follows: • c for s, followed by cycles of • c for s, • c for s, and • c for s. dissociation curves were generated to analyze the individual pcr products after cycles. the expression levels of fmdv mrna genes were normalized against those of porcine β-actin mrna. the analyses of the relative gene expression data were performed using the −∆∆ct method [ ] . for western blot analysis, the cells were lysed with pierce ripa, and the cell lysates were resolved and separated by % sds-page and transferred to polyvinylidene fluoride membrane. the membranes were probed with primary antibodies against fmdv vp protein (dilution, : ) to detect virus replication. a monoclonal antibody to β-actin (dilution, : ) was used as a loading control. the membranes were incubated with goat anti-mouse and anti-rabbit conjugated with horseradish peroxidase for h at room temperature and examined by pierce™ ecl western blotting substrate. to determine the antiviral activity of mizoribine, indirect immunofluorescence assay (ifa) was performed as previously described with minor modifications [ ] . the ibrs- cells were seeded into a -well plate at a concentration of × cells/well and incubated for one day. subsequently, the cells were incubated with tcid fmdv for h, and mizoribine diluted at indicated concentrations was added to the cells after removal of the viral inoculum. after h of incubation, the ibrs- cells were washed thrice with pbs and fixed with % paraformaldehyde for min. then, paraformaldehyde was removed and absolute methanol was added to the cells and incubated for min. subsequently, the cells were washed with pbs and blocked using blocking buffer (pbs supplemented with . % triton x- and % fbs). rabbit hyperimmune serum raised against type o fmdv (o/my /by/ ) was used to probe type o fmdv, and peroxidase-conjugated goat anti-rabbit igg (h + l) was employed as secondary antibody. finally, the cells were counterstained with , -diamidino- -phenylindole (dapi) and viewed under fluorescence microscope (nikon eclipse ts fluorescence microscope, yokagawa electric corporation, tokyo, japan). specific-pathogen-free three-day-old kunming suckling mice were inoculated with µg of mizoribine dissolved in µm dmso, % tween , and . ml of pbs by subcutaneous neck injection. the negative control was inoculated with pbs. after h, viral challenge was performed by intradermal injection with % lethal dose (ld ) fmdv serotype o o/my /by/ into the subcutaneous neck region of the mice. the animals were monitored for five days, and a log-rank test was performed for statistical analysis using graphpad software. at h post-infection, the suckling mice were euthanized and processed for histological and immunohistochemical investigations. for histopathological analysis, the heart tissues were fixed in % paraformaldehyde solution, embedded in paraffin, and cut into µm thick sections for standard hematoxylin and eosin (h & e) staining. with regard to immunohistochemical (ihc) studies, the fmdv antigen was detected as follows: µm thick paraffin-embedded tissue sections were deparaffinized and treated with methanol-hydrogen peroxide for min before heat-induced antigen retrieval in . m sodium citrate buffer (ph = ) for min. rabbit hyperimmune serum raised against type o fmdv (o/my /by/ ) was used as primary antibody. the tissue sections were processed with a splink detection kit for min and stained with dab for min at room temperature. after washing, the tissue sections were counterstained, mounted, examined under a digital microscope (ba digital, motic, xiamen, china), and photographed. the optical density of each tissue section was determined by image-pro plus . software (media cybernetics, rockville, md, usa). all the data are expressed as mean ± standard deviation (sd) for at least three independent experiments. one-way anova was used to analyze the difference between mizoribine and control groups using graphpad prism (graphpad software, inc., la jolla, ca, usa), version . , and significant differences were defined at p < . . selective index (si) = cc /ec . mutagenesis versus inhibition in the efficiency of extinction of foot-and-mouth disease virus antiviral activity of ovine interferon tau against foot-and-mouth disease virus development of vaccines toward the global control and eradication of foot-and-mouth disease foot and mouth disease vaccine strain selection: current approaches and future perspectives vaccination against foot-and-mouth disease virus confers complete clinical protection in days and partial protection in days: use in emergency outbreak response mizoribine and mycophenolate mofetil mode of action and effects in clinical use effects of cyclosporine, azathioprine, mizoribine, and prednisolone on replication of human cytomegalovirus recent progress in antiviral chemotherapy for respiratory syncytial virus infections kurane, i. inhibitory effect of mizoribine and ribavirin on the replication of severe acute respiratory syndrome (sars)-associated coronavirus inhibitors of the impdh enzyme as potential anti-bovine viral diarrhoea virus agents studies on bredinin. i. isolation, characterization and biological properties comparative inhibitory effects of various nucleoside and nonnucleoside analogues on replication of influenza virus types a and b in vitro and in ovo potentiating effect of mizoribine on the anti-herpes virus activity of acyclovir effects of mizoribine on mhc-restricted exogenous antigen presentation in dendritic cells inhibition of bovine viral diarrhea virus (bvdv) by mizoribine: synergistic effect of combination with interferon-alpha enhanced inhibition of foot-and-mouth disease virus by combinations of porcine interferon-alpha and antiviral agents in vitro inhibition of caprine herpesvirus by acyclovir and mizoribine antiviral activity of bovine type iii interferon against foot-and-mouth disease virus detection of all seven serotypes of foot-and-mouth disease virus by real-time, fluorogenic reverse transcription polymerase chain reaction assay analysis of relative gene expression data using real-time quantitative pcr and the (-delta delta c(t)) method. methods a novel type i interferon, interferon alphaomega, shows antiviral activity against foot-and-mouth disease virus in vitro the authors declare no conflict of interest. the funders had no role in the design of the study; in the collection, analyses, or interpretation of data; in the writing of the manuscript; or in the decision to publish the results. key: cord- - uscua y authors: cerda-cavieres, christopher; quiroz, gabriel; iturriaga-vásquez, patricio; rodríguez-lavado, julio; alarcón-espósito, jazmín; saitz, claudio; pessoa-mahana, carlos d.; chung, hery; araya-maturana, ramiro; mella-raipán, jaime; cabezas, david; ojeda-gómez, claudia; reyes-parada, miguel; pessoa-mahana, hernán title: synthesis, docking, -d-qsar, and biological assays of novel indole derivatives targeting serotonin transporter, dopamine d receptor, and mao-a enzyme: in the pursuit for potential multitarget directed ligands date: - - journal: molecules doi: . /molecules sha: doc_id: cord_uid: uscua y a series of compounds of general structure , -dihydro-benzo[ , ]oxazin- -yl)- -{ -[ -( h- indolyl)-propyl]- -piperazinyl}-ethanamides, series i: (a–o) and ( -{ -[ -( h- -indolyl)-propyl]- -piperazinyl}-acetylamine)-n-( -morfolin- -yl-ethyl)-fluorinated benzamides series ii: (a–l) were synthesized and evaluated as novel multitarget ligands towards dopamine d( ) receptor, serotonin transporter (sert), and monoamine oxidase-a (mao-a) directed to the management of major depressive disorder (mdd). all the assayed compounds showed affinity for sert in the nanomolar range, with five of them displaying ki values from to nm. compounds k, ki = . ± . nm, and c, ki = . ± . nm, showed the highest potencies. the affinities for d( ) ranged from micro to nanomolar, while mao-a inhibition was more discrete. nevertheless, compounds m and n showed affinities for the d( ) receptor in the nanomolar range ( n: ki = ± nm and m: ki = ± nm). compound n was the only derivative displaying comparable affinities for sert and d( ) receptor (d( )/sert ratio = . ) and could be considered as a multitarget lead for further optimization. in addition, docking studies aimed to rationalize the molecular interactions and binding modes of the designed compounds in the most relevant protein targets were carried out. furthermore, in order to obtain information on the structure–activity relationship of the synthesized series, a -d-qsar comfa and comsia study was conducted and validated internally and externally (q( ) = . , . for comfa and comsia and r( )(ncv) = . , . for comfa and comsia, respectively). the fluorinated benzamides derivatives a-d were finally connected to indolylpropylpiperazines a-c to achieve the expected compounds a-l, with yields ranging from % to % (scheme ). in summary, compounds were synthesized for series i in yields ranging from % to %. the synthetic pathway of this series involved the fluorinated benzamide derivatives a-d, which were obtained from commercially available isomeric fluoro nitrobenzoic acids in a three-step sequence with good to excellent yields as shown in scheme . in summary, compounds were synthesized for series i in yields ranging from % to %. the synthetic pathway of this series involved the fluorinated benzamide derivatives a-d, which were obtained from commercially available isomeric fluoro nitrobenzoic acids in a three-step sequence with good to excellent yields as shown in scheme . the fluorinated benzamides derivatives a-d were finally connected to indolylpropylpiperazines a-c to achieve the expected compounds a-l, with yields ranging from % to % (scheme ). the fluorinated benzamides derivatives a-d were finally connected to indolylpropylpiperazines a-c to achieve the expected compounds a-l, with yields ranging from % to % (scheme ). molecules , table summarizes the affinity of compounds a-o for sert, d receptor, and mao-a. most compounds were potent and clearly selective as sert ligands, showing in all cases affinities in the nanomolar range, whereas the affinities for d and mao-a ranged from micromolar to much higher values, respectively. a detailed analysis of sert activities indicates that a c- substitution of the indole ring with halogens (fluorine or bromine; compounds g or m) leads to more potent compounds than the unsubstituted derivative ( a). on the other hand, the presence of a halogen atom at c- of the benzoxazine ring increased the affinity (e.g., c, d, and e vs. a). accordingly, the most potent compounds were those exhibiting a dual halogen substitution pattern ( i, j, and k), with ki values below nm. the c- halogen substitution on the benzoxazine ring gave no consistent effects, slight increases ( b and h) or decreases ( n) of affinity were observed, as compared with the corresponding c- unsubstituted compounds ( a, g, and m, respectively). the d receptor affinity for this series indicates that no conclusive structure-activity relationships can be extracted for these compounds. nevertheless, it is apparent that dihalogenated derivatives, bearing one halogen atom at the c- of the indole ring and the other at either the c- or c- of the benzoxazine moiety ( h- k, m- o), resulted in more potent compounds than their corresponding monohalogenated or unsubstituted counterparts ( a- e, g). moreover, the presence of a methoxyl group at the c- of the benzoxazine ring has almost no effect on the affinity of the compounds for d receptor. it is worth mentioning that the dihalogenated compound n was the only derivative displaying comparable affinities for sert and d receptor (d /sert ratio = . ) and could be considered as a potential leader in the search of more potent multitarget compounds. scheme . synthesis of series ii derivatives a-l. reagents and conditions: k co , ch cn, • c, yield ( - %). table summarizes the affinity of compounds a-o for sert, d receptor, and mao-a. most compounds were potent and clearly selective as sert ligands, showing in all cases affinities in the nanomolar range, whereas the affinities for d and mao-a ranged from micromolar to much higher values, respectively. a detailed analysis of sert activities indicates that a c- substitution of the indole ring with halogens (fluorine or bromine; compounds g or m) leads to more potent compounds than the unsubstituted derivative ( a). on the other hand, the presence of a halogen atom at c- of the benzoxazine ring increased the affinity (e.g., c, d, and e vs. a). accordingly, the most potent compounds were those exhibiting a dual halogen substitution pattern ( i, j, and k), with ki values below nm. the c- halogen substitution on the benzoxazine ring gave no consistent effects, slight increases ( b and h) or decreases ( n) of affinity were observed, as compared with the corresponding c- unsubstituted compounds ( a, g, and m, respectively). the d receptor affinity for this series indicates that no conclusive structure-activity relationships can be extracted for these compounds. nevertheless, it is apparent that dihalogenated derivatives, bearing one halogen atom at the c- of the indole ring and the other at either the c- or c- of the benzoxazine moiety ( h- k, m- o), resulted in more potent compounds than their corresponding monohalogenated or unsubstituted counterparts ( a- e, g). moreover, the presence of a methoxyl group at the c- of the benzoxazine ring has almost no effect on the affinity of the compounds for d receptor. it is worth mentioning that the dihalogenated compound n was the only derivative displaying comparable affinities for sert and d receptor (d /sert ratio = . ) and could be considered as a potential leader in the search of more potent multitarget compounds. table . affinities, measured as ki values at the serotonin transporter (sert), d receptor, and percent of monoamine oxidase-a (mao-a) inhibition (at µm) of indolepiperazinyl benzoxazine derivatives (series i). molecules , , x for peer review of considering the pharmacological results, docking studies aimed to rationalize the molecular interactions and binding modes of the designed compounds were carried out only in the human sert (hsert) and in selected cases at the d receptor. the most potent compounds g, h, i, and k showed a common docking pose (figure ), which favors the following stabilizing interactions: a π-π interaction between the indole ring and the πdonor aromatic residue tyr , a coulombic interaction between the protonated piperazine n- with the asp residue, and a π-cation interaction for the protonated piperazine with tyr . furthermore, aromatic interactions were also observed for the benzoxazine ring with the residues phe and considering the pharmacological results, docking studies aimed to rationalize the molecular interactions and binding modes of the designed compounds were carried out only in the human sert (hsert) and in selected cases at the d receptor. the most potent compounds g, h, i, and k showed a common docking pose (figure ), which favors the following stabilizing interactions: a π-π interaction between the indole ring and the π-donor aromatic residue tyr , a coulombic interaction between the protonated piperazine n- with the asp residue, and a π-cation interaction for the protonated piperazine with tyr . furthermore, aromatic interactions were also observed for the benzoxazine ring with the residues phe and phe . these drug-target interactions are in agreement with those described in the crystal structure of the hsert in complex with the inhibitor (s)-citalopram [ , ] . the relevance of the fluorinated substitution on the indole ring is clearly evidenced by comparison of compounds f and l. both derivatives share the same substitution pattern in the benzoxazine ring, differing only by the presence of a fluorine atom at the indole moiety, which induces a different docking pose for f. thus, the least potent compound of the series ( f) adopted a binding mode in which both indole and piperazine ring interactions are clearly less favored than the c- fluorinated counterpart ( figure ). compounds showing intermediate affinities ( a- e and m- o) exhibited docking poses between the most and least favorable binding modes (not shown). the relevance of the fluorinated substitution on the indole ring is clearly evidenced by comparison of compounds f and l. both derivatives share the same substitution pattern in the benzoxazine ring, differing only by the presence of a fluorine atom at the indole moiety, which induces a different docking pose for f. thus, the least potent compound of the series ( f) adopted a binding mode in which both indole and piperazine ring interactions are clearly less favored than the c- fluorinated counterpart ( figure ). compounds showing intermediate affinities ( a- e and m- o) exhibited docking poses between the most and least favorable binding modes (not shown). phe . these drug-target interactions are in agreement with those described in the crystal structure of the hsert in complex with the inhibitor (s)-citalopram [ , ] . the relevance of the fluorinated substitution on the indole ring is clearly evidenced by comparison of compounds f and l. both derivatives share the same substitution pattern in the benzoxazine ring, differing only by the presence of a fluorine atom at the indole moiety, which induces a different docking pose for f. thus, the least potent compound of the series ( f) adopted a binding mode in which both indole and piperazine ring interactions are clearly less favored than the c- fluorinated counterpart ( figure ). compounds showing intermediate affinities ( a- e and m- o) exhibited docking poses between the most and least favorable binding modes (not shown). docking simulations showed that compounds of this series adopt, at the d receptor, a binding mode similar to that experimentally determined for the atypical antipsychotic risperidone [ ] . thus, the indole moiety appears located into the deep hydrophobic sub-pocket of the orthosteric site, lined by cys , thr , ser , phe , and trp , while the protonated piperazine n- locates in a favorable position to establish a coulombic interaction with asp ( figure ) . furthermore, the benzoxazine portion extends to the additional hydrophobic sub-pocket lined by val , trp , phe , and tyr , in a similar fashion to that observed in the crystal structure for the pyrimidinone moiety of risperidone. interestingly, this general binding mode was observed for both the most and the least potent compounds of this series ( a, b, and m, n), respectively ( figure a ,b). therefore, it is tempting to speculate that the higher affinity showed by brominated derivatives ( m and n) is due to the formation of a halogen bond between the bromine and a hydroxyl group of an adjacent residue (e.g., ser ). as observed (figure ), this could also change the position of the benzoxazine moiety, favoring its interactions at the more external hydrophobic sub-pocket. docking simulations showed that compounds of this series adopt, at the d receptor, a binding mode similar to that experimentally determined for the atypical antipsychotic risperidone [ ] . thus, the indole moiety appears located into the deep hydrophobic sub-pocket of the orthosteric site, lined by cys , thr , ser , phe , and trp , while the protonated piperazine n- locates in a favorable position to establish a coulombic interaction with asp ( figure ) . furthermore, the benzoxazine portion extends to the additional hydrophobic sub-pocket lined by val , trp , phe , and tyr , in a similar fashion to that observed in the crystal structure for the pyrimidinone moiety of risperidone. interestingly, this general binding mode was observed for both the most and the least potent compounds of this series ( a, b, and m, n), respectively ( figures a and b) . therefore, it is tempting to speculate that the higher affinity showed by brominated derivatives ( m and n) is due to the formation of a halogen bond between the bromine and a hydroxyl group of an adjacent residue (e.g., ser ). as observed (figure ), this could also change the position of the benzoxazine moiety, favoring its interactions at the more external hydrophobic sub-pocket. table summarizes the affinity of series ii compounds a- l for sert, d receptor, and mao-a. as in the case of indole benzoxazine derivatives (series i), most indole morpholine ethylbenzamides (series ii) were potent sert ligands, showing much lower affinities for d receptor and virtually no effect upon mao-a activity. regarding sert activity, and in agreement with our previous studies, halogen substitution at c- of the indole ring with fluorine or bromine (compounds a-g) conducted an increase in affinity as compared with the unsubstituted analogues i-l, with the fluoro derivatives a-d being the most potent of the series. on the other hand, when the acetanilide portion, connected to the indolylpropylpiperazinyl fragment, was functionalized with a fluorine atom (at c- ) and a morpholino ethylcarboxamide, the best affinities were obtained when the bulkier substituent was located at meta position (compounds c, g, and k). table summarizes the affinity of series ii compounds a- l for sert, d receptor, and mao-a. as in the case of indole benzoxazine derivatives (series i), most indole morpholine ethylbenzamides (series ii) were potent sert ligands, showing much lower affinities for d receptor and virtually no effect upon mao-a activity. regarding sert activity, and in agreement with our previous studies, halogen substitution at c- of the indole ring with fluorine or bromine (compounds a-g) conducted an increase in affinity as compared with the unsubstituted analogues i-l, with the fluoro derivatives a-d being the most potent of the series. on the other hand, when the acetanilide portion, connected to the indolylpropylpiperazinyl fragment, was functionalized with a fluorine atom (at c- ) and a morpholino ethylcarboxamide, the best affinities were obtained when the bulkier substituent was located at meta position (compounds c, g, and k). similar to the analysis of series i and considering the pharmacological results, docking studies were carried out only in hsert. in this series, seven compounds exhibited ki values between and nm ( a, b, c, f, g, k, and l) . docking simulations showed that compounds with the lowest ki values ( c and g) share a common binding mode into the s site of the sert, which is similar to that described for compounds of series i ( figure a ). thus, the piperazine n- can establish ionic and π-cation interactions with asp and tyr , respectively, while the indole moiety can participate in aromatic interactions with tyr and phe . interestingly, the ethylmorpholinic chain extends towards the extracellular vestibule (also known as the s site). on the other hand, for the compounds with the lowest affinities ( e and i), docking simulations showed that the piperazine n- was located farther away from asp and tyr , making the possible ionic interactions with these residues unlikely or much weaker ( figure b ). the analysis of the docking poses indicates that the most potent compounds, i.e., those having a , -substitution pattern ( c, g, and k) exhibited an extended conformation at the binding site, while the least potent similar to the analysis of series i and considering the pharmacological results, docking studies were carried out only in hsert. in this series, seven compounds exhibited ki values between and nm ( a, b, c, f, g, k, and l). docking simulations showed that compounds with the lowest ki values ( c and g) share a common binding mode into the s site of the sert, which is similar to that described for compounds of series i ( figure a ). thus, the piperazine n- can establish ionic and π-cation interactions with asp and tyr , respectively, while the indole moiety can participate in aromatic interactions with tyr and phe . interestingly, the ethylmorpholinic chain extends towards the extracellular vestibule (also known as the s site). on the other hand, for the compounds with the lowest affinities ( e and i), docking simulations showed that the piperazine n- was located farther away from asp and tyr , making the possible ionic interactions with these residues unlikely or much weaker ( figure b ). the analysis of the docking poses indicates that the most potent compounds, i.e., those having a , -substitution pattern ( c, g, and k) exhibited an extended conformation at the binding site, while the least potent compounds ( e and i, showing a , -substitution pattern) adopted a more constrained binding mode, impairing the most relevant interactions. molecules , , x for peer review of compounds ( e and i, showing a , -substitution pattern) adopted a more constrained binding mode, impairing the most relevant interactions. to systematize the structure-activity relationship of the synthesized molecules, we carried out a -d-qsar study of the comfa and comsia type. the complete series of molecules was divided into training ( compounds) and test sets ( compounds) in a ratio of : , selecting the test set compounds at random to avoid bias. the q values for the best models were . and . for comfa and comsia, respectively while the r ncv values were . and . for comfa and comsia, respectively. the statistical summary, as well as the tables of affinities for both models and their respective graphs, are incorporated in the supplementary material. the steric contour map of comfa ( figure a ) shows a green polyhedron on the bromine atom of compound k, the most active of the series. this means that the insertion of bulky atoms or groups in this position is favorable for biological activity. this is consistent with docking studies showing that compounds of series i place halogen into the void space close to lipophilic residues like trp and tyr . in the case of compounds of series ii, the meta-substituted benzamides placed the chain towards the green region, not the ortho-substituted ones, so it is preferable that the chains are in the meta-position. this is confirmed in the docking of these compounds, in which better accommodation is observed in the sert binding site. on the other hand, the electrostatic contour map ( figure b ) shows three blue polyhedra of significant size. this means that the presence of positively charged atoms in these positions would be favorable for affinity. such polyhedra are located on the carbon atom bonded to the halogen in the case of series i, suggesting that the presence of electronegative atoms bonded to the aforementioned carbon is favorable. the second blue polyhedron is localized on the oxygen atom of the carbonyl group belonging to the ortho-substituted series ii amidecompounds. therefore, oxygen atom remotion would be favorable for affinity. finally, the third polyhedron is observed on the oxygen atom of the morpholine ring in the ortho-substituted compounds for series ii, indicating that changing the morpholine by a piperazine or piperidine ring should lead to better affinities. furthermore, alkyl chains substitutions at the ortho-position in the benzamide ring resulted in less favorable affinities compared to meta substitutions as was experimentally corroborated. the hydrophobic contour map of comsia ( figure c ) showed a gray polyhedron at position c of the indole ring, meaning that the presence of hydrophilic groups is favorable for affinity. in fact, the c fluorine-substituted indoles displayed the best affinities of the series. other polar groups like a b figure . docking poses in sert obtained for compounds c in cyan and g in purple (a), and e in light blue, and i in orange (b). nearby residues < Å (grey sticks) and na + atoms (pink spheres) are shown. dotted lines represent ionic interactions, orange lines represent π-cation interactions, and aromatic interactions are shown with green lines. to systematize the structure-activity relationship of the synthesized molecules, we carried out a -d-qsar study of the comfa and comsia type. the complete series of molecules was divided into training ( compounds) and test sets ( compounds) in a ratio of : , selecting the test set compounds at random to avoid bias. the q values for the best models were . and . for comfa and comsia, respectively while the r ncv values were . and . for comfa and comsia, respectively. the statistical summary, as well as the tables of affinities for both models and their respective graphs, are incorporated in the supplementary material. the steric contour map of comfa ( figure a ) shows a green polyhedron on the bromine atom of compound k, the most active of the series. this means that the insertion of bulky atoms or groups in this position is favorable for biological activity. this is consistent with docking studies showing that compounds of series i place halogen into the void space close to lipophilic residues like trp and tyr . in the case of compounds of series ii, the meta-substituted benzamides placed the chain towards the green region, not the ortho-substituted ones, so it is preferable that the chains are in the meta-position. this is confirmed in the docking of these compounds, in which better accommodation is observed in the sert binding site. on the other hand, the electrostatic contour map ( figure b ) shows three blue polyhedra of significant size. this means that the presence of positively charged atoms in these positions would be favorable for affinity. such polyhedra are located on the carbon atom bonded to the halogen in the case of series i, suggesting that the presence of electronegative atoms bonded to the aforementioned carbon is favorable. the second blue polyhedron is localized on the oxygen atom of the carbonyl group belonging to the ortho-substituted series ii amide-compounds. therefore, oxygen atom remotion would be favorable for affinity. finally, the third polyhedron is observed on the oxygen atom of the morpholine ring in the ortho-substituted compounds for series ii, indicating that changing the morpholine by a piperazine or piperidine ring should lead to better affinities. furthermore, alkyl chains substitutions at the ortho-position in the benzamide ring resulted in less favorable affinities compared to meta substitutions as was experimentally corroborated. red polyhedron on the halogen atom at position of the indole ring means that the presence of electron-rich atoms is favorable for affinity. it is interesting to note that the blue polyhedron intersecting the carbon atom of indole at position is complementary to the red polyhedron. in consequence, the presence of a positive charge on the indole ring is favorable for activity. other potential electron-withdrawing groups to be explored are cn, no , and cor. docking studies showed π-stacking interaction between the π-deficient indole ring with tyr , phe , and trp residues. the hydrophobic contour map of comsia ( figure c ) showed a gray polyhedron at position c of the indole ring, meaning that the presence of hydrophilic groups is favorable for affinity. in fact, the c fluorine-substituted indoles displayed the best affinities of the series. other polar groups like oh, nh , or nr would also be interesting to evaluate at this position. similarly, a yellow polyhedron located on the bromine atom of the benzoxazine framework (compound k) means that the presence of lipophilic groups is favorable for activity. in concordance, halogens like cl, br, and i would be the most appropriated substituents and groups, such as aromatic rings, alkyl, and/or alkoxy chains, could also be explored. in the case of compounds of series ii, a yellow polyhedron is located on the amide group of the meta-substituted compounds; therefore, the replacement of the amide by a less-polar function, such as a ketone or ester, would be an interesting option to explore. on the other hand, the electrostatic contour map of comsia ( figure d ) showed two polyhedra around compound k. a red polyhedron on the halogen atom at position of the indole ring means that the presence of electron-rich atoms is favorable for affinity. it is interesting to note that the blue polyhedron intersecting the carbon atom of indole at position is complementary to the red polyhedron. in consequence, the presence of a positive charge on the indole ring is favorable for activity. other potential electron-withdrawing groups to be explored are cn, no , and cor. docking studies showed π-stacking interaction between the π-deficient indole ring with tyr , phe , and trp residues. melting points were determined on a hot-stage apparatus and were uncorrected. the h and c-nmr spectra were obtained on a bruker drx- spectrometer ( and mhz, respectively) in cdcl , dmso-d , and cd cocd -d . chemical shifts were recorded in ppm (δ) relative to tms as an internal standard. j values are given in hz. micro-analyses were carried out on a fisons ea analyzer. high-resolution mass spectra were recorded on a dsa-tofaxion tof ms (perkin elmer, shelton, ct, usa), positive mode. silica gel merck ( - mesh) and aluminum sheets coated with silica gel f were used for column and tlc chromatography, respectively. to a solution containing -chloro- -( , -dihydrobenzo[b] [ , ] oxazin- -yl) ethanamide a ( . g; . mmol) in dry ch cn ( ml), n-boc-piperazine ( mg; . mmol) and anhydrous k co ( mg; . mmol) were added. the mixture was stirred at • c for h. after this time, the mixture was diluted with water ( ml) and the solution extracted with etoac ( ml × ), dried over anhydrous na so , and concentrated under reduced pressure. the organic crude was purified by silica gel column chromatography with etoac as eluent, to provide a ( mg; % yield) as a white solid. m. [ , ] oxazin- -yl)-ethanamide c ( . g; . mmol), n-boc-piperazine ( mg; . mmol), and anhydrous k co ( mg; . mmol), to afford c ( mg; % yield) as a white solid. m. [ , ] oxazin- -yl)- -oxo-ethyl]- -piperazinyl] tert-butylcarbamate a ( g; . mmol) in dry ch cl ( ml) and trifluoroacetic acid ( ml) was stirred at • c, for h. after this time, dry ch cl ( ml) was added and neutralized with solid nahco ( g) to later filter on celite. the mixture was finally diluted with a saturated solution of nahco ( ml), extracted with etoac ( × ml), dried over anhydrous na so , and concentrated under vacuum to obtain pure a ( mg; % yield) as an unstable yellow light solid, highly hygroscopic; to a solution of -( -fluoro- h- -indolyl)-propyl- -methylbencensulfonate b ( mg; . mmol) in ch cn ( ml), -( -fluoro- , -dihydro-benzo[b] [ , ] [ , ] oxazin- -yl) ethanamide e ( mg; . mmol), and anhydrous k co ( mg; . mmol) were added. the mixture was heated at • c for h. after this time, the resulting mixture was poured into water ( ml) and extracted with etoac ( × ml), dried over anhydrous na so , and concentrated under reduced pressure. the organic crude was purified by column chromatography etoac/meoh . , . , . , . , . , . , . , . , . , . , . , . , . , . to a mixture containing water-acetic acid-ethanol ( : : ), -fluoro-n-( -morpholin- -yl-ethyl)- nitro-benzamide a ( g; . mmol) and iron powder ( mg; . mmol) were added. the resulting mixture was heated and stirred for h at • c. after this time, the mixture was filtered to remove excess metallic iron, transferred to a flask containing a mixture of etoac/h o ( ml, : ), and neutralized with nahco ( gr). the aqueous phase was extracted with etoac ( ml × ). the organic layer was dried over anhydrous na so and concentrated under vacuum to give a crude, which was purified by column chromatography with etoac/meoh ( : ) to give a ( mg; % yield) as a yellow light solid. m.p.: . - . to determine the binding of all compounds at sert, competitive binding assays were performed according to previously reported procedures with some modifications [ ] . briefly, assays were carried out in a total volume of . ml containing µg protein of membrane from a clonal cell line hek- that overexpresses sert, mm tris buffer, ph . , mm nacl, mm kcl, nm [ h]-paroxetine (specific activity . ci/mmol, perkinelmer), and the compounds to be tested at different concentrations ( − - − m). after h at • c, incubations were stopped by rapid filtration through whatman gf/c filters presoaked in . % polyethyleneimine, which were washed five times with ml of ice-cold buffer, dried, and put in eppendorf tubes with scintillation liquid. radioactivity was counted by a liquid scintillation counter (microbeta microplate counter, perkinelmer). control curve was performed with fluoxetine in the same experimental conditions. non-specific binding was determined with µm fluoxetine. to determine the binding of all compounds at d receptor, competitive binding assays were performed according to provider indications with some modifications. briefly, assays were carried out in a total volume of . ml containing µg protein of membrane from a cho-k clonal cell line that overexpresses d receptor, mm tris buffer, ph . , mm nacl, mm kcl, mm mgcl , mm edta, . nm [ h]-methylspiperone (specific activity . ci/mmol, perkinelmer), and the compounds to be tested at different concentrations ( − - − m). after h at • c, incubations were stopped by rapid filtration through whatman gf/c filters presoaked in . % polyethyleneimine, which were washed five times with ml of ice-cold wash buffer ( mm tris buffer, ph . , mm nacl), dried, and put in eppendorf tubes with scintillation liquid. radioactivity was counted as described before. control curve was performed with haloperidol in the same conditions. non-specific binding was determined with µm haloperidol. analysis of data: all curves were fitted using the sigmoidal dose-response inhibition curve (variable slope) equation built into graphpad prism . (graphpad software inc., san diego, ca, usa). the analysis gives the ic value (i.e., the drug concentration inhibiting specific binding by %) to calculate ki (affinity constant) by the cheng-prussof equation (ki = ic /( + ([radioligand]/kd (radioligand))). the kd values used correspond to . nm to [ h] paroxetine on sert [ ] , and . nm to [ h]-methylspiperone on d (provided by the manufacturer). the ic and ki values correspond to the results of three independent experiments, each in triplicate. all data are expressed as the mean ± sem. all experimental procedures were approved by the ethics committee of the university of santiago de chile and the science council (fondecyt) of chile and followed internationally accepted guidelines (nih guide for the care and use of laboratory animals). the effects of the compounds on rat mao-a activity were studied following a previously reported methodology [ , ] , using a crude rat brain mitochondrial suspension as a source of enzyme. serotonin ( µm) was used as the selective substrate for mao-a. this compound and its metabolite were detected by hplc with electrochemical detection. as an exploratory evaluation, the percentage of mao-a inhibition in the presence of µm of the different compounds was determined, with the idea of evaluating in detail those compounds showing an inhibitory activity in the range of - %. molecular docking studies for the two families of compounds were performed on two different protein targets (sert and d receptor). all dockings were carried out at ph . in the crystal structures of human sert (hsert pdb: i ) [ ] and human d receptor [hd pdb: cm ) [ ] . all compounds were modelled using the spartan' software (wavefunction, inc. irvine, ca) and geometry optimization calculations were carried out using the software package at the hartree-fock level using the - g* basis set. docking studies were performed using autodockv . [ ] software suite with autodock tools adt . . [ , ] following the standard docking procedure for rigid proteins. grid maps were calculated using the autogrid option with a grid volume of × × points with a grid spacing of . Å and centered on the coordinates x, y, z: . . ; . − . ; and . . for the sert and d receptor respectively. docking simulations were performed with a lamarckian genetic algorithm (lga) and binding energies were estimated according to the internal scoring function implemented by the program; independent runs per ligand were carried out with an initial population of individuals. default settings were used for all other parameters. the lowest free-energy resulting complexes were selected and further analyzed using the visual molecular dynamic (vmd) visualization program [ ] . validation of the docking protocol was performed using the co-crystallized ligands (s)-citalopram and risperidone for sert and d , respectively. comfa and comsia studies were performed with sybyl x- . software [ ] installed in a windows environment on a pc with an intel core i cpu. the geometric optimization, field calculation, and charges calculation were performed as previously reported [ ] ( figure s and table s in supplementary material) [ ] . the internal validation of the models was done by calculating the cross-validation coefficient q [ ] . the models with the highest value of q were selected and then subjected to external validation [ ] [ ] [ ] (table s ). in all cases, the best models passed the validation limits [ ] (table s ). the regression graphs of each model and the tables of experimental versus calculated values are in the supplementary material (table s , figure s ). according to these results, the design of hybrid or bifunctional compounds, i.e., molecules that incorporate two pharmacophores known to act at different receptors into a single chemical entity, is an attractive approach for the development of agents having a targeted polypharmacological profile [ , , ] . in the present work, we attempted to combine sert effects previously demonstrated for indolylalkylpiperazine derivatives, functionalizing the parent scaffold with structural fragments of drugs with known activity upon d receptor or mao-a. unexpectedly, the synthesized compounds did not show, in most cases, a multitarget profile, since they exhibited a high affinity for sert while showing almost no effect at d receptor or mao-a. this indicates that this strategy, although plausible, requires a very fine design of the fragments to be connected and how these are going to be linked. beyond these considerations, our results highlight the remarkable stability of the indolylpropylpiperazine skeleton as sert ligand, which exhibits a high affinity by this target, apparently regardless of the type of the associated moiety [ ] [ ] [ ] . we think that this represents an important feature for the design of polypharmacological molecules, in which an effect upon sert is pursued. docking and qsar results allowed us to rationalize the high sert affinity observed for compounds in both studied families. thus, the presence of a halogen at the c- position of the indole ring and fluorine atoms at the benzoxazine (series i) or acetanilide (series ii) moieties probably induces electronic deprotection of the corresponding aromatic rings, favoring stronger π-π interactions of these frameworks with donor aromatic residues at the binding site. interestingly, one of the compounds ( n) showed a promissory multitarget profile, being the only derivative showing a relatively high and comparable affinity for sert and d receptor (ki = . and nm, respectively). even though at this time it is difficult to determine the molecular aspects underlying this pharmacological promiscuity, it is clear that for polypharmacological drugs, a similar affinity for different receptors is the most relevant characteristic, and therefore n stands as a very attractive lead for further optimization. figure s . the superimposed structures of all compounds used in the comfa/comsia models. figure s . plots of experimental versus predicted pki values for the training and test set molecules for comfa (a, b) and comsia (c, d) models. figure s . hsert affinity curves for compounds of series i ( a, b, c, d, e, f, g, h, i, j, k, l, m, n, o , and fluoxetine), displaying ic values. each determination was made in triplicate and the data were expressed as the mean ± sd. figure s . d affinity curves for compounds of series i ( a, b, c, d, e, f, g, h, i, j, k, l, m, n, o , and haloperidol), displaying ic values. each determination was made in triplicate and the data were expressed as the mean ± sd. figure s . hsert affinity curves for compounds of series ii ( a, b, c, d, e, f, g, h, i, j, k, l , and fluoxetine), displaying ic values. each determination was made in triplicate and the data were expressed as the mean ± sd. figure s . d affinity curves for compounds of series ii ( a, b, c, d, e, f, g, h, i, j, k, l , and haloperidol), displaying ic values. each determination was made in triplicate and the data expressed as the mean ± sd. hrms: (ei) calculated for c h brfn o (m + ) = -morpholin- -ylethyl) benzamide ( f) -bromo- -( -piperazin- -yl-propyl)- h-indole c ( mg; . mmol), -( -chloro-acetylamino)- -fluoro-n-( -morpholin- -yl-ethyl)-benzamide b ( mg; . mmol), and anhydrous k co ( mg; . mmol), to afford f ( mg mmol), -( -chloro-acetylamino)- -fluoro-n-( -morpholin- -yl-ethyl)-benzamide d ( mg; . mmol), and anhydrous k co ( mg; . mmol), to afford g ( mg (dd, h, h- , j o = -morpholin- -ylethyl) benzamide ( h) -bromo- -( -piperazin- -yl-propyl)- h-indole c ( mg; . mmol), -( -chloro-acetylamino)- -fluoro-n-( -morpholin- -yl-ethyl)-benzamide c (s, h, h- ), . - . (m, h, h- ), . (t, h, h- hrms: (ei) calculated for c h brfn o (m + ) = piperazin- -yl-propyl)- h-indole a ( mg; . mmol), -( -chloro-acetylamino)- -fluoro-n-( -morpholin- -yl-ethyl)-benzamide a ( mg; . mmol), and anhydrous k co dmso-d ): δ . (m, h, h- ), . - . (m, h, h- , h- , h- and h- ), . (m, h, h- ) hz), . , . (d, j c-f = hz), . , and . ppm. hrms: (ei) calculated for c h piperazin- -yl-propyl)- h-indole a ( mg; . mmol), -( -chloro-acetylamino)- -fluoro-n-( -morpholin- -yl-ethyl)-benzamide b ( mg; . mmol), and anhydrous k co (s, h, h- ), . - . (m, h, h- ), . (t, h, h- , j = . hz), . (td, h, h- or h- hz), . , . (d, j c-f = hz), . , and . ppm. hrms: (ei) calculated for c h piperazin- -yl-propyl)- h-indole a ( mg; . mmol), -( -chloro-acetylamino)- -fluoro-n-( -morpholin- -yl-ethyl)-benzamide d ( mg; . mmol), and anhydrous k co (s, h, h- ), . (q, h, h- , j = . hz), . (t, h, h- , j = . hz), . (t, h, h- or h- , j = . hz), . (t, h, h- or h- piperazin- -yl-propyl)- h-indole a ( mg; . mmol), -( -chloro-acetylamino)- -fluoro-n-( -morpholin- -yl-ethyl)-benzamide c ( mg (m, h, h- ), . (t, h, h- , j = . hz), . (td, h, h- or h- ci/mmol; code net ) ci/mmol; net ), membrane from clonal cell line hek- that overexpresses sert (code: rbhstm ua), and membrane from cho-k clonal cell line that overexpresses d receptor (code: rbhd cm ua) were purchased from perkin-elmer depression fact sheet depression, mania and self-reported creativity in bipolar disorder characterizing neurocognitive markers of familial risk for depression using multi-modal imaging, behavioral and self-report measures uncovering the genetic architecture of major depression the social cost of major depression grand challenges in global mental health history and evolution of the monoamine hypothesis of depression new approaches to antidepressant drug discovery: beyond monoamines pathophysiology of depression: do we have any solid evidence of interest to clinicians? world psychiatry chapter novel therapeutic targets for major depressive disorder the failure of the antidepressant drug discovery process is systemic developmental changes in serotonin signaling: implications for early brain function, behavior and adaptation the pharmacological basis of the serotonin system: application to antidepressant response psychopharmacology of anxiety and depression: historical aspects, current treatments and perspectives circadian regulation of depression: a role for serotonin a brief history of the development of antidepressant drugs: from monoamines to glutamate perspectives in designing multifunctional molecules in antipsychotic drug discovery benzo-and thienobenzo-diazepines: multi-target drugs for cns disorders the development of novel polypharmacological agents targeting the multiple binding sites of nicotinic acetylcholine receptors triple reuptake inhibitors as potential therapeutics for depression and other disorders: design paradigm and developmental challenges the role of dopamine and its dysfunction as a consequence of oxidative stress the debate over dopamine's role in reward: the case for incentive salience learning and motivation dopamine system dysregulation in major depressive disorders the role of dopamine in schizophrenia from a neurobiological and evolutionary perspective: old fashioned, but still in vogue the role of dopamine in the brain-lessons learned from parkinson's disease the role of dopamine in the pathophysiology of depression molecular role of dopamine in anhedonia linked to reward deficiency syndrome (rds) and anti-reward systems major depression as a complex dynamic system design, synthesis and evaluation of vilazodone-tacrine hybrids as multitarget-directed ligands against depression with cognitive impairment multitarget selective antidepressants design: latest developments, opportunities and challenges polypharmacy" and multi-target agents, complementary strategies for improving the treatment of depression: a comparative appraisal synthesis, in vitro evaluation and molecular docking of a new class of indolylpropyl benzamidopiperazines as dual ache and sert ligands for alzheimer's disease synthesis and docking of novel -indolylpropyl derivatives as new polypharmacological agents displaying affinity for -ht( a) synthesis, docking and pharmacological evaluation of novel homo-and hetero-bis -piperazinylpropylindole derivatives at sert and -ht a receptor synthesis and biological screening of novel indolalkyl arenes targeting the serotonine transporter synthesis, -hydroxytryptamine a receptor affinity and docking studies of -[ -( -aryl- -piperazinyl)-propyl]- h-indole derivatives inhibition of monoamine oxidase by indole and benzofuran derivatives synthesis and structure-activity relationship in a class of indolebutylpiperazines as dual -ht( a) receptor agonists and serotonin reuptake inhibitors potent and highly selective dual-targeting monoamine oxidase-b inhibitors: fluorinated chalcones of morpholine versus imidazole structure-guided development of dual β adrenergic/dopamine d receptor agonists gmeiner, p. β-arrestin biased dopamine d receptor partial agonists: synthesis and pharmacological evaluation proof of concept study for designed multiple ligands targeting the dopamine d , serotonin -ht a, and muscarinic m acetylcholine receptors synthesis, conformational analysis and antidepressant activity of moclobemide new analogues moclobemide: therapeutic use and clinical studies synthesis and biological evaluation of potential acetylcholinesterase inhibitors based on a benzoxazine core x-ray structures and mechanism of the human serotonin transporter structural basis for action by diverse antidepressants on biogenic amine transporters structure of the d dopamine receptor bound to the atypical antipsychotic drug risperidone monoamine oxidase inhibitory properties of some methoxylated and alkylthio amphetamine derivatives: structure-activity relationships pharmacological profile of antidepressants and related compounds at human monoamine transporters autodock and autodocktools : automated docking with selective receptor flexibility python: a programming language for software integration and development vmd: visual molecular dynamics . ; tripos international three-dimensional quantitative structure-activity relationships ( d-qsar) on a series of piperazine-carboxamides fatty acid amide hydrolase (faah) inhibitors as a useful tool for the design of new cannabinoid ligands molecular similarity indices in a comparative analysis (comsia) of drug molecules to correlate and predict their biological activity validation of the general purpose tripos . force field beware of q ! best practices for qsar model development, validation, and exploitation some case studies on application of "r(m) " metrics for judging quality of quantitative structure-activity relationship predictions: emphasis on scaling of response data polypharmacology of dopamine receptor ligands multitarget opioid ligands in pain relief: new players in an old game this article is an open access article distributed under the terms and conditions of the creative commons attribution the authors declare no conflict of interest. key: cord- -tmcm kxn authors: nakamura, shingo; ishihara, masayuki; sato, yoko; takayama, tomohiro; hiruma, sumiyo; ando, naoko; fukuda, koichi; murakami, kaoru; yokoe, hidetaka title: concentrated bioshell calcium oxide (biscao) water kills pathogenic microbes: characterization and activity date: - - journal: molecules doi: . /molecules sha: doc_id: cord_uid: tmcm kxn bioshell calcium oxide (biscao) exhibits deodorizing properties and broad microbicidal activity. in this study, we examined possible utility of biscao water for that purpose. biscao water was prepared by adding wt% biscao to clean water and gently collecting the supernatant in a bottle. the same volume of clean water was gently poured onto the biscao precipitate and the supernatant was gently collected in a bottle; this process was repeated fifty times. the produced biscao water contained nanoparticles (about – nm) composed of smaller nanoparticles ( – nm), and was colorless and transparent, with a ph > . . in vitro assays demonstrated that biscao water eliminated more than . % of influenza a (h n ) and feline calicivirus, escherichia coli such as nbrc and o- :h , pseudomonas aeruginosa, salmonella, and staphylococcus aureus within min. we compared biscao water with the other microbicidal reagents such as ethanol, biscao, bisca(oh)( ) suspensions, povidone iodine, naclo, biscao dispersion and colloidal dispersion with respect to deodorization activity and microbicidal efficacy. the results showed that biscao water was a potent reagent with excellent deodorization and disinfection activities against pathogenic bacteria and viruses (including both enveloped and nonenveloped viruses). calcium oxide (cao) and calcium hydroxide (ca(oh) ) produced from limestone are readily available and important inorganic compounds used in various industries as adsorbents, toxic-waste remediation agents, and alkalization agents. however, both reagents contain harmful impurities, and cao is especially dangerous, because it easily generates high heat in response to hydration [ , ] . although scallop shells have been used as food additives and are a readily available source of cao and ca(oh) , most are discarded as industrial waste. these wasted shells accumulate on the shores of harvesting districts in japan, causing serious problems such as offensive odors and soil pollution due to release of harmful materials from the shells [ ] . the harmful materials in scallop shells can be removed by heating at a high temperature, and by grinding and sieving. most commercially available spraying biscao water, which is colorless and transparent with a ph > . , on a smooth metal or plastic surface, followed by drying, provides a white powder coating. scanning electron microscope (sem) images of dried powders of biscao water are shown in figure a ,b, and cryo-sem [ , ] images of biscao water are shown in figure c ,d. after drying biscao water, the biscao particles become larger ( - µm) compared to those in biscao water. the microparticles were connected with each other. nanoscale cao particles ( - nm) in biscao water aggregate to generate larger biscao particles ( - nm). furthermore, when the dried powder was suspended into clean water, the powder was insoluble and ph in the supernatant was below . , suggesting the microparticles were caco produced by interaction of ca + and co . molecules , , of spraying biscao water, which is colorless and transparent with a ph > . , on a smooth metal or plastic surface, followed by drying, provides a white powder coating. scanning electron microscope (sem) images of dried powders of biscao water are shown in figure -a,b, and cryo-sem [ , ] images of biscao water are shown in figure -c,d. after drying biscao water, the biscao particles become larger ( - μm) compared to those in biscao water. the microparticles were connected with each other. nanoscale cao particles ( - nm) in biscao water aggregate to generate larger biscao particles ( - nm). furthermore, when the dried powder was suspended into clean water, the powder was insoluble and ph in the supernatant was below . , suggesting the microparticles were caco produced by interaction of ca + and co . scanning electron microscopy (sem) images of drying biscao water and cryo-sem images of biscao water. the particle surface structure of drying biscao water at -fold magnification (a) and at , -fold magnification (b) was observed with sem images taken with a field-resolved scanning electron microscope. cryo-sem observations were performed on biscao water at -fold magnification (c) and , -fold magnification (d). arrows indicate nanoparticles comprising assemblies of smaller nanoparticles. bactericidal activities of biscao against escherichia coli (e. coli) nbrc , e. coli o- :h , p. aeruginosa, salmonella, and staphylococcus aureus (s. aureus) were evaluated by the japan food research laboratory (jfrl; tokyo, japan) ( table ). the levels (colony forming units; cfu) of e. coli, p. aeruginosa, and salmonella were reduced to below the detection limit within min, % of e. coli o- :h was eliminated after min, and % of s. aureus (a gram positive bacterium) was eliminated after min and reduced to below the detection limit after min. scanning electron microscopy (sem) images of drying biscao water and cryo-sem images of biscao water. the particle surface structure of drying biscao water at -fold magnification (a) and at , -fold magnification (b) was observed with sem images taken with a field-resolved scanning electron microscope. cryo-sem observations were performed on biscao water at -fold magnification (c) and , -fold magnification (d). arrows indicate nanoparticles comprising assemblies of smaller nanoparticles. bactericidal activities of biscao against escherichia coli (e. coli) nbrc , e. coli o- :h , p. aeruginosa, salmonella, and staphylococcus aureus (s. aureus) were evaluated by the japan food research laboratory (jfrl; tokyo, japan) ( table ). the levels (colony forming units; cfu) of e. coli, p. aeruginosa, and salmonella were reduced to below the detection limit within min, % of e. coli o- :h was eliminated after min, and % of s. aureus (a gram positive bacterium) was eliminated after min and reduced to below the detection limit after min. the virucidal activities of biscao water against influenza a (h n ), an enveloped virus, and feline calicivirus, a nonenveloped virus, were evaluated using the fifty-percent tissue culture infectious dose (tcid ) method [ , ] , which was performed by jfrl (table ) . feline calicivirus treated with biscao water was reduced to below the detection limit within min, and influenza a was reduced from . to . (tcid /ml) at min compared with the control, and then reduced to below the detection limit after min. we evaluated and compared the deodorization efficacy of biscao water, biscao and bisca(oh) suspensions, and biscao dispersion and colloidal dispersion using tainted pork meat as a malodorous material. tainted pork meat was mixed with each deodorant, then placed on petri dishes and sealed in plastic bags for h. the odor intensity was then measured using a handheld odor meter. all deodorants tested exhibited concentration-dependent deodorization effects. biscao water had the highest deodorization efficacy at each concentration tested; bisca(oh) suspensions were less efficient. biscao suspension, dispersion, and colloidal dispersion exhibited intermediate efficacies ( figure ). efficacies of biscao water, suspension, dispersion, and colloidal dispersions for deodorizing contaminated minced pork. each deodorant was added to tainted pork meat on petri dishes, then sealed in plastic bags for h. the odor intensity was measured using a handheld odor meter. the experiments were repeated four times. we investigated the microbicidal efficacy of biscao water, suspension, dispersion, and colloidal dispersion against a contaminated suspension comprising normal bacterial flora, compared to other antiseptics/disinfectants. equal volumes of each disinfectant and the contaminated suspension were mixed well and incubated at room temperature for min, then the number of colony forming units (cfu/ml) per sample was determined. during incubation of bathtub water with % dulbecco's modified eagle's medium (dmem) + bovine serum albumin (bsa) ( . wt%) at °c for h, the tc and cf values increased from ± cfu/ml and ± cfu/ml to . ± . (× ) cfu/ml and . ± . (× ) cfu/ml, respectively. the cfu/ml for tc and cf following treatment with undiluted (final -fold diluted) and -fold diluted (final -fold diluted) biscao water, and . and . wt% (final . wt% and . wt%) of biscao dispersion and colloidal dispersion were below the detection limit, whereas low counts of tc and cf remained viable following treatment with . wt% of biscao water, dispersion, and colloidal dispersion ( figure ). in contrast, some tc and cf remained viable following treatment with undiluted ethanol, -fold diluted, and -fold diluted (final -fold diluted, -fold diluted, and -fold diluted) ethanol, and . , . , and . wt% (final . , . , and . wt%) povidone iodine. the microbicidal activities of biscao and bisca(oh) (suspension) and naclo against tc and cf were intermediate between that of biscao water and povidone iodine, and no cfu were detectable following treatment with high concentrations of . wt% (final . wt%). plating and counting were performed as a set of technical replicates (n = ). efficacies of biscao water, suspension, dispersion, and colloidal dispersions for deodorizing contaminated minced pork. each deodorant was added to tainted pork meat on petri dishes, then sealed in plastic bags for h. the odor intensity was measured using a handheld odor meter. the experiments were repeated four times. we investigated the microbicidal efficacy of biscao water, suspension, dispersion, and colloidal dispersion against a contaminated suspension comprising normal bacterial flora, compared to other antiseptics/disinfectants. equal volumes of each disinfectant and the contaminated suspension were mixed well and incubated at room temperature for min, then the number of colony forming units (cfu/ml) per sample was determined. during incubation of bathtub water with % dulbecco's modified eagle's medium (dmem) + bovine serum albumin (bsa) ( . wt%) at • c for h, the tc and cf values increased from ± cfu/ml and ± cfu/ml to . ± . (× ) cfu/ml and . ± . (× ) cfu/ml, respectively. the cfu/ml for tc and cf following treatment with undiluted (final -fold diluted) and -fold diluted (final -fold diluted) biscao water, and . and . wt% (final . wt% and . wt%) of biscao dispersion and colloidal dispersion were below the detection limit, whereas low counts of tc and cf remained viable following treatment with . wt% of biscao water, dispersion, and colloidal dispersion ( figure ). in contrast, some tc and cf remained viable following treatment with undiluted ethanol, -fold diluted, and -fold diluted (final -fold diluted, -fold diluted, and -fold diluted) ethanol, and . , . , and . wt% (final . , . , and . wt%) povidone iodine. the microbicidal activities of biscao and bisca(oh) (suspension) and naclo against tc and cf were intermediate between that of biscao water and povidone iodine, and no cfu were detectable following treatment with high concentrations of . wt% (final . wt%). plating and counting were performed as a set of technical replicates (n = ). the number of colony forming units (cfu/ml) per sample was determined after disinfecting contaminated wood pieces with tc values of . ± . (× ) and cf values of . ± . (× ). the cfu/ml following treatment with undiluted and -fold diluted biscao water and . and . wt% of naclo, biscao dispersion, and colloidal dispersion were below the detection limit for both tc and cf, whereas low counts of tc and cf remained viable following treatment with -fold diluted biscao water and . wt% of naclo, biscao dispersion, and colloidal dispersion ( figure ). low counts of tc and cf remained viable following treatment with undiluted, -fold diluted, and -fold diluted ethanol; . , . , and . wt% bisca(oh) ; and povidone iodine. biscao (suspension) had a microbicidal activity against tc and cf intermediate between that of biscao water and povidone iodine: the tc and cf counts were below the detection limit following treatment with a high concentration of . wt% biscao (suspension). the number of colony forming units (cfu/ml) per sample was determined after disinfecting contaminated wood pieces with tc values of . ± . (× ) and cf values of . ± . (× ). the cfu/ml following treatment with undiluted and -fold diluted biscao water and . and . wt% of naclo, biscao dispersion, and colloidal dispersion were below the detection limit for both tc and cf, whereas low counts of tc and cf remained viable following treatment with -fold diluted biscao water and . wt% of naclo, biscao dispersion, and colloidal dispersion ( figure ). low counts of tc and cf remained viable following treatment with undiluted, -fold diluted, and -fold diluted ethanol; . , . , and . wt% bisca(oh) ; and povidone iodine. biscao (suspension) had a microbicidal activity against tc and cf intermediate between that of biscao water and povidone iodine: the tc and cf counts were below the detection limit following treatment with a high concentration of . wt% biscao (suspension). a suspension ( μl) containing . ± . (× ) cfu/ml of tc and . ± . (× ) cfu/ml of cf was inoculated on nonwoven surgical masks and dried or left wet, then biscao water was sprayed on some of the masks and air dried. the tc and cf of sprayed and unsprayed masks were about (× ) and , and (× ) and (× ), respectively ( figure ) . thus, dry white-powdercoated nonwoven surgical masks had strong antimicrobial activity. furthermore, the spraying of biscao water on the contaminated surface of a surgical mask resulted in almost total disinfection. no change in quality was observed in the surgical mask after spraying biscao ten times. this result suggested that surgical masks may be reused multiple times following disinfection with biscao a suspension ( µl) containing . ± . (× ) cfu/ml of tc and . ± . (× ) cfu/ml of cf was inoculated on nonwoven surgical masks and dried or left wet, then biscao water was sprayed on some of the masks and air dried. the tc and cf of sprayed and unsprayed masks were about (× ) and , and (× ) and (× ), respectively ( figure ) . thus, dry white-powder-coated nonwoven surgical masks had strong antimicrobial activity. furthermore, the spraying of biscao water on the contaminated surface of a surgical mask resulted in almost total disinfection. no change in quality was observed in the surgical mask after spraying biscao ten times. this result suggested that surgical masks may be reused multiple times following disinfection with biscao water. scallop shell is composed of caco and is converted to cao when heated above °c. according to the manufacturer, biscao is prepared by heating shell powder at °c for h to obtain over . % cao, grinding using a dry grinder, followed by cooling in a vacuum chamber and vacuum packing. the produced fine cao powder, biscao, has an average particle diameter of about μm [ ] . both biscao and bisca(oh) are poorly water-soluble under alkaline conditions. the generated precipitates in suspensions of high concentrations of biscao and bisca(oh) can result in a significant loss of cao and the plugging of spray nozzles. a methodology is therefore needed to prepare biscao and bisca(oh) dispersions without precipitates. we previously reported that the addition of phosphate compounds such as phosphoric acid (h po ), trisodium phosphate (na po ), na hpo , or sodium dihydrogen phosphate (nah po ) to biscao or bisca(oh) suspensions results in the formation of dispersions [ ] . furthermore, biscao and bisca(oh) colloidal dispersions can be prepared by mixing with na-polypo or na-tripo as flocculent agents. two layers quickly form a supernatant, and flocculants/precipitates composed of polymeric colloidal calcium phosphate [ ] . the present study showed that biscao water (ph > . ), in addition to biscao dispersion and colloidal dispersion, has microbicidal activity against various pathogenic bacteria, including s. aureus (a gram-positive bacterium) ( table ) and viruses such as influenza a (h n ) as an enveloped virus and feline calicivirus as a nonenveloped virus (table ) . biscao water, which is colorless and transparent with a ph > . , was sprayed and dried on smooth metal or plastic surfaces and provided a white powder coating. scanning electron microscopy (sem) images of drying biscao water showed microparticles ( - μm) connected with each other (figure -a,b) . cryo-sem observations indicated that cao nanoparticles ( - nm) aggregated to generate larger biscao particles ( - nm) in biscao water (figure -c,d) . we examined the deodorizing and bactericidal activities of biscao water, dispersion, and scallop shell is composed of caco and is converted to cao when heated above • c. according to the manufacturer, biscao is prepared by heating shell powder at • c for h to obtain over . % cao, grinding using a dry grinder, followed by cooling in a vacuum chamber and vacuum packing. the produced fine cao powder, biscao, has an average particle diameter of about µm [ ] . both biscao and bisca(oh) are poorly water-soluble under alkaline conditions. the generated precipitates in suspensions of high concentrations of biscao and bisca(oh) can result in a significant loss of cao and the plugging of spray nozzles. a methodology is therefore needed to prepare biscao and bisca(oh) dispersions without precipitates. we previously reported that the addition of phosphate compounds such as phosphoric acid (h po ), trisodium phosphate (na po ), na hpo , or sodium dihydrogen phosphate (nah po ) to biscao or bisca(oh) suspensions results in the formation of dispersions [ ] . furthermore, biscao and bisca(oh) colloidal dispersions can be prepared by mixing with na-polypo or na-tripo as flocculent agents. two layers quickly form a supernatant, and flocculants/precipitates composed of polymeric colloidal calcium phosphate [ ] . the present study showed that biscao water (ph > . ), in addition to biscao dispersion and colloidal dispersion, has microbicidal activity against various pathogenic bacteria, including s. aureus (a gram-positive bacterium) ( table ) and viruses such as influenza a (h n ) as an enveloped virus and feline calicivirus as a nonenveloped virus (table ) . biscao water, which is colorless and transparent with a ph > . , was sprayed and dried on smooth metal or plastic surfaces and provided a white powder coating. scanning electron microscopy (sem) images of drying biscao water showed microparticles ( - µm) connected with each other (figure a,b) . cryo-sem observations indicated that cao nanoparticles ( - nm) aggregated to generate larger biscao particles ( - nm) in biscao water (figure c,d) . we examined the deodorizing and bactericidal activities of biscao water, dispersion, and colloidal dispersion against tc and cf under various conditions such as particle size and concentration. biscao water, dispersion, and colloidal dispersion containing smaller particles and at higher concentration showed higher deodorization and microbicidal activities against tc and cf, probably due to higher brunauer-emmett-teller (bet)-specific surface areas (data not shown). we anticipate that differences in the bet-specific surface areas of biscao water, dispersion, and colloidal dispersion influenced their deodorizing and microbicidal activities. cryo-sem showed that the nanoparticles in biscao dispersion, colloidal dispersion, and biscao water were - nm [ ] , - nm [ ] , and - nm in diameter, respectively. the hydration of cao generates a strong base and is the primary mechanism for the deodorization and microbicidal activities of biscao dispersion. the cao content of biscao is much higher than that of bisca(oh) , and this suggested that biscao water, suspension, dispersion, and colloidal dispersion showed higher deodorizing and microbicidal activities than bisca(oh) because of the higher ph. however, our preliminary study showed that biscao exhibited higher activity than naoh solution at the same ph (data not shown). this suggests that alkalinity alone is not responsible for the deodorizing and microbicidal property of biscao. rather, we suggest that the microbicidal action is due to the reducing activity of biscao, and that the high disinfection activity of biscao is due to the oh− concentration of the thin water layer formed around biscao particles being higher than in the bulk solvent [ , ] . furthermore, active radical species generated from magnesium oxide and biscao may also contribute to strong disinfection activity [ , ] , as supported by a multiparameter flow cytometry study conducted by hewitt et al. [ ] . although high ph is certainly the main contributor to the deodorizing and microbicidal activity of biscao, active radical species generated from biscao may be an alternative microbicidal factor. the recent worldwide epidemic of coronavirus disease (covid- ) due to a newly discovered coronavirus is causing a crisis [ , ] . the world health organization (who) recommends "to ensure that environmental cleaning and disinfection procedures are followed consistently and correctly. thoroughly cleaning environmental surfaces with water and detergent and applying commonly used hospital-level disinfectants such as naclo are effective and sufficient procedures." [ ] . some antiseptics/disinfectants, such as ethanol and naclo, show significant activity towards sars-cov- by breaking the envelope of virus. however, they are cytotoxic to cellular and organic components, and high concentrations are required for antiseptic/disinfection activity [ ] [ ] [ ] . furthermore, chlorine-derived compounds are ineffective in the presence of organic materials [ , ] . therefore, antiseptics/disinfectants that can decrease the bacterial bioburden without harmful side effects and environmental disruption are essential for environmental hygiene and public health. the character of biscao water for having virucidal activity against an enveloped-type virus ( table ) may be valuable for the limitation of the spread of respiratory viruses such as covid- , although it is necessary to study additional microbicidal activity including coronavirus sars-cov- . recommendations for personal protective equipment (ppe), including face masks such as surgical masks or n respirators, are necessary for the protection of health-care personnel [ ] . recommendations on face masks vary across countries, and the use of masks increases substantially once local epidemics begin, including the use of masks in community settings. however, this increased use of face masks by the general public exacerbates the global supply shortage of face masks, sending prices soaring [ ] . in this study, spraying biscao water on the contaminated surface of a surgical mask resulted in almost complete elimination of the test microbes without changing the quality of the mask. there is little possibility that dry microparticle-aggregates blow up and/or suck in, since nanoscale cao particles in biscao water aggregate to generate larger biscao particles and the microparticles were composed of caco produced by interaction of ca + and co . this result suggested that face masks may be reused multiple times by sterilization with biscao water, which could contribute to their safe and economical reuse, rather than disposal after a single use. however, our preliminary experiments show that the potency of biscao water does not have a long-term effect on the spraying surface, therefore the spray may need to be performed both at the start of use and after use ( figure ). according to the manufacturer, scallop shell powders were heated at • c for h, then ground using a dry super grinder (nano jetmizer nj- -d; aishin nano technologies co. ltd., saitama, japan), followed by cooling in a vacuum chamber. this provided biscao dry powder with particle diameters of - µm (average µm). this was purchased from plus lab corp., kanagawa, japan. according to the manufacturer, the content of cao in this biscao preparation is . %. bisca(oh) was obtained from scallow, kohkin inst. co. ltd., tochigi, japan and had a dry-powder-particle diameter of - µm (average µm). the cao and ca(oh) contents were < % and > %, respectively. according to the manufacturer, biscao water was prepared by adding g of biscao to ml chilled clean water (< • c), gently mixing, and standing for min. the supernatant ( ml) was collected and transferred to a -l water tank. another chilled ml of clean water was gently poured and mixed onto the remaining biscao precipitate and the supernatant was collected in the tank. this process was repeated fifty times. the produced biscao water (total l) was colorless and transparent with a ph of about . , and the biscao water is now commercially available from plus lab corp. scanning electron microscope (sem) images of dry powder were obtained by osmium metal coating using a neo-osmium coater (neoc-stb; meiwafosis co., ltd., tokyo). the surface structure of each dry powder was observed using a field-resolved scanning electron microscope (jsm- f; jeol ltd. tokyo, japan). for cryo-sem, samples were frozen in liquid nitrogen, then knife-cut and observed using a jeol jsm f sem (jeol ltd., tokyo, japan) under vacuum conditions at − • c. the accelerating voltage was kv, and the detection signal was a backscattered electron image. we evaluated the bactericidal activities of biscao water towards e. coli (nbrc ), e. coli o- :h (atcc ), p. aeruginosa (nbrc ), salmonella (nbrc ), and s. aureus (nbrc ). each bacterial suspension was prepared in soybean-casein digest broth with lecithin and polysorbate (scdlp) liquid and agar medium. the assays were performed by the japan food research laboratory (jfrl; tokyo, japan). briefly, . ml of each bacterial suspension ( - cells/ml) was added to ml of biscao water. the mixture was stirred and then left at room temperature for , , and min to allow the bacteria to interact with biscao. at each time point, the mixture was ten-fold diluted with scdlp medium to terminate the interaction. the mixtures were subjected to ten-fold serial dilution with phosphate-buffered saline (pbs) onto scdlp agar plates ( . cm). the number of cfu was determined after incubating at • c for h. to evaluate the virucidal activities of biscao water, feline calicivirus (f- ; atcc vr- ) and human influenza a virus (h n ; atcc vr- ) were used and assayed using scdlp medium and the tcid method. the assays were performed by jfrl. briefly, viral suspension in pbs ( µl) was added to -ml biscao water. the mixture was stirred and then left at room temperature for , , and min to allow the virus to interact with biscao. at each time point, the mixture was ten-fold diluted with % fetal bovine serum containing dmem to terminate the interaction. the mixtures were subjected to two-fold serial dilution with pbs in a -well cell culture plate sown with crandell feline kidney (crfk) cells for feline calicivirus and madin-darby canine kidney (mdck) cells for influenza a virus. the antiviral activity of biscao water was estimated as the tcid ratio of the biscao-treated sample to the control (treated with clean water). the addition of . , . , and . g of biscao and bisca(oh) to ml of clean water, followed by rotary mixing, generated . , . , and . wt% biscao and bisca(oh) suspensions, respectively. next, . , . , and . wt% of na hpo (fujifilm wako pure chemical corp., osaka, japan) for biscao dispersion and na-polypo (fujifilm wako pure chemical corp.) for biscao colloidal dispersion were added to . , . , and . wt% biscao water suspension, respectively, then rotary mixed to prepare each biscao dispersion and colloidal dispersion. ethanol ( . %; fujifilm wako pure chemical corp.) was used and diluted with clean water. five grams of tainted pork meat was mixed with ml of each deodorant, then placed on petri dishes and sealed in plastic bags ( × cm) for h. the odor intensity was measured using a handheld odor meter (omx-srm; shinyei technology co. ltd., hyogo, japan). the microbicidal efficacy of biscao water against suspensions and wood pieces contaminated with high normal bacterial flora was evaluated and compared with the actions of ethanol, biscao and bisca(oh) suspensions, povidone iodine, naclo, biscao dispersion, and colloidal dispersion. various concentrations of povidone iodine and naclo were prepared by the dilution of -wt% isodine (meiji seika pharma co., ltd., tokyo, japan) and -wt% naclo (yoshida pharmaceutical corp., tokyo, japan) with clean water. the concentrations of naclo were confirmed as residual chlorine levels using clo (hclo and clo -) -selective test papers (high concentration, - ppm; low concentration, - ppm; kyoritu check laboratory corp., tokyo, japan). suspensions contaminated with normal bacterial flora were prepared by incubating bathtub water with % dmem + bsa ( . wt%) at • c for h [ , ] . ten milliliters of each disinfectant were added to ml of the contaminated suspension, mixed well, and incubated at room temperature for min. to prepare contaminated wood pieces, five wood pieces ( . cm × . cm × . cm) were added to ml of the contaminated suspension and incubated at • c for h, then rinsed with clean water [ , ] . for the disinfection assay of contaminated wood pieces, a wood piece was added to ml of each disinfectant, vortexed gently for min, and then tc and cf were released from the contaminated wood piece in ml of clean water by vigorous vortexing for min. to count the number of cfu, aliquots ( ml of each mixture) were gently poured into individual petri dishes containing prealiquoted portions of simple and easy dry medium for tc or cf (nissui pharmaceutical co., ltd., tokyo, japan) [ , , , ] , and the plates were incubated for h in a • c incubator (a ; ikuta sangyo co., ltd., ueda, nagano, japan). plating and counting were performed as a set of technical replicates (n = ). suspension contaminated with . ± . (× ) cfu/ml of tc and . ± . (× ) cfu/ml of cf ( µl total) was inoculated and dried for min at room temperature on a round area ( -cm diameter) of the surface of a surgical mask (surgical mask st; utsunomiya seisaku co. ltd. osaka, japan). "spray before use" was conducted by spraying about . ml of biscao water on the round area and drying for h at room temperature. "spray after use" was conducted by spraying about . ml of biscao water on the inoculated round area and drying for h at room temperature. the inoculated area of each mask was resected, and then tc and cf were released from the contaminated sections into ml of clean water by vigorous vortexing for min. to count the cfu, aliquots ( ml of each mixture) were gently poured into individual petri dishes containing prealiquoted portions of simple and easy dry medium for tc or cf, and the plates were incubated for h in a • c incubator. plating and counting were performed as a set of technical replicates (n = ). biscao water contains nanoparticles ( - nm) and is colorless and transparent, with a ph > . . biscao water completely eliminated various pathogenic bacteria within min and viruses within min in in vitro assays. furthermore, biscao water exhibited higher deodorization of tainted pork meat and higher microbicidal efficacy using suspensions contaminated with normal bacteria compared to ethanol, biscao and bisca(oh) suspensions, povidone iodine, naclo, biscao dispersion and colloidal dispersion. the results showed that biscao water has excellent deodorization and disinfection activities for pathogenic microbes, potentially for covid- measures. funding: this study was partially supported by jsps kakenhi (no. k for y.s.). the authors declare no conflict of interest, and the founding sponsor had no role in the design of the paper. an overview- a novel method to make regenerable core-shell calcium-based sorbants antimicrobial characteristics of heated scallop shell powder and its application antibacterial characteristics of heated scallop-shell nano-particles inactivation of avian influenza virus, newcastle disease virus and goose parvovirus using solution of nano-sized scallop shell powder sporicidal kinetics of baccillus subtilis spores by heated scallop shell powder comparison of antifungal activities of scallop shell, oyster shell and their pyrolyzed products disinfection treatment of heated scallop-shell powder on biofilm of escherichia coli atcc surrogated for e. coli o :h ability of heated acallop-shell powder to disinfect staphylococcus aureus biofilm heated scallop-shell powder treatment for deactivation and removal of listeria sp. biofilm formed at a low temperature comparison of various disinfectants on bactericidal activity under organic matter contaminated water skin cleansing technique with disinfectant using improved high-velocity steam-air micro mist jet spray chronic wounds and bacteria. clinical relevance, detection and therapy evidence-based practice: tap water cleansing of leg ulcers in the community a systematic review of wound cleansing for pressure ulcers stability of weak acidic hypochlorous acid solution with microbicidal activity effects of a low concentration hypochlorous acid nasal irrigation solution on bacteria, fungi, and virus disinfection by hypochlorous acid for pseudomonas aeruginosa-infected wounds in diabetic db/db mice development of antimicrobial biomaterials produced from chitin-nanofiber sheet/silver nanoparticle composites adsorption of silver nanoparticles onto different surface structures of chitin/chitosan and correlations with antimicrobial activities synthesis and application of silver nanoparticles (ag nps) for the prevention of infection in healthcare workers healing of pseudomonas aeruginosa-infected wounds in diabetic db/db mice by weakly acidic hypochlorous acid cleansing and silver nanoparticle/chitin nanofiber sheet covering bioshell calcium oxide (biscao) for cleansing and healing of pseudomonas aeruginosa-infected wounds in hairless rats cleaning technique using high-velocity steam-air micromist jet spray preparation and application of bioshell calcium oxide (biscao) nanoparticles-dispersions with bactericidal activity application of colloidal dispersions of bioshell calcium oxide (biscao) for disinfection persistence of coronaviruses on inanimate surfaces and their inactivation with biocidal agents simple and environmentally friendly preparation and size control of silver nanoparticles using an inhomogeneous system with silver-containing glass powder an evaluation of the antibacterial action of ceramic powder slurries using multi-parameter flow cytometry human coronavirus: insights into environmental resistance and its influence on the development of new antiseptic strategies a toxicity index of skin and wound cleaners used on in vitro fibroblasts and keratinocytes in vitro toxicity of topical antimicrobial agents to human fibroblasts cytotoxicity of silver nanoparticle and chitin-nanofiber sheet composites caused by oxidative stress covid- coronavirus: recommended personal protective equipment for the orthopaedic and trauma surgeon rational use of face masks in the covid- pandemic key: cord- - gk nmi authors: proskurnina, elena v.; izmailov, dmitry yu.; sozarukova, madina m.; zhuravleva, tatiana a.; leneva, irina a.; poromov, artem a. title: antioxidant potential of antiviral drug umifenovir date: - - journal: molecules doi: . /molecules sha: doc_id: cord_uid: gk nmi free radical reactions play an important role in biological functions of living systems. the balance between oxidants and antioxidants is necessary for the normal homeostasis of cells and organisms. experimental works demonstrate the role of oxidative stress that is caused by influenza virus as well as the toxic effects of some antiviral drugs. therefore, antiviral drugs should be characterized by its pro- and antioxidant activity, because it can affect its therapeutic efficiency. the aim of the study was to quantify the antioxidant capacity and propose the mechanism of the antioxidant effect of the antiviral drug umifenovir (arbidol(®)). the kinetic chemiluminescence with the , ’-azobis ( -amidinopropane) dihydrochloride + luminol system was used to quantify the antioxidant capacity of umifenovir relative to the standard compound trolox. with computer simulation, the reaction scheme and rate constants were proposed. the antioxidant capacity of . μm umifenovir (maximum concentration of umifenovir in blood after oral administration of mg) was as high as . ± . μm of trolox. thus, the total antioxidant capacity of umifenovir is comparable to the antioxidant capacity of trolox. unlike trolox, umifenovir reacts with free radicals in two stages. for trolox, the free radical scavenging rate constant was k = nm(− ) min.(− ), for umifenovir k( ) = nm(− )min.(− ), k( ) = nm(− )min.(− ). slower kinetics of umifenovir provides the prolonged antioxidant effect when compared to trolox. this phenomenon can make a serious contribution to the compensation of oxidative stress that is caused by a viral disease and the therapeutic effect of the drug. free radical reactions play an important role in the biological functions of living systems. the balance between prooxidants and antioxidants is necessary for the normal homeostasis of cells and organisms. oxidative stress plays an important role in many pathological processes, including viral infections, such as influenza [ ] . excessive reactive oxygen species (ros) cause oxidative damage of lipid membranes and mitochondrial respiratory chain. mitochondria act as a platform for antiviral innate immunity. mitochondrial antiviral signaling involves the activation of the retinoic acid-inducible gene i-like receptors, which requires oxidative phosphorylation activity. the cells with respiratory defects exhibited severely impaired virus-induced induction of interferons and proinflammatory cytokines. mice with respiratory chain defects were highly susceptible to viral infection and exhibited significant lung inflammation [ ] . oxidative and nitrosative stress may also contribute to reduced all clinical isolates of influenza a viruses that were obtained before and during therapy were susceptible to umifenovir with % effective concentration ranging from . +/− . to . +/− . µm without the development of drug-resistant variants [ ] . experimental studies at concentrations of - mg/ml proved the direct antiviral activity of umifenovir on the early viral replication of severe acute respiratory syndrome (sars) virus in the cultured gmk-ah- cells [ ] . umifenovir (arbidol) is licensed and widely used in russia for the prophylaxis and/or treatment of influenza. the clinical trials of umifenovir were performed in the former ussr during - and post-marketing phase iv trial in - influenza seasons. in a double-blind, randomized, placebo-controlled clinical study arbitr (clinicaltrials.gov identifier: nct ) investigating the efficacy and safety of arbidol among adults that was carried out in russia, umifenovir ( mg daily for five days) significantly (p < . ) reduced the duration of fever ( h in umifenovir group and . in placebo group), muscle pain ( . vs . ), and weakness ( . vs . ) in umifenovir-treated group when compared to control untreated patients, and reduced the risk of complications, namely influenza with low respiratory tract infections ( % vs . %) [ ] . post-marketing surveillance of the efficiently of umifenovir in clinical use was made by retrospective analyzed of patients with influenza and other arvi in hospitals from regions of the russian federation. in patients that were treated with umifenovir (in the first h after disease onset), the duration of fever and frequency of pneumonia proved to be lower than those in the patients who did not receive antiviral therapy. umifenovir therapy substantially reduces the duration of fever and risk of complications, especially in patients with laboratory-confirmed influenza infection [ ] . the prophylactic effect of arbidol was also studied in a randomized placebo-controlled trial in children. the study was conducted in by pasteur institute in st. petersburg and it included children who received arbidol twice a week for three weeks before the peak of influenza morbidity. arbidol prophylaxis . - -fold reduced the overall morbidity (depending on the age group), and the duration of illness was also decreased by . - . days [ ] . arbidol effectiveness for preventing and treating influenza might be due to the co-existence of its two actions: ( ) specific antiviral activity against influenza and other respiratory viruses; ( ) interferon-inducing and immune-modulating properties, which was shown on cultured cells, animals [ ] , and humans [ ] . arbidol-treated patients with lower baseline immunity showed improvement in immunological parameters (number of cd , cd lymphocytes and b lymphocytes, and concentration of serum immunoglobulins). the administration of a single oral dose of umifenovir . g by healthy adult volunteers resulted in the induction of serum interferon up to - interferon units/ml. for children, the administration of . g/kg/day leads to the . -fold increase in the endogenous interferon production in % of subjects [ ] . there is some evidence that umifenovir helps the phagocytic action of macrophages [ ] . the presence of not only direct antiviral action, but also other indirect effects was the reason for the investigation of antioxidant activity of umifenovir. it is a free hydroxyl group that provides the antioxidant activity of umifenovir ( figure ). previously, the antioxidant effect of umifenovir to prevent lipid peroxidation was studied in vitro [ ] . the authors proved this substance to be a lipid antioxidant. however, lipid peroxidation does not restrict oxidative stress. the assessment of antioxidant potential in relation to ros-reactions in the aqueous phase is no less important, because non-lipid ros may cause oxidative modification of proteins and dna. more studies are needed to characterize comprehensively the antioxidant properties of umifenovir. the conventional approach is based on the quantitative comparison with a reference compound to assess the antioxidant capacity of a substance. this parameter reflects the ability of the substance to scavenge free radicals. for example, the trap method (total radical-trapping antioxidant potential) is widely used, which is based on scavenging free radicals that formed by thermolabile azo-compounds [ ] . however, this method does not take the physicochemical characteristics of the antioxidant into account. another strategy is based on the evaluation of the rate constants of the interaction of the antioxidant with the radicals [ , ] . molecules , , of in this work, we studied the antioxidant properties of umifenovir with the modified trap protocol [ ] . while using computer simulation of chemical kinetics, the scheme of antioxidant reactions and the rate constants were proposed. in the endogenous interferon production in % of subjects [ ] . there is some evidence that umifenovir helps the phagocytic action of macrophages [ ] . the presence of not only direct antiviral action, but also other indirect effects was the reason for the investigation of antioxidant activity of umifenovir. it is a free hydroxyl group that provides the antioxidant activity of umifenovir ( figure ). previously, the antioxidant effect of umifenovir to prevent lipid peroxidation was studied in vitro [ ] . the authors proved this substance to be a lipid antioxidant. however, lipid peroxidation does not restrict oxidative stress. the assessment of antioxidant potential in relation to ros-reactions in the aqueous phase is no less important, because non-lipid ros may cause oxidative modification of proteins and dna. more studies are needed to characterize comprehensively the antioxidant properties of umifenovir. the steady-state production of free radicals was provided by the decomposition of , -azobis ( -amidinopropane) dihydrochloride (abap) at • c. luminescence decreases to the background level almost instantly when trolox was added to the abap + luminol mixture. after the certain interval (a latent period), trolox was completely exhausted, and luminescence returns sharply until the initial level (figure a where c is trolox concentration, nm. the conventional approach is based on the quantitative comparison with a reference compound to assess the antioxidant capacity of a substance. this parameter reflects the ability of the substance to scavenge free radicals. for example, the trap method (total radical-trapping antioxidant potential) is widely used, which is based on scavenging free radicals that formed by thermolabile azo-compounds [ ] . however, this method does not take the physicochemical characteristics of the antioxidant into account. another strategy is based on the evaluation of the rate constants of the interaction of the antioxidant with the radicals [ , ] . in this work, we studied the antioxidant properties of umifenovir with the modified trap protocol [ ] . while using computer simulation of chemical kinetics, the scheme of antioxidant reactions and the rate constants were proposed. the steady-state production of free radicals was provided by the decomposition of , ′-azobis ( -amidinopropane) dihydrochloride (abap) at °c . luminescence decreases to the background level almost instantly when trolox was added to the abap + luminol mixture. after the certain interval (a latent period), trolox was completely exhausted, and luminescence returns sharply until the initial level ( figure а ). the area of the luminescence depression reflects the total amount of scavenged radicals. it linearly depended on trolox concentration ( figure b ). the calibration equation is: where c is trolox concentration, nm. for umifenovir, the chemiluminograms were of another type. the depression was incomplete, and after depression, the stationary level was lower that the initial one ( figure a) . therefore, the mechanisms of free-radical scavenging of trolox and umifenovir are different. according to these curves, the scavenging effect of umifenovir involves at least two phases: "fast" and "slow". the area of depression s ("fast" part only) linearly depends on the umifenovir concentration ( figure b ): for umifenovir, the chemiluminograms were of another type. the depression was incomplete, and after depression, the stationary level was lower that the initial one ( figure a) . therefore, the molecules , , of mechanisms of free-radical scavenging of trolox and umifenovir are different. according to these curves, the scavenging effect of umifenovir involves at least two phases: "fast" and "slow". molecules , , x for peer review of where c is umifenovir concentration, nm. (a) (b) the trolox-equivalent antioxidant capacity of a substance can be determined using trap or tar (total antioxidant reactivity) methodology [ ] . the tar index is obtained from the instantaneous decrease in luminescence after adding the antioxidant, while the trap index is calculated from the latent period. however, for umifenovir, both methods are not applicable, as they do not take the "slow" part of the scavenging effect into account. we propose to use the area of total depression s as a measure of the antioxidant capacity of umifenovir ( figure ). the area of depression is defined as an integral of the difference between the blank and analytical plots. it is a sum of the area of "fast" depression s and "slow" depression s . the areas were calculated while using the specially designed features of powergraph . software. calculation of the total antioxidant potential s of umifenovir as a sum of s and s , where s is the area of the "fast" part of chemiluminescence depression, s is the area of the "slow" part of chemiluminescence depression. the plots are obtained for nm that is a maximum concentration of umifenovir in blood after oral administration of mg. antioxidant capacity in μm of trolox, mean ± standard deviation, n = where c is umifenovir concentration, nm. the trolox-equivalent antioxidant capacity of a substance can be determined using trap or tar (total antioxidant reactivity) methodology [ ] . the tar index is obtained from the instantaneous decrease in luminescence after adding the antioxidant, while the trap index is calculated from the latent period. however, for umifenovir, both methods are not applicable, as they do not take the "slow" part of the scavenging effect into account. we propose to use the area of total depression s as a measure of the antioxidant capacity of umifenovir ( figure ). the area of depression is defined as an integral of the difference between the blank and analytical plots. it is a sum of the area of "fast" depression s and "slow" depression s . the areas were calculated while using the specially designed features of powergraph . software. the trolox-equivalent antioxidant capacity of a substance can be determined using trap or tar (total antioxidant reactivity) methodology [ ] . the tar index is obtained from the instantaneous decrease in luminescence after adding the antioxidant, while the trap index is calculated from the latent period. however, for umifenovir, both methods are not applicable, as they do not take the "slow" part of the scavenging effect into account. we propose to use the area of total depression s as a measure of the antioxidant capacity of umifenovir (figure ). the area of depression is defined as an integral of the difference between the blank and analytical plots. it is a sum of the area of "fast" depression s and "slow" depression s . the areas were calculated while using the specially designed features of powergraph . software. figure . calculation of the total antioxidant potential s of umifenovir as a sum of s and s , where s is the area of the "fast" part of chemiluminescence depression, s is the area of the "slow" part of chemiluminescence depression. the plots are obtained for nm that is a maximum concentration of umifenovir in blood after oral administration of mg. antioxidant capacity in µm of trolox, mean ± standard deviation, n = . µm s = . ± . ("fast" capacity) s = . ± . ("slow" capacity) s = . ± . (total capacity) . µm (maximal concentration of umifenovir in blood after taking mg per os) s = . ± . ("fast" capacity) s = . ± . ("slow" capacity) s = . ± . (total capacity) computer simulation was used to estimate the rate constants of antioxidant reaction of umifenovir (the antioxidant activity). for the supposed reactions and given initial concentrations of the reactants, the rate constants were varied to achieve maximum fitting of the calculated and experimental plots. figures a and a show the experimental curves that were obtained for nm trolox and umifenovir used for the computer simulation, respectively. to simulate a blank stationary level of chemiluminescence, a simple model that consists of two reactions was proposed: ( ) the reaction of free radical generation and ( ) the chemiluminescence reaction: where lum is a luminol molecule, r• is a free radical or a reaction product in the excited state with which the antioxidant reacts, p is a stable product of the free-radical reaction (here and below). to simulate the effect of antioxidants, a reaction for trolox was added to the set: where in is an antioxidant (inhibitor). two reactions should be added for umifenovir: ( ') r• + in → p (k in ) ( ) r• + in → p (k in ) figure a shows the calculated data for trolox and umifenovir, figure b shows the combined calculated and the experimental plots for umifenovir. computer simulation was used to estimate the rate constants of antioxidant reaction of umifenovir (the antioxidant activity). for the supposed reactions and given initial concentrations of the reactants, the rate constants were varied to achieve maximum fitting of the calculated and experimental plots. figures a and a show the experimental curves that were obtained for nm trolox and umifenovir used for the computer simulation, respectively. to simulate a blank stationary level of chemiluminescence, a simple model that consists of two reactions was proposed: ) the reaction of free radical generation and ) the chemiluminescence reaction: ) abap + lum → r• (rate constant kr) ) r• → p + light, (klum) where lum is a luminol molecule, r• is a free radical or a reaction product in the excited state with which the antioxidant reacts, p is a stable product of the free-radical reaction (here and below). to simulate the effect of antioxidants, a reaction for trolox was added to the set: ) r• + in → p (kin), where in is an antioxidant (inhibitor). two reactions should be added for umifenovir: ') r• + in → p (kin ) ) r• + in → p (kin ) figure a shows the calculated data for trolox and umifenovir, figure b shows the combined calculated and the experimental plots for umifenovir. for trolox, the rate constant of free radical scavenging were estimated: k in = nm − min − . for umifenovir, the rate constants were estimated: k in = nm − min − , k in = nm − min − . it was previously shown that the effect of any antioxidant on the chemiluminescence kinetics can be described by a single reaction between the antioxidant and a free radical [ ] : r• + in→ p (k in ) in this case, the chemiluminescence kinetics strongly depends on the rate constant k in . depending on the k in value and the chemiluminescence system used, all of the antioxidants can be classified as strong, medium, and weak (figure a ). strong antioxidants are characterized by high rate constants and quench the chemiluminescence sharply and completely for a fixed time interval, which is referred to a "latent period". the complete exhaustion of the antioxidant following with a sharp increase in the luminescence causes the end of the latent period, which returns the previous stationary level. as an example, fig. a presents a simulated chemuliminogram for an antioxidant with k in = nm − min. − . weak antioxidants with low rate constants (for example, k in = nm − min − in figure a ) do not give the latent period. instead, the luminescence decreases slightly. the effect of weak antioxidants is prolonged, as they react very slowly. the effect of medium antioxidants (k in = nm − min. − in figure a ) is between the effects of strong and weak antioxidants. it was previously shown that the effect of any antioxidant on the chemiluminescence kinetics can be described by a single reaction between the antioxidant and a free radical [ ] : r• + in→ p (kin) in this case, the chemiluminescence kinetics strongly depends on the rate constant kin. depending on the kin value and the chemiluminescence system used, all of the antioxidants can be classified as strong, medium, and weak (figure a ). strong antioxidants are characterized by high rate constants and quench the chemiluminescence sharply and completely for a fixed time interval, which is referred to a "latent period". the complete exhaustion of the antioxidant following with a sharp increase in the luminescence causes the end of the latent period, which returns the previous stationary level. as an example, fig. a presents a simulated chemuliminogram for an antioxidant with kin = nm − min. − . weak antioxidants with low rate constants (for example, kin = nm − min − in figure a ) do not give the latent period. instead, the luminescence decreases slightly. the effect of weak antioxidants is prolonged, as they react very slowly. the effect of medium antioxidants (kin = nm − min. − in figure a ) is between the effects of strong and weak antioxidants. the addition of trolox to the chemiluminescence system results in the complete depression of chemiluminescence, which is typical for strong antioxidants (figure a) . a one-stage scheme with kin = nm − min. − appeared to be adequate for the successful computer simulation. trolox can be classified as a strong antioxidant from the kinetics point of view. for umifenovir, the chemiluminescence kinetics is more complicated (figure a) . first, immediately after the addition of the antioxidant, a significant but incomplete decrease in intensity occurs, which is typical for antioxidants of medium strength. second, there is a stage of a slight decrease of the intensity of chemiluminescence with a slow and long rise of the luminescence to a stationary level, which is typical for weak antioxidants. thus, a two-stage model is needed, which consists of two consequent antioxidant reactions with different rate constants. figure b gives the examples of simulated plots for abstract antioxidants. the computer simulation confirmed the twostage mechanism of the antioxidant effect of umifenovir. the found rate constants nm − min. − and nm − min. − are characteristic for medium and weak antioxidant. hence, from the kinetics point of view, umifenovir can be classified as a medium antioxidant. the difference between the mechanisms of action for trolox and umifenovir makes it impossible to trap and tar methodologies for the quantification of antioxidant capacity of umifenovir. as a measure of the addition of trolox to the chemiluminescence system results in the complete depression of chemiluminescence, which is typical for strong antioxidants (figure a) . a one-stage scheme with k in = nm − min. − appeared to be adequate for the successful computer simulation. trolox can be classified as a strong antioxidant from the kinetics point of view. for umifenovir, the chemiluminescence kinetics is more complicated (figure a) . first, immediately after the addition of the antioxidant, a significant but incomplete decrease in intensity occurs, which is typical for antioxidants of medium strength. second, there is a stage of a slight decrease of the intensity of chemiluminescence with a slow and long rise of the luminescence to a stationary level, which is typical for weak antioxidants. thus, a two-stage model is needed, which consists of two consequent antioxidant reactions with different rate constants. figure b gives the examples of simulated plots for abstract antioxidants. the computer simulation confirmed the two-stage mechanism of the antioxidant effect of umifenovir. the found rate constants nm − min. − and nm − min. − are characteristic for medium and weak antioxidant. hence, from the kinetics point of view, umifenovir can be classified as a medium antioxidant. the difference between the mechanisms of action for trolox and umifenovir makes it impossible to trap and tar methodologies for the quantification of antioxidant capacity of umifenovir. as a measure of antioxidant capacity, we propose using the total area of depression of the chemiluminescence, including "fast" and "slow" parts of the chemiluminograms. for . µm of umifenovir, which corresponds to the maximal concentration in blood after oral administration of mg, the trolox-equivalent antioxidant capacity was as high as . µm. hence, from the thermodynamic point of view, umifenovir is a more effective antioxidant than trolox in . / . = . times. note that the effect of umifenovir lasts for about an hour (figure ) , while the effect of . µm trolox lasts for about minutes. thus, umifenovir acts five-fold longer than trolox. let us compare our results to the data that were obtained by vasil'eva at al. [ ] . using a chemiluminescence system consisting of phospholipid liposomes, feso , and coumarin c in tris buffer solution, they determined the antioxidant effect of umifenovir on lipid peroxidation as a concentration of % depression (c %) of amplitude of slow flash. this value ( µm) was two orders of magnitude lower than c % of α-tocopherol ( . µm). the authors attribute this fact to a poor solubility of umifenovir in lipids. note that, because of the comprehensive mechanism of lipid peroxidation, the amplitude of slow flash correlates with the antioxidant capacity in a complex way and it can only be used for semi-quantitative assessment. measurements of the amplitude of the fast flash would be more adequate, but it was not possible with the chemiluminometer used by the authors. based on the latent time of the slow flash, the authors proposed that the antioxidant effect of umifenovir was due to the reaction with free radicals, but not with iron. however, these conclusions require confirmation by computer simulation, which may be the aim of the next study. in this study, we used , '-azobis( -amidinopropane)hydrochloride, a well-known water soluble radical initiator, which has been successfully applied as an initiator of lipid peroxidation [ ] . this substance forms the aliphatic carbon-centered radical, which is transformed into more reactive peroxyl radical in the presence of oxygen. it is considered to be a good model substance for ozone and uv-b exposure [ ] . abap, as a radical generator, offers a useful tool for studying oxidative stress in biochemical and biological models and antioxidant properties of substances due to a simple chemical mechanism of the degradation and possibility to work at the physiological temperature. however, such studies can only be performed in aqueous phase, not in lipids. summing up, umifenovir has potent antioxidant effect in relation to organic radicals in the aqueous phase due to two-stage mechanism. this phenomenon can make a serious contribution to the compensation of oxidative stress that is caused by a viral disease and to the therapeutic effect of the drug. further studies on the therapeutic effect of umifenovir on animal influenza models in comparison with its antioxidant potential will be of special interest. the enhanced chemiluminescence protocol quantified the antioxidant activity of umifenovir. the chemiluminescent system consisted of a source of free radicals , '-azobis ( -amidinopropane) dihydrochloride (abap, sigma, saint louis, mo, usa) and a chemiluminescent probe luminol (sigma). the method was described elsewhere [ ] . a luminol solution of mm (sigma, saint louis, mo, usa) and abap solution of mmol/l was prepared by dissolving the weighed samples in phosphate buffer solution ( mm kh po , ph . , sigma, saint louis, mo, usa). the total volume in a cuvette was . ml. a mixture of abap and luminol (final concentrations were . mm and µm, respectively) was added to a buffer solution (ph . ) at • c. the chemiluminescence was recorded until a stationary level had been achieved, and then an aliquot of the antioxidant solution of trolox or umifenovir was added. the registration was performed until the new steady-state level was achieved (figure a) . as a reference compound, trolox (sigma, saint louis, mo, usa), a water-soluble analogue of vitamin e, was used. a stock solution of mm trolox was prepared in phosphate buffer solution. working solutions were prepared by the dilution of the stock solution with the buffer solution. umifenovir (erregierre s.p.a., san paolo d'argon, bg, italy, m = . g/mol) was dissolved in acetone ( . % panreac, castellar del vallès, barcelona, spain). the measurements were carried out with a -channel lum- chemiluminometer (disoft, moscow, russia). the chemiluminometer provides the detection of visible light within a range from to nm. no light filters were used in our experiments. signal processing were performed with powergraph . professional software (disoft, moscow, russia). the statistical processing of the data was performed with statistica v. . software (statsoft inc., tulsa, ok, usa). in contrast to trap and tar methodologies, in determining the total antioxidant potential of umifenovir, we calculated the total area of depression s as a sum of s and s , where s is the area of depression of the "fast" phase of scavenging free radicals, s is the area of depression of the "slow" phase of scavenging free radicals (the shaded area in figure ). the areas of depression were calculated with powergraph . . professional software. the computer simulation was carried out with the specially designed computer program "kinetic analyzer" (d.yu. izmailov). for a set of the predetermined reactions and the initial concentrations of the reactants, the rate constants were selected providing the maximal fitting of experimental and calculated plots. as a criterion for maximal fitting, the minimum sum of squared residuals was calculated with the originpro software (originlab, northampton, ma, usa). the role of oxidative stress in influenza virus infection lr-mediated antiviral innate immunity requires oxidative phosphorylation activity oxidative and nitrosative stress affect antiviral, inflammatory and apoptotic signaling of cultured thymocytes evaluation of the role of the antioxidant silymarin in modulating the in vivo genotoxicity of the antiviral drug ribavirin in mice identification of an antioxidant small-molecule with broad-spectrum antiviral activity an evaluation of the antioxidant and antiviral action of extracts of rosemary and provencal herbs antioxidant and antiviral activities of euphorbia thymifolia l antiviral and antioxidant activity of flavonoids and proanthocyanidins from crataegus sinaica in vitro antiviral, anti-inflammatory, and antioxidant activities of the ethanol extract of mentha piperita l antioxidant impact on specific antiviral activity of human recombinant interferon alpha- b with respect to herpes simplex in cell culture antiviral and antioxidant properties of echinochrome a antiviral activity of silymarin against mayaro virus and protective effect in virus-induced oxidative stress oxygen radicals in influenza-induced pathogenesis and treatment with pyran polymer-conjugated sod the pathology of influenza virus infection pharmacokinetic properties and bioequivalence of two formulations of arbidol: an open-label, single-dose, randomized-sequence, two-period crossover study in healthy chinese male volunteers characteristics of arbidol-resistant mutants of influenza virus: implications for the mechanism of anti-influenza action of arbidol antiviral activity of arbidol, a broad-spectrum drug for use against respiratory viruses, varies according to test conditions clinical efficacy of arbidol (umifenovir) in the therapy of influenza in adults: preliminary results of the multicenter double-blind randomized placebo-controlled study arbitr study of the antiviral activity of domestic anti-influenza chemotherapy in cell culture and animal models umifenovir (arbidol) efficacy in experimental mixed viral and bacterial pneumonia of mice arbidol (umifenovir): a broad-spectrum antiviral drug that inhibits medically important arthropod-borne flaviviruses umifenovir susceptibility monitoring and characterization of influenza viruses isolated during arbitr clinical study antiviral activity of arbidol and its derivatives against the pathogen of severe acute respiratory syndrome in the cell cultures clinical efficacy of umifenovir in influenza and arvi (study arbitr) pharmacoepidemiological study of influenza and other acute respiratory viral infections in risk groups the antiviral activity of arbidol hydrochloride against herpes simplex virus type ii (hsv- ) in a mouse model of vaginitis mechanisms of arbidole's immunomodulating action arbidol for preventing and treating influenza in adults and children antioxidant properties of arbidol and its structural analogs evaluation of total antioxidant potential (trap) and total antioxidant reactivity from luminol-enhanced chemiluminescence measurements. free radic photochemiluminescent study of the antioxidant activity in biological systems. mathematical modeling kinetic chemiluminescence as a method for study of free radical reactions determination of antioxidants by sensitized chemilumine scence using , '_azo_bis( _amidinopropane) determination of antioxidant activity by measuring the chemiluminescence kinetics. fotobiologiya i fotomeditsina effect of phytyl side chain of vitamin e on its antioxidant activity plant defense metabolism is increased by the free radical-generating compound aaph. free radic in this section you can acknowledge any support given which is not covered by the author contribution or funding sections. this may include administrative and technical support, or donations in kind (e.g., materials used for experiments). the authors declare no conflict of interest. key: cord- - g uo rd authors: tomani, jean claude didelot; kagisha, vedaste; tchinda, alembert tiabou; jansen, olivia; ledoux, allison; vanhamme, luc; frederich, michel; muganga, raymond; souopgui, jacob title: the inhibition of nlrp inflammasome and il- production by hibiscus noldeae baker f. derived constituents provides a link to its anti-inflammatory therapeutic potentials date: - - journal: molecules doi: . /molecules sha: doc_id: cord_uid: g uo rd the activation of nod-, lrr-, and pyrin domain-containing protein (nlrp ) inflammasome and/or its components is associated with the physio-pathogenesis of many respiratory diseases including asthma, copd (chronic obstructive pulmonary disease), sars cov- (severe acute respiratory syndrome coronavirus ), and in several autoimmune diseases. hibiscus noldeae baker f. has been widely reported to be traditionally used in the treatment of different ailments, some of which are of inflammatory background such as asthma, wounds, headache, etc. however, the claims have not been supported by evidence at the molecular and functional levels. here, we report on the bio-guided fractionation of h. noldeae and assessment of the inhibitory properties of some fractions and purified compounds on nlrp inflammasome and interleukin (il- ). the activation of the nlrp inflammasome was determined by detecting the activity of caspase- and the production of interleukin β (il- β) in lipopolysaccharide (lps) and atp-stimulated tamm-horsfall protein (thp- ) macrophages, while the production of il- was studied in lps-stimulated raw . mouse macrophages. it was observed that hexane and ethyl acetate fractions of the crude extract of the aerial parts of h. noldeae, as well as caffeic acid, isoquercetin, and er . and er . fractions revealed significant inhibitory effects on caspase- activities, and on il- β and il- production. the er . and er . fractions downregulated the production of il- β and il- , in a similar range as the caspase- inhibitor ac-yvad-cho and the drug dexamethasone, both used as controls, respectively. overall, our work does provide the very first scientific based evidence for hibiscus noldeae anti-inflammatory effects and widespread use by traditional healers in rwanda for a variety of ailments. respiratory diseases, with asthma being the most important, are classified among the major categories of non-communicable diseases and their prevalence accounted for more than billion patients worldwide in [ , ] . asthma is a chronic inflammatory disease resulting from the malfunction of the interaction between the airways and the immune cells [ , ] . inflammation is considered to be a physiological defense mechanism which helps the body to protect itself from microbial infections, burn, toxic chemicals, allergens, or other harmful stimuli. depending on the number or the degree of events and mediators linked to that reaction, inflammatory reaction can trigger, maintain, or aggravate many diseases [ ] . sometimes the delicate balance between immune-mediated clearance and noxious stimuli is troubled and this correlates with inadvertent self-harm from chronic inflammation. as a result, the immune system inappropriately leads to inflammation against its own cells, resulting in different chronic inflammatory diseases including respiratory diseases such as asthma and chronic obstructive pulmonary disease (copd) [ ] . recently, the complicated form of sars cov- (severe acute respiratory syndrome coronavirus ) viral pneumonia outbreak was reported to be associated with a plethora of cytokines wreaking havoc throughout the body often vividly referred to as "cytokine storm", which triggers a violent inflammatory immune response that contributes to respiratory problems, multiple organ failure, and finally death in severe cases of sars-cov- infection [ ] [ ] [ ] [ ] . several studies have reported the role of different inflammatory cells and mediators in the pathogenesis of asthma where the volume and nature of involved inflammatory mediators determine the severity and exacerbations of the disease. depending on those inflammatory patterns, there are two main subtypes of asthma, the eosinophilic (diagnosed by the expression of th -derived cytokines) and the neutrophilic (diagnosed by the expression of th -derived cytokines and chemokines) [ ] . the latter asthma subset is characterized by a high proportion of neutrophils recruited by inflammatory cytokines like interleukin β (il- β), interleukin (il- ), and tumor necrosis factor alpha (tnf-α). it is well known that inflammasomes, multiprotein cytoplasmic complexes activating inflammatory responses, are involved in the upregulation of these cytokines, particularly the il- β through the activation of caspase- [ , ] . among these inflammasomes, nod-, lrr-, and pyrin domain-containing protein (nlrp ) is the most widely studied and its properties and functions are the best characterized [ ] . in fact, in response to a microbial infection or injury, the nlrp inflammasome is activated and involved in the host protection by inducing immune responses to clear injury or microbial invasion [ , ] . the activation of nlrp inflammasome requires two distinct signals. the first is a pro-inflammatory priming step, mediated by toll-like receptors (tlr), that induces the upregulation of the nf-κb transcription factor and promotes the expression of pro-il- β and the nlrp inflammasome component. the second signal is an activation step that induces the assembly of the nlrp / apoptosis-associated speck-like protein containing a caspase recruitment domain (asc)/pro-caspase- complex, ending up in the proximity auto-cleavage and maturation of caspase- which, in turn, induces a caspase- -mediated maturation of pro-il- β and release of active il- β [ ] . several pieces of evidence from research suggest that inflammasomes and/or their components and effectors are associated with the physio-pathogenesis of asthma and other respiratory diseases including copd, sars cov- , etc. [ ] [ ] [ ] [ ] . in fact, it has been reported that the excessive release of active il- β and asc (apoptosis-associated speck-like protein containing a caspase recruitment domain) specks leads to the induction of neutrophil-dominated inflammatory responses in the airways that contributes to the pathogenesis of copd and neutrophilic asthma [ , ] . the neutrophilic asthma is the most severe and has been reported to be refractory to the corticoid-based treatment [ ] . despite this resistance, the use of corticosteroids remains the basic treatment for several respiratory diseases involving cytokine storms. a recent clinical trial report revealed that the treatment with dexamethasone significantly alleviated covid- disease severity of hospitalized patients who were under invasive mechanical ventilation or oxygen [ ] . regarding the neutrophilic asthma, drugs that alleviate disease severity include specific monoclonal antibodies against key mediators of the inflammatory pathway [ ] but the associated treatment cost is not affordable for low and middle income countries (lmics). hence, urgent efforts are needed to identify new, effective, affordable, and safe anti-inflammatory drugs. a reliable therapeutic source which remains poorly explored is the traditional pharmacopeia. previously, our investigations on the anti-asthmatic activities of herbal recipes from rwandese traditional healers revealed a candidate recipe with significant inhibitory properties on the activation of caspase- using caspase-glo inflammasome assays [ ] . hibiscus noldeae, from the hibiscus genus in the malvaceae family, was one of the plants composing the recipe. plants from the same hibiscus genus, including h. acetosella, h. sabdariffa, and h. vitifolius have been reported to have anti-inflammatory potentials and some of the responsible molecules, such as gossypin, caffeoylhydroxycitric acid, delphinidin- -sambubioside, and neochlorogenic acid have been isolated [ ] [ ] [ ] . in contrast, no such studies has been reported on h. noldeae, although used by traditional healers in the treatment of asthma, urogenital infections, postpartum abdominal pain, swelling of the legs, cleaning of babies, facilitation of delivery, sprains, headache, wounds, women infertility, abortion prevention, etc. [ ] . in this study, we report on the bio-guided fractionation of h. noldeae and assessment of the inhibitory properties of some fractions and purified compounds on nlrp inflammasome and il- . the activation of the nlrp inflammasome was determined by detecting the activity of caspase- and the production of il- β in lipopolysaccharide (lps)-and atp-stimulated thp- macrophages, while the production of il- was studied in lps-stimulated raw . mouse macrophages, a well-known standard method. it was observed that hexane and ethyl acetate fractions of the aerial parts of h. noldeae, as well as caffeic acid, isoquercetin, and er . and er . fractions revealed the highest inhibitory effects on caspase- activities, and on il- β and il- production. the er . and er . , our fractions of most interest, downregulated the production of il- β and il- , in a similar range as the caspase- inhibitor ac-yvad-cho and the drug dexamethasone all used as controls, respectively. overall, our findings pave the way for the first time on the scientific based evidence on hibiscus noldeae activities underlying its potential role in the treatment of inflammatory diseases and asthma in rwanda. hibiscus noldeae (h. noldeae) has been studied by virtue of its attribute as one of the medicinal plants used by the traditional healers in rwanda to manufacture their remedies. in the context of asthma treatment, the recipe, containing different plants, that revealed the best anti-inflammatory activity [ ] comprised h. noldeae in major proportion ( %). we therefore hypothesized that this medicinal plant may harbor the therapeutic constituents. a bio-guided fractionation approach was employed and as shown in figure , a complex chemical profile for hexane and ethylacetate fractions was observed in the high performance liquid chromatography with diode-array detection (hplc-dad) analysis, indicating the presence of different major substances of different polarities. many peaks eluted between and min, a fact that demonstrates the presence of medium polarity molecules, most of which are major compounds. the transposition of the analytical method to the preparative hplc allowed for the isolation of five of the seven major compounds from ethylacetate and hexane fractions of h. noldeae. three of the five isolated compounds were pure and were identified by chromatographic and/or spectroscopic comparison with data from literature. this led to the elucidation of one flavone glycoside, namely quercetin -o-glucopyranoside, along with two polyphenols, caffeic acid and caffeoyl-hydrocitric acid (supplementary table s and figures s -s ). noldeae. an in-house hplc method used to screen the major components of crude extract was used. analytical separation was carried out on a hypersil ods ® rp column ( × . µm; particle size µm; thermofisher scientific, waltham, ma, usa). all samples were dissolved in methanol hplc-grade, filtered through a . µm pore size filter membrane and analyzed on an agilent hplc machine. samples were eluted with a nonlinear gradient method with acetonitrile and . % trifluoroacetic acid in ultra-pure water. the column temperature was maintained at °c. then µl of each sample were injected into the hplc-uv/dad (diode-array detection) system and the analysis, performed at a flow rate of . ml/min, was monitored at , , , and nm. prior to the investigation of anti-inflammatory properties of the compounds of interest, their effect on the cell viability on both thp- monocytes and derived macrophages was first determined. cells were treated with different concentrations ( , , , and µg/ml) for h, and the cell viability was determined using celltiter-glo luminescent cell viability assay kit (promega co., madison, wi, usa). up to µg/ml, all tested compounds did not exhibit cytotoxicity to both thp- and raw . cell lines ( figures s and s ). our anti-inflammatory activities were therefore determined within this range. to investigate whether the isolated compounds and fractions of interest possessed potential nlrp inflammasome inhibitory effects, their influence on the nlrp inflammasome activation, in lps and atp-stimulated thp- -derived macrophages was assessed. the levels of caspase- activities in the culture media were determined using the caspase-glo ® inflammasome assay (promega co., madison, wi, usa). as shown in figure below, it was observed as expected that lps and atp treatment significantly (*** p < . ) increased caspase- activities in the cell culture supernatant when compared with the untreated cells. however, when cells were pretreated with the isolated compounds and fractions of interest in graded concentrations ranging from to µg/ml, the caspase- activities were reduced by at least %. the tested compounds revealed their maximal inhibitory activities at µg/ml, except for the er . fraction that showed a dose-dependent inhibition of the caspase- activation (figure ) . noldeae. an in-house hplc method used to screen the major components of crude extract was used. analytical separation was carried out on a hypersil ods ® rp column ( × . µm; particle size µm; thermofisher scientific, waltham, ma, usa). all samples were dissolved in methanol hplc-grade, filtered through a . µm pore size filter membrane and analyzed on an agilent hplc machine. samples were eluted with a nonlinear gradient method with acetonitrile and . % trifluoroacetic acid in ultra-pure water. the column temperature was maintained at • c. then µl of each sample were injected into the hplc-uv/dad (diode-array detection) system and the analysis, performed at a flow rate of . ml/min, was monitored at , , , and nm. prior to the investigation of anti-inflammatory properties of the compounds of interest, their effect on the cell viability on both thp- monocytes and derived macrophages was first determined. cells were treated with different concentrations ( , , , and µg/ml) for h, and the cell viability was determined using celltiter-glo luminescent cell viability assay kit (promega co., madison, wi, usa). up to µg/ml, all tested compounds did not exhibit cytotoxicity to both thp- and raw . cell lines ( figures s and s ). our anti-inflammatory activities were therefore determined within this range. to investigate whether the isolated compounds and fractions of interest possessed potential nlrp inflammasome inhibitory effects, their influence on the nlrp inflammasome activation, in lps and atp-stimulated thp- -derived macrophages was assessed. the levels of caspase- activities in the culture media were determined using the caspase-glo ® inflammasome assay (promega co., madison, wi, usa). as shown in figure below, it was observed as expected that lps and atp treatment significantly (*** p < . ) increased caspase- activities in the cell culture supernatant when compared with the untreated cells. however, when cells were pretreated with the isolated compounds and fractions of interest in graded concentrations ranging from to µg/ml, the caspase- activities were reduced by at least %. the tested compounds revealed their maximal inhibitory activities at µg/ml, except for the er . fraction that showed a dose-dependent inhibition of the caspase- activation (figure ). then, µl of the culture supernatant were used to determine the caspase- activity using caspase-glo inflammasome assay (promega co., madison, wi, usa). the results are presented as the mean ± sd from independent experiments (n = ), each done in triplicate. a one-way anova, followed by benforroni's multiple comparison test, was used to compare the sample groups. *** means caspase- fold activation changes with a p < . . next, the production of il- β was investigated using the human il- β (enzyme linked immunosorbent assay) elisa kit (invitrogen/thermofisher, waltham, ma, usa). as controls, two distinct reference inhibitors, namely ac-yvad-cho (yvad) which specifically acts on caspase and dexamethasone (dex) which downregulates the nf-κb pathway were used. as results, it was observed as expected that lps and atp treatment significantly (*** p < . ) increased the production of il- β in the cell culture supernatant when compared with the untreated cells ( figure a ). when cells were first treated with h. noldeae constituents or the controls, the production il- β was significantly inhibited. reductions of more than % could be revealed in some cases ( figure a ). moreover, it was observed that, at equivalent dose of µg/ml, er . fraction and dexamethasone showed similar inhibitory effects, while at the same concentration other tested constituents of h. noldeae revealed inhibitory activities comparable to the caspase reference inhibitor yvad. isoquercetin was observed to have the lowest inhibitory effect on il- β production ( figure a ). cells were then stimulated by atp ( mm) for additional h. the supernatants were harvested and kept at − • c before analysis. then, µl of the culture supernatant were used to determine the caspase- activity using caspase-glo inflammasome assay (promega co., madison, wi, usa). the results are presented as the mean ± sd from independent experiments (n = ), each done in triplicate. a one-way anova, followed by benforroni's multiple comparison test, was used to compare the sample groups. *** means caspase- fold activation changes with a p < . . next, the production of il- β was investigated using the human il- β (enzyme linked immunosorbent assay) elisa kit (invitrogen/thermofisher, waltham, ma, usa). as controls, two distinct reference inhibitors, namely ac-yvad-cho (yvad) which specifically acts on caspase and dexamethasone (dex) which downregulates the nf-κb pathway were used. as results, it was observed as expected that lps and atp treatment significantly (*** p < . ) increased the production of il- β in the cell culture supernatant when compared with the untreated cells ( figure a ). when cells were first treated with h. noldeae constituents or the controls, the production il- β was significantly inhibited. reductions of more than % could be revealed in some cases ( figure a ). moreover, it was observed that, at equivalent dose of µg/ml, er . fraction and dexamethasone showed similar inhibitory effects, while at the same concentration other tested constituents of h. noldeae revealed inhibitory activities comparable to the caspase reference inhibitor yvad. isoquercetin was observed to have the lowest inhibitory effect on il- β production ( figure a) . concentrations of µm yvad and µg/ml dex were used as controls. cells were then stimulated by atp ( mm) for additional h. supernatants were harvested and kept at − °c before analysis. for the elisa analysis, samples were diluted times in the assay diluent. the concentration of il- β in the culture supernatant was measured using a human il- β enzyme linked immunosorbent assay (elisa) kit (invitrogen/thermofisher, waltham, ma, usa) according to the manufacturer's instructions. for each experiment, the amount of il- β produced without any pretreatment was considered as % and the remaining treatments were calculated as (treatment/lps) × . the results are presented as the mean ± sd of a triplicate from a representative of three independent experiments (n = ). a one-way anova, followed by benforroni's multiple comparison test, was used to compare the sample groups. *** stands for p < . . (b) immunoblotting of cell lysates by anti-proil- β (cell signaling technology, danvers, ma, usa) or anti-pro caspase- (abcam, cambridge, uk) was done twice according to the manufacturer's instructions. membranes were then stripped, reprobed with anti-β-actin antibodies and exposed again to detect the internal β-actin standard. it is well-known that the activation of the nlrp inflammasome induces the activation of caspase- that promotes the production of gasdermin d. the n-terminal domain of gasdermin d oligomerizes to form cytolytic pores and leads to the programmed cell death called "pyroptosis" [ ] . therefore, we investigated the effect of our compounds and fractions of interest on inflammasome activation-induced cell death by measuring the release of lactate dehydrogenase (ldh) in the culture concentrations of µm yvad and µg/ml dex were used as controls. cells were then stimulated by atp ( mm) for additional h. supernatants were harvested and kept at − • c before analysis. for the elisa analysis, samples were diluted times in the assay diluent. the concentration of il- β in the culture supernatant was measured using a human il- β enzyme linked immunosorbent assay (elisa) kit (invitrogen/thermofisher, waltham, ma, usa) according to the manufacturer's instructions. for each experiment, the amount of il- β produced without any pretreatment was considered as % and the remaining treatments were calculated as (treatment/lps) × . the results are presented as the mean ± sd of a triplicate from a representative of three independent experiments (n = ). a one-way anova, followed by benforroni's multiple comparison test, was used to compare the sample groups. *** stands for p < . . (b) immunoblotting of cell lysates by anti-proil- β (cell signaling technology, danvers, ma, usa) or anti-pro caspase- (abcam, cambridge, uk) was done twice according to the manufacturer's instructions. membranes were then stripped, reprobed with anti-β-actin antibodies and exposed again to detect the internal β-actin standard. it is well-known that the activation of the nlrp inflammasome induces the activation of caspase- that promotes the production of gasdermin d. the n-terminal domain of gasdermin d oligomerizes to form cytolytic pores and leads to the programmed cell death called "pyroptosis" [ ] . therefore, we investigated the effect of our compounds and fractions of interest on inflammasome activation-induced cell death by measuring the release of lactate dehydrogenase (ldh) in the culture supernatants. as expected, the pretreatment of cells by our test compounds significantly inhibited atp-induced pyroptosis ( figure s ). nlrp inflammasome-associated il- β production is a two-step process: a priming signal that promotes pro-il- β and nlrp synthesis and a secondary signal that induces the inflammasome assembly and the subsequent caspase- activation [ ] . we investigated the effect of the crude extract and fractions of interest on the expression of pro-il- β and pro-caspase- by western blot (wb). we observed as shown in figure b that cells pretreatment with er . and er . fractions led to the reduced pro-caspase- amount marked by the faint wb-signals. this suggest that our fractions of interest inhibited the expression of this protein by impacting the first priming step of the nlrp inflammasome activation or induced its degradation by a mechanism to be clarified. moreover, the pro-il- β production appeared induced as marked by the width of the wb-signals ( figure b ). knowing the critical role of caspase- in the inflammasome activation, these observations suggest that the reduced expression of the pro-caspase by both er . and er . fractions may in fact be acting to inhibit the upregulation of caspase- , hence block the pro-il- β cleavage. in addition, treatment with dexamethasone, the crude extract and the etoac fraction revealed reduced pro-il- β wb-signals. while the effect of dexamethasone is associated with the inhibition of the nf-κb activities leading to the repression of pro-il- b production, the effects of the crude extract and etoac fraction could results from the dilution of the er . and er . fractions or likely their effects on nf-kb activities by their constituents to be identified. interleukin (il- ) like il- β is a biomarker of inflammasome activation. hence, the effect of the fractions and purified compounds from h. noldeae on the production of il- in raw . macrophages supernatants by elisa was investigated. dexamethasone at concentration of µg/ml was used as control. treatment of raw . cells with lps alone resulted in a significant increase in this cytokine production as compared to the control group. as expected, a pre-treatment of cells with dexamethasone reduced about % of the production of il- . also, it was observed that the levels of il- production were significantly (*** p < . ) decreased upon pre-treatment of cells with h. noldeae constitutuents. moreover, while a saturation of inhibitory effects was already observed at either or µg/ml with the er . fraction or with the crude extract and the etoac fraction, a dose-dependent inhibition of il- production was revealed by the caffeic acid and er . fraction pre-treatment. at µg/ml, the inhibitory effect of er . fraction was similar to that of dexamethasone ( figure ). inhibition of il- production by h. noldeae constituents. cells were pre-treated with crude extract (a), etoac fraction (b) and partially purified er . (d) and er . (e) fractions, and caffeic acid (c) for h and stimulated by lps ( µg/ml) for h. dexamethasone (dex) at µg/ml was used as a control. cell-free supernatants were harvested and stored at − °c until analysis. the concentration of il- in the culture supernatant was measured using il- elisa kit (bd biosciences) according to the manufacturer's instructions. for each experiment, the amount of il- produced was considered as % and the remaining treatments was calculated as (treatment/lps) × . the results are presented as the mean ± sd from a triplicate of the representative experiment. a one-way anova, followed by benforroni's multiple comparison test, was used to compare the sample groups. *** stands for a p < . . an important body of studies have proven the associations between neutrophilic asthma and increased il- β responses induced by the excessive nlrp inflammasome activation. this raises the possibility of targeting different inflammasome components as substantial therapeutic targets in neutrophil-dominated forms of severe, steroid-refractory asthma [ ] . the analysis of inflammasome formation is mostly investigated through the monitoring of downstream processes and outcomes, the presence of cleaved caspase- enzyme being the most widely used method, as well as the caspase- activity on substrates such as pro-il- β, pro-il- , and pro-gasdermin-d [ ] . in view of determining the inhibitory properties of the h. noldeae constituents of interest on nlrp inflammasome, their effect on either caspase- activation using the caspase glo assays (promega, madison, wi, usa) and/or il- β production as hallmarks of the nlrp inflammasome was evaluated. in fact, it is well known that the inflammasome activation induces a cascade of events including the recruitment of pro-caspase- and its cleavage into a corresponding active caspase- enzyme that also cleaves pro-il- β into its mature form, il- β [ ] . in our previous study, among the plant recipes evaluated, r sz was the most potent in inhibiting caspase- activity [ ] . hibiscus noldeae, one of the plant's components of the recipe, has been cited by three different traditional healers during our investigation, indicating its possible importance in the treatment of asthma. in addition, few studies reported on the traditional use of the . inhibition of il- production by h. noldeae constituents. cells were pre-treated with crude extract (a), etoac fraction (b) and partially purified er . (d) and er . (e) fractions, and caffeic acid (c) for h and stimulated by lps ( µg/ml) for h. dexamethasone (dex) at µg/ml was used as a control. cell-free supernatants were harvested and stored at − • c until analysis. the concentration of il- in the culture supernatant was measured using il- elisa kit (bd biosciences) according to the manufacturer's instructions. for each experiment, the amount of il- produced was considered as % and the remaining treatments was calculated as (treatment/lps) × . the results are presented as the mean ± sd from a triplicate of the representative experiment. a one-way anova, followed by benforroni's multiple comparison test, was used to compare the sample groups. *** stands for a p < . . an important body of studies have proven the associations between neutrophilic asthma and increased il- β responses induced by the excessive nlrp inflammasome activation. this raises the possibility of targeting different inflammasome components as substantial therapeutic targets in neutrophil-dominated forms of severe, steroid-refractory asthma [ ] . the analysis of inflammasome formation is mostly investigated through the monitoring of downstream processes and outcomes, the presence of cleaved caspase- enzyme being the most widely used method, as well as the caspase- activity on substrates such as pro-il- β, pro-il- , and pro-gasdermin-d [ ] . in view of determining the inhibitory properties of the h. noldeae constituents of interest on nlrp inflammasome, their effect on either caspase- activation using the caspase glo assays (promega, madison, wi, usa) and/or il- β production as hallmarks of the nlrp inflammasome was evaluated. in fact, it is well known that the inflammasome activation induces a cascade of events including the recruitment of pro-caspase- and its cleavage into a corresponding active caspase- enzyme that also cleaves pro-il- β into its mature form, il- β [ ] . in our previous study, among the plant recipes evaluated, r sz was the most potent in inhibiting caspase- activity [ ] . hibiscus noldeae, one of the plant's components of the recipe, has been cited by three different traditional healers during our investigation, indicating its possible importance in the treatment of asthma. in addition, few studies reported on the traditional use of the plant in the treatment of different ailments, some of which are of inflammatory background such as asthma, wounds, headache, etc. [ , [ ] [ ] [ ] . an important question was then to determine whether h. noldeae, would have inhibitory properties on inflammasome or on il- production, a well-known downstream target of il- β. a bioguided fractionation approach to address this question was employed. many of the isolated compounds ( figure and table s ) were polyphenols, secondary plant metabolites proven to exert various biological properties including anti-bacterial, anti-inflammatory and anti-allergic activities [ ] [ ] [ ] . as far as inflammation is concerned, cell death could also lead to the activation of the inflammatory pathway in a manner similar to the inflammasome activation [ , ] . therefore, plant crude extracts, fractions and purified compouds were first tested for eventual cytotoxicity allowing to select the range of working sample concentrations in such a way that their pharmacological effects could not be biased. within a plant extract, there can be hundreds of molecules, some of which are active individually or act synergistically against a given disease. in most of the cases, major compounds are responsible of the activity of the extract though it is not always the case [ , ] . in this study, an important question was to determine whether the inhibition of nlrp inflammasome resulted from a single compound or from a combined effect of different compounds. we opted first to investigate the effect of cell pre-treatment with our various samples on the caspase- activities and observed an average reduction of about one-third which formed a plateau at the tested concentration ranges as shown in (figure ). such reduction suggested the presence of an inhibitor of caspase- in the plant extract. indeed, caffeic acid, purified from h. noldeae in this work, was reported to interact with and inhibits caspase- and asc activation [ ] . the plateau could be explained by the fact that a saturated quantity of caffeic acid is already in the lowest sample concentration used ( µg/ml). the findings that our plant products of interest had inhibitory effects on the caspase- activities prompted us to assess the production of il- β in the supernatant from the different samples. consistent with the inhibition of the caspase- activities all the tested crude extract, fractions and purified constituents of h. noldeae revealed significant inhibition of il- β production ( figure a ). to ensure that h. noldeae constituents acted on the inflammasome activation pathway, we further investigated their effects on pyroptosis. in fact, the activation of the nlrp inflammasome induces the activation of caspase- that promotes the production of gasdermin d and the latter is associated to the programmed cell death called "pyroptosis" [ ] . in contrast, compared to the production of il- β, the effect of dexamethasone was less than that of er . and er . fractions ( figure s ). this may be explained by the fact that dexamethasone is able to act at the transcriptional level or to selectively induce the degradation of il- β transcripts after their production [ ] . thus, the additional effect of these both mechanisms justifies its enhanced inhibition of il- β production in the supernatant. however, at the highest concentration tested ( µg/ml) an unexplained ease of the inhibitory effect was observed with both fractions. such unexpected reactions deserve further investigation during the characterization of er . and er . fractions. nlrp inflammasome-associated il- β production is a two-step process. our findings on both pyroptosis assays ( figure s ) and il- β production ( figure a) suggest that h. noldeae derived products interfere with the nlrp inflammasome signaling, to block cell death (pyroptosis) and il- β production. however, the western blot results showed that er . and er . fractions repressed the expression of pro-caspase- ( figure b ). capsase- is a critical component of the inflammasome and the reduced expression of this enzyme by fractions from h. noldeae indicates that er . and er . may in fact be acting to inhibit signal (priming step) of inflammasome activation by specifically blocking upregulation of caspase- . in our future research, the mode of action of the molecules contained in these fractions will be elucidated. several studies have reported in vivo anti-inflammatory properties of caffeic acid and isoquercetin as well as several corresponding derivatives [ , , ] . in this study, as suggested by our results, several polyphenolic compounds detected in the plant extracts of h. noldeae likely contribute to the inhibitory property of the plant on the inflammasome expression. however, the activity of the plant remains inferior to the activity of purified caffeic acid as well as the concentrated fractions er . and er . . this suggests that the fractionation of the plant extracts led to the enhancement of their inflammasome inhibition potency. this is in accordance with previous studies that reported the beneficial effect of fractionation in concentrating polyphenolic compounds, leading to enhanced biological activities [ , ] . we thus argue that the bioactive compounds in fraction er . , er . together with the well-known caffeic acid and isoquercetin are the main compounds involved in the regulation of the nlrp inflammasome. in the present study, the effect of our plant samples on the production of il- in raw . , a murine cell line widely used for the in vitro production of il- , was also investigated. this cytokine is a known downstream target of il- β and is consistently increased in serum from patients with nlrp inflammasome-mediated conditions. in fact, il- β has been reported to be one of the most potent activators of il- production, both in vitro and in vivo, suggesting that il- could be a therapeutic target in the treatment of il- β-mediated inflammation [ ] . in addition, abnormal expression of il- has been associated with the pathogenesis of a variety of human diseases, including asthma, cancers and autoimmune diseases such as rheumatoid arthrosis, and recently with covid- [ , ] . in asthma, it has been reported that il- is linked with mixed eosinophilic-neutrophilic bronchitis during exacerbations of the disease and worse pulmonary function in humans [ ] . moreover, cytokine-targeting therapies are gaining an increasing popularity in the treatment of inflammatory diseases including asthma and other respiratory diseases. with the covid- outbreak pandemic, it has been reported that dexamethasone and monoclonal antibody drugs targeting il- (tocilizumab) reduced the mortality rate [ , , ] . in the light of the above-mentioned role of il- in different pathologies, we investigated the effect of the h. noldeae constituents of interest on its production in raw . cell line. as shown in figure , results obtained indicate that fractions and partially purified er . and er . compounds from the plant exhibited similar trends as on il- β production ( figure a ). these findings allowed to argue that this medicinal plant is an important drug target for 'cytokine storm'-related diseases such as the neutrophilic asthma and the covid- pandemic. aerial parts of hibiscus noldeae were collected from the arboretum of ruhande, in huye district, southern province in rwanda. the plant species was identified with reference to herbarium specimens and various literature resources by nshutiyayesu samuel (from the university of rwanda, huye, rwanda) and the authentication and id verification by dr. olivier lachenaud (from the botanic garden meise/meise, belgium) as previously reported [ ] . the aerial parts of h. noldeae were cleaned of dust and debris and washed gently with tap water, and air-dried under shade. the plant material was then pulverized with a grinder. a total amount of g of the powder was extracted with the mixture of methanol: dichloromethane ( : ) as previously described [ ] . the dried crude extract ( g) was triturated in distilled water and then subjected to a liquid-liquid partitioning process using n-hexane and ethyl acetate successively. in each case, the mixture to be partitioned was vigorously shaken for min, rested for min, and then separated in a separatory funnel. each partition was conducted three times and the same fractions were combined and dried-up using rotary evaporator. at the end, the non-soluble part was dissolved in methanol to make the methanol fraction. major compounds from two fractions showing the highest inhibitory activity on nlrp inflammasome components, namely hexane and ethylacetate fractions, were further isolated on preparative hplc. to this end, an in-house hplc method used to screen the major components of crude extracts was used. in this regard, analytical separation was carried out on a hypersil ods ® rp column ( × . µm; particle size µm; thermofisher scientific, waltham, ma, usa). all samples were dissolved in methanol hplc-grade, filtered through a . µm pore size filter membrane and analyzed on an agilent (santa clara, ca, usa) hplc machine. samples were eluted with a nonlinear gradient method with acetonitrile (solvent a) and . % trifluoroacetic acid in ultra-pure water (solvent b) ( table ). the column temperature was maintained at • c. then, µl of each sample was injected into the hplc-uv/dad system and the analysis, performed at a flow rate of . ml/min, was monitored at , , , and nm. preparative hplc analysis was carried out on a variant prepstar machine. all extracts ( mg) were dissolved in methanol and then diluted with water. the amount of methanol was not allowed to exceed %. samples were filtered through a . µm filter membrane before injection. the mobile phase consisted of trifluoroacetic acid (tfa) . % in ultrapure water (a) and acetonitrile (b). the table gives the gradient used to separate the major compounds identified with hplc analytical method described above. this gradient was obtained by transposing the analytical hplc methods to preparative hplc using hplc calculator. the flow rate was ml/min and the separation was monitored at and nm. structural identification of isolated compounds was performed by d and d nuclear magnetic resonance (nmr) and infrared spectroscopy (ir). the detection was followed at and nm. human leukemia thp- monocyte and raw . murine cell lines were kindly received from the institute for medical immunology/biopark/ulb and were cultured in rpmi (life technologies, waltham, ma, usa) and dmem, respectively. all media were supplemented with % heat-inactivated fetal bovine serum (fbs; life technologies), % glutamine, and % of penicillin-streptomycin (life technologies) and % sodium pyruvate (for raw . only) and maintained at • c under % co in a humidified atmosphere. cells were passaged at least times before any experiment. for the determination of cell densities, the cell counting was done using a neubauer counting chamber with trypan blue ( . % in pbs; ph = . ; life technologies) staining. before investigating the anti-inflammatory activity of different fractions, their effect on the cell viability (thp- , monocytes and derived-macrophages, and raw . ) was screened as previously reported [ ] . the evaluation of caspase- activity was done using the caspase glo inflammasome assay (promega co. madison, wi, usa). briefly, thp- cells were grown for days in -well plates to a density of × cells/well in the presence of nm pma at • c in a humidified % co incubator. pma-supplemented medium was then discarded and macrophages were pretreated with fresh medium containing fractions at different concentrations for h. dexamethasone at a concentration of µg/ml or µm ac-yvad-cho treatments were used as controls. cells were further incubated with lps (sigma, saint-louis, mo, usa) (at a final concentration of µg/ml) for h and then with µm atp (invitrogen/thermofisher, waltham, ma, usa) for additional h. the caspase- activity was measured using the caspase-glo inflammasome assay (promega co., madison, wi, usa) according to the manufacturer's instructions. the -well plate containing treated cells was removed from the incubator and µl of the supernatant in each well were transferred to the corresponding well of a new white -well plate. an aliquot of µl of the caspase-glo reagent was then added to each well and gently mixed on a plate shaker at rpm for s. the mixture was then incubated for h at room temperature before measuring the luminescence on a berthold technologies luminometer. the culture supernatant of stimulated the thp- macrophage was used for the measurement of the cleaved form of il- β. the concentration of il- β in the culture supernatant was measured using a human il- β elisa kit (invivogen/thermofisher, waltham, ma, usa) according to the manufacturer's instructions. in all experiments, raw . cells were sub-cultured in a -well plate and incubated for h at • c and % co to acclimate before treatment. they were then pretreated with test compounds, at indicated concentrations, for h prior to h stimulation with µg/ml lps. cell-free supernatants were harvested and stored at − • c until analysis. the concentration of il- in the culture supernatant was measured using an elisa kit (bd biosciences, franklin lakes, nj, usa) according to the manufacturer's instructions. the evaluation of pyroptosis inhibition was done using the ldh-glo tm cytotoxicity assay (promega co. madison, wi, usa). briefly, thp- cells were cultured for days in -well plates to a density of × cells/well in the presence of nm pma at • c in a humidified % co incubator. pma-supplemented medium was then discarded and macrophages were pretreated with fresh medium containing fractions at different concentrations for h. dexamethasone at a concentration of µg/ml or µm ac-yvad-cho treatments were used as controls. cells were further incubated with lps (sigma, saint-louis, mo, usa) (at a final concentration of µg/ml) for h and then with µm atp (invitrogen/thermofisher, waltham, ma, usa) for additional h. then, µl of % triton x- was added to µl of vehicle-treated cells control for min before collecting the samples for ldh detection. supernatants were harvested and -fold diluted in the ldh storage buffer and kept at - • c until analysis. the ldh activity was measured using the ldh-glo tm cytotoxicity assay (promega co., madison, wi, usa) according to the manufacturer's instructions. the % cytotoxicity = × (experimental ldh release − medium background)/(triton x- ldh release control − medium background). for western blot analysis, thp- cells ( × cells/well) were differentiated in a -well plate by nm pma for days and then treated as stated above. cells were washed with µl of an ice-cold pbs, and directly lysed with a reporter lysis buffer (promega) and then centrifuged at , rpm (round per minute) for min at • c. the supernatants were kept at − • c until analysis. the protein quantitation in the supernatant was quantified by the bradford assay. an equivalent amount ( µg of protein) of each sample was separated by sodium dodecyl sulfate polyacrylamide gel electrophoresis (sds-page) and electro-transferred onto nitrocellulose membranes. the membranes were immunoblotted by anti-proil- b (cell signaling technology, danvers, ma, usa) or anti-pro caspase- (abcam) according to the manufacturer's instructions. membranes were then stripped, reprobed with anti-β-actin antibodies (cell signaling technology) and exposed again to detect the internal β-actin standard. immunoblots were visualized using a chemiluminescence odyssey imaging system (odyssey ® fc, bad homburg, germany). to scan and quantify the immunoblots, we used the image studio lite ver . software (li-cor biotechnology-gmbh, bad homburg, germany). each test was done in triplicates and all the experiments were reproduced at least two times independently. for each elisa experiment, the amount of il- β or il- produced was considered as % and the remaining treatments was calculated as (treatment/lps) × . the results are presented as the mean ± sd from two or three independent experiments (n = ), each done in triplicate. data were analyzed using graphpad prism and statistical analysis data expressed as the mean ± standard deviation. comparisons of data between groups were performed by one-way anova followed by benforroni's multiple comparison test. the p-values less than . were considered to be statistically significant. considering the importance of inflammasome components in the severity of respiratory diseases in general and in asthma in particular, our preliminary results at the molecular and functional levels suggest that h. noldeae and/or its constituents may play a role in the management of asthma and other respiratory diseases if the ex vivo results obtained can be reproduced in vivo. additional purification and characterization of er . and er . fractions are of prime interest for our future study. supplementary materials: the following are available online. table s : the h-nmr chemical shifts for the purified compounds from h. noldeae. figure s : h-nmr spectrum of quercetin- -o-glucoside (isoquercetin). figure s : c spectrum of quercetin- -o-glucoside (isoquercetin) (dmso-d , mhz). figure s : h, c-hmbc of quercetin- -o-glucoside (isoquercetin) (dmso-d , mhz). figure s : effect of different h. noldeae's constituents on the thp- cell viability. figure s : effect of different h. noldeae constituents on the raw . cell viability. figure s : crude extract and the ethylacetate fraction as well as the caffeic acid and semi-purified fractions (er . and er . ), thereof exhibited significant inhibition of pyroptosis in thp- derived macrophages. the authors gratefully acknowledge the technical support of allison marotte, olivier bonnet, delphine etienne, dansala welba, and alain nyirimigabo. we are especially grateful to suzanne, a traditional healer at mamba/huye, for her cooperation and to have availed the plant material. the study could not have been completed without the cooperation of the university of rwanda's authorities, particularly the college of science and technology. the authors declare no conflict of interest. respiratory diseases in the world. realities of today-opportunities for tomorrow inflammasome-il- -th response in allergic lung inflammation the immunology of asthma and allergic rhinitis. rhinosinusitis characterization of the anti-inflammation mechanism for the ao herbal extract interleukin- β targeted therapy in severe persistent asthma (spa) and chronic obstructive pulmonary disease (copd): proposed similarities between biphasic pathobiology of spa/copd and ischemia-reperfusion injury molecular immune pathogenesis and diagnosis of covid- the epidemiology and pathogenesis of coronavirus disease (covid- ) outbreak cytokine storm intervention in the early stages of covid- pneumonia covid- : consider cytokine storm syndromes and immunosuppression inflammasomes in the lung caspase- activation by nlrp inflammasome dampens il- -dependent house dust mite-induced allergic lung inflammation pharmacological use of nlrp inflammasome inhibitors: novel intervention strategies in diabetes-associated vascular complications caspase- : the inflammasome and beyond targeting nlrp inflammasome activation in severe asthma the il- cytokine family and its role in inflammation and fibrosis in the lung covid- : pathogenesis, cytokine storm and therapeutic potential of interferons dexamethasone in hospitalized patients with covid- -preliminary report an ethnobotanical survey and inhibitory e ff ects on nlrp in fl ammasomes/caspase- of herbal recipes' extracts traditionally used in rwanda for asthma treatment biologically active compounds from the genus hibiscus comparison of metabolic profiles and bioactivities of the leaves of three edible congolese hibiscus species anti-inflammatory activity and molecular mechanism of delphinidin -sambubioside, a hibiscus anthocyanin nlrp inflammasome activation and gasdermin d-driven pyroptosis are immunopathogenic upon gastrointestinal norovirus infection the nlrp inflammasome: an overview of mechanisms of activation and regulation contribution to the inventory of medicinal plants from the bushi area ethnopharmacological survey of plants used for the treatment of female infertility in baham, cameroon ethnobotanical survey of medicinal plants used for pregnant womens health conditions in menoua division-west cameroon medicinal plants: traditions of yesterday and drugs of tomorrow potential effects of flavonoids on the etiology of vascular disease review of the biology of quercetin and related bioflavonoids a and cell death-driven inflammation cell death and inflammation-a vital but dangerous liaison handbook of medicinal plants: a comprehensive review of their traditional medical uses and scientific justifications comparative suppression of nlrp inflammasome activation with lps-induced inflammation by blueberry extracts (vaccinium spp glucocorticoids selectively inhibit the transcription of the interleukin ,f gene and decrease the stability of interleukin , mrna targeting asc in nlrp inflammasome by caffeic acid phenethyl ester: a novel strategy to treat acute gout quercetin as drug to treat asthma-what is missing? phenolic content, antioxidant effect and cytotoxic activity of leea indica leaves caffeic acid derivatives: in vitro and in vivo anti-inflammatory properties cutting edge: il- is a marker of inflammation with no direct role in inflammasome-mediated mouse models detectable serum severe acute respiratory syndrome coronavirus viral load (rnaemia) is closely correlated with drastically elevated interleukin level in critically ill patients with coronavirus disease therapeutic potential of anti-il- therapies for granulocytic airway inflammation in asthma anti-inflammatory agents: present and future tocilizumab in patients with severe covid- : a retrospective cohort study key: cord- -q b lm authors: cao, ying-li; wang, ying; guo, rong; yang, fan; zhang, yun; wang, shu-hui; liu, li title: identification and characterization of three novel small interference rnas that effectively down-regulate the isolated nucleocapsid gene expression of sars coronavirus date: - - journal: molecules doi: . /molecules sha: doc_id: cord_uid: q b lm nucleocapsid (n) protein of severe acute respiratory syndrome-associated coronavirus (sars-cov) is a major pathological determinant in the host that may cause host cell apoptosis, upregulate the proinflammatory cytokine production, and block innate immune responses. therefore, n gene has long been thought an ideal target for the design of small interference rna (sirna). sirna is a class of small non-coding rnas with a size of - nt that functions post-transcriptionally to block targeted gene expression. in this study, we analyzed the n gene coding sequences derived from different isolates, and found that nucleotide deletions and substitutions are mainly located at the first nt sequence. combining previous reports and the above sequence information, we create three novel sirnas that specifically target the conserved and unexploited regions in the n gene. we show that these sirnas could effectively and specifically block the isolated n gene expression in mammal cells. furthermore, we provide evidence to show that n gene can effectively up-regulate m gene mediated interferon β (ifnβ) production, while blocking n gene expression by specific sirna significantly reduces ifnβ gene expression. our data indicate that the inhibitory effect of sirna on the isolated n gene expression might be influenced by the sequence context around the targeted sites. severe acute respiratory syndrome (sars) that spread worldwide in is caused by a novel type of coronavirus called sars-associated coronavirus (sars-cov). sars-cov is an enveloped and positively single-stranded rna virus with a typical genome size  . kb [ ] [ ] [ ] . the viral envelope is a lipid bilayer enbedded with three viral transmembrane proteins: the matrix (m), envelope (e) and spike (s) proteins. the genomic rna of sars-cov is protected and packaged by the viral nucleocapsid (n) protein by forming a helical structure of ribonucleoprotein complex within the viral envelope. in addition, the physical interaction between the n and m proteins might be necessary for the assembly of coronavirus [ ] [ ] [ ] . the n gene of sars-cov is located at a region proximal to the ' end of the viral genome with a size of nt long that encodes for a aa basic protein. the main functions of n protein are two folds: ) it binds to viral genomic rna for the formation of viral nucleocapsid; ) it also selfassociates into polymer that might be critical for the helical structure formation [ ] . protein structural study reveals that the sars-cov n protein contains two structural domains flanked by intrinsically disordered (id) regions [ ] . the n terminal domain (ntd, amino acid residues - ) can bind nonspecifically to a variety of nucleotide substrates [ ] and functions as a putative rna binding domain associated with the viral rna genome [ ] , whereas the c terminal domain (ctd, amino acid residues - ) is thought to be responsible for the dimerized self-association [ , ] . however, recent studies show that the c terminal sequence of n protein also promotes higher ordered oligomerization and is able to interact with nucleic acids as well [ , ] . moreover, chang demonstrated that all id sequences are able to bind rna [ ] , indicating that the multisite nucleic acid binding property of sars-cov n protein may have an inherent advantage to promote the formation of viral helical nucleocapsid core. sars-cov infection is a life-threatening disease that often develops with acute lung injury and acute respiratory distress syndrome. the inflammation and immune responses in the host are often induced or damaged by viral gene products such as n gene products. sars-cov n protein possesses multifarious activities and may be actively involved in sars-cov induced pathogenesis [ ] . for instance, n protein induces the pro-inflammatory responses by activating the promoter activity of either cyclooxygenase- (cox- ) or il- by directly interacting with the nfb binding element [ , ] . overexpression of n protein in serum starved cell lines such as monkey kidney cos- cells [ ] or human pulmonary fibroblast hpf cells [ ] induces apoptosis. more importantly, sars-cov n protein can function as an antagonist to counteract the host innate immune response by inhibiting the activity of irf and nfkb, and subsequently blocking interferon  production [ ] . sars-cov n protein is believed to be one of the major pathological determinants in the host cells [ ] . therefore, down-regulating n protein expression by the small interference rna (sirna) approach might be good strategy to reverse the virus-induced damage to the host and may help development of a more effective therapeutic means to control viral transmission. sirna is a type of rna molecule with - nt in length that functions post-transcriptionally to down-regulate the targeted gene expression. sars-cov n gene specific sirnas have been designed and investigated by a number of groups, and at least targeted sites of n gene specific sirnas have been selected and tested [ ] [ ] [ ] [ ] [ ] . sequence analysis indicates that all these known sirnas are located at / of the ' n gene sequence (within first nt out of the total nt). nucleotide substitution and deletion mutations can be frequently recovered from the first nt sequence of n gene. therefore, these sequence alterations may have a potential to weaken the effect of sirna mediated gene silencing. in the current study, we compared the n gene sequences derived from different isolates of sars-cov and selected three novel sirna targeting sites in the n gene, including one targeting the ' terminus of the gene. functional analysis indicates that all three novel sirnas are effective in downregulating n gene expression. moreover, our study demonstrates that sars-cov n gene is able to up-regulate the m gene mediated interferon  production, while the n gene specific sirna can effectively reduce this up-regulation. sars-cov n protein is a multi-functional protein that contributes greatly to viral induced pathogenesis and often serves as the therapeutic targeting site for the development of anti-viral drugs against sars-cov infection, including sirna. sars-cov is a positive single-stranded rna virus frequently associated with sequence alterations during viral transmission. a better sirna should be designed to target the conserved region in the targeted sequence. to this end, we randomly selected the n genes derived from different isolates of sars-cov: hku- (ay ), tor (ay ), bj (ay ), hzs -fb (ay ), zj (ay ), sin (ay ), shanghaiqxc (ay ), shanghaiqxc (ay ), cuhk-ag (ay ), pumc (ay ), gz-b (ay ), tc (ay ), gz-c (ay ), zs-c (ay ), lc (ay . ) and lc (ay . ). clustalw analysis revealed that a total of five sequence alterations (two sequence deletions and three nucleotide substitutions) occurred in the n gene in these isolates ( figure ). interestingly, sequence alterations in the n gene are frequently uncovered in the ' portion of the gene (within the first nt), which is similar to our previous observation on sars-cov m gene [ ] . however, different from the m gene mutation, which is associated with single nucleotide substitution, the n gene mutation tends to be larger in size, such as twelve nucleotide deletions (nt - ) and di-nucleotide substitutions (nt - , figure ). the n gene also possesses two additional single nucleotide substitutions (nt and nt ) and one single nucleotide deletion (nt ). previously, a number of n gene-specific sirnas based on random selection have been reported by other groups [ ] [ ] [ ] [ ] [ ] . sequence analysis revealed that all these sirnas are located within the first nt sequence of the n gene, and no targeted site has been selected for the last nt sequence. considering the nucleotide substitutions in n gene as well as the previous reports, we chose three unexploited regions that were well conserved in the n genes among the isolates of sars-cov. the selected targeted sites were at + + nt, + + nt and + + nt relative to the ' atg initiation codon, and their respective sirnas were named as si-n , si-n and si-n ( figure ). a known sirna (named si-n# , previous name no. in reference [ ] ) that targeted the ' terminus (+ + nt) of n gene was also constructed for control purposes. three novel sirnas (si-n , si-n and si-n ) targeting at  nt,  nt and  nt, respectively, are underlined. a known si-n# (no. ) targeting at  nt reported by shi et al. [ ] is also indicated. the full length of n gene cdna was amplified from sars-cov strain hku- infected vero e cells. after confirmation by sequencing analysis, the n gene was then subcloned into eukaryotic expression vector pcmv-myc to generate a pcmv-myc-np plasmid construct. the expression of n gene was confirmed by rt-pcr and western blot analysis (figure a and b) . to visualize the subcellular localization of n protein, n gene was also fused with egfp to make a pegfp-np fusion gene construct. after transfection into hek cells, egfp-np fusion protein was mainly distributed in the cytoplasm (figure c ). alternatively, hek cells transfected with pcmv-myc-np were subjected to immunostaining analysis. in agreement with the above result, as well as a previous report [ ] , the myc-tagged np gene products were indeed predominantly expressed in the cytoplasm ( figure d ). to test if the selected sirnas have an inhibitory effect on n gene expression, pegfp-np was cotransfected with increased doses of either si-n or si-n into hek cells. figure demonstrates that both si-n and si-n inhibited pegfp-np expression post-transcriptionally and functioned in a dose-dependent manner, indicating that both sirnas are effective in inhibiting the targeted mrna expression. pegfp-np was co-transfected with the increased dose of either pbs/u -si-n or pbs/u -si-n into hek cells. total rnas were isolated from the transfected cells and subjected to standard rt-pcr by using n specific primers. beta actin expression was served as internal control. the effect of sirna on n gene repression was further confirmed by western blot analysis. figure a demonstrates a significant reduction in n protein expression as the ratio of n to si-n increased. quantitation of the band intensity revealed about fold reduction in n protein expression when cotransfection with higher doses of si-n (figure b) . the specificity of si-n on n gene expression was further confirmed by using a non-specific sirna, si-m , as a negative control. si-m has been shown to be a potent inhibitor to sars-cov m gene expression [ , ] . higher doses of si-n but not si-m dramatically inhibited egfp-np gene expression, indicating the specificity of si-n mediated n gene repression (figure c ). quantitative analysis by flow cytometric approach further demonstrated that si-n could specifically and markedly reduce egfp-np gene expression (figure d) . similarly, si-n , which targeted at the ' half of n gene, also dramatically inhibited n protein expression by about four-fold when the molar ratio of si-n : n reached : (figure a and b) . the specificity of si-n mediated n gene repression was demonstrated by the fact that higher doses of si-n , but not si-m , dramatically inhibited egfp-np gene expression (figure c ). the results were further confirmed by measuring the mean fluorescent intensity (mfi) of the transfected cells cotransfected with egfp-np and the indicated sirnas (figure d ). sequence analysis reveals that all the n gene sirnas reported by other groups have their own recognition sites, mainly located within the first nt sequence of the n gene [ ] [ ] [ ] [ ] [ ] . therefore, to our knowledge, the last nt sequence of n gene has not been designed for sirna targeting. to this end, we created the third sirna that targets to the ' terminus (+ + nt) of n gene, and named it si-n . western blot analysis demonstrated that si-n markedly inhibited myc-tagged n protein expression in a dose dependent manner (figure a and b ). in addition, pegfp-np was also co-transfected with increased doses of either si-m or si-n into targeted cells. the result shown in figure c clearly demonstrated that higher doses of si-n , but not si-m , dramatically inhibited egfp-np gene expression. the result was further confirmed by flow cytometric analysis by measuring the mean fluorescent intensity (mfi) of the transfected cells co-transfected with egfp-np and the indicated sirnas (figure d ). overall, the above results provide strong evidence to show that all three novel sirnas (si-n , si-n and si-n ) are specific and effective inhibitors to block the isolated sars-cov n gene expression. to assess the strength of si-n , si-n and si-n mediated n gene repression, we chose a known sirna, si-n# (no. ) , which has been shown to be a potent n gene inhibitor [ ] as a positive control. about . g of pcmv-myc-np was co-transfected with . g of each plasmid pbs/u , si-n# , si-n , si-n and si-n into targeted cells. the real time qrt-pcr result showed that si-n# , si-n , si-n and si-n all induced significant inhibition on the isolated n gene expression as compared with that of the vector control (figure ) . the quantitative analysis showed that the n gene inhibitions induced by si-n# , si-n , si-n and si-n were about . , . , . and . -fold, respectively (figure ) , indicating that si-n might be a more potent inhibitor on n gene expression. previously, we demonstrated that sars-cov m gene could upregulate inf gene expression in a transient transfection system [ ] . also, it has been shown that inf gene expression and transcription could be inhibited by sars-cov infection, probably due to the presence of n gene products [ ] . to detect the influence of n gene on m mediated inf production, n and m were co-transfected into hek cells. interestingly, we found that the n gene was not able to inhibit, but rather enhanced m gene mediated inf mrna production (figure a ). addition of si-n , si-n or si-n in the co-transfection system significantly reduced inf mrna production (figure a) . the semiquantitative rt-pcr result was further confirmed by using real time qrt-pcr analysis (figure b) , indicating that the identified n gene specific sirna could functionally counteract the n gene mediated cellular processes. human embryonic kidney cell line (hek ) cells were obtained from the cell culture center of institute of basic medical sciences, chinese academy of medical sciences. african green monkey kidney epithelial cell line vero e was provided by dr. k.y. yuen from the university of hong kong. cells were cultured in dulbecco's modified eagle medium (hyclone, south logan, ut, usa) supplemented with % fetal calf serum and incubated in a c incubator containing % co . anti-myc and anti-actin antibodies were purchased from santa cruz biotechnology (santa cruz, ca, usa). horseradish peroxidase (hrp)-labeled goat anti mouse igg and enhanced chemiluminescence (ecl) detection kit were also derived from santa cruz biotechnology. the sars-cov n gene was isolated from sars-cov strain hku- [ ] (provided by dr. ky yuen, the university of hong kong) by standard rt-pcr with a pair of primers: n : -tatagaatt ctgtctgataatggaccccaat-  and n : -tataggtaccttatgcctgagttgaatcag- . the amplified full length of the n gene was first subcloned into pgem-t easy vector. after confirmation by sequencing analysis, the n gene products were released by ecori/kpni double digestion and then subcloned into the respective sites of pcmv-myc to generate pcmv-myc-n. the n gene products were also inserted into the ecori/kpni sites of pegfp-n vector to form pegfp-np fusion gene construct. for construction of n gene specific sirnas, three novel targeted sites ( - nt, - nt and - nt) in the n gene coding sequence were selected. two oligos for each targeted site were synthesized as sin- -f, '-gggcgttccaatcaacaccaaaagcttttggtgttgattggaacgccctttttg- ' and sin- -re, '-aattcaaaaagggcgttccaatcaacaccaaaagcttttggtgttgattggaacgccc- '; sin- -f, -gggaccaagacctaa tcagacaagcttgtctgattaggtcttggtccctttttg-  and sin- -re, -aattcaaaaagggaccaagacctaatcagacaagcttgt ctgattaggtcttggtccc- ; sin- -f, -ggagcttctgctgattcaactaagcttagttgaatcagcagaagctcctttttg-  and sin- -re, -aattcaaaaaggagcttctgctgattcaactaagcttagttgaatcagcagaagctcc- . the oligos sin- -f and sin- -re, sin- -f and sin- -re, sin- -f and sin- -re were annealed pair-wisely to form duplexes. to construct the sirna targeting to the  terminus of n gene (named as si-no that targets to - nt) as described by shi et al. [ ] , two synthesized oligos -gataatggaccccaatcaaacaagcttgtttgattggggtccattatctttttg-  and -aattcaaaaagataatggaccccaatcaaacaa gcttgtttgattggggtccattatc-  were also annealed. the duplex products were then subcloned into pbs/u [ ] (provided by dr. yang shi, harvard medical school) to form pbs/u -sin , pbs/u -sin , pbs/u -sin and pbs/u -no , respectively. total rnas were extracted from the cultured cells with trizol (invitrogen, carlsbad, ca). all primers used in the rt-pcr reactions were listed in table . one g of total rnas was first reverse transcribed using amv reverse transcriptase (promega, usa). about l of the transcribed cdnas was subjected to standard pcr reaction using n gene specific primers. one-step real-time quantitative rt-pcr (qrt-pcr) (takara biotechnology, dalian, china) was also performed to monitor the targeted gene expression. real time qrt-pcr was carried out with iq real-time pcr detection system (bio-rad laboratories) at the following conditions: °c for min and °c for sec; °c for sec and °c for sec and repeated for cycles. the dissociation of the reaction products was conducted from °c to °c as the temperature rose at . °c per ten seconds. cell cultured in -mm dishes were transiently transfected with the indicated plasmid dnas using profection ® mammalian transfection systems (promega, usa) according to the manual instruction. briefly, transfected dnas were first mixed with l of m cacl and brought to total l with sterile and deionized water. then the dna-cacl mixture was added into equal volume of ×hbs drop by drop accompanying with gentle vortexing. after minutes incubation, the reaction mixture was evenly distributed into the cell culture medium and incubated for hours before harvesting. the transfected cells were lysed with a lysis buffer containing % np- , mm tris-hcl (ph . ), mm nacl, μm navo , g/ml leupeptin, g/ml aprotinin, and m pmsf. about g of cell lysate for each sample was resolved onto % sds-page. after separation, the separated proteins were transferred onto hybond nitrocellular membrane (pharmacia). the transferred membrane was first probed with a primary antibody. then, a secondary antibody labeled with horseradish peroxidase was added to the reaction and finally visualized with an ecl kit. sars-cov n gene has long been selected as one of the major targets for sirna design. however, genomic alterations such as nucleotide deletion and substitution frequently occur in sars-cov. this type of change has the potential to weaken the inhibitory effect induced by sirna. in the current study we analyzed the n gene sequences derived from different isolates and found that in addition to single nucleotide substitutions, the n gene also possesses a longer nucleotide deletion and dinucleotide substitution. previous studies on m gene specific sirnas indicate that the sirna targeting site and its surrounding sequences may influence the inhibitory effect [ ] . the current study further support this observation by showing that si-n# , which targets a twelve nucleotide deletion region in the n gene of one viral strain generates a less inhibitory effect than that of either si-n or si-n (figure b ). although n gene specific sirna has been intensively studied, no sirna was reported for the last nt sequence. in this study, we created and tested a third sirna (si-n ) which was effective to target the ' terminal sequence of n gene and subsequently in downregulating n gene expression. interestingly, the inhibitory effect induced by si-n was similar to that of si-n# , implying that sirnas targeting at both the  and  terminal sequences of the isolated n gene might induce less inhibition. we also notice that there might be a discrepancy between our study and a previous report by shi et al. in which they demonstrated that si-n# (no. ) was the strongest inhibitor among the eleven n gene sirnas tested [ ] . this discrepancy might be due to: ) the potential structural difference between vector expressed n mrna and virus derived subgenomic n mrnas and/or ) the differences in the expression system used by us (pbs/u ) versus shi's (chemical synthesized sirnas). finally, we provide evidence to show that sars-cov n gene products were able to up-regulate m gene mediated inf production, while n gene specific sirna could functionally reduce this enhancement. however, the mechanism underlining n gene mediated inf production remains an interesting point to be further addressed. the genome sequence of the sars-associated coronavirus characterization of a novel coronavirus associated with severe acute respiratory syndrome comparative full-length genome sequence analysis of sars coronavirus isolates and common mutations associated with putative origins of infection dissection and identification of regions required to form pseudoparticles by the interaction between the nucleocapsid (n) and membrane (m) proteins of sars coronavirus a major determinant for membrane protein interaction localizes to the carboxy-terminal domain of the mouse coronavirus nucleocapsid protein characterization of the coronavirus m protein and nucleocapsid interaction in infected cells structure of the sars coronavirus nucleocapsid protein rna-binding dimerization domain suggests a mechanism for helical packaging of viral rna modular organization of sars coronavirus nucleocapsid protein biochemical and immunological studies of nucleocapsid proteins of severe acute respiratory syndrome and e human coronaviruses structure of the n-terminal rna-binding domain of the sars cov nucleocapsid protein crystal structure of the severe acute respiratory syndrome (sars) coronavirus nucleocapsid protein dimerization domain reveals evolutionary linkage between corona-and arteriviridae carboxyl terminus of severe acute respiratory syndrome coronavirus nucleocapsid protein: self-association analysis and nucleic acid binding characterization multiple nucleic acid binding sites and intrinsic disorder of severe acute respiratory syndrome coronavirus nucleocapsid protein: implications for ribonucleocapsid protein packaging the sars-cov nucleocapsid protein: a protein with multifarious activities nucleocapsid protein of sars-cov activates the expression of cyclooxygenase- by binding directly to regulatory elements for nuclear factor-kappa b and ccaat/enhancer binding protein nucleocapsid protein of sars-cov activates interleukin- expression through cellular transcription factor nf-kappab the sars coronavirus nucleocapsid protein induces actin reorganization and apoptosis in cos- cells in the absence of growth factors m and n proteins of sars coronavirus induce apoptosis in hpf cells severe acute respiratory syndrome coronavirus open reading frame (orf) b, orf , and nucleocapsid proteins function as interferon antagonists prophylactic and therapeutic effects of small interfering rna targeting sars-coronavirus inhibition of genes expression of sars coronavirus by synthetic small interfering rnas small interfering rna inhibits sars-cov nucleocapsid gene expression in cultured cells and mouse muscles kinetics and synergistic effects of sirnas targeting structural and replicase genes of sars-associated coronavirus inhibition of sars-cov gene expression by adenovirus-delivered small hairpin rna small interfering rna effectively inhibits the expression of sars coronavirus membrane gene at two novel targeting sites sirnas targeting terminal sequences of the sarsassociated coronavirus membrane gene inhibit m protein expression through degradation of m mrna inhibition of beta interferon induction by severe acute respiratory syndrome coronavirus suggests a two-step model for activation of interferon regulatory factor the complete genome sequence of severe acute respiratory syndrome coronavirus strain hku- (hk- ) a dna vector-based rnai technology to suppress gene expression in mammalian cells this article is an open access article distributed under the terms and conditions of the creative commons attribution license key: cord- - sqizkio authors: mowaka, shereen; ashoush, nermeen; tadros, mariam; el zahar, noha; ayoub, bassam title: enhanced extraction technique of omarigliptin from human plasma—applied to biological samples from healthy human volunteers date: - - journal: molecules doi: . /molecules sha: doc_id: cord_uid: sqizkio enhancing drug extraction from human plasma is a challenging approach that critically affects pharmacokinetic and any further clinical studies based on the drug c(min) and c(max) values. it also has a serious impact on the sensitivity and the lower limit of quantification (lloq) value of the bio-analytical methods. an advanced liquid chromatography tandem mass spectrometry (lc-ms/ms) bio-analytical method of omarigliptin ( – nm) was established in human plasma using one-step liquid-liquid extraction. alogliptin was used as an internal standard (is) to attain good recovery and reproducibility while reducing the effects of the matrix. enhanced plasma extraction of omarigliptin was successfully achieved with tertiary butyl methyl ether—diethyl ether (tbme-dee) mixture as the extracting solvent, while using acetonitrile as the diluent solvent for the is to effectively decrease the formed emulsion. multiple reaction monitoring (mrm) of the transition pairs of m/z . to . for omarigliptin and m/z . to . for alogliptin was employed in positive electro spray ionization (esi) mode. human plasma samples were collected after . h (t(max)) of marizev(®) ( . mg) tablets administration to healthy human volunteers showing average concentration of . nm. validation results were all satisfactory including successful stability studies with bias below %. the proposed study will be valuable for ethnicity comparison studies that will be commenced on omarigliptin in egypt by the authors in prospective study, following the fda recommends, to evaluate possible sub-group dissimilarities that include pharmacokinetic parameters. diabetes mellitus prevalence increases constantly worldwide. moreover, it is worthy to mention that many recent covid- studies reported that diabetes mellitus was a major contributing factor either for non-survivals and/or hospitalization. diabetic patients represented % of non-survivors in a study and represented % of the hospitalized patients in another study [ , ] . generally, people with diabetes mellitus are most likely to suffer from different complications when infected with virus either for non-survivals and/or hospitalization. diabetic patients represented % of non-survivors in a study and represented % of the hospitalized patients in another study [ , ] . generally, people with diabetes mellitus are most likely to suffer from different complications when infected with virus ranging from mild to severe. unmanaged levels of blood glucose elevate the risk and the severity of any common respiratory attack [ , ] . uncontrolled diabetes mellitus leads to a weak immunity response, as the body becomes no more able to fight the infection [ , ] . hence, it becomes imperative to find better therapeutic strategies for glycemic control. gliptins are considered to be effective agents for the treatment of type diabetes mellitus. omariglitpin (otn), figure , is a long-acting once weekly administered antidiabetic drug acting as dipeptidyl peptidase- (dpp- ) inhibitor [ ] [ ] [ ] . it has been licensed for use in japan since but its phase iii development in us has been halted for undisclosed commercial reasons [ ] . it improved the glycated hemoglobin a c (hba c) as it reversibly inhibits dpp- enzyme, which prolongs the circulating half-life of glucagon-like peptide- (glp- ) increasing insulin secretion. contrary to the once-daily administered dpp- inhibitors available in market, once-weekly administered otn can improve patients' compliance to the treatment protocol [ ] [ ] [ ] [ ] [ ] . literature review reveals that otn pharmacokinetics parameters were studied with renal impairment [ ] , in repositioning studies [ ] and in healthy japanese volunteers [ ] . otn pharmacokinetics were investigating the supra-therapeutic dose of otn [ ] and the effect of age, sex and obesity [ ] . clinical studies were also concerned with the effects of multiple and chronic dosage regimens [ , ] . although, these many articles deal with the pharmacokinetics and/or bioanalysis of otn [ ] [ ] [ ] [ ] [ ] [ ] [ ] , most of the reported studies did not discuss the full details of the validated bio-analytical procedure that may be attributed to using the same methods for many clinical studies by the same common authors [ ] [ ] [ ] [ ] [ ] [ ] . they were emphasizing the findings of the main pharmacokinetic parameters rather than developing or validating a bio-analytical procedure. one liquid chromatography tandem mass spectrometry (lc-ms/ms) method was discussed in full details for otn bio-analysis [ ] but that method was related to rats' plasma only and the authors used only direct precipitation with acetonitrile for the extraction of otn from spiked and biological rats' plasma samples. the authors of the current study modified the previously mentioned rats' plasma method [ ] in another repositioning publication [ ] but with direct precipitation with acetonitrile. the lower limit of quantification (lloq) was ng/ml which is very high in comparison to the lloq in the current work with the enhanced extraction technique. moreover, the same authors literature review reveals that otn pharmacokinetics parameters were studied with renal impairment [ ] , in repositioning studies [ ] and in healthy japanese volunteers [ ] . otn pharmacokinetics were investigating the supra-therapeutic dose of otn [ ] and the effect of age, sex and obesity [ ] . clinical studies were also concerned with the effects of multiple and chronic dosage regimens [ , ] . although, these many articles deal with the pharmacokinetics and/or bio-analysis of otn [ ] [ ] [ ] [ ] [ ] [ ] [ ] , most of the reported studies did not discuss the full details of the validated bio-analytical procedure that may be attributed to using the same methods for many clinical studies by the same common authors [ ] [ ] [ ] [ ] [ ] [ ] . they were emphasizing the findings of the main pharmacokinetic parameters rather than developing or validating a bio-analytical procedure. one liquid chromatography tandem mass spectrometry (lc-ms/ms) method was discussed in full details for otn bio-analysis [ ] but that method was related to rats' plasma only and the authors used only direct precipitation with acetonitrile for the extraction of otn from spiked and biological rats' plasma samples. the authors of the current study modified the previously mentioned rats' plasma method [ ] in another repositioning publication [ ] but with direct precipitation with acetonitrile. the lower limit of quantification (lloq) was ng/ml which is very high in comparison to the lloq in the current work with the enhanced extraction technique. moreover, the same authors reported in another uplc study that the uv detector failed to detect otn in human plasma due to interference [ ] . enhancing the extraction techniques for new drugs enrich the literature and opens the door for further studies. hence, the purpose of our study is to focus on the bio-analysis of otn using lc-ms/ms by means of an enhanced one-step liquid-liquid extraction of otn from human plasma followed by vacuum evaporation and then reconstitution. the bioanalytical method was validated as per fda guidelines to include linearity, selectivity, accuracy, precision, stability and matrix factor [ ] . six different batches of blank human plasma, otn raw material ( . %), alogliptin raw material, internal standard (is) ( . %), marizev ® ( . mg) tablets, tertiary butyl methyl ether (tbme), diethyl ether (dee) were kindly donated by the british university in egypt (cdrd, bue) based on previous research collaborations (repositioning cdrd-bue project). acetonitrile, methanol, and water (all of hplc grade) and formic acid were obtained from (sigma, st. louis, mo, usa). some of the coming conditions were adopted from the authors' previous lc-ms/ms work (published repositioning study on otn and is using direct precipitation for rats' plasma [ ] ). otn standard stock solution in methanol was prepared as ( mm) then working solutions with different concentrations were prepared in methanol ( . , , , , , , and µm). µl of each one of the prepared working solutions was used to spike µl blank plasma to prepare calibrators and qc samples as nm (lloq), , , , nm (medium quality control sample, mqc) and , (high quality control sample, hqc) and nm. an aliquot of µl of each plasma sample was spiked with µl of the is in acetonitrile ( nm), liquid-liquid extracted by . ml of a mixture ( : , v/v) of tbme and dee for min centrifugation at , rpm and then frozen at − • c. . ml of the upper organic level was vacuum evaporated till dryness, reconstituted with µl methanol and injected into the auto-sampler. peak area ratios to is was used against concentrations to predict the calibration curve and the regression equation. fda guidelines [ ] were followed for calibrators to predict the linearity while lloq, mqc, hqc levels (n = ) were used to evaluate both accuracy and precision five times a day and on three successive days. the bias value, standard deviation (s.d.) and % rsd were calculated. six different batches of blank plasma (from different sources) donated form (cdrd-bue) were checked for interference as a measure of selectivity. injection of high values of concentration directly after the blank samples was used to ensure the absence of a significant carry over. matrix factor was estimated molecules , , of by calculating the ratio of the peak area in the presence of the matrix components of human plasma (blank matrix as human plasma sample spiked after extraction with the drug under investigation, otn), to the peak area in absence of the human plasma matrix components (solution of the drug, otn). comparing the area ratios under the peak of the post extracted samples to the neat standards was used to calculate the matrix factor while comparing the area ratios under the peak of the underlying extracted samples to the post extracted samples was used to calculate the extraction recovery. stability of lloq and hqc was estimated based on different bio-assays after leaving the samples h in the auto-sampler or leaving them h at room temperature (bench top stability) or analyzing them after three complete cycles of both freeze and thaw. finally, stability for the long term was investigated by checking the samples after two weeks while freezing at − • c. after the approval of the british university in egypt ethical committee (code: cl/ , march ) and after submission of the proposed lc-ms/ms study to clinicaltrials.gov (id: nct ), one ml blood samples from volunteers were collected after . h of the oral administration of the drug (marizev ® as . mg of otn). then, centrifugation at rpm was performed to extract the plasma. this experimental trial was performed in order to employ the developed method for the determination of otn in human plasma. the proposed study will be valuable for ethnicity comparison studies that will be commenced on otn in egypt by the authors in a prospective study. this study will cover one week bio-analytical assays after its administration as the fda recommends [ ] and will compare results across pharmacokinetic studies to evaluate possible sub-group dissimilarities. ethnic difference has been taken in consideration in recent years. typically, global companies start clinical development in japan after the united states and europe, some genetic factors could explain a significant proportion of dose variability of many drugs between different ethnic groups [ , ] . the recognition of racial differences in disease outcomes and many investigations have identified genetic factors that explain a significant proportion of dose variability. this evidence underpins the prevailing hypothesis that genotype guided therapy should improve dosing accuracy [ , ] that requires validated bioanalytical studies to be used for the comparative pharmacokinetic studies. therefore, an advanced analytical technique and enhanced extraction of drugs from human plasma become a challenging approach that greatly affects pharmacokinetics, other clinical studies based on the drug c min and c max values and the bio-analytical methods sensitivity. in our study, effective liquid-liquid extraction based on the use of tbme-dee mixture as the extracting solvent and vacuum evaporation followed by reconstitution, was established for the extraction of otn from human plasma. enhanced extraction was successfully achieved after using acetonitrile as the diluent solvent for the is decreasing the emulsion formed due to the addition of the aqueous immiscible organic solvent tbme [ ] . therefore, using an appropriate mixture of the extracting solvent accompanied with high volume of acetonitrile; decreased the formed emulsion especially after freezing, which enabled the accurate withdrawing of up to . ml from the upper clear organic layer without interference from the emulsion intermediate layer. adding acetonitrile (that contained the is) was crucial as it decreased the usual resulting emulsion from mixing the immiscible extracting organic solvent and the plasma sample. changing the ratio of the tbme-dee mixture to ( - %) or ( - %), respectively did not affect the readings in the preliminary trials. lc-ms/ms bio-assay of otn ( - nm) was achieved in human plasma using alogliptin as is. mrm function of the transition pairs of m/z . to . for otn and m/z . to . for alogliptin was employed utilizing positive esi mode, as shown in figures - . the transition pairs were also used to verify the identity of the analyte whereas no other qualifying ion transitions were detected or found significant to be used. the use of glass tubes was avoided throughout the investigation especially for vacuum evaporation, centrifugation and reconstitution to avoid the reported gliptins' adhesion [ ] based on previous work by the authors on different gliptins in lab. the authors mentioned in the current study modified the rats' plasma method [ ] in another repositioning publication [ ] but with direct precipitation with acetonitrile, the lloq was ng/ml which is very high in comparison to the lloq in the current work with the enhanced extraction technique ( . ng/ml equivalent to nm). the lloq in the current investigation is less than five times of the previous lloq [ ] . moreover, the same authors in another uplc study [ ] , showed that the uv detector failed to detect otn in human plasma due to interference from plasma endogenous components with the uv detector. they recommended lc-ms/ms accompanied with liquid-liquid extraction/vacuum evaporation to enhance the extraction procedure which is the case in the current study. a comparison between all the previously reported methods for otn extraction and determination in plasma either rats' or human plasma, as shown in table , was implemented. in spite of some common authors reported lower lloq ( ng/ml) in human plasma using ethyl acetate (after ph adjustment) in liquid-liquid extraction [ ] [ ] [ ] [ ] [ ] [ ] , they did not mention the full validation study for the bioanalytical procedure. no data regarding stability studies, selectivity, carry over, extraction recovery, method development, full detailed chromatographic procedure and/or matrix effect was reported. furthermore, the authors of the current investigation did not prefer to use the ph adjustment step that was reported in the previous lc-ms/ms method [ ] [ ] [ ] [ ] [ ] [ ] as it did not show significant difference after using tbme-dee mixture with the satisfying full validation bioanalytical results. molecules , , x for peer review of calibration ( - nm) and full detailed validation outcomes were satisfactory with fda bioanalytical guidelines [ ] . adequate method selectivity from six different batches of blank plasma was designated where no significant interference was observed among the mrm channels in blank ( figure ), zero samples with is ( figure ) and reasonable outcomes at the lloq level of nm (figure ) and no significant carry over was detected. the authors used lc-ms/ms technique as a detection technique rather than the separation strategy and based on their experience with chromatography, they ensured, while developing the lc method, that both the drug and the is did not elute on the dead volume time before applying the sequence order in the instrument. the equation of the calibration curve was; y = . x − . , r = . showing the good linearity of the applied method. accuracy (n = ) and precision (n = ) were within ± % as shown in (table ) . extraction recovery was . % for the lloq and . % for the hqc sample. matrix factor denoting the effect of the matrix on the signal response and the ionization efficiency through matrix enhancement and/or suppression was evaluated. it was ranged from . % to . % indicating ion suppression for all concentrations of otn in plasma. stability measurements mentioned under table . comparison between previously reported methods for omariglitpin (otn) extraction and determination in plasma either rats' plasma or human plasma. extraction lloq application reference liquid-liquid using tbme-dee nm ( . ng/ml) human plasma underlying investigation lc-ms/ms direct precipitation using acetonitrile ng/ml rats' plasma [ ] lc-ms/ms liquid-liquid using ethyl acetate after ph adjustment ng/ml human plasma [ ] [ ] [ ] [ ] [ ] [ ] lc-ms/ms direct precipitation using acetonitrile ng/ml rats' plasma [ ] uplc-uv liquid-liquid using dee . µg/ml rats' plasma [ ] calibration ( - nm) and full detailed validation outcomes were satisfactory with fda bio-analytical guidelines [ ] . adequate method selectivity from six different batches of blank plasma was designated where no significant interference was observed among the mrm channels in blank ( figure ), zero samples with is ( figure ) and reasonable outcomes at the lloq level of nm (figure ) and no significant carry over was detected. the authors used lc-ms/ms technique as a detection technique rather than the separation strategy and based on their experience with chromatography, they ensured, while developing the lc method, that both the drug and the is did not elute on the dead volume time before applying the sequence order in the instrument. the equation of the calibration curve was; y = . x − . , r = . showing the good linearity of the applied method. accuracy (n = ) and precision (n = ) were within ± % as shown in (table ). extraction recovery was . % for the lloq and . % for the hqc sample. matrix factor denoting the effect of the matrix on the signal response and the ionization efficiency through matrix enhancement and/or suppression was evaluated. it was ranged from . % to . % indicating ion suppression for all concentrations of otn in plasma. stability measurements mentioned under methods showed recoveries more than % from the time-zero control which indicates otn stability can be maintained through the sample treatment and storage. eventually, otn validated bioanalytical method was efficaciously applied to analyze human plasma samples. they were collected after . h (tmax) of marizev ® ( . mg) tablets administration to healthy human volunteers (as shown in figure ) showing average concentration of . nm, standard deviation of . nm and percent relative standard deviation of . %. working on anti-diabetic drugs' bio-analysis is of great importance in the current days. it opens the door for ethnicity comparisons related to more pharmacokinetic bioequivalence studies and drugs' approval in more countries leading to better hba c control and other outcomes. bio-analysis of otn ( - nm) using lc-ms/ms was established as per fda guidelines. liquid-liquid extraction based on tbme-dee mixture without the need of ph adjustment and vacuum evaporation followed by reconstitution, was implemented. then, enhanced extraction of otn from human eventually, otn validated bioanalytical method was efficaciously applied to analyze human plasma samples. they were collected after . h (t max ) of marizev ® ( . mg) tablets administration to healthy human volunteers (as shown in figure ) showing average concentration of . nm, standard deviation of . nm and percent relative standard deviation of . %. eventually, otn validated bioanalytical method was efficaciously applied to analyze human plasma samples. they were collected after . h (tmax) of marizev ® ( . mg) tablets administration to healthy human volunteers (as shown in figure ) showing average concentration of . nm, standard deviation of . nm and percent relative standard deviation of . %. working on anti-diabetic drugs' bio-analysis is of great importance in the current days. it opens the door for ethnicity comparisons related to more pharmacokinetic bioequivalence studies and drugs' approval in more countries leading to better hba c control and other outcomes. bio-analysis of otn ( - nm) using lc-ms/ms was established as per fda guidelines. liquid-liquid extraction based on tbme-dee mixture without the need of ph adjustment and vacuum evaporation followed by reconstitution, was implemented. then, enhanced extraction of otn from human working on anti-diabetic drugs' bio-analysis is of great importance in the current days. it opens the door for ethnicity comparisons related to more pharmacokinetic bioequivalence studies and drugs' approval in more countries leading to better hba c control and other outcomes. bio-analysis of otn ( - nm) using lc-ms/ms was established as per fda guidelines. liquid-liquid extraction based on tbme-dee mixture without the need of ph adjustment and vacuum evaporation followed by reconstitution, was implemented. then, enhanced extraction of otn from human plasma was achieved successfully after using acetonitrile as the diluent solvent for the is to decrease the formed emulsion. the effective liquid-liquid extraction showed more sensitive results than direct precipitation. the lloq in the current investigation is less than five times of the previous lloq reported by the same authors. enhancing the extraction techniques for new drugs enrich the literature and opens the door for further pharmacokinetic and bioequivalence studies. clinical course and outcomes of critically ill patients with sars-cov- pneumonia in wuhan, china: a single-centered, retrospective, observational study clinical considerations for patients with diabetes in times of covid- epidemic plasma glucose levels and diabetes are independent predictors for mortality and morbidity in patients with sars spectrum of clinical and radiographic findings in patients with diagnosis of h n and correlation with clinical severity omarigliptin for the treatment of type diabetes the efficacy and safety of once-weekly dpp- inhibitor omarigliptin in patients with type diabetes mellitus a systemic review and meta-analysis omarigliptin: first global approval pharmacological action and clinical results of omarigliptin (marizev ® tablet), a novel dipeptidyl peptidase- inhibitor for once-weekly treatment of type diabetes. folia pharmacol pharmacokinetic-pharmacodynamic (dipeptidyl peptidase- inhibition) model to support dose rationale in diabetes patients, including those with renal impairment, for once-weekly administered omarigliptin repositioning of omarigliptin as a once-weekly intranasal anti-parkinsonian agent single and multiple dose pharmacokinetics and pharmacodynamics of omarigliptin, a novel, once-weekly dipeptidyl peptidase- inhibitor, in healthy japanese men thorough qtc study confirms early pharmacokinetics/qtc modeling: a supratherapeutic dose of omarigliptin, a once-weekly dpp- inhibitor, does not prolong the qtc interval effects of age, sex, and obesity on the single-dose pharmacokinetics of omarigliptin in healthy subjects pharmacokinetic and pharmacodynamic effects of multiple-dose administration of omarigliptin, a once-weekly dipeptidyl peptidase- inhibitor, in obese participants with and without type diabetes mellitus pharmacokinetics and pharmacodynamics of omarigliptin, a once-weekly dipeptidyl peptidase- (dpp- ) inhibitor, after single and multiple doses in healthy subjects absorption, metabolism and excretion of [ c] omarigliptin, a once-weekly dpp- inhibitor, in humans ultra-high pressure liquid chromatography-tandem mass spectrometry method for the determination of omarigliptin in rat plasma and its application to a pharmacokinetic study in rats analysis and bio-analysis of omarigliptin, trelagliptin and alogliptin: applied to biological samples and degradation kinetic study department of health and human services food and drug administration center for drug evaluation and research (cder) center for veterinary medicine (cvm) ethnic differences in pharmacokinetics in new drug applications and approved doses in japan race influences warfarin dose changes associated with genetic factors a simple -well liquid-liquid extraction with a mixture of acetonitrile and methyl t-butyl ether for the determination of a drug in human plasma by high-performance liquid chromatography with tandem mass spectrometry a concise review of the bioanalytical methods for the quantitation of sitagliptin, an important dipeptidyl peptidase- (dpp ) inhibitor, utilized for the characterization of the drug this article is an open access article distributed under the terms and conditions of the creative commons attribution funding: this research was partially funded by the british university in egypt "grant number yirg- " granted to dr. shereen hassib mowaka from bue as the pi with valuable contribution of all the other authors as researchers. moreover, the apc was kindly funded by the british university in egypt. the authors declare no conflict of interest. key: cord- -teahway authors: eleftheriou, phaedra; amanatidou, dionysia; petrou, anthi; geronikaki, athina title: in silico evaluation of the effectivity of approved protease inhibitors against the main protease of the novel sars-cov- virus date: - - journal: molecules doi: . /molecules sha: doc_id: cord_uid: teahway the coronavirus disease, covid- , caused by the novel coronavirus sars-cov- , which first emerged in wuhan, china and was made known to the world in december turned into a pandemic causing more than , deaths worldwide up to april th, . it has . % sequence identity with sars-cov- and the same strategy for host cell invasion through the ace- surface protein. since the development of novel drugs is a long-lasting process, researchers look for effective substances among drugs already approved or developed for other purposes. the d structure of the sars-cov- main protease was compared with the d structures of seven proteases, which are drug targets, and docking analysis to the sars-cov- protease structure of thirty four approved and on-trial protease inhibitors was performed. increased d structural similarity between the sars-cov- main protease, the hcv protease and α-thrombin was found. according to docking analysis the most promising results were found for hcv protease, dpp- , α-thrombin and coagulation factor xa known inhibitors, with several of them exhibiting estimated free binding energy lower than − . kcal/mol and better prediction results than reference compounds. since some of the compounds are well-tolerated drugs, the promising in silico results may warrant further evaluation for viral anticipation. dpp- inhibitors with anti-viral action may be more useful for infected patients with diabetes, while anti-coagulant treatment is proposed in severe sars-cov- induced pneumonia. the coronavirus sars-cov- , responsible for the pandemic which started in wuhan, china, during the latter part of , has spread to countries, areas and territories according to the who [ , ] by april th and continues to spread, infecting approximately , and causing the death of approximately people daily. although young people usually develop mild symptoms, it can cause severe lower respiratory disease affecting mostly the elderly and individuals with other co-morbidities such as cardiovascular problems, pre-existing respiratory disease, diabetes, hypertension or cancer [ ] . sars-cov- belongs to the βcov genera of the coronavirinae subfamily of the order of nidovirals [ ] . compared to sars and mers-coronavirus strains that caused the two most recent epidemics, in - and , respectively-it mostly resembles sars-cov- ( - ), with . % sequence identity and the same mechanism for host cell entrance, via the angiotensin-converting enzyme- (ace- ) surface protein of the cell [ ] . as far as cleavage recognition motifs are concerned, the sars-cov- main protease cleaves the viral polyprotein at no less than sites, recognizing the sequence leu-gln ↓ ser/ala/gly [ ] . the hiv- protease recognizes nine cleavage sites in its polyprotein substrate, with sequence differences that keep the feature of cleavage, most commonly, between phe and pro. alternatively, it cleaves between amino acids with hydrophobic or aromatic side chains (tyr/met/leu/phe ↓ pro/phe/tyr/ala/leu/met) [ ] . the hcv protease cleaves its polyprotein substrate at four sites, recognizing a glu/asp-glu/asp-xxx-cys ↓ ser general motif [ ] . the human organism protease dpp- preferentially cleaves at x ↓ pro or x ↓ ala sites [ ] , whereas thrombin preferentially cleaves at the gly/ala/val/ile/pro-arg ↓ ser/ala/gly/thr/(note/d)-x(note/d)-arg/(note) motif [ ] . the coagulation factor xa cleaves after arg, recognizing a motif val/pro/ile-gln/asp/glu-phe/gly-arg ↓ ser-leu [ ] . renin recognizes the sequence ile-his-pro-phe-his-leu↓ [ ] , while angiotensin-converting enzyme (ace) recognizes the angiotensin i sequence asp-arg-val-tyr-ile-his-pro-phe ↓ his-leu [ ] . similarities between proteases at the catalytic amino acids of the active site and in the nature of amino acids of the substrate may be an indication for efficient inhibition of the proteases by the same inhibitors. however, the nature of surrounding amino acids of the active site and the d structure of the active center are also of great importance. in the present study, the d structure of the sars-cov- main protease was compared with the d structures of proteases which are drug targets with approved inhibitors. in addition, a number of approved inhibitors of the above proteases were evaluated by docking analysis for their ability to inhibit the sars-cov- main protease, interacting with the active site of the enzyme. since the d structure of the active site of the enzyme is crucial for catalytic activity, we proceeded to a comparison of the sars-cov- main protease, mpro, with the hiv- protease, the hcv protease (ns protein) and the human proteases dpp- , thrombin, factor xa, renin and ace, which constitute known drug targets with approved inhibitors. significant d similarity was found between the sars-cov- and the hcv protease (p = . × − ) as well as between the sars-cov- protease and α-thrombin (p = . × − ). interestingly, the protein parts with increased structural alignment included the catalytic area of the enzyme. the lowest similarity was observed in the case of angiotensin-converting enzyme and coagulation factor xa (figures and ) . the structural similarity between the sars-cov- protease and some of the selected proteases, in combination with the existence of the same amino acids at certain positions of the substrate cleavage site, such as ser at the p ' position of the recognition sequence of the hcv protease and thrombin are promising features in the effort to identify effective sars-cov- protease inhibitors among the approved drugs of the selected proteases. to investigate this possibility further, we proceeded to docking analysis of randomly selected approved or on-trial inhibitors of the selected proteases (table ). an exception from the random selection was made for the hiv- inhibitors where lopinavir and ritonavir were selected because they have already been proposed for the treatment of sars-cov- . the rest of the drugs that were selected are the anti-hcv drugs telaprevir and boceprevir, the dpp- inhibitor sitagliptin, the renin inhibitor alisliren, the ace inhibitor captopril, the thrombin direct inhibitors argatroban and dabigatran and the coagulation factor xa inhibitor rivaroxaban. docking analysis was performed using the sars-cov- structure lu . the results were promising for most of the compounds, with the exception of the ace and renin inhibitors, captopril and aliskiren, for which the calculated free binding energy was − . and − . kcal mol − , respectively ( table ). according to our previous experience and to the literature, a free binding energy higher than − . kcal mol − is an indication that the compound is inactive [ , ] . the best results were obtained for the hcv protease inhibitors telaprevir and boceprevir, with free binding energies of − . and − . kcal mol − , respectively, followed by the thrombin inhibitor argatropan and the dpp- inhibitor, sitagliptin, with free binding energies of − . and − . kcal mol − , respectively. the hiv- protease inhibitors ritonavir and lopinavir showed a little lower predicted activity, with free binding energies of − . and − . kcal mol − , respectively. docking analysis to the whole enzyme indicated that the enzyme active site is the second preferred binding site of lopinavir, ritonavir and boceprevir. according to this observation, the expected inhibitory action of these compounds is to be lower than that predicted based on their binding energy to the active site [ ] . rivaroxamban and dabigatran are also expected to be active, with calculated free binding energies of − . and − . kcal mol − , respectively. since the evaluation of the randomly selected hcv protease, dpp- , α-thrombin and coagulation factor xa inhibitors showed promising results, we proceeded to the evaluation of a more extended sample of approved drugs of these categories, including a number of drugs currently in phase iii clinical trial (table , table ). for better estimation of the probable inhibitory effect of the selected compounds, a second protein structure of the sars-cov- protease was also used (pdb id: m n). the whole enzyme, including both protease subunits, was selected for the process. for comparison reasons, the estimated binding energy of the compounds to their initial targets were also calculated. for the same reasons, for some of the most promising sars-cov- candidate inhibitors, the estimated binding energy to the hiv- rvj protease structure was calculated. docking analysis of both sars-cov- protease structures gave similar results. interestingly, out of the compounds which were selected for evaluation exhibited lower estimated binding energy than the initial inhibitor of the m n structure (− . kcal mol − ) [ ] and of the n inhibitor (− . kcal mol − ) [ ] . docking of the compounds to the target proteases for which they were designed showed a generally higher binding probability (lower estimated binding energy) for the initial targets compared to the sars-cov- protease but the most active compounds showed comparable results for the two targets. for example, faldaprevir exhibited an estimated binding energy of − . kcal mol − for the sars-cov- protease and − . kcal mol − for the hcv protease, while teneligliptin showed − . kcal mol − for the new protease and − . kcal mol − for its initial target. table . estimated binding energies of approved or under-trial iii protease inhibitors to the structure of the sars-cov- main protease structures (pdb code: lu and pdb code: m n), to their initial protease target (hcv protease structure wf , dpp- structure ffw, a-thrombin structure dwe, factor xa structure bti) and to the hiv- protease structure rvj. according to the estimated free binding energies, all evaluated drugs could act as sars-cov- protease inhibitors. among the hcv protease inhibitors, faldaprevir exhibited the lowest free binding energy. teneligliptin and gemigliptin are the most promising drugs among the dpp- inhibitors. in the group of anti-coagulants, inogartan and argatroban of the first randomly selected compounds were the most promising among a-thrombin inhibitors and edoxaban among factor xa inhibitors. docking analysis of the whole enzyme indicated that the enzyme active site is the first preferred binding site for all the hcv protease and dpp- inhibitors presented in table but the third preferred site for inogatran and the second for edoxaban. consequently, the expected inhibitory action of inogatran and edoxaban are to be lower than that predicted based on their relative binding energy to the active site. in conclusion, faldaprevir, teneligliptin and gemigliptin are the compounds expected to exhibit the highest activity against the sars-cov- main protease. the sars-cov- protease preferentially cleaves its substrate after gln which follows leu and before a ser or ala or gly amino acid (leu-gln ↓ ser/ala/gly). the gln amino acid, at the p position of the substrate, is an amino acid with a relatively long side chain leading to a polar amide group with hydrogen donor/acceptor possibility. as shown by studies of complexes of the mpro enzyme with substrate analogues [ ] , gln is placed in vicinity to cys , at the subsite s of the active center, surrounded by the side chains of the amino acids asn , glu , his , and his and the main chains of phe and leu . the p amino acid, leu, has a relatively long, hydrophobic but not bulky or aromatic side chain. the side chain of leu is inserted at the s subsite, which consists of the side chains of the amino acids his , met , tyr and met placed in vicinity to asp . the p amino acid of the peptide interacts with the amino acids met , leu , phe , and gln and the main chain of gln . in the case of the substrate analogue n , in addition to polar or hydrophobic interactions, a hydrogen bond (hb) is formed between the his side chain and o of the lactam ring which serves as a mimetic of the gln amino acid of the natural substrate [ ] . a second hb is formed between the h of the closest to the lactam ring nh group of the backbone of the ligand and the co of the main chain of his of the enzyme, while the nh group of the backbone of leu amino acid of the ligand interacts with the co of the backbone of gln of the enzyme. the lactam ring as a mimetic of the gln amino acid of the substrate is also present at the structure of the a-ketomide inhibitors designed by zang et al. [ ] . in this case, the h of the nh group of the lactam ring forms two hydrogen bonds with the co of the main chain of phe and the carboxylate of glu , while the co oxygen of the lactam moiety forms a hydrogen bond with the imidazole of his . in the most active compound of this series, the p leu of the natural substrate has been replaced by a cyclopropane, while the pyridon ring at the p position participates in complex stabilization through hydrogen bonds with the o of the side chain of gln and the nh of the main chain of glu . the two above substrates constitute peptidomimetics with peptide bonds between amino acid mimetic moieties. in both cases, the catalytic amino acid cys forms a covalent bond with the ligand. most of the approved drugs proposed as sars-cov- protease inhibitors according to in silico studies are not peptidomimetics. according to docking analysis, various groups may be placed at the s subsite such as the -methoxy- -methyl-quinoline moiety of simeprevir [ ] or the oxadiazole group of raltegravir, which adopts a curved form within the binding pocket [ ] . interaction with thr , thr and ser also seems to play an important role in complex stabilization of many compounds including raltegravir and ribavirin [ ] . most inhibitors used in this study do not contain the classic peptide bonds but share with peptide substrates the characteristic of many polar, hd/a groups. in gemigliptin, the tetrahydropyridine ring and the -amino- -oxobutyl bridge are placed at the s subsite between glu and cys . a hydrogen bond between the co group of the backbone of glu and the nh group of the -amino- -oxobutyl bridge contributes to complex stabilization, while cys participates in hydrophobic interactions with carbons of the bicyclic moiety ( figure ). the trifluoromethyl pyrimidine moiety interacts with his of the s subsite which stabilizes the complex by halogen bond formation with the f atoms and pi interactions with the pyrimidine ring. halogen bonds are also formed between the f atoms of the fluoropiperidinone ring and amino acids val , arg and thr . in linagliptin, the n of the piperidine ring participates in hydrogen bond interactions with the o of the co group of the main chain of glu of the s subsite, while the nh substituent of the ring forms hb with pro . the his of the s subsite participates in complex stabilization by polar interaction with the n atom of the methylquinazoline and by hydrophobic and pi interactions with the carbons of this moiety, while met also forms hydrophobic interactions. additional stabilization is achieved by the interaction of met with the dihydropurine moiety ( figure ). in faldaprevir, the cyclopentyl carbamate moiety participates in hydrogen bond formation with the asn of the s subsite, while hydrogen bonds are also formed between the -ethenylcyclopropane- -carboxylic acid group and the amino acids thr and ser (figure ). in the case of melagatran, a curved conformation is adopted. the catalytic amino acid, cys , forms hydrophobic interactions with cyclohexan, while asn of the s subsite strongly contributes to complex stabilization via polar interaction with the n atom of the aminoacetyl acid group. at the s subsite, the nh group of the side chain of his participates in hydrogen interaction with the o of the methyl carbamoyl bridge, while met is involved in hydrophobic interactions with the phenyl ring of the molecule. in addition, the oh group of the nearby ser participates in hydrogen bond formation with the nh of the benzimidamide group ( figure ) . in betrixaban, the chloropyridin moiety is placed at the s subsite, interacting via halogen bonds with the amino acids glu , phe and leu and via hydrophobic interactions with cys . in addition, a hydrogen bond is formed between cys and the n of the amide bridge connecting the chloropyridin- yl and the methoxybenzyl rings of the molecule. his of the s subsite forms pi interactions with the carbons of the methoxyphenyl ring. the n,n-dimethylbenzimidamide moiety, placed in the area of the s subsite, strongly contributes to complex stabilization via the hydrophobic interaction between met and the phenyl ring as well as by hydrogen bond formation between the amino acids arg , thr and gln and the n of the n,n-dimethylbenzimidamide moiety ( figure ). in general, there is a great variety between the groups placed at the s or s subsites of the enzyme, as concluded by our results and the literature [ , , ] . most of the amino acids involved in complex stabilization of the natural substrate and the peptide analogues [ , ] participate in interactions with the studied compounds as well. however, the substantial interaction with his in peptidomimetic complexes is not observed in the interaction pattern of the most potent compounds. different structures may offer the possibility of interaction with the crucial amino acids of the sars-cov- active site contributing to complex stability. however, the right distances between polar, hd/a, hydrophobic/aromatic moieties constitute important features of each structure. according to docking analysis, all active molecules are long enough to interact simultaneously with amino acids surrounding glu , cys and amino acids in the vicinity of thr and his and contain several hydrophobic and aromatic moieties as well as hydrogen bond donor/acceptor groups capable of hydrogen bond formation and polar interactions. the enzyme amino acids involved in hydrogen bond formation are glu , pro , gln , arg , thr , thr , thr , his , cys , ser , asn , gly , cys , his , thr , and glu (figures - ) . apart from the polar and hydrophobic interactions, at least two hydrogen bonds are formed in the case of the most active compounds, while interactions with phe were also involved in complex stabilization of argatroban. the most promising compounds of all categories contain multiple hydrophobic moieties as well as polar/hydrogen donor/acceptor moieties. the most active molecules of the categories of gliptines and previrs are relatively long molecules and adopt linear conformations. hydrophobic moieties at distances of approximately Å, Å, Å and Å are present in all potent compounds. met , his , cys , cys , leu of the mpro active site are mainly involved in hydrophobic and pi interactions, with a maximum distance between them of approximately Å (figures - ) . polar or hydrogen donor/acceptor moieties are present at multiple distances, with a distance of Å being common in active compounds. glu and his , at a distance of approximately Å, participate in hydrogen bond and polar interactions of the most potent gliptins (figures and ) , while ser , thr and asn , at a distance of approximately Å, participate in hydrogen bond formation of faldaprevir. in the sars-cov- protease, amino acids involved in hydrophobic and polar interactions are distributed in all the active sites, although pi interactions are mostly observed at the region of his . distances of approximately Å, Å, Å and Å are observed between amino acids participating in polar and hydrophobic interactions. amino acids with hydrophobic or polar/hydrogen donor acceptor properties exist at similar distances at the active sites of the initial target of these compounds, dpp- and the hcv protease, respectively. for example, in the case of the gliptins' initial target dpp- , a great number of tyrosine amino acids are present at the active site. as shown in the case of teneligliptin, figure , the amino acids tyr , phe , tyr , tyr , ty , and tyr are involved in hydrophobic and pi interactions with distances among them varying between Å and Å. the amino acids arg , glu , glu , ser , tyr and asn are involved in polar interactions and hydrogen bond formation. all hydrophobic interactions originate from amino acids oriented at the same moiety of the active site, while the amino acids participating in polar interaction are placed in the other moiety. distances, between and Å are observed between amino acids of the active site participating in polar and hydrophobic interactions. the length of the molecules, the great number of polar and hydrophobic groups, the flexibility of the molecules and the appropriate distances between characteristic groups seem to favor binding of gliptins at the mpro active site. a higher binding energy was estimated for the smaller molecule alogliptin (figure ) . in the case of thrombin inhibitors, a number of hydrogen bonds and hydrophobic interactions mainly stabilize the complex. his , ser and met participate in hydrogen bond formation between melagatran and mpro enzyme, while leu , his , met and cys are involved in hydrophobic interactions. although these interactions lead to a substantially low estimated binding energy of the compound, the significantly higher hydrogen bonds, pi bonds, polar and hydrophobic interactions may be responsible for the much better prediction results for binding to the initial protease target. ala , ala , gly , cys , cys , trp , his and leu seem to be involved in binding to thrombin. the evaluated compounds were approved drugs, currently on the market with the exception of four compounds which were under phase iii trial (table ). there are several compounds among the most promising ones, which are characterized as well-tolerated or having no side effects, such as danoprevir and sovaprevir of the hcv protease inhibitors, teneligliptin and gemigliptin but also trelagliptin, evogliptin and gosogliptin among the dpp- inhibitors, inogatran and melagatran of α-thrombin inhibitors. the dpp- inhibitors developed for the treatment of diabetes type ii as well as the α-thrombin and factor xa inhibitors, used as anti-coagulants, may not be appropriate for patients who do not suffer such problems. however, they may be worth consideration for patients already being treated for type ii diabetes or with anti-coagulants. moreover, higher platelet count and increased coagulation features are observed in covid- patients with severe pneumonia compared to patients with non sars-cov- -induced pneumonia. furthermore, according to recent results, anti-coagulant treatment by heparin administration reduced mortality of covid- patients with severe pneumonia and markedly elevated d-dimers [ ] . in general, the results can be considered as promising for some of the evaluated drugs and could be useful for scientists working in the field. in vitro evaluation is also within our future targets if the recombinant sars-cov- protease becomes available in the market. the d alignment was performed between the d structure of the sars-cov docking analysis was carried out on a molecular docking server using autodock [ , ] , as previously described [ ] . the lamarckian genetic algorithm (lga) and the solis and wets local search method [ ] were used for the docking simulation. during docking, all rotatable torsions were released. autodock parameter set-and distance-dependent dielectric functions were used in the calculation of the van der waals and the electrostatic terms, respectively- different runs that were set to terminate after a maximum of , , energy evaluations were performed in each docking experiment. the population size was set to . a translational step of . Å, and quaternion and torsion steps of were applied during the search. docking analysis of the hcv protease and the hiv- protease presented in table was performed using autodock . . via ligandscout. among the values calculated by the program is the estimated free binding energy, which is an indicator of the probability of the compounds to form a stable complex with the selected enzyme target and consequently the probability to be effective enzyme inhibitors. the protein structures chosen from the protein data bank and the docking center and docking box chosen for each docking analysis were as follows: for docking analysis of the sars-cov- main protease enzyme, the structure lu of the enzyme in a complex with the competitive inhibitor n for active compounds, the docking was repeated, extending the docking box to cover the whole enzyme, to ensure that the compound does not preferentially bind to other sites of the enzyme instead of the active site [ ] . for better estimation of the probable inhibitory effect of the selected compounds, a second protein structure of the sars-cov- protease complexed with the inhibitor , , -trihydroxy- -phenyl- h-chromen- -one was also used (pdb id: m n). in this structure, both subunits were included in the pdb file and the whole enzyme constructed by the two identical subunits was used. the use of the two subunit form was preferred as it could enable the evaluation of a probable interference of the second subunit in binding of the inhibitors, since glu which commonly participates in complex stabilization interacts with ser of the other protein chain. the docking center was set at x = − . , y = − . , z = . and the docking box was set at x = , y = , z = . for comparison reasons, the estimated binding energy of the compounds to their initial targets was also calculated. the structures chosen were the hcv structure wf in complex with asunaprevir (center: for some of the most promising sars-cov- candidate inhibitors, the estimated binding energy to the hiv- protease rvj structure (crystallized in complex with amprenavir) was evaluated (center: x = − . , y = . , z = . , box x = , y = , z = ). in all cases, during enzyme preparation, the inhibitor of the initial enzyme complex was abstracted, and the ph was set at . for ligand preparation. for the evaluation of the docking method, the initial ligand was docked to the enzyme and the position was compared with its initial position at the complex. examples of the similarity of the two positions are shown at figure . stomach ache, abnormal results of blood tests that measure liver function, anemia [ ] otamixaban -ended at phase iii sanofi - figure . docking of the initial ligand ( , , -trihydroxy- -phenyl- h-chromen- -one) of the m n complex to the sars-cov- protease structure m n and of the initial ligand (sitagliptin) to the ffw structure of dpp- . the position of the docked ligand (green) is very close to that of the initial ligand (magenta). the d structure alignment of the sars-cov- main protease with the hiv- and hcv viral proteases as well as with the human proteases dpp- , thrombin, coagulation factor xa, renin and ace showed greater similarity with the hcv protease and with thrombin. this finding, in combination with the fact that these two proteases preferentially cleave before ser like sars-cov- protease, enhances the possibility of finding effective sars-cov- inhibitors among the approved anti-hcv protease and anti-thrombin inhibitors. docking analysis of approved or phase iii trial protease inhibitors to the sars-cov- protease revealed several drugs that may act as sars-cov- protease inhibitors, within the classes of the hcv protease, dpp- , α-thrombin and coagulation factor xa inhibitors. twenty five of them exhibited estimated binding energy lower than the reference inhibitor n , and twenty one lower than − . kcal mol − . several of the most promising compounds are approved drugs characterized as well tolerated in subjects. compared to lopinavir and ritonavir, most of the evaluated compounds showed better results, as characterized by lower predicted free binding energy and high preference for binding to the active site compared to other sites of the enzyme. these promising in silico results encourage further evaluation for in vitro and in vivo anti-viral activity. the dpp- inhibitors developed for the treatment of type ii diabetes, as well as α-thrombin and factor xa inhibitors used as anti-coagulants, may not be appropriate for patients who do not suffer such problems but may be useful for patients who need such treatment. it is worth mentioning that diabetic patients belong to the high-risk group for this virus, while anti-coagulant therapy has been proposed for covid- patients with severe pneumonia. the authors declare no conflict of interest. rna-dependent rna polymerase dpp- dipeptidyl peptidase- covid- ) outbreak situation covid- coronavirus pandemic. available online genomic characterization of the novel human pathogenic coronavirus isolated from a patient with atypical pneumonia after visiting wuhan a pneumonia outbreak associated with a new coronavirus of probable bat origin therapeutic options for the novel coronavirus ( -ncov) coronaviruses-drug discovery and therapeutic options hydroxychloroquine, a less toxic derivative of chloroquine, is effective in inhibiting sars-cov- infection in vitro apeiron biologics to trial coronavirus drug candidate in china rapid identification of potential inhibitors of sars-cov- main protease by deep docking of . billion compounds potential inhibitors against -ncov coronavirus m protease from clinically approved medicines in silico screening of natural compounds against covid- by targeting mpro and ace using molecular docking virtual screening and repurposing of fda approved drugs against covid- main protease targeting sars-cov- : a systematic drug repurposing approach to identify promising inhibitors against c-like proteinase and -o-ribose methyltransferase ul-haq, z. identification of chymotrypsin-like protease inhibitors of sars-cov- via integrated computational approach molecular investigation of sars-cov- proteins and their interactions with antiviral drugs putative inhibitors of sars-cov- main protease from a library of marine natural products: a virtual screening and molecular modeling study an investigation into the identification of potential inhibitors of sars-cov- main protease using molecular docking study potential inhibitors of coronavirus -chymotrypsin-like protease ( cl pro ): an in silico screening of alkaloids and terpenoids from african medicinal plants potential anti-sars-cov- drug candidates identified through virtual screening of the chembl database for compounds that target the main coronavirus protease. febs open bio emerging principles in protease-based drug discovery the use of stems in the selection of international nonproprietary names (inn) for pharmaceutical substances; world health organization crystal structure of sars-cov- main protease provides a basis for design of improved α-ketoamide inhibitors comparative studies on retroviral proteases: substrate specificity new details of hcv ns / a proteinase functionality revealed by a high-throughput cleavage assay dipeptidyl peptidase iv inhibition for the treatment of type diabetes: potential importance of selectivity over dipeptidyl peptidases and factor xa active site substrate specificity with substrate phage display and computational molecular modeling the extended cleavage specificity of human thrombin molecular recognition and regulation of human angiotensin-i converting enzyme (ace) activity by natural inhibitory peptides the his-pro-phe motif of angiotensinogen is a crucial determinant of the substrate specificity of renin direct thrombin inhibitors an ensemble view of thrombin allostery docking analysis targeted to the whole enzyme: an application to the prediction of inhibition of ptp b by thiomorpholine and thiazolyl derivatives prediction of enzyme inhibition and mode of inhibitory action based on calculation of distances between hydrogen bond donor/acceptor groups of the molecule and docking analysis: an application on the discovery of novel effective ptp b inhibitors structure of mpro from covid- virus and discovery of its inhibitors difference of coagulation features between severe pneumonia induced by sars-cov and non-sars-cov mmdb and vast+: tracking structural similarities between macromolecular complexes application of the pm semi-empirical method to modeling proteins enhances docking accuracy of auto-dock automated docking using a lamarckian genetic algorithm and an empirical binding free energy function minimization by random search techniques a phase study in combination with bms- and bms- in japanese hepatitis c virus (hcv) patients. available online daclatasvir plus asunaprevir for chronic hcv genotype b infection pharmacokinetics, safety, and tolerability of faldaprevir in patients with renal impairment management of adverse events during the treatment of chronic hepatitis c infection patients with hcv genotype infection: safety, antiviral activity, resistance, and pharmacokinetics fda drug safety communication: fda adds warnings about heart failure risk to labels of type diabetes medicines containing saxagliptin and alogliptin multiple-dose pharmacokinetics and pharmacodynamics of evogliptin (da- ), a novel dipeptidyl peptidase iv inhibitor, in healthy volunteers addition of dipeptidyl peptidase- inhibitors to sulphonylureas and risk of hypoglycaemia: systematic review and meta-analysis inhibitors for type diabetes: drug safety communication-may cause severe joint pain sitagliptin phosphate monograph for professionals available online this article is an open access article distributed under the terms and conditions of the creative commons attribution key: cord- -h bu jrq authors: kim, sarah; min, sein; chae, heelim; jeong, hye jin; namgoong, sung keon; oh, sangwon; jeong, keunhong title: hyperpolarization of nitrile compounds using signal amplification by reversible exchange date: - - journal: molecules doi: . /molecules sha: doc_id: cord_uid: h bu jrq signal amplification by reversible exchange (sabre), a hyperpolarization technique, has been harnessed as a powerful tool to achieve useful hyperpolarized materials by polarization transfer from parahydrogen. in this study, we systemically applied sabre to a series of nitrile compounds, which have been rarely investigated. by performing sabre in various magnetic fields and concentrations on nitrile compounds, we unveiled its hyperpolarization properties to maximize the spin polarization and its transfer to the next spins. through this sequential study, we obtained a ~ -fold enhancement for several nitrile compounds, which is the highest number ever reported for the nitrile compounds. our study revealed that the spin polarization on hydrogens decreases with longer distances from the nitrile group, and its maximum polarization is found to be approximately g with μl of substrates in all structures. interestingly, more branched structures in the ligand showed less effective polarization transfer mechanisms than the structural isomers of butyronitrile and isobutyronitrile. these first systematic sabre studies on a series of nitrile compounds will provide new opportunities for further research on the hyperpolarization of various useful nitrile materials. nuclear magnetic resonance (nmr) is a powerful tool for identifying the (in)organic and biological structures of molecules in various states. the study of the dynamics of molecules is a key subject in the area of nmr studies with specific pulse sequences. one of the prominent applications of this nmr method is magnetic resonance imaging (mri), which is a precise, non-invasive body-scanning technique. the signals in nmr depend on the population differences in magnetic-field-induced zeeman splitting, which is proportional to the external field, and follows a boltzmann distribution. hence, a strong magnetic field is needed. this results in high costs in purchasing a strong magnet as well as maintaining the superconducting magnet. to overcome these shortcomings, hyperpolarization (hp) techniques have been developed to increase the signal strength [ ] [ ] [ ] [ ] . along with other well-known hyperpolarization techniques, parahydrogen-induced hyperpolarization, which uses a higher proportion of parahydrogen gas than orthohydrogen, has been proposed as a potential cost-efficient and rapid technique, with processing in non-harsh conditions. hyperpolarization can be achieved using spin-state energy transfer from polarized antiparallel spins of parahydrogen [ , ] . its benefits are particularly significant in focusing on various molecular structures and medical molecules , , of applications such as mri. parahydrogen-induced polarization (phip) uses the hydrogenation reaction on an unsaturated bond, which induces hyperpolarization through additional covalent bonding on carbon [ ] [ ] [ ] . this process results in structural changes after the hydrogenation reaction. while signal amplification by reversible exchange (sabre), which is another way of using parahydrogen, employs a non-hydrogenation parahydrogen-based hyperpolarization technique [ ] [ ] [ ] , sabre relies on the temporary bonding of the substrate at the metal center (iridium) to enable hyperpolarization creation on the nucleus spins of the substrates. with these powerful characteristics, most studies using sabre have focused on specific structures with sp nitrogen atoms, such as pyridine, purines, diazirines, triazole, or their derivatives [ ] [ ] [ ] [ ] [ ] [ ] . previous studies helped in setting up the polarization mechanism, and its trend in the polarization for the structures has been studied over the last few decades [ ] [ ] [ ] [ ] [ ] . on the other hand, newly discovered structures lack these perspectives. hence, reports on detecting small concentrated materials and reaction mechanisms that have the potential for application in the near future are important [ ] [ ] [ ] [ ] . in this context, polarization with nitrile is quite new and studies focusing on nitrile compounds have not been conducted systemically, other than for the aromatic and nonaromatic nitrile materials with low enhancement factors [ , ] . furthermore, n and c polarization transfer via nitrile groups (short range coupling) has recently attracted interest due to its potentially long relaxation time (t ) [ , ] . therefore, its systematic study with nitrile groups is expected to render more insights into the polarization transfer mechanism, such as sabre-sheath (sabre in shield enables alignment transfer to heteronuclei), for a heteronuclear system in the future. the nitrile is an organic compound that has a −c≡n functional group in the structure. nitriles are found in many useful organics including nitrile gloves, nitrile-containing polymers used in laboratory work, and methyl cyanoacrylate, which is used in super glue. more importantly, over nitrile-containing pharmaceuticals are now prescribed all over the world [ , ] . the core structure, i.e., the nitrile group, is robust, and it is not well metabolized. it can stay for a long time in the body [ , ] . overall, this material is widely used not only in industry but also in fundamental research and needs to be investigated more systematically and extensively. here, we present a systematic hyperpolarization study on nitriles using sabre to obtain the maximum hyperpolarization in different magnetic fields and concentrations. our study unveiled some unprecedented results, which showed much more signal enhancement than that ever reported before and polarization trends with different carbon attachments. after the activation of an ir-catalyst ( . µmol) in bubbling parahydrogen for min, different volumes of acetonitrile were added to the solution (meoh-d µl). the polarization transfer from parahydrogen to acetonitrile was performed in the solenoid coil, which was matched with different magnetic fields. subsequent experiments with other nitrile compounds were performed using the same procedure ( figure ). it is worth noting that the enhancement factor of more than -fold in the hyperpolarization is much bigger than any previously reported result. for example, it is only -fold in the case of acetonitrile. however, it can be enhanced as much as times by adding pyridine-d ( times for pyridine), which was introduced as a co-substrate. further studies on this co-substrate with more nitrile compounds would be needed in the future [ ] . furthermore, its polarization is dependent on the concentration and is maximized with μl of acetonitrile in μl of solvent, which is normally it is worth noting that the enhancement factor of more than -fold in the hyperpolarization is much bigger than any previously reported result. for example, it is only -fold in the case of acetonitrile. however, it can be enhanced as much as times by adding pyridine-d ( times for pyridine), which was introduced as a co-substrate. further studies on this co-substrate with more nitrile compounds would be needed in the future [ ] . furthermore, its polarization is dependent on the concentration and is maximized with μl of acetonitrile in μl of solvent, which is normally it is worth noting that the enhancement factor of more than -fold in the hyperpolarization is much bigger than any previously reported result. for example, it is only -fold in the case of acetonitrile. however, it can be enhanced as much as times by adding pyridine-d ( times for pyridine), which was introduced as a co-substrate. further studies on this co-substrate with more nitrile compounds would be needed in the future [ ] . furthermore, its polarization is dependent on the concentration and is maximized with µl of acetonitrile in µl of solvent, which is normally attributed to the chemical exchange dynamics with optimized molar concentrations [ ] . this is the main reason behind the polarization enhancement, and its efficiency is also related to the relaxation effect induced by the ir-catalyst including the proton t time. its phase trend ( figure ) is changed from the earth magnetic field (~ . g) and reversed again by increasing the magnetic field. this is different from the previously reported phenomenon [ ] and seems to be slightly different in terms of the polarization transfer mechanism from parahydrogen and needs further investigation. it is speculated to be because of the different matching condition of the magnetic field with different j-j coupling from both hydrides in the ir-catalyst. we found that the concentration showing the maximum polarization number at the same magnetic field was different. in the earth field, a small amount of acetonitrile showed the biggest polarization, whereas there was a trend of a higher concentration of substrate leading to larger polarization in the higher magnetic field. this indicates that the polarization efficiency depends not only on the magnetic field but also on the concentration of the substrate in acetonitrile sabre. molecules , , x of attributed to the chemical exchange dynamics with optimized molar concentrations [ ] . this is the main reason behind the polarization enhancement, and its efficiency is also related to the relaxation effect induced by the ir-catalyst including the proton t time. its phase trend (figure ) is changed from the earth magnetic field (~ . g) and reversed again by increasing the magnetic field. this is different from the previously reported phenomenon [ ] and seems to be slightly different in terms of the polarization transfer mechanism from parahydrogen and needs further investigation. it is speculated to be because of the different matching condition of the magnetic field with different j-j coupling from both hydrides in the ir-catalyst. we found that the concentration showing the maximum polarization number at the same magnetic field was different. in the earth field, a small amount of acetonitrile showed the biggest polarization, whereas there was a trend of a higher concentration of substrate leading to larger polarization in the higher magnetic field. this indicates that the polarization efficiency depends not only on the magnetic field but also on the concentration of the substrate in acetonitrile sabre. the different aspect of propionitrile compared to acetonitrile is that one more methyl group is attached than in acetonitrile, which can be polarized by the polarization transfer from the protons from h- . therefore, it causes less polarization on the h- than the h- , which is supported by our result (figure ) . the maximum enhancement factor is approximately -fold, which is shown in the g in µl and g in µl of propionitrile, which has almost the same trend as acetonitrile (the concentration for each volume is indicated in supplementary materials). however, more interestingly, the polarization in the earth field is bigger than g in all the h- protons, whereas the h- showed the same trend as the acetonitrile case. this can be attributed to the polarization transfer through the long range j-j coupling [ ] , which can be matched with a low magnetic field. this is supported by the smaller polarization enhancement than of the protons in h- . however, the protons in h- can be hyperpolarized in higher magnetic fields, and its polarization is transferred into the h- . even though the polarization transfer is active from h- to h- , the enhancement factor of h- in propionitrile is similar to that of acetonitrile. the different aspect of propionitrile compared to acetonitrile is that one more methyl group is attached than in acetonitrile, which can be polarized by the polarization transfer from the protons from h- . therefore, it causes less polarization on the h- than the h- , which is supported by our result (figure ) . the maximum enhancement factor is approximately -fold, which is shown in the g in μl and g in μl of propionitrile, which has almost the same trend as acetonitrile (the concentration for each volume is indicated in supplementary material). however, more interestingly, the polarization in the earth field is bigger than g in all the h- protons, whereas the h- showed the same trend as the acetonitrile case. this can be attributed to the polarization transfer through the long range j-j coupling [ ] , which can be matched with a low magnetic field. this is supported by the smaller polarization enhancement than of the protons in h- . however, the protons in h- can be hyperpolarized in higher magnetic fields, and its polarization is transferred into the h- . even though the polarization transfer is active from h- to h- , the enhancement factor of h- in propionitrile is similar to that of acetonitrile. these two structures are structural isomers with differently branched structures. the largest enhanced signals from protons were on h- in both structures-with an approximately -fold enhancement, which showed almost the same trend with propionitrile and acetonitrile. in both cases, μl was the optimal volume for obtaining the highest polarization ( figure ). for the polarizationtransfer efficiency, polarization is transferred to the other protons from h- in butyronitrile. however, polarization transfer in isobutyronitrile is much less compared to that in butyronitrile case. this indicates that the polarization efficiency is closely related to the polarization-transferring proton number (two protons in butyronitrile vs. one proton in isobutyronitrile) and adjacent numbers of protons (polarization-transferred protons) that are coupled (two protons in butyronitrile vs. six protons in isobutyronitrile). this reveals that a linear type of nitrile or other functional groups that these two structures are structural isomers with differently branched structures. the largest enhanced signals from protons were on h- in both structures-with an approximately -fold enhancement, which showed almost the same trend with propionitrile and acetonitrile. in both cases, µl was the optimal volume for obtaining the highest polarization ( figure ). for the polarization-transfer efficiency, polarization is transferred to the other protons from h- in butyronitrile. however, polarization transfer in isobutyronitrile is much less compared to that in butyronitrile case. this indicates that the polarization efficiency is closely related to the polarization-transferring proton number (two protons in butyronitrile vs. one proton in isobutyronitrile) and adjacent numbers of protons (polarization-transferred protons) that are coupled (two protons in butyronitrile vs. six protons in isobutyronitrile). this reveals that a linear type of nitrile or other functional groups that are chelated with the ir-catalyst would be a better option for achieving higher polarization-transfer efficiency. molecules , , x of are chelated with the ir-catalyst would be a better option for achieving higher polarization-transfer efficiency. we additionally performed sabre on valeronitrile, which contains one more methyl group in the nitrile than butyronitrile. its optimal polarization factor with different magnetic fields and concentrations also showed the same trend as the butyronitrile case-an approximately -fold we additionally performed sabre on valeronitrile, which contains one more methyl group in the nitrile than butyronitrile. its optimal polarization factor with different magnetic fields and concentrations also showed the same trend as the butyronitrile case-an approximately -fold enhancement in g with a µl amount of valeronitrile. it is hard to discern two hyperpolarized signals from the methyl group of h- in the spectrum, which have a small chemical shift difference. therefore, we calculated the enhancement factor on all five hydrogens from h- of valeronitrile's structure. almost all the trends were similar to those for butyronitrile, except strong hyperpolarization on all protons in the g for µl of substrate, which may be attributed to the change in the chelating strength caused by the increased weight and coupling strength. (figure ) however, this needs to be studied further. molecules , , x of enhancement in g with a μl amount of valeronitrile. it is hard to discern two hyperpolarized signals from the methyl group of h- in the spectrum, which have a small chemical shift difference. therefore, we calculated the enhancement factor on all five hydrogens from h- of valeronitrile's structure. almost all the trends were similar to those for butyronitrile, except strong hyperpolarization on all protons in the g for μl of substrate, which may be attributed to the change in the chelating strength caused by the increased weight and coupling strength. (figure ) however, this needs to be studied further. mhz h spectra were obtained with spinsolve ultra (magritek, abingdon, uk). this device was automated with a % d o sample in shimming, and the nmr signal was checked using a wilkinson's catalyst. the parahydrogen generator was activated by a home-built instrument [ ] . hydrogen gases (a spin isomer mixture of ortho-h and para-h ) were passed through heat exchangers filled with fe(oh)o catalysts in a liquid nitrogen dewar to obtain up to % p-h . an ir-catalyst, (ir(cod)(imes)cl) (cod=η - , -cyclooctadiene and imes= , -bis( , , trimethylphenyl)imidazole- -ylidene) [ , ] and nitrile compound for each concentration ( , , , and μl of acetonitrile, corresponding to , , , and μmol, respectively; all concentration amounts are tabulated in supplementary material) were added to the nmr tube. after dissolving in μl of deuterated methanol, the solution was bubbled through the tube with para-hydrogen for min [ ] . the mixture was foamed for min at each magnetic field before being moved to the nmr spectrometer. the spectrum was obtained at mhz. nmr data were processed and analyzed using the mnova software ( . . , masterlab research, s.l, abingdon, uk). in this study, we applied the promising hyperpolarization technique sabre on a series of nitrile compounds with an additional methyl group attached. by performing sabre with various magnetic fields and substrate concentrations, we unveiled its hyperpolarization characteristics and properties to maximize the polarization and its transfer efficiency. through this sequential study, we could conclude that the polarization on hydrogen decreases with increasing distance from the nitrile group and its highest polarization is observed at approximately g and with μl of substrates in all mhz h spectra were obtained with spinsolve ultra (magritek, abingdon, uk). this device was automated with a % d o sample in shimming, and the nmr signal was checked using a wilkinson's catalyst. the parahydrogen generator was activated by a home-built instrument [ ] . hydrogen gases (a spin isomer mixture of ortho-h and para-h ) were passed through heat exchangers filled with fe(oh)o catalysts in a liquid nitrogen dewar to obtain up to % p-h . an ir-catalyst, (ir(cod)(imes)cl) (cod=η - , -cyclooctadiene and imes= , -bis( , , trimethylphenyl)imidazole- -ylidene) [ , ] and nitrile compound for each concentration ( , , , and µl of acetonitrile, corresponding to , , , and µmol, respectively; all concentration amounts are tabulated in supplementary materials) were added to the nmr tube. after dissolving in µl of deuterated methanol, the solution was bubbled through the tube with para-hydrogen for min [ ] . the mixture was foamed for min at each magnetic field before being moved to the nmr spectrometer. the spectrum was obtained at mhz. nmr data were processed and analyzed using the mnova software ( . . , masterlab research, s.l, abingdon, uk). in this study, we applied the promising hyperpolarization technique sabre on a series of nitrile compounds with an additional methyl group attached. by performing sabre with various magnetic fields and substrate concentrations, we unveiled its hyperpolarization characteristics and properties to maximize the polarization and its transfer efficiency. through this sequential study, we could conclude that the polarization on hydrogen decreases with increasing distance from the nitrile group and its highest polarization is observed at approximately g and with µl of substrates in all structures. furthermore, we obtained the best enhancement factor of more than -fold for various nitrile compounds using percent of parahydrogen. therefore, we can expect enhanced polarization numbers in the future using a higher concentration of parahydrogen. this study has only been performed in methanol, which is not a biological solvent. to be applicable in biological systems, a hyperpolarization study in biological solvent systems or new strategy for obtaining hyperpolarization in an aqueous solvent would be needed [ ] [ ] [ ] . interestingly, the more branched structures in the ligand showed less effective polarization-transfer mechanisms than the butyronitrile and isobutyronitrile sabre results. furthermore, upon investigating several cases, the h- protons from nitrile compounds showed higher polarization, which is contrary to previously reported cases. it is noteworthy that the effective polarization transfer may be possible using low or ultralow magnetic fields, which are normally of approximately g. these first-ever systematic sabre studies on a series of nitrile compounds will certainly widen the new possibilities for more hyperpolarizable chemical materials using sabre in the future. increase in signal-to-noise ratio of > , times in liquid-state nmr design and implementation of c hyper polarization from para-hydrogen, for new mri contrast agents transformation of symmetrization order to nuclear-spin magnetization by chemical reaction and nuclear magnetic resonance dynamic nuclear polarization at high magnetic fields parahydrogen induced polarization in scalar coupled systems: analytical solutions for spectral patterns and their field dependence. zeitschrift für phys. chemie parahydrogen and synthesis allow dramatically enhanced nuclear alignment nmr studies of the reaction path of the o-h /p-h spin conversion catalyzed by vaska's complex in the solid state para-hydrogen induced polarization (phip) para hydrogen induced polarization in hydrogenation reactions reversible interactions with para-hydrogen enhance nmr sensitivity by polarization transfer a theoretical basis for spontaneous polarization transfer in non-hydrogenative parahydrogen-induced polarization fine-tuning the efficiency of para-hydrogen-induced hyperpolarization by rational n-heterocyclic carbene design trace analysis by low-field nmr: breaking the sensitivity limit toward biocompatible nuclear hyperpolarization using signal amplification by reversible exchange: quantitative in situ spectroscopy and high-field imaging optimization of sabre for polarization of the tuberculosis drugs pyrazinamide and isoniazid iridium n-heterocyclic carbene complexes as efficient catalysts for magnetization transfer from para-hydrogen the search for new hydrogenation catalyst motifs based on n-heterocyclic carbene ligands reaction monitoring using sabre-hyperpolarized benchtop ( t) nmr spectroscopy parahydrogen-induced polarization in the hydrogenation of lignin-derived phenols using wilkinson's catalyst signal amplification by reversible exchange for covid- antiviral drug candidates demonstration of heterogeneous parahydrogen induced polarization using hyperpolarized agent migration from dissolved rh(i) complex to gas phase chemical kinetics and spin dynamics of the formation of hyperpolarization hyperpolarized nmr spectroscopy: d-dnp, phip, and sabre techniques detecting low concentrations of unsaturated c-c bonds by parahydrogen-induced polarization using an efficient home-built parahydrogen generator monitoring of hydrogenation by benchtop nmr with parahydrogen-induced polarization squid-based ultralow-field mri of a hyperpolarized material using signal amplification by reversible exchange organic reaction monitoring of a glycine derivative using signal amplification by reversible exchange-hyperpolarized benchtop nuclear magnetic resonance spectroscopy strategies for the hyperpolarization of acetonitrile and related ligands by sabre iridium(iii) hydrido n-heterocyclic carbene-phosphine complexes as catalysts in magnetization transfer reactions hyperpolarization induced by parahydrogen in reversible exchange the absence of quadrupolar nuclei facilitates efficient c hyperpolarization via reversible exchange with parahydrogen nitrile-containing pharmaceuticals: efficacious roles of the nitrile pharmacophore identification of selective, nonpeptidic nitrile inhibitors of cathepsin s using the substrate activity screening method investigation of ketone warheads as alternatives to the nitrile for preparation of potent and selective cathepsin k inhibitors a novel class of nonpeptidic biaryl inhibitors of human cathepsin k investigating pyridazine and phthalazine exchange in a series of iridium complexes in order to define their role in the catalytic transfer of magnetisation from para-hydrogen low cost and portable parahydrogen generator for the phip spontaneous transfer of parahydrogen derived spin order to pyridine at low magnetic field achieving % nmr polarization in water in less than min using sabre parahydrogen-based hyperpolarization for biomedicine nmr detection in biofluid extracts at sub-µm concentrations: via para-h induced hyperpolarization key: cord- - vorrnq authors: vivek-ananth, r.p.; rana, abhijit; rajan, nithin; biswal, himansu s.; samal, areejit title: in silico identification of potential natural product inhibitors of human proteases key to sars-cov- infection date: - - journal: molecules doi: . /molecules sha: doc_id: cord_uid: vorrnq presently, there are no approved drugs or vaccines to treat covid- , which has spread to over countries and at the time of writing was responsible for over , deaths worldwide. recent studies have shown that two human proteases, tmprss and cathepsin l, play a key role in host cell entry of sars-cov- . importantly, inhibitors of these proteases were shown to block sars-cov- infection. here, we perform virtual screening of , phytochemicals produced by indian medicinal plants to identify natural product inhibitors of tmprss and cathepsin l. autodock vina was used to perform molecular docking of phytochemicals against tmprss and cathepsin l. potential phytochemical inhibitors were filtered by comparing their docked binding energies with those of known inhibitors of tmprss and cathepsin l. further, the ligand binding site residues and non-covalent interactions between protein and ligand were used as an additional filter to identify phytochemical inhibitors that either bind to or form interactions with residues important for the specificity of the target proteases. this led to the identification of inhibitors of tmprss and inhibitors of cathepsin l among phytochemicals of indian medicinal plants. further, we have performed molecular dynamics (md) simulations to analyze the stability of the protein-ligand complexes for the three top inhibitors of tmprss namely, qingdainone, edgeworoside c and adlumidine, and of cathepsin l namely, ararobinol, (+)-oxoturkiyenine and α, α-cinchophylline. interestingly, several herbal sources of identified phytochemical inhibitors have antiviral or anti-inflammatory use in traditional medicine. further in vitro and in vivo testing is needed before clinical trials of the promising phytochemical inhibitors identified here. in december , a new respiratory disease with unknown cause with clinical symptoms of fever, cough, shortness of breath, fatigue and pneumonia was first reported in wuhan, (china) [ ] [ ] [ ] . while most cases of this new disease show mild to moderate symptoms, a small fraction of cases, especially those with comorbid conditions like diabetes and hypertension, can develop fatal conditions such as acute respiratory distress syndrome (ards) due to severe lung damage [ ] . in january , a novel betacoronavirus, initially named -ncov, was discovered to be the etiological agent of this also showed that tmprss is more essential for s protein priming and sars-cov- entry. in parallel, ou et al. [ ] used specific inhibitors of cathepsin l and cathepsin b to show that cathepsin l rather than cathepsin b is essential for s protein priming of sars-cov- and membrane fusion in lysosomes. these studies highlight at least two alternate pathways for host cell entry of sars-cov- . on the one hand, after sars-cov- attachment to ace , the membrane fusion and cytoplasmic entry can occur at the plasma membrane provided the cell surface protease tmprss is available to carry out s protein priming. on the other hand, after sars-cov- attachment to ace , the virus can be internalized as part of endosomes in the endocytic pathway, and later, the membrane fusion and cytoplasmic entry will occur in lysosomes provided the lysosomal protease cathepsin l is available to carry out s protein priming [ ] [ ] [ ] . depending on the target cell and associated expression of host cell proteases, sars-cov- may use one of the alternative pathways for host cell entry. importantly, the above-mentioned studies also showed that known inhibitors camostat mesylate and nafamostat mesylate of tmprss and e- d and pc- (sid ) of cathepsin l can block or significantly reduce the host cell entry of sars-cov- [ ] [ ] [ ] . in conclusion, human proteases tmprss and cathepsin l are key factors for host cell entry and are important targets for anti-covid drugs [ ] [ ] [ ] ] . to expedite this search for anti-covid drugs, several computational studies have used homology modeling or published crystal structures of sars-cov- proteins, molecular docking and diverse small molecule libraries, to predict potential inhibitors of sars-cov- proteins including among existing approved drugs for repurposing and natural compounds (see e.g., [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] ). in comparison, fewer computational studies [ , ] have focussed on identification of potential inhibitors of host factors. in this work, we perform virtual screening of a large phytochemical library specific to indian medicinal plants to identify potential natural product inhibitors of tmprss and cathepsin l. plant-based natural products have made immense contributions to drug discovery [ ] . specifically, % of the small-molecule drugs approved to date by the us food and drug administration (fda) are either natural products or natural product derivatives. recently, there are reports from china of successful use of traditional chinese medicine and associated herbs in treatment of covid- patients [ ] . on similar lines, there have been suggestions to tap the rich legacy of traditional indian medicine and information on phytochemicals of indian herbs in the search for anti-covid drugs [ ] . previously, some of us have built imppat, the largest resource to date on phytochemicals of indian herbs [ ] . in this work, we perform molecular docking using a large library of , phytochemicals compiled mainly from imppat to identify potential natural product inhibitors of tmprss and cathepsin l. subsequently, we have also performed molecular dynamics (md) simulations to investigate the stability of protein-ligand complexes for the top phytochemical inhibitors of tmprss and cathepsin l identified in this work. this computational study aims to predict potential phytochemical inhibitors of human proteases, tmprss and cathepsin l, that are important for priming of s protein and cell entry of sars-cov- [ ] [ ] [ ] . briefly, the workflow for this virtual screening is as follows (figure ). in the first stage, we prepared the ligands for molecular docking with target proteases. we compiled a library of , phytochemicals produced by medicinal plants used in traditional indian medicine, and the main source of this compilation was imppat [ ] , the largest resource on phytochemicals of indian herbs to date (see section ). next, the standard drug-likeness measure, lipinski's rule of five (ro ) [ ] , was used to filter a subset of , drug-like phytochemicals. next, the filtered phytochemicals were prepared for docking by retrieving their d structures from pubchem [ ] followed by energy-minimization using openbabel [ ] (section ). in the second stage, we prepared the target proteins for docking with prepared ligands. for tmprss , the d structure is yet to be determined experimentally, and thus, we built a homology model of tmprss using swiss-model [ ] which was used for docking after energy-minimization using ucsf chimera [ ] (figure ; section ). figure displays the tmprss model structure with the catalytic triad s , h and d and the substrate binding residue d in the s subsite. for cathepsin l, the crystal structure (pdb mqy) with . Å resolution was used for docking after energy-minimization using ucsf chimera (figure ; section ). figure displays the cathepsin l structure with the catalytic dyad c and h in s subsite, and other important residues in s and s subsites. molecules , , x of model of tmprss using swiss-model [ ] which was used for docking after energyminimization using ucsf chimera [ ] (figure ; section ). figure displays the tmprss model structure with the catalytic triad s , h and d and the substrate binding residue d in the s subsite. for cathepsin l, the crystal structure (pdb mqy) with . Å resolution was used for docking after energy-minimization using ucsf chimera (figure ; section ). figure displays the cathepsin l structure with the catalytic dyad c and h in s subsite, and other important residues in s and s ′ subsites. in the third stage, we performed protein-ligand docking using autodock vina [ ] . for proteinligand docking, an appropriate grid box was manually determined for tmprss and cathepsin l (section ). to decide on a stringent binding energy cut off for the identification of potential inhibitors, docking was first performed for known inhibitors of target proteins (section ). based on the docking binding energies of the known inhibitors, camostat and nafamostat, to tmprss , we decided on a stringent criteria of binding energy ≤− . kcal/mol for the best docked pose of screened ligands to identify potential inhibitors of tmprss (section ). similarly, based on the docking binding energies of the known inhibitors, e- d and pc- , to cathepsin l, we decided on a stringent criteria of binding energy ≤− . kcal/mol for the best docked pose of screened ligands to identify potential inhibitors of cathepsin l (section ). thereafter, docking was performed for the prepared ligands in the phytochemical library against the prepared structures of tmprss and cathepsin l (section ). lastly, we filtered the subset of phytochemicals whose binding energy in the best docked pose with tmprss (respectively, cathepsin l) is ≤− . kcal/mol (respectively, ≤− . in the third stage, we performed protein-ligand docking using autodock vina [ ] . for proteinligand docking, an appropriate grid box was manually determined for tmprss and cathepsin l (section ). to decide on a stringent binding energy cut off for the identification of potential inhibitors, docking was first performed for known inhibitors of target proteins (section ). based on the docking binding energies of the known inhibitors, camostat and nafamostat, to tmprss , we decided on a stringent criteria of binding energy ≤− . kcal/mol for the best docked pose of screened ligands to identify potential inhibitors of tmprss (section ). similarly, based on the docking binding energies of the known inhibitors, e- d and pc- , to cathepsin l, we decided on a stringent criteria of binding energy ≤− . kcal/mol for the best docked pose of screened ligands to identify potential inhibitors of cathepsin l (section ). thereafter, docking was performed for the prepared ligands in the phytochemical library against the prepared structures of tmprss and cathepsin l (section ). lastly, we filtered the subset of phytochemicals whose binding energy in the best docked pose with tmprss (respectively, cathepsin l) is ≤− . kcal/mol (respectively, ≤− . kcal/mol). moreover, the best docked pose with tmprss or cathepsin l of each filtered phytochemical was separated from autodock vina output file, and then, combined with the target protein structure to obtain the docked protein-ligand complex in .pdb format (section ). at the end of third stage, we obtained phytochemicals whose binding energy in the best docked pose with tmprss is ≤− . kcal/mol and phytochemicals whose binding energy in the best docked pose with cathepsin l is ≤− . kcal/mol. kcal/mol). moreover, the best docked pose with tmprss or cathepsin l of each filtered phytochemical was separated from autodock vina output file, and then, combined with the target protein structure to obtain the docked protein-ligand complex in .pdb format (section ). at the end of third stage, we obtained phytochemicals whose binding energy in the best docked pose with tmprss is ≤− . kcal/mol and phytochemicals whose binding energy in the best docked pose with cathepsin l is ≤− . kcal/mol. (a) cartoon representation of the homology model structure of tmprss which has been energy-minimized using ucsf chimera. the figure zooms into the region containing the catalytic triad ser (s ), his (h ) and asp (d ), and the substrate binding residue asp (d ) in the s subsite of the enzyme. (b) alignment of protein sequences for tmprss and hepsin (pdb z g) which was used as a template to model the structure of tmprss . (c) general ramachandran plot of the energy-minimized model structure of tmprss , which displays the torsional angles, phi (φ) and psi (ψ), of the amino acid residues in the protein. cartoon representation of the crystal structure of human cathepsin l (pdb mqy). the figure zooms into the region containing the catalytic residues cys (c ) and his (h ) in the s subsite, residues asp (d ), met (m ), ala (a ), met (m ) and leu (l ) in the s subsite, trp (w ) at the centre of s ′ subsite and the conserved residue gly (g ) in the s subsite of the enzyme. (a) cartoon representation of the homology model structure of tmprss which has been energy-minimized using ucsf chimera. the figure zooms into the region containing the catalytic triad ser (s ), his (h ) and asp (d ), and the substrate binding residue asp (d ) in the s subsite of the enzyme. (b) alignment of protein sequences for tmprss and hepsin (pdb z g) which was used as a template to model the structure of tmprss . (c) general ramachandran plot of the energy-minimized model structure of tmprss , which displays the torsional angles, phi (ϕ) and psi (ψ), of the amino acid residues in the protein. molecules , , x of kcal/mol). moreover, the best docked pose with tmprss or cathepsin l of each filtered phytochemical was separated from autodock vina output file, and then, combined with the target protein structure to obtain the docked protein-ligand complex in .pdb format (section ). at the end of third stage, we obtained phytochemicals whose binding energy in the best docked pose with tmprss is ≤− . kcal/mol and phytochemicals whose binding energy in the best docked pose with cathepsin l is ≤− . kcal/mol. (a) cartoon representation of the homology model structure of tmprss which has been energy-minimized using ucsf chimera. the figure zooms into the region containing the catalytic triad ser (s ), his (h ) and asp (d ), and the substrate binding residue asp (d ) in the s subsite of the enzyme. (b) alignment of protein sequences for tmprss and hepsin (pdb z g) which was used as a template to model the structure of tmprss . (c) general ramachandran plot of the energy-minimized model structure of tmprss , which displays the torsional angles, phi (φ) and psi (ψ), of the amino acid residues in the protein. cartoon representation of the crystal structure of human cathepsin l (pdb mqy). the figure zooms into the region containing the catalytic residues cys (c ) and his (h ) in the s subsite, residues asp (d ), met (m ), ala (a ), met (m ) and leu (l ) in the s subsite, trp (w ) at the centre of s ′ subsite and the conserved residue gly (g ) in the s subsite of the enzyme. in the fourth stage, the structure of docked protein-ligand complex in .pdb format for each filtered phytochemical from third stage was used to determine the ligand binding site residues in the target protein and different non-covalent interactions such as hydrogen bond, halogen bond, hydrophobic interactions, etc. between ligand and target protein (section ). in case of tmprss , the specificity of this trypsin-like protease is determined by the conserved substrate binding residue d in the s pocket [ ] (section ), and therefore, a potent inhibitor should either bind to or form non-covalent interactions with d . in case of cathepsin l, the specificity of this cysteine protease is dependent on the catalytic residues, c and h , in the s subsite (section ), and therefore, a potent inhibitor should either bind to or form non-covalent interactions with the catalytic residues. in this work, we consider a phytochemical to be a potential inhibitor of tmprss (respectively, cathepsin l) only if the ligand binding energy in the best docked pose is ≤− . kcal/mol (respectively, ≤− . kcal/mol) and the ligand binds to or forms non-covalent interactions with the residue d in tmprss (respectively, residues c and h in cathepsin l). at the end of fourth stage, we obtained phytochemicals (labelled t -t ; figure ; supplementary table s ) as potential inhibitors of tmprss and phytochemicals (labelled c -c ; figure ; supplementary table s ) as potential inhibitors of cathepsin l. using imppat [ ] , we provide a list of indian medicinal plants that can produce the identified phytochemical inhibitors of tmprss and cathepsin l (tables and ; supplementary table s ) . furthermore, we have also compiled information on potential antiviral or anti-inflammatory use in traditional medicine of the herbal sources of the identified phytochemical inhibitors of tmprss and cathepsin l (tables and ; supplementary table s ). in supplementary tables s and s , we list the ligand binding site residues and non-covalent protein-ligand interactions for the identified phytochemical inhibitors of tmprss and cathepsin l. we have also predicted the physicochemical and admet properties of the identified phytochemical inhibitors of tmprss and cathepsin l (section ; supplementary tables s and s ) . finally, in the fifth stage, for the top three inhibitors of tmprss namely, t (qingdainone), t (edgeworoside c) and t (adlumidine), and of cathepsin l namely, c (ararobinol), c ((+)-oxoturkiyenine) and c ( α, α-cinchophylline), their respective protein-ligand complexes were analyzed using ns md simulation (section ; figures and ; supplementary figures s and s ) and their interaction binding energy was computed using mm-pbsa method (section ; table ). as mentioned above, we have identified potential natural product inhibitors of tmprss by computational screening of , phytochemicals produced by indian medicinal plants, and these compounds labelled t -t are listed in supplementary table s along with their pubchem identifier, common name, iupac name and structure in smiles format. in this section, we provide a detailed discussion of the top nine phytochemical inhibitors (labelled as t -t ) whose binding energies in the best docked poses with tmprss are ≤− . kcal/mol. figure displays the structure of these top phytochemical inhibitors of tmprss and table provides a list of indian medicinal plants that can produce them. phytochemical t , qingdainone, has a binding energy of − . kcal/mol. t is a quinazoline alkaloid produced by strobilanthes cusia, a herb with antiviral activity [ ] . figure a shows the tmprss residues that form hydrogen bonds or π-π stacking interactions with t . tmprss residue d forms c-h· · · o type hydrogen bond with t whereas residue a forms c-h· · · n type hydrogen bond with t . further, t forms hydrophobic contacts with residues i , s , t , e , n , a , d , c and a . phytochemical t , edgeworoside c, also has a binding energy of − . kcal/mol. t is a coumarin produced by edgeworthia gardneri, a medicinal plant consumed as a herbal tea in tibet [ ] . in traditional medicine, edgeworthia gardneri has been used to treat metabolic disorders including diabetes [ , ] . figure b shows the tmprss residues that form hydrogen bonds or π-π stacking interactions with t . t forms hydrogen bonds with residues a , n , a , v , d and d of tmprss . the phenolic hydroxyl group of t acts as both acceptor and donor forming o-h· · · o and n-h· · · o type hydrogen bonds with the substrate binding residue d and c-h· · · o type hydrogen bond with residue v . the hydroxyl groups attached to the pyran ring of t form hydrogen bonds with residues a , n and d . further, t forms hydrophobic contacts with residues e , i , a , n , and a . phytochemical t , adlumidine, also has a binding energy of − . kcal/mol. t is produced by fumaria indica, a herb used in traditional medicine to treat fever, cough, skin ailments and urinary diseases [ ] . adlumidine has also been reported to be an inhibitor of gaba receptor [ ] . figure c shows the tmprss residues that form hydrogen bonds or π-π stacking interactions with t . the two , -dioxole groups present in t facilitate the formation of an extensive hydrogen bond network with e , e , s , c and a . t also forms c-h· · · s type hydrogen bond [ ] with c . further, t forms hydrophobic contacts with residues e , i , s , t , n , a and a . molecules , , x of h···o and n-h···o type hydrogen bonds with the substrate binding residue d and c-h···o type hydrogen bond with residue v . the hydroxyl groups attached to the pyran ring of t form hydrogen bonds with residues a , n and d . further, t forms hydrophobic contacts with residues e , i , a , n , and a . phytochemical t , adlumidine, also has a binding energy of − . kcal/mol. t is produced by fumaria indica, a herb used in traditional medicine to treat fever, cough, skin ailments and urinary diseases [ ] . adlumidine has also been reported to be an inhibitor of gaba receptor [ ] . figure c shows the tmprss residues that form hydrogen bonds or π-π stacking interactions with t . the two , -dioxole groups present in t facilitate the formation of an extensive hydrogen bond network with e , e , s , c and a . t also forms c-h···s type hydrogen bond [ ] with c . further, t forms hydrophobic contacts with residues e , i , s , t , n , a and a . phytochemicals t (pseudo-α-colubrine), t (strychnine n-oxide), t (α-colubrine) and t ( hydroxy- -methoxystrychnine) have binding energies of − . kcal/mol, − . kcal/mol, − . kcal/mol phytochemicals t (pseudo-α-colubrine), t (strychnine n-oxide), t (α-colubrine) and t ( -hydroxy- -methoxystrychnine) have binding energies of − . kcal/mol, − . kcal/mol, − . kcal/mol and − . kcal/mol, respectively. these four phytochemicals are monoterpenoid indole alkaloids produced by strychnos nux-vomica. the herb strychnos nux-vomica is used in traditional indian medicine and its alkaloids have been shown to exhibit anti-inflammatory, anti-oxidant, antitumor and hepatoprotective activities [ ] . note that strychnos nux-vomica is a poisonous plant whose seeds are extensively used in ayurveda only after proper detoxification procedure called shodhana described in ayurvedic texts [ ] . figure d shows the tmprss residues that form hydrogen bonds or π-π stacking interactions with t . t forms c-h· · · o type hydrogen bonds with residues a , n , d (substrate binding residue), c and d . further, t forms hydrophobic contacts with residues e , i , s , t , n , a , v , d and a . figure f shows the tmprss residues that form hydrogen bonds or π-π stacking interactions with t . t forms a c-h· · · n type hydrogen bond with residue n . the substrate binding residue d also forms a c-h· · · o type hydrogen bond with t . further, t forms hydrophobic contacts with residues n , a , v and a . supplementary figure s a shows the tmprss residues that form hydrogen bonds or π-π stacking interactions with t . t forms five c-h· · · o type hydrogen bonds with residues n , d (substrate binding residue), c and d . further, t forms hydrophobic contacts with residues e , t , n , a , a , v and a . supplementary figure s c shows the tmprss residues that form hydrogen bonds or π-π stacking interactions with t . the phenolic hydroxyl group of t forms hydrogen bonds with residues s and a . the substrate binding site d also forms a c-h· · · o type hydrogen bond with t . further, t forms hydrophobic contacts with residues e , n , a , a and v . phytochemical t , bicuculline, has a binding energy of − . kcal/mol, and it is a stereoisomer of t . t is an isoquinoline alkaloid and is produced by herbs corydalis govaniana, nerium oleander and fumaria indica. bicuculline has also been reported to be a gaba receptor inhibitor [ ] . figure e shows the tmprss residues that form hydrogen bonds or π-π stacking interactions with t . the two , -dioxole groups present in t facilitate the formation of an extensive hydrogen bond network with residues e , e and a . the furan- -one ring also forms a c-h· · · o type hydrogen bond with c . further, t forms hydrophobic contacts with residues e , t , e , n , a and a . phytochemical t , egenine, has a binding energy of − . kcal/mol. t is an isoquinoline alkaloid produced by fumaria vaillantii. in traditional medicine, fumaria vaillantii has been reported to exhibit antifungal, anti-inflammatory and anti-psychotic activities [ ] . supplementary figure s b shows the tmprss residues that form hydrogen bonds or π-π stacking interactions with t . the two , -dioxole groups present in t form hydrogen bonds with g , e , e and a . one of the hydroxyl groups present in t forms c-h· · · o type hydrogen bond with residue c . further, t forms hydrophobic contacts with residues t , a , e , n , e , and a . the carbon atoms of the ligand are shown in green colour while the carbon atoms of the amino acid residues in tmprss are shown in cyan colour. tmprss residues interacting with the ligand atoms via hydrogen bonds or π-π stacking are labelled with the corresponding single letter residue code along with their position in the protein sequence. the hydrogen bonds and π-π stacking are displayed using yellow and red dotted lines, respectively. as mentioned above, we have identified nine potential natural product inhibitors of cathepsin l by computational screening of , phytochemicals produced by indian medicinal plants, and these compounds labelled c -c are listed in supplementary table s along with their pubchem identifier, common name, iupac name and structure in smiles format. figure displays the structure of these top nine phytochemical inhibitors of cathepsin l and table provides a list of indian medicinal plants that produce them. t . the carbon atoms of the ligand are shown in green colour while the carbon atoms of the amino acid residues in tmprss are shown in cyan colour. tmprss residues interacting with the ligand atoms via hydrogen bonds or π-π stacking are labelled with the corresponding single letter residue code along with their position in the protein sequence. the hydrogen bonds and π-π stacking are displayed using yellow and red dotted lines, respectively. as mentioned above, we have identified nine potential natural product inhibitors of cathepsin l by computational screening of , phytochemicals produced by indian medicinal plants, and these compounds labelled c -c are listed in supplementary table s along with their pubchem identifier, common name, iupac name and structure in smiles format. figure displays the structure of these top nine phytochemical inhibitors of cathepsin l and table provides a list of indian medicinal plants that produce them. phytochemical c , ararobinol, has a binding energy of − . kcal/mol. c is a bianthraquinone produced by herb senna occidentalis used in ayurveda. in traditional medicine, senna occidentalis has been reported for antibacterial, antifungal, anti-inflammatory, anti-diabetic and anti-cancer activities [ ] . interestingly, there is a chinese patent application [ ] on potential use of ararobinol to treat human influenza virus infections, however, this suggests only a potential antiviral activity of c not specific to sars-cov- which further needs to be verified through in vitro and in vivo experimental studies. figure a shows the cathepsin l residues that form hydrogen bonds or π-π stacking interactions with c . residues q and a form c-h· · · n and c-h· · · o type of hydrogen bonds, respectively, with c . also, the residue w forms both face-to-edge and face-to-face type of π-π stacking interaction with c . further, c forms hydrophobic contacts with residues c , g , l , h and w . phytochemical c , (+)-oxoturkiyenine, has a binding energy of − . kcal/mol. c is an isoquinolinederived alkaloid produced by the herb hypecoum pendulum [ ] . figure b shows the cathepsin l residues that form hydrogen bonds or π-π stacking interactions with c . the , -dihydro-furan ring present in c forms two n-h· · · o type hydrogen bonds with residue q and w . the catalytic residue h forms n-h· · · o type hydrogen bond with c . also, the residue w forms a face-to-edge type of π-π stacking interaction with c . further, c forms hydrophobic contacts with residues g , h , h and w . phytochemical c , α, α-cinchophylline, has a binding energy of − . kcal/mol. c is a cinchophyllinetype of alkaloid produced by the herb cinchona calisaya. the cinchona alkaloids have been reported for their antimicrobial, antiparasitic and anti-inflammatory activities [ ] . figure c shows the cathepsin l residues that form hydrogen bonds or π-π stacking interactions with c . c forms hydrogen bonds with residues of cathepsin l. one of the catalytic residue c forms c-h· · · s and n-h· · · s type hydrogen bonds with c . the other catalytic residue h forms c-h· · · n and n-h· · · n type hydrogen bonds with c . further, one of the two pyrrole rings present in c forms hydrogen bond with residue g . lastly, m forms a c-h· · · s type hydrogen bond with c . further, c forms hydrophobic contacts with residues q , c , l , m , a and w . phytochemical c , rugosanine b, has a binding energy of − . kcal/mol. c is a cyclopeptide alkaloid produced by the bark of ziziphus rugosa [ ] . various parts of ziziphus rugosa have been reported for their antibacterial, antifungal, anti-inflammatory and analgesic activities [ ] . figure d shows the cathepsin l residues that form hydrogen bonds or π-π stacking interactions with c . the pyrrole ring present in c forms a n-h· · · o type hydrogen bond with residue d . moreover, c forms c-h· · · o type hydrogen bonds with a , d and l . also, the residue w forms a face-to-edge type of π-π stacking interaction with c . further, c forms hydrophobic contacts with residues g , c , g , m , a , a , d , h , g , w and a . phytochemical c , trichotomine, has a binding energy of − . kcal/mol. c is a bisindole alkaloid present in clerodendrum trichotomum. clerodendrum trichotomum has been reported for its use to treat cold, hypertension, rheumatism, dysentery and other inflammatory diseases [ ] . figure e shows the cathepsin l residues that form hydrogen bonds or π-π stacking interactions with c . the carboxylic acid group present in c forms hydrogen bonds with residues q and h . the indole ring of c forms a n-h· · · n type hydrogen bond with residue g . further, c forms hydrophobic contacts with residues g , c , g , g , l and y . phytochemical c , tectol, has binding energy of − . kcal/mol. c is a naphthoquinone derivative [ ] present in tectona grandis and tecomella undulata. tectona grandis has been reported to have anti-inflammatory and antiparasitic activities [ ] . tecomella undulata has been used to treat syphilis and also reported to have anti-inflammatory and anti-hiv activities [ ] . additionally, tectol has been reported to inhibit farnesyltransferase enzyme [ ] . figure f shows the cathepsin l residues that form hydrogen bonds or π-π stacking interactions with c . the pyran group of c is involved in a c-h· · · o type hydrogen bond with residue l . the catalytic residue c forms a c-h· · · s type hydrogen bond with c . the other catalytic residue h forms a n-h· · · o type hydrogen bond with c . also, the residue w forms both face-to-face and face-to-edge type of π-π stacking interaction with c . further, c forms hydrophobic contacts with g , l and w . phytochemical c , silymonin, has a binding energy of − . kcal/mol. c is a flavanolignan [ ] present in silybum marianum. silybum marianum has been used as a hepatoprotective agent and is reported to have anti-oxidant and anti-inflammatory activities [ ] . supplementary figure s a shows the cathepsin l residues that form hydrogen bonds or π-π stacking interactions with c . c has four hydroxyl groups which help in the formation of an extensive network of hydrogen bonds with residues q , a and g . c also forms two n-h· · · o type hydrogen bonds with h and w . also, the residue w forms a face-to-edge type of π-π stacking interaction with c . further, c forms hydrophobic contacts with residues g , a , l , h and w . phytochemical c , picrasidine m, has a binding energy of − . kcal/mol. c is a β-carboline alkaloid present in picrasma quassioides. picrasma quassioides has been reported to have antiviral and antifungal activities [ ] . supplementary figure s b shows the cathepsin l residues that form hydrogen bonds or π-π stacking interactions with c . the carboxylic group of residue d forms two c-h· · · o type hydrogen bonds with c . also, residues m and g form hydrogen bonds of type c-h· · · s and c-h· · · o, respectively, with c . also, the residue w forms a face-to-edge type of π-π stacking interaction with c . further, c forms hydrophobic contacts with residues g , l , d and w . phytochemical c , silymonin, has a binding energy of − . kcal/mol. c is a flavanolignan [ ] present in silybum marianum. silybum marianum has been used as a hepatoprotective agent and is reported to have anti-oxidant and anti-inflammatory activities [ ] . supplementary figure s a shows the cathepsin l residues that form hydrogen bonds or π-π stacking interactions with c . c has four hydroxyl groups which help in the formation of an extensive network of hydrogen bonds with residues q , a and g . c also forms two n-h···o type hydrogen bonds with h and w . also, the residue w forms a face-to-edge type of π-π stacking interaction with c . further, c forms hydrophobic contacts with residues g , a , l , h and w . phytochemical c , picrasidine m, has a binding energy of − . kcal/mol. c is a β-carboline alkaloid present in picrasma quassioides. picrasma quassioides has been reported to have antiviral and antifungal activities [ ] . supplementary figure s b shows the cathepsin l residues that form hydrogen bonds or π-π stacking interactions with c . the carboxylic group of residue d forms two c-h···o type hydrogen bonds with c . also, residues m and g form hydrogen bonds of phytochemical c , trisjuglone, has a binding energy of − . kcal/mol. c is a naphthoquinone present in juglans regia (i.e., walnut). in traditional medicine, juglans regia has been reported to have anti-inflammatory, antifungal and antimicrobial activities [ ] . supplementary figure s c shows the cathepsin l residues that form hydrogen bonds or π-π stacking interactions with c . the benzoquinone moiety present in c forms two c-h· · · o type hydrogen bonds with residue q and one n-h· · · o type hydrogen bond with w . in contrast, the other catalytic residue h forms a n-h· · · o type hydrogen bond with c . also, the residue w forms a face-to-edge type of π-π stacking interaction with c . further, c forms hydrophobic contacts with residues q , g , c , l and w . in order to investigate the stability of the protein-ligand complexes of the identified inhibitors, md simulation of ns was performed for top three inhibitors of tmprss namely, qingdainone (t ), edgeworoside c (t ) and adlumidine (t ), and of cathepsin l namely, ararobinol (c ), (+)-oxoturkiyenine (c ) and α, α-cinchophylline (c ). specifically, we have performed six ns md runs for protein-ligand complexes (tmprss -t , tmprss -t , tmprss -t , cathepsin l-c , cathepsin l-c and cathepsin l-c ) and two ns md runs for free tmprss and cathepsin l proteins (section ). to access the stability of the six protein-ligand complexes, we have computed radius of gyration (r g ) of the protein, root mean square deviation (rmsd) of the cα atoms of the protein, root mean square fluctuations (rmsf) of the cα atoms of the protein, rmsd of the ligand and finally distance of the center of mass of the ligand from the center of mass of the catalytic residues or substrate binding residues of the protein in complex with the ligand (figures and ) . the r g value of tmprss in complex with t , t and t remains largely stable throughout the md simulation (figure a ). this implies that the top inhibitors of tmprss namely, t , t and t do not induce any major structural changes to tmprss and tmprss remains structurally stable in complex with these inhibitors. tmprss in complex with t , t and t has an average r g value of . ± . nm, . ± . nm and . ± . nm, respectively. further, rmsd value of the cα atoms of tmprss in complex with t , t and t become stable after ns (figure b ). over the ns to ns time interval, tmprss in complex with t , t and t has an average rmsd value of . ± . nm, . ± . nm and . ± . nm, respectively. lastly, figure c shows the rmsf value per residue for tmprss in complex with t , t and t . r g , rmsd and rmsf values of tmprss in complex with t , t and t closely follow r g , rmsd and rmsf values of tmprss free protein (figure a figure s a -c show the superimposed snapshots at ns, ns and ns of tmprss -t , tmprss -t and tmprss -t complexes, respectively. to further quantify the stability of the inhibitors t , t and t bound to tmprss , we have computed the rmsd of t , t and t (figure d ) and distance of the center of mass of t , t and t from the center of mass of the substrate binding residue d in tmprss (figure e ). both rmsd of t , t and t bound with tmprss and distance of the center of mass of t , t and t from the center of mass of the substrate binding residue d becomes largely stable after ns of the md simulation (figure d,e) . also, r g value of cathepsin l in complex with c , c and c is stable throughout the md simulation implying c , c and c do not induce any major structural changes to cathepsin l and cathepsin l remains structurally stable in complex with these inhibitors (figure a ). cathepsin l in complex with c , c and c has an average r g value of . ± . nm, . ± . nm and . ± . nm, respectively. similarly, rmsd value of the cα atoms of cathepsin l in complex with c , c and c become largely stable after ns (figure b) . over the ns to ns time interval, cathepsin l in complex with c , c and c has an average rmsd value of . ± . nm, . ± . nm and . ± . nm, respectively. figure c shows the rmsf value per residue for cathepsin l in complex with c , c and c . as in the case of tmprss , r g , rmsd and rmsf values of cathepsin l in complex with c , c and c closely follow r g , rmsd and rmsf values of cathepsin l free protein (figure a c has a largely stable rmsd after ns of the md simulation, c has the lowest and most stable rmsd in comparison with c and c , and c shows a largely stable rmsd from ns to ns and from ns to ns of the md simulation (figure d) . distance of the center of mass of c , c and c from the center of mass of the catalytic residues c and h in cathepsin l also remains largely consistent after ns of the md simulation (figure e,f) . molecular mechanics poisson-boltzmann surface area (mm-pbsa) is a widely used method to compute the binding energy of small molecules with biological macromolecules such as proteins [ ] . notably, the protein-ligand binding energy obtained using mm-pbsa method has been found to be more accurate than that obtained from protein-ligand docking [ ] . therefore, we have computed the binding energy for the top three inhibitors of tmprss and cathepsin l using mm-pbsa method. from the ns md simulation of the six protein-ligand complexes (tmprss -t , tmprss -t , tmprss -t , cathepsin l-c , cathepsin l-c and cathepsin l-c ), snapshots were obtained between ns to ns of the simulation at an interval of ns along the trajectory, and thereafter, c has a largely stable rmsd after ns of the md simulation, c has the lowest and most stable rmsd in comparison with c and c , and c shows a largely stable rmsd from ns to ns and from ns to ns of the md simulation (figure d) . distance of the center of mass of c , c and c from the center of mass of the catalytic residues c and h in cathepsin l also remains largely consistent after ns of the md simulation (figure e ,f). molecular mechanics poisson-boltzmann surface area (mm-pbsa) is a widely used method to compute the binding energy of small molecules with biological macromolecules such as proteins [ ] . notably, the protein-ligand binding energy obtained using mm-pbsa method has been found to be more accurate than that obtained from protein-ligand docking [ ] . therefore, we have computed the binding energy for the top three inhibitors of tmprss and cathepsin l using mm-pbsa method. from the ns md simulation of the six protein-ligand complexes (tmprss -t , tmprss -t , tmprss -t , cathepsin l-c , cathepsin l-c and cathepsin l-c ), snapshots were obtained between ns to ns of the simulation at an interval of ns along the trajectory, and thereafter, the snapshots were used to compute the binding energy using g_mmpbsa (section ) [ , ] . the final binding energy is the sum of contributions from van der waals, electrostatic, polar solvation, and solvent accessible surface area (sasa) energy components. the contribution of each of the above components to the binding energy of the top inhibitors is shown in table . (table ) . moreover, in comparison to binding energy obtained from docking using autodock vina, binding energy for the tmprss and cathepsin l complexes with their top inhibitors computed using mm-pbsa method were found to be -fold to -fold lower, signifying even stronger binding (tables - ) previously, some of us have built the indian medicinal plants, phytochemistry and therapeutics (imppat) database [ ] which is the largest resource on phytochemicals of indian herbs to date. for this study, we compiled a ligand library of , phytochemicals by augmenting the information in imppat [ ] with additional information compiled from other literature sources [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] . thereafter, the widely used drug-likeness measure, lipinski's rule of five (ro ) [ ] , was employed to filter the potential drug-like molecules within the ligand library of , phytochemicals. specifically, , phytochemicals passed the r drug-likeness filter. we then retrieved the three-dimensional ( d) structures of these phytochemicals from pubchem [ ] . next the d structures of the drug-like phytochemicals were energy-minimized using obminimize within the openbabel toolbox [ ] . finally, the energy-minimized d structures of ligands in .sdf format were converted to .pdb format using openbabel. tmprss is a trypsin-like serine protease whose catalytic site consists of the triad ser (s ), his (h ) and asp (d ) [ ] . it is well established that trypsin-like serine proteases cleave peptide bonds following positively charged amino acid residues such as arginine or lysine, and this specificity of the enzyme is determined by a negatively charged aspartate residue located at the bottom of its s pocket [ ] . in tmprss , this specificity is determined by the conserved negatively charged residue asp (d ) at the bottom of the s pocket [ ] . to date the d structure of tmprss has not been experimentally determined, and thus, we have used swiss-model [ ] webserver (https://swissmodel.expasy.org/interactive) to perform homology modeling of tmprss . we submitted the tmprss protein sequence (ncbi reference sequence np_ . ) to swiss-model and selected the crystal structure of human protein hepsin (pdb z g) [ ] as the template to build the model structure (figure a) . note that hepsin (pdb z g) is also a type ii transmembrane trypsin-like serine protease, and it shares % sequence similarity with tmprss (np_ . ) (figure b) . subsequently, ucsf chimera [ ] was used to minimize the energy of the tmprss model structure obtained from swiss-model. thereafter, the energy-minimized tmprss model structure was assessed using the structure assessment tool within swiss-model. in the tmprss model, . % of the amino acid residues were found to be in the ramachandran favoured regions in the ramachandran plot (figure c ) and the model structure has a molprobity [ ] score of . . we use the crystal structure (pdb mqy) [ ] of human cathepsin l with . Å resolution obtained from protein data bank for virtual screening. ucsf chimera was used to minimize the energy of the cathepsin l structure. figure displays the cathepsin l structure with important residues in s , s and s subsites of the enzyme [ ] . previous research has also revealed that s and s subsites of cathepsin l are important for the specificity of the enzyme [ , ] . in cathepsin l, the catalytic site consists of cys (c ) and his (h ) in the s subsite, and trp (w ) is at the center of the s subsite [ ] (figure ) . in cathepsin l, the s subsite with important residues asp (d ), met (m ), ala (a ), met (m ) and leu (l ) forms a deep hydrophobic pocket, and lastly, the conserved residue gly (g ) is at the center of the s subsite [ , ] (figure ). autodock vina [ ] was used to perform the molecular docking of energy-minimized d structures of ligands with energy-minimized structure of target proteins. accordingly, the d structures of prepared ligands in .pdb format were converted to .pdbqt format using the python script prepare_ligand .py from autodocktools [ ] . similarly, the energy-minimized structure of tmprss and cathepsin l in .pdb format were converted to .pdbqt format using the python script prepare_receptor .py from autodocktools [ ] . for protein-ligand docking, the appropriate grid box specified by the search space centre and search space size for tmprss and cathepsin l was manually determined by considering the key residues in target proteins such as the catalytic residues and substrate binding residues, which are important for the function and specificity of the considered proteases as reported in the literature. for tmprss , the grid box was defined by the search space centre ( . , − . , . ) and search space size of Å × Å × Å. for cathepsin l, the grid box was defined by the search space centre ( . , . , . ) and search space size of Å × Å × Å. for both target proteins, the molecular docking of prepared ligands was performed using autodock vina with the exhaustiveness set as . the top conformation of the docked ligand with lowest binding energy, i.e., the best docked pose, was obtained from the output of autodock vina using the associated script vina_split. subsequently, a combined structure file in .pdb format of the docked protein-ligand complex (with ligand in the best docked pose) was prepared using a custom script and pdb-tools [ ] . we searched the combined structure file of the docked protein-ligand complex for ligand binding residues in protein and different non-covalent interactions that can facilitate the binding of the ligand with the protein. these non-covalent protein-ligand interactions were identified using different geometric criteria which are specific to different types of interactions: binding site residue. ligand binding site residues are defined as amino acids in protein which have at least one non-hydrogen atom in the proximity of at least one non-hydrogen atom of the ligand. the distance cut off to determine this proximity between non-hydrogen atoms of protein and ligand is taken to be the sum of their van der waals radius plus . Å [ ] . hydrogen bonds. the accepted geometric criteria for hydrogen bonds of type d-h· · · a are as follows. firstly, the distance between the hydrogen (h) and acceptor (a) atom should be less than the sum of their van der waals radii. secondly, the angle formed by donor (d), h and a atoms should be > • (supplementary figure s a) . moreover, carbon (c), nitrogen (n), oxygen (o) or sulfur (s) atoms can be donors while n, o or s atoms can be acceptors [ , , ] . chalcogen bonds. in contrast to hydrogen bonds, chalcogen bonds are of type c-y· · · a, where y can be a s or selenium (se) atom and a can be a n, o or s atom. the accepted geometric criteria for chalcogen interactions are as follows. firstly, the distance between y and a should be less than the sum of their van der waals radii. secondly, the angle formed by the triad, that is ∠c-y· · · a, should lie in the range • to • (supplementary figure s b) [ ] . halogen bonds. halogen bonds are of type c-y· · · a-b, where halogen y can be a fluorine (f), chlorine (cl), bromine (br) or iodine (i) atom and a can be a n, o or s atom. the formation of the halogen bond is favoured when the distance between y and a is ≤ . Å and the angle θ of the a atom relative to the c-y bond, and the angle θ of the halogen y relative to the a-b bond should be ≥ • (supplementary figure s c) [ ] . π-π stacking. this interaction occurs between two aromatic rings and can be majorly classified into two types, namely, face-to-face and face-to-edge. in the case of face-to-face type of π-π interaction, the distance between the centroids of the two participating aromatic rings should be < . Å and the angle between their ring planes should be < • . in the case of face-to-edge type of π-π interaction, the distance between the centroids of the two participating aromatic rings should be < . Å and the angle formed by the ring planes should be in the range • to • (supplementary figure s d,e) . hydrophobic interactions. the geometric criteria for the formation of hydrophobic interactions between atoms in protein and ligand are as follows [ ] . the distance between a carbon atom in protein or ligand and a carbon, halogen or sulfur atom in ligand or protein, respectively, should be ≤ Å. furthermore, we ensure that the involved atoms in a hydrophobic interaction between protein and ligand do not form hydrogen, chalcogen or halogen bonds between them [ ] . in order to detect the above-mentioned protein-ligand interactions, an in-house python program was written to enable batch processing of combined structure files containing docked protein-ligand complexes for our large phytochemical library. in order to identify potent phytochemical inhibitors of target proteins, we decided to compare the binding energy of the best docked pose of ligands with binding energies of the best docked pose of known inhibitors of tmprss and cathepsin l obtained from autodock vina. recent experiments have shown that both camostat mesylate and nafamostat mesylate, which are approved for human use in japan, can block the tmprss -dependent cell entry of sars-cov- [ , ] . by docking these two inhibitors to tmprss using autodock vina with exhaustiveness set at , the predicted binding energies of camostat and nafamostat was found to be − . kcal/mol and − . kcal/mol, respectively. supplementary figure s a ,b show the best docked poses of nafamostat and camostat with tmprss , and it is seen that both molecules form hydrogen bonds with the substrate binding residue d . importantly, in comparison to camostat mesylate, nafamostat mesylate in a recent experiment was shown to inhibit the tmprss -dependent cell entry with -fold higher efficiency and an ec value in lower nanomolar range [ ] , and thus, the docked binding energies of these two known inhibitors are in line with experiments. based on above observations, we decided on a stringent criteria of docked binding energy ≤− . kcal/mol for screened ligands to be identified as potential inhibitors of tmprss . recent experiments have shown that the small molecules e- d and pc- (sid ) can block the cathepsin l-dependent cell entry of sars-cov- [ ] [ ] [ ] . note that cathepsin l is one of cysteine cathepsin proteases encoded by the human genome, and the cathepsins share a high sequence similarity to papain, a non-specific plant protease [ ] . e -d is a broad spectrum inhibitor which can inhibit proteases cathepsins b, h, l and calpain, while pc- is a specific inhibitor of cathepsin l [ ] . moreover, a recent study [ ] used the specific inhibitor pc- of cathepsin l to conclude that cathepsin l rather than cathepsin b is important for cell entry of sars-cov- . as both cathepsin l and cathepsin b are expressed in several mammalian tissues, it is important to design specific inhibitors of cathepsin l [ , ] to avoid any off-target toxicity. along with the above-mentioned two inhibitors of cathepsin l, we have also considered the co-crystallized inhibitor gh present in the crystal structure of cathepsin l (pdb mqy) as another reference inhibitor of cathepsin l. we remark that while recent experiments [ ] [ ] [ ] have shown that e- d and pc- can block the cathepsin l-dependent cell entry of sars-cov- , similar experimental data specific to sars-cov- infection is lacking for cathepsin l inhibitor gh . by docking these-three known inhibitors to cathepsin l using autodock vina with exhaustiveness set at , the predicted binding energies of e- d, pc- and gh were found to be − . kcal/mol, − . kcal/mol and − . kcal/mol, respectively. notably, the docked binding energies are in line with known specificity of e- d and pc- to cathepsin l. supplementary figure s c -e show the best docked poses of pc- , e- d and gh with cathepsin l. it is seen that both e- d and pc- form hydrogen bonds with both catalytic residues c and h . based on above observations, we decided on a stringent criteria of docked binding energy ≤− . kcal/mol for screened ligands to be identified as potential inhibitors of cathepsin l. we have also compared the docked pose of gh obtained from autodock vina with the pose of gh in the co-crystallized structure of cathepsin l, and the rmsd between the heavy atoms in the two poses was found to be . Å. supplementary figure s shows the superimposed structures of the docked pose of gh from autodock vina and the pose of gh in the co-crystallized structure of cathepsin l. apart from pc- and e- d, seven other cathepsin-l inhibitors namely, dec-rvkr-cmk, k , small molecule , mdl , ssaa e , est and oxocarbazate, have been reported in literature to exhibit ant-coronavirus activity [ ] . using gromacs . . [ ] along with gromos a force field [ ] , we have performed md simulations for the top inhibitors of tmprss and cathepsin l to assess the stability of their protein-ligand complexes. the automated topology builder (atb) version . (https://atb.uq.edu.au/) was used to generate topology for the top inhibitors [ ] . the protein-ligand complex was placed in the center of a cubic periodic box and was solvated by the addition of simple point charge (spc) waters. then net charge of the system was neutralized by addition of na + and cl − ions. energy minimization was performed using the steepest descent algorithm. then the system was heated to k during a constant number of particles, volume, and temperature (nvt) simulation of ps with fs time step. then the pressure was equilibrated to bar during a constant number of particles, pressure, and temperature (npt) simulation of ps with fs time step. in both the above simulations, protein and ligand were position restrained. thereafter, the position restraint was removed and a production md run was performed for a period of ns with fs time step. during the md simulation, the structural coordinates were saved after every ps. temperature was maintained at k using the v-rescale temperature [ ] . pressure was maintained at bar using parrinello-rahman pressure coupling method [ ] . time constant for the temperature and pressure coupling were kept at . ps and ps, respectively. the short-range interactions were computed for the atom pairs within the cutoff of . nm, whereas the long-range electrostatic interactions were calculated using particle mesh ewald (pme) method with fourth order cubic interpolation and . nm grid spacing. all bonds were constrained using the lincs method. the trajectories obtained from the md simulations were then used to compute and analyze the radius of gyration of the protein (r g ), root mean square deviation (rmsd) of the protein backbone cα atoms and root mean square fluctuation (rmsf) of the protein backbone cα atoms using gromacs scripts. molecular mechanics poisson-boltzmann surface area (mm-pbsa) method was used to compute the binding energy of the interactions between the protein-ligand complexes for the top inhibitors of tmprss and cathepsin l. from the ns trajectory obtained from the md simulation, snapshots were obtained between ns to ns of the simulation at an interval of ns along the trajectory, and thereafter, the binding energy was computed using g_mmpbsa [ , ] . g_mmpbsa computes the gibbs free energy of binding (∆g binding ) using mm-pbsa method as described by the following equation: where g complex, g protein and g ligand represent the total binding energy of the protein-ligand complex, the free protein and the unbounded ligand, respectively. in order to validate the model structure of tmprss built using swiss-model, we have used two approaches. firstly, we have run a ns md simulation of the tmprss free protein and have computed radius of gyration (r g ), rmsd and rmsf for the tmprss free protein from its md trajectory (supplementary figure s a-c) . the r g of the tmprss free protein remains largely stable throughout the ns md simulation (supplementary figure s a) . this signifies that the tmprss model structure remains structurally intact and stable during the md simulation. the rmsd of the tmprss free protein becomes stable after ns (supplementary figure s b) . secondly, we have used the md trajectory of tmprss in complex with the top three potential inhibitors (t , t or t ) from ns to ns to validate the tmprss model structure. the md trajectory of tmprss in complex with the inhibitors from ns to ns was used for validation, ns to ns was used for equilibration, and from ns to ns for finding the binding energy of t , t or t with tmprss using mm-pbsa method. to validate the tmprss model structure, the binding energy computed using mm-pbsa method of t , t or t with tmprss during ns to ns was compared with binding energy of t , t or t with tmprss during ns to ns of the md simulation. we find that t , t and t have average binding energies of − . ± . kcal/mol, − . ± . kcal/mol and − . ± . kcal/mol, respectively, with tmprss during ns to ns of the md simulation. the above reported binding energies of t , t and t with tmprss during ns to ns are very close to the binding energies computed for the same inhibitors in complex with tmprss during ns to ns of the md simulation as reported in table . these results further validate the tmprss model structure used for binding energy computations during ns to ns of the md simulations. in order to assess the pharmacokinetic properties and potential toxicity of the inhibitors of tmprss and cathepsin l predicted from this study, we have also computed the absorption, distribution, metabolism, excretion and toxicity (admet) properties of the inhibitors using swissadme [ ] and vnn-admet [ ] . the current covid- pandemic is a serious threat to humankind. as of july , covid- has led to more than , deaths worldwide. due to the absence of approved therapeutics or vaccines against sars-cov- , several countries have been forced to implement partial or complete lockdown measures to restrict infection spread; however, such measures have in turn resulted in an economic catastrophe. consequently, there is an urgent need to develop antivirals and vaccines against sars-cov- to protect humankind. in this direction, protein-ligand docking and md simulation are powerful computational methods to expedite the search for anti-covid drugs by rapid identification of promising lead molecules. here, we have used molecular docking and md simulations in the search of natural compound inhibitors of two human proteases, tmprss and cathepsin l, that are key host factors in sars-cov- infection [ ] [ ] [ ] . since early civilization, humans have used medicinal plants in different systems of traditional medicine to treat various ailments [ ] . specifically, traditional systems of indian medicine including ayurveda, siddha and unani have over centuries acquired invaluable knowledge on medicinal plants spanning the rich biodiversity of the subcontinent for treating various ailments including viral infections [ ] . as plant-based natural products have been an indomitable source of lead molecules in the course of modern drug discovery [ ] , it is worthwhile to search for potential anti-covid drugs among phytochemicals of indian medicinal plants. in this direction, some of us have built imppat [ ] , the largest resource on phytochemicals of indian medicinal plants to facilitate natural product-based drug discovery. in this work, we have performed virtual screening of , phytochemicals that are produced by indian medicinal plants to identify potential inhibitors of key host factors, tmprss and cathepsin l, for sars-cov- infection. for the identified top inhibitors of tmprss and cathepsin l, we have performed md simulation, and thereafter, employed mm-pbsa method to compute binding energies of the protein-ligand complexes. we have predicted potential phytochemical inhibitors of tmprss , of which the top three candidates, namely, qingdainone (t ), edgeworoside c (t ) and adlumidine (t ), have binding energy value of − . ± . kcal/mol, − . ± . kcal/mol and − . ± . kcal/mol, respectively (table ) . we have also predicted nine potential phytochemical inhibitors of cathepsin l, of which the top three candidates, namely, ararobinol (c ), (+)-oxoturkiyenine (c ) and α, α-cinchophylline (c ), have binding energy value of − . ± . kcal/mol, − . ± . kcal/mol and − . ± . kcal/mol, respectively (table ) . furthermore, most of the herbal sources of the identified phytochemical inhibitors of tmprss and cathepsin l have been reported to have antiviral or anti-inflammatory use in traditional medicine (tables and ) . additional in vitro and in vivo testing of the identified phytochemical inhibitors of tmprss and cathepsin l is needed before these molecules can enter clinical trials against covid- . in conclusion, we expect the natural product inhibitors identified in this computational study for tmprss and cathepsin l will likely inform future research toward natural product-based anti-covid therapeutics. figure s : superimposition of the snapshots at ns, ns and ns of (a) tmprss -t complex, (b) tmprss -t complex, (c) tmprss -t complex, (d) cathepsin l-c complex, (e) cathepsin l-c complex and (f) cathepsin l-c complex obtained from their respective md simulation trajectories, figure s : cartoon representation of the protein-ligand interactions of the phytochemical inhibitors of tmprss . interactions of tmprss residues with atoms of (a) t , (b) t , and (c) t . the carbon atoms of the ligand are shown in green colour while the carbon atoms of the amino acid residues in tmprss are shown in cyan colour. tmprss residues interacting with the ligand atoms via hydrogen bonds or π-π stacking are labelled with the corresponding single letter residue code along with their position in the protein sequence. the hydrogen bonds and π-π stacking are displayed using yellow and red dotted lines, respectively, figure s : cartoon representation of the protein-ligand interactions of the phytochemical inhibitors of cathepsin l. interactions of cathepsin l residues with atoms of (a) c , (b) c , and (c) c . the carbon atoms of the ligand are shown in green colour while the carbon atoms of the amino acid residues in cathepsin l are shown in cyan colour. cathepsin l residues interacting with the ligand atoms via hydrogen bonds or π-π stacking are labelled with the corresponding single letter residue code along with their position in the protein sequence. the hydrogen bonds and π-π stacking are displayed using yellow and red dotted lines, respectively, figure s : geometric criteria for the identification of protein-ligand interactions. (a) hydrogen bond, (b) chalcogen bond, (c) halogen bond, (d) face-to-face π-π stacking, and (e) face-to-edge π-π stacking, figure s : cartoon representation of the protein-ligand interactions of the known inhibitors of tmprss and cathepsin l. interactions of tmprss residues with atoms of (a) nafamostat, and (b) camostat. interactions of cathepsin l residues with atoms of (c) pc- , (d) e- d, and (e) gh . the carbon atoms of the ligand are shown in green colour while the carbon atoms of the amino acid residues in tmprss or cathepsin l are shown in cyan colour. tmprss or cathepsin l residues interacting with the ligand atoms via hydrogen bonds or π-π stacking or halogen bonds are labelled with the corresponding single letter residue code along with their position in the protein sequence. the hydrogen bonds, π-π stacking and halogen bonds are displayed using yellow, red and black dotted lines, respectively, figure s : superimposition of the docked pose of gh with cathepsin l obtained from autodock vina (shown in green colour) and the pose of gh in the co-crystallized structure with cathepsin l (shown in red colour), table s : top list of phytochemical inhibitors of tmprss . for each phytochemical, the table gives the symbol, docked binding energy, pubchem identifier, common name, iupac name and smiles, table s : top list of phytochemical inhibitors of cathepsin l. for each phytochemical, the table gives the symbol, docked binding energy, pubchem identifier, common name, iupac name and smiles, table s : herbal sources of top phytochemical inhibitors of tmprss . for each phytochemical, the table gives the symbol, docked binding energy, common name and plant source. plant sources which have been reported to have anti-viral or anti-inflammatory therapeutic use in traditional medicine literature are shown in bold and marked with an [*] sign, table s : the table lists the ligand binding site residues and non-covalent protein-ligand interactions namely, hydrogen bond interactions, π-π stacking interactions of face-to-face type and face-to-edge type and hydrophobic interactions that were identified from the docked protein-ligand complexes of the top phytochemical inhibitors of tmprss , table s : the table lists the ligand binding site residues and non-covalent protein-ligand interactions namely, hydrogen bond interactions, π-π stacking interactions of face-to-face type and face-to-edge type and hydrophobic interactions that were identified from the docked protein-ligand complexes of the top phytochemical inhibitors of cathepsin l, table s : admet properties of the top list of potential phytochemical inhibitors of tmprss . for each phytochemical, the table gives the symbol, pubchem identifier; several physicochemical properties such as molecular weight in g/mol, logp (partition coefficient), molarrefractivity, tpsa (topological surface area) in Å , number of hydrogen bond acceptors, number of hydrogen bond donors, total number of atoms, number of rotatable bonds, shape complexity (fraction of carbon atoms that are sp hybridized), stereochemical complexity (fraction of carbon atoms which are stereogenic); several drug-likeness properties such as lipinski ro filter (lipinski's rule of filter), qedw (weighted quantitative estimate of drug-likeness), qeduw (unweighted quantitative estimate of drug-likeness), leadlikeness-number of violations predicted by swissadme, synthetic accessibility predicted by swissadme; several absorption and distribution properties such as solubility class predicted by swissadme using esol method, solubility class predicted by swissadme using method by ali et al., solubility class predicted by swissadme using silicos-it, gasterointestinal absorption predicted by swissadme, skin permeation predicted by swissadme (log kp cm/s), blood brain barrier permeation predicted by swissadme, blood brain barrier permeation predicted by vnnadmet, bioavailability score predicted by swissadme; several metabolism properties such as p-glycoprotein substrate predicted by swissadme, p-glycoprotein substrate predicted by vnnadmet, p-glycoprotein inhibitor predicted by vnnadmet, cytochrome p a inhibitor predicted by swissadme, cytochrome p a inhibitor predicted by vnnadmet, cytochrome p c inhibitor predicted by swissadme, cytochrome p c inhibitor predicted by vnnadmet, cytochrome p c inhibitor predicted by swissadme, cytochrome p c inhibitor predicted by vnnadmet, cytochrome p d inhibitor predicted by swissadme, cytochrome p d inhibitor predicted by vnnadmet, cytochrome p a inhibitor predicted by swissadme, cytochrome p a inhibitor predicted by vnnadmet, human liver microsomal stability predicted by vnnadmet (hlm); several toxicity properties such as pains-number of alerts predicted by swissadme, brenk-number of alerts predicted by swissadme, drug-induced liver injury predicted by vnnadmet (dili), cytotoxicity predicted by vnnadmet, herg blocker predicted by vnnadmet, mitochondrial toxicity predicted by vnnadmet (mmp) and mutagenecity predicted by vnnadmet (ames), table s : admet properties of the top list of potential phytochemical inhibitors of cathepsin l. for each phytochemical, the table gives the symbol, pubchem identifier; several physicochemical properties such as molecular weight in g/mol, logp (partition coefficient), molarrefractivity, tpsa (topological surface area) in Å , number of hydrogen bond acceptors, number of hydrogen bond donors, total number of atoms, number of rotatable bonds, shape complexity (fraction of carbon atoms that are sp hybridized), stereochemical complexity (fraction of carbon atoms which are stereogenic); several drug-likeness properties such as lipinski ro filter (lipinski's rule of filter), qedw (weighted quantitative estimate of drug-likeness), qeduw (unweighted quantitative estimate of drug-likeness), leadlikeness-number of violations predicted by swissadme, synthetic accessibility predicted by swissadme; several absorption and distribution properties such as solubility class predicted by swissadme using esol method, solubility class predicted by swissadme using method by ali et al., solubility class predicted by swissadme using silicos-it, gasterointestinal absorption predicted by swissadme, skin permeation predicted by swissadme (log kp cm/s), blood brain barrier permeation predicted by swissadme, blood brain barrier permeation predicted by vnnadmet, bioavailability score predicted by swissadme; several metabolism properties such as p-glycoprotein substrate predicted by swissadme, p-glycoprotein substrate predicted by vnnadmet, p-glycoprotein inhibitor predicted by vnnadmet, cytochrome p a inhibitor predicted by swissadme, cytochrome p a inhibitor predicted by vnnadmet, cytochrome p c inhibitor predicted by swissadme, cytochrome p c inhibitor predicted by vnnadmet, cytochrome p c inhibitor predicted by swissadme, cytochrome p c inhibitor predicted by vnnadmet, cytochrome p d inhibitor predicted by swissadme, cytochrome p d inhibitor predicted by vnnadmet, cytochrome p a inhibitor predicted by swissadme, cytochrome p a inhibitor predicted by vnnadmet, human liver microsomal stability predicted by vnnadmet (hlm); several toxicity properties such as pains-number of alerts predicted by swissadme, brenk-number of alerts predicted by swissadme, drug-induced liver injury predicted by vnnadmet (dili), cytotoxicity predicted by vnnadmet, herg blocker predicted by vnnadmet, mitochondrial toxicity predicted by vnnadmet (mmp) and mutagenecity predicted by vnnadmet (ames). clinical features of patients infected with novel coronavirus in a novel coronavirus outbreak of global health concern a novel coronavirus from patients with pneumonia in china a familial cluster of pneumonia associated with the novel coronavirus indicating person-to-person transmission: a study of a family cluster bats are natural reservoirs of sars-like coronaviruses a pneumonia outbreak associated with a new coronavirus of probable bat origin recent insights into emerging coronaviruses therapeutic options for the novel coronavirus ( -ncov) genome composition and divergence of the novel coronavirus ( -ncov) originating in china sars-cov- cell entry depends on ace and tmprss and is blocked by a clinically proven protease inhibitor characterization of spike glycoprotein of sars-cov- on virus entry and its immune cross-reactivity with sars-cov cell entry mechanisms of sars-cov- structural basis of receptor recognition by sars-cov- structural basis for the recognition of sars-cov- by full-length human ace nafamostat mesylate blocks activation of sars-cov- : new treatment option for covid- analysis of therapeutic targets for sars-cov- and discovery of potential drugs by computational methods remdesivir, sofosbuvir, galidesivir, and tenofovir against sars-cov- rna dependent rna polymerase (rdrp): a molecular docking study a molecular modeling approach to identify effective antiviral phytochemicals against the main protease of sars-cov- virtual screening of natural products against type ii transmembrane serine protease (tmprss ), the priming agent of coronavirus (sars-cov- ) in silico studies on therapeutic agents for covid- : drug repurposing approach identification of phytochemical inhibitors against main protease of covid- using molecular modeling approaches potential covalent drugs targeting the main protease of the sars-cov- coronavirus high throughput virtual screening to discover inhibitors of the main protease of the coronavirus sars-cov- prediction of small molecule inhibitors targeting the severe acute respiratory syndrome coronavirus- rna-dependent rna polymerase natural products as sources of new drugs over the years from to traditional chinese medicine for covid- treatment covid- : a promising cure for the global panic imppat: a curated database of indian medicinal plants experimental and computational approaches to estimate solubility and permeability in drug discovery and development settings pubchem update: improved access to chemical data open babel: an open chemical toolbox swiss-model: homology modelling of protein structures and complexes ucsf chimera-a visualization system for exploratory research and analysis autodock vina: improving the speed and accuracy of docking with a new scoring function, efficient optimization, and multithreading cloning of the tmprss gene, which encodes a novel serine protease with transmembrane, ldlra, and srcr domains and maps to q . coumarins with α-glucosidase and α-amylase inhibitory activities from the flower of edgeworthia gardneri in vitro evaluation of dual agonists for pparγ/β from the flower of edgeworthia gardneri (wall.) meisn the flower of edgeworthia gardneri (wall.) meisn. suppresses adipogenesis through modulation of the ampk pathway in t -l adipocytes a review on ethnobotany, phytochemistry and pharmacology of fumaria indica (fumitory). asian pac inhibition of [ h]gaba binding to rat brain synaptic membranes by bicuculline related alkaloids the prodigious hydrogen bonds with sulfur and selenium in molecular assemblies, structural biology, and functional materials effect of shodhana (processing) on kupeelu (strychnos nux-vomica linn.) with special reference to strychnine and brucine content bicuculline and gabazine are allosteric inhibitors of channel opening of the gabaa receptor indian medicinal plants: an illustrated dictionary a review on its ethnobotany, phytochemical and pharmacological profile application of anthraqinone derivative in resisting influenza virus and bird flu virus h n +)-oxoturkiyenine: an isoquinoline-derived alkaloid from hypecoum pendulum spectrum of biological properties of cinchona alkaloids: a brief review cyclopeptide alkaloids from zizyphus rugosa bark analgesic and anti-inflammatory activities of zizyphus rugosa root barks traditional uses and pharmacological properties of clerodendrum phytochemicals identification of anti-wood rot compounds in teak (tectona grandisl.f.) sawdust extract traditional uses, phytochemistry and pharmacology of tecomella undulata-a review discovery and preliminary structure-activity relationship studies on tecomaquinone i and tectol as novel farnesyltransferase and plasmodial inhibitors effect of the flavanolignans of silybum marianum l. on lipid peroxidation in rat liver microsomes and freshly isolated hepatocytes silybum marianum: beyond hepatoprotection. j. evid. -based complementary altern the mm/pbsa and mm/gbsa methods to estimate ligand-binding affinities electrostatics of nanosystems: application to microtubules and the ribosome open source drug discovery consortium g_mmpbsa-a gromacs tool for high-throughput mm-pbsa calculations the wealth of india the wealth of india: a dictionary of indian raw materials & industrial products the wealth of india the wealth of india: a dictionary of indian raw materials & industrial products the wealth of india the wealth of india: a dictionary of indian raw materials & industrial products the wealth of india the wealth of india: a dictionary of indian raw materials & industrial products the wealth of india the wealth of india: a dictionary of indian raw materials & industrial products drug research perspectives drug research perspectives drug research perspectives drug research perspectives drug research perspectives substrate specificity of trypsin investigated by using a genetic selection hepatocyte growth factor is a preferred in vitro substrate for human hepsin, a membrane-anchored serine protease implicated in prostate and ovarian cancers molprobity: all-atom structure validation for macromolecular crystallography prospective evaluation of free energy calculations for the prioritization of cathepsin l inhibitors the crystal structure of human cathepsin l complexed with e- cysteine cathepsins: from structure, function and regulation to new frontiers structural basis for the recognition and cleavage of histone h by cathepsin l autodock and autodocktools : automated docking with selective receptor flexibility pdb-tools: a swiss army knife for molecular structures assessment of ligand-binding residue predictions in casp o hydrogen bonds in protein-ligand complexes: strong and weak interactions in molecular recognition geometric characteristics of hydrogen bonds involving sulfur atoms in proteins chalcogen bonding in protein−ligand complexes: pdb survey and quantum mechanical calculations halogen bonding in complexes of proteins and non-natural amino acids a systematic analysis of atomic protein-ligand interactions in the pdb † †electronic supplementary information (esi) available review: lysosomal cysteine proteases: facts and opportunities cathepsin l-selective inhibitors: a potentially promising treatment for covid- patients high performance molecular simulations through multi-level parallelism from laptops to supercomputers definition and testing of the gromos force-field versions a and b automated topology builder version . : prediction of solvation free enthalpies in water and hexane molecular dynamics with coupling to an external bath polymorphic transitions in single crystals: a new molecular dynamics method swissadme: a free web tool to evaluate pharmacokinetics, drug-likeness and medicinal chemistry friendliness of small molecules the traditional medicine and modern medicine from natural products this article is an open access article distributed under the terms and conditions of the creative commons attribution funding: this research has received no specific external funding. the authors declare no conflict of interest. key: cord- - nz qioh authors: carabineiro, sónia alexandra correia title: applications of gold nanoparticles in nanomedicine: recent advances in vaccines † date: - - journal: molecules doi: . /molecules sha: doc_id: cord_uid: nz qioh nowadays, gold is used in (nano-)medicine, usually in the form of nanoparticles, due to the solid proofs given of its therapeutic effects on several diseases. gold also plays an important role in the vaccine field as an adjuvant and a carrier, reducing toxicity, enhancing immunogenic activity, and providing stability in storage. an even brighter golden future is expected for gold applications in this area. it is well known that gold is inert in the bulk stable and is only active as a catalyst in the form of nanoparticles [ ] [ ] [ ] [ ] [ ] [ ] [ ] . as the name suggests, nanoparticles have small (nano) size and corresponding large surface/volume ratio. examples are shown in figure . due to these characteristics, au nanoparticles can have an important role, not only in catalysis, but also in nanomedicine, suitable for several biological applications. in fact, it has been argued that au nanoparticles could be used in almost all medical applications, from diagnostics to therapy, prevention, and hygiene [ ] [ ] [ ] [ ] [ ] [ ] [ ] . gold nanoparticles have remarkable properties and can also play an important role in vaccine research for several diseases, as showed in several recent reviews [ , , [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] . vaccines have been a significant advancement in public health, preventing the death of many millions of people every year [ ] . the main purpose of vaccination is to introduce an antigen from a vaccines have been a significant advancement in public health, preventing the death of many millions of people every year [ ] . the main purpose of vaccination is to introduce an antigen from usually nanoparticles are synthesized in colloidal solution by reduction of chloroauric acid (haucl ) by variations of the so-called turkevich method [ ] . their characterization is carried out by ultraviolet-visible spectroscopy, their diameters determined by transmission electron microscopy. then, they can be further functionalized with the desired molecules, according to their specific needs. one example is given in scheme , showing a procedure carried out by saha et al. [ ] . gold nanoparticles were synthesized in aqueous medium by an ultra-sound assisted green method using black pepper extract and chitosan as reducing agent and a stabilizing agent, respectively. the biopolymer-inspired biogenic method (scheme ) was found to be stable and with enhanced bioactivity. the developed nanomaterial boosts the production of reactive oxygen species and misbalances the antioxidant parameters of detoxification enzymes of filarial parasites, such as gst, which besides its immune prophylactic potency, is also a promising candidate vaccine as shown in s. cervi and w. bancrofti [ ] . gold nanoparticles, in general, have remarkably high surface-to-volume ratio, are biocompatible and inert, and can be easily functionalized with several molecules; thus, they can also play an important role in the vaccine field as adjuvants, reducing toxicity, enhancing immunogenic activity, and providing stability of vaccine in storage, and have great potential as carriers for the development of a great diversity of fully synthetic vaccines [ ] [ ] [ ] [ ] [ ] , , , , [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] . their shape and size can affect immunological responses in vivo and in vitro [ , [ ] [ ] [ ] [ ] . moreover, they are able to penetrate blood vessels and tissue barriers and to be targeted to a specific cell by means of specifically functionalized molecules [ ] . moreover, gold nanoparticles can be packaged inside virus-like particles generated by heterologous expression of viral structural genes that are powerful tools in vaccine development [ ] . anionic gold nanoparticles increased the half-life of a green fluorescent protein expressing adenovirus from similar to h to days at °c (from to > days at room temperature) [ ] . promising cd + t cell results were obtained with polyelectrolyte multilayers assembled on gold nanoparticles [ , ] . a positively charged antigen and a negatively charged immuno-adjuvant on gold nanoparticles resulted in a new vaccine platform. usually nanoparticles are synthesized in colloidal solution by reduction of chloroauric acid (haucl ) by variations of the so-called turkevich method [ ] . their characterization is carried out by ultraviolet-visible spectroscopy, their diameters determined by transmission electron microscopy. then, they can be further functionalized with the desired molecules, according to their specific needs. one example is given in scheme , showing a procedure carried out by saha et al. [ ] . gold nanoparticles were synthesized in aqueous medium by an ultra-sound assisted green method using black pepper extract and chitosan as reducing agent and a stabilizing agent, respectively. the biopolymer-inspired biogenic method (scheme ) was found to be stable and with enhanced bioactivity. the developed nanomaterial boosts the production of reactive oxygen species and misbalances the antioxidant parameters of detoxification enzymes of filarial parasites, such as gst, which besides its immune prophylactic potency, is also a promising candidate vaccine as shown in s. cervi and w. bancrofti [ ] . gold nanoparticles, in general, have remarkably high surface-to-volume ratio, are biocompatible and inert, and can be easily functionalized with several molecules; thus, they can also play an important role in the vaccine field as adjuvants, reducing toxicity, enhancing immunogenic activity, and providing stability of vaccine in storage, and have great potential as carriers for the development of a great diversity of fully synthetic vaccines [ ] [ ] [ ] [ ] [ ] , , , , [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] . their shape and size can affect immunological responses in vivo and in vitro [ , [ ] [ ] [ ] [ ] . moreover, they are able to penetrate blood vessels and tissue barriers and to be targeted to a specific cell by means of specifically functionalized molecules [ ] . moreover, gold nanoparticles can be packaged inside virus-like particles generated by heterologous expression of viral structural genes that are powerful tools in vaccine development [ ] . anionic gold nanoparticles increased the half-life of a green fluorescent protein expressing adenovirus from similar to h to days at • c (from to > days at room temperature) [ ] . promising cd + t cell results were obtained with polyelectrolyte multilayers assembled on gold [ ] ). an immunostimulatory nanocomposite (cpg-au@hbc vlp), designed by self-assembling engineered virus-like particles encapsulating cpg-gold nanoparticle conjugates through electrostatic interactions, showed an increase in cd + and cd + t cell numbers, inducing important cellular and humoral immune response [ ] . ultrasmall graphene oxide-supported gold nanoparticles (usgo-au) were obtained from reduction of chloroauric acid using usgo and then decorated with ovalbumin antigen (ova) through physical adsorption to obtain usgo-au@ova and used immune adjuvants [ ] . it was shown that usgo-au@ova can efficiently stimulate raw . cells to secrete tumor necrosis factor (tnf-), a mediator for cellular immune response. usgo-au@ova can also promote robust ova specific antibody response, cd + t cells proliferation, and secretion of different cytokines. fluorescent au nanoclusters were synthesized using ovalbumin-cpg oligodeoxynucleotides (odns) conjugates as templates [ ] . the nanoclusters can act as self-vaccines to assist in generation of high immunostimulatory activity. gold-based nanovaccines were synthesized using a self-assembling conjugation method [ ] . dendritic cells uptake gold nanovaccines with minimal toxicity and are able to process the vaccine peptides on the particles to stimulate cytotoxic t lymphocytes (ctls). these high-peptide density au nanovaccines can stimulate ctls better than free peptides and have great potential as carriers for various vaccine types [ ] . hydrophilic gold nanodots were used to control lipopolysaccharide assembly to ease the formation of stable endotoxin nanovesicles, which are stable precursors of cubosomes and hexosomes with specific immunological effects that might be useful in vaccine development [ ] . needle-free vaccine delivery systems efficiently transport powdered or particulate dna and protein vaccines into the epidermal tissue [ ] [ ] [ ] [ ] . it can be used to directly transfect antigen presenting cells by formulating dna or protein vaccines onto - µm gold particles (particle-mediated immunization). a solid-in-oil dispersion of gold nanorods can also enhance transdermal protein delivery and skin vaccination [ ] . plasmid dna (pdna)-coated gold nanoparticles were successfully delivered into ex vivo murine and porcine skin at low inlet pressures using parallel arrays of microchannels [ ] . it was shown that full-length pdna was preserved after each particle preparation and jetting procedures. below, several examples are given on the role of gold nanoparticles in the attempts of producing vaccines for several diseases. cancer is one of the main causes of death worldwide, that can affect people at all ages, even small children and foetuses, the risk usually increasing with age [ , ] . the present treatments consist of surgery, chemotherapy, or radiotherapy, which can have adverse side effects, due to a lack of specificity for tumors [ ] . an ideal treatment should aim only at the target tumor cells and have limited detrimental effects on normal cells [ , ] . [ ] ). an immunostimulatory nanocomposite (cpg-au@hbc vlp), designed by self-assembling engineered virus-like particles encapsulating cpg-gold nanoparticle conjugates through electrostatic interactions, showed an increase in cd + and cd + t cell numbers, inducing important cellular and humoral immune response [ ] . ultrasmall graphene oxide-supported gold nanoparticles (usgo-au) were obtained from reduction of chloroauric acid using usgo and then decorated with ovalbumin antigen (ova) through physical adsorption to obtain usgo-au@ova and used immune adjuvants [ ] . it was shown that usgo-au@ova can efficiently stimulate raw . cells to secrete tumor necrosis factor (tnf-), a mediator for cellular immune response. usgo-au@ova can also promote robust ova specific antibody response, cd + t cells proliferation, and secretion of different cytokines. fluorescent au nanoclusters were synthesized using ovalbumin-cpg oligodeoxynucleotides (odns) conjugates as templates [ ] . the nanoclusters can act as self-vaccines to assist in generation of high immunostimulatory activity. gold-based nanovaccines were synthesized using a self-assembling conjugation method [ ] . dendritic cells uptake gold nanovaccines with minimal toxicity and are able to process the vaccine peptides on the particles to stimulate cytotoxic t lymphocytes (ctls). these high-peptide density au nanovaccines can stimulate ctls better than free peptides and have great potential as carriers for various vaccine types [ ] . hydrophilic gold nanodots were used to control lipopolysaccharide assembly to ease the formation of stable endotoxin nanovesicles, which are stable precursors of cubosomes and hexosomes with specific immunological effects that might be useful in vaccine development [ ] . needle-free vaccine delivery systems efficiently transport powdered or particulate dna and protein vaccines into the epidermal tissue [ ] [ ] [ ] [ ] . it can be used to directly transfect antigen presenting cells by formulating dna or protein vaccines onto - µm gold particles (particle-mediated immunization). a solid-in-oil dispersion of gold nanorods can also enhance transdermal protein delivery and skin vaccination [ ] . plasmid dna (pdna)-coated gold nanoparticles were successfully delivered into ex vivo murine and porcine skin at low inlet pressures using parallel arrays of microchannels [ ] . it was shown that full-length pdna was preserved after each particle preparation and jetting procedures. below, several examples are given on the role of gold nanoparticles in the attempts of producing vaccines for several diseases. cancer is one of the main causes of death worldwide, that can affect people at all ages, even small children and foetuses, the risk usually increasing with age [ , ] . the present treatments consist of surgery, chemotherapy, or radiotherapy, which can have adverse side effects, due to a lack of specificity for tumors [ ] . an ideal treatment should aim only at the target tumor cells and have limited detrimental effects on normal cells [ , ] . it was first postulated by coley in that the immune system was able to recognize and set a response against tumors [ ] . later, it was shown that immunization of mice with mutated tumor cells could induce a protective anti-tumor immune response against non-immunogenic tumor [ ] . this led to cancer immunotherapy research and to the development of cancer vaccines capable of generating an active tumor-specific immune response. cancer vaccines have high specificity for tumor cells and long-lasting immunological memory that may safeguard against recurrences, and can be used to either prevent (prophylactic) or treat established cancer (therapeutic) [ , ] . therapeutic cancer vaccines (also called active immunotherapy) work to enable the immune system of a patient to eradicate cancer cells [ ] . identification of tumor-associated antigens (taas) and tumor-specific antigens (tsas) has led to increased efforts to develop vaccination strategies. vaccines may be composed of whole cells or cell extracts, genetically modified tumor cells, dendritic cells (dcs) loaded with taas, immunization with soluble proteins or synthetic peptides, recombinant viruses or bacteria encoding tumor-associated antigens, and plasmid dna-encoding tsas or taas in conjunction with appropriate immunomodulators [ , ] . all of these vaccination approaches aim to induce specific immunological responses and are localized into taas, destroying only the tumor cells and leaving the majority of other cells of the body undamaged. an efficient delivery to cells is needed and gold nanoparticles have proved to be excellent carriers [ ] [ ] [ ] [ ] [ ] [ ] [ ] . gold nanoparticles enabled efficient antigen delivery to dendritic cells and then activated the cells to facilitate cross-presentation, inducing antigen-specific cytotoxic t-lymphocyte responses for effective cancer therapy [ ] . moreover, they show several advantages over other nanoparticulate-based carriers, such as the ease with which they control their size for different applications, the variety of antigens and adjuvants that can be easily linked to and displayed on their surface, the fact that they can be detected using non-invasive imaging techniques, providing clinicians with information on where or whether the vaccines have been delivered, which helps to predict or evaluate therapeutic efficacy, and the biocompatibility and non-toxicity of gold [ , , ] . au nanoparticle immunotherapies are well suited for synergistic combination therapy with existing cancer therapies such as photothermal ablation [ , ] . all these features suggest that gold nanoparticle-based antigen delivery systems may be a useful vaccine technology that is able to prevent and/or treat cancer. the several functionalities of gold nanoparticles make them promising vehicles for immune therapies, especially for combinatorial treatment approaches that target multiple immune pathways (figure ) . a polymerizable version of the tn-antigen glycan was synthesized and the polymers were conjugated to gold nanoparticles, producing nanoscale glycoconjugates [ ] . these nanomaterials generated a strong and long-lasting production of antibodies, selective to the tn-antigen glycan and cross-reactive toward mucin proteins displaying tn, and thus represent a simple approach to the synthesis of anticancer vaccines. gold nanoparticles obtained by a modified turkevich method were functionalized with mucin- (muc- ) glycoprotein using clealand's reagent [ ] . (muc- is involved in fundamental biological processes that can be found over-expressed and with a clearly changed glycan pattern on epithelial tumor cells, and thus is a promising target structure in the search for effective carbohydrate-based cancer vaccines and immunotherapeutics [ ] ). the obtained au-muc- nano-construction has been shown to be an important macrophage activator, leading to the release of cytokines such as tnf-alpha, il- , il- , and il- on peritoneal macrophages isolated from mice [ ] . au nanoparticle immunotherapies are well suited for synergistic combination therapy with existing cancer therapies such as photothermal ablation [ , ] . all these features suggest that gold nanoparticle-based antigen delivery systems may be a useful vaccine technology that is able to prevent and/or treat cancer. the several functionalities of gold nanoparticles make them promising vehicles for immune therapies, especially for combinatorial treatment approaches that target multiple immune pathways (figure ) . a polymerizable version of the tn-antigen glycan was synthesized and the polymers were conjugated to gold nanoparticles, producing nanoscale glycoconjugates [ ] . these nanomaterials generated a strong and long-lasting production of antibodies, selective to the tn-antigen glycan and cross-reactive toward mucin proteins displaying tn, and thus represent a simple approach to the synthesis of anticancer vaccines. gold nanoparticles obtained by a modified turkevich method were functionalized with mucin- (muc- ) glycoprotein using clealand's reagent [ ] . other authors also reported on the synthesis of muc -glycopeptide antigens and their coupling to gold nanoparticles of different sizes and developed a new dot-blot immunoassay to test the potential antigen-antibody binding [ ] . a glycopeptide sequence derived from muc- glycoprotein and the t-cell epitope p sequence were immobilized gold nanoparticles attached to polyethylene glycol chains [ ] . after immunization, mice showed significant mhc-ii mediated immune responses, and their antisera were able to recognize human mcf- breast cancer cells. gold nanoparticles designed this way have the potential to be used in the development of anticancer vaccines. gold nanoparticles proved to be efficient in facilitating the delivery of the ovalbumin (ova) peptide antigen and the cpg adjuvant and enhance their therapeutic effect in a b -ova tumor model. gold nanoparticle-mediated ova peptide delivery can have important therapeutic benefits without the need of an adjuvant, showing that gold nanoparticles are effective peptide vaccine carriers, having the potential to allow the use of lower and safer adjuvant doses during vaccination [ ] . the use of gold nanoparticles in hiv vaccine development has been recently reviewed by several authors [ ] . after more than years since the discovery of hiv in , no effective vaccine is yet available [ , [ ] [ ] [ ] . some histocompatibility complex molecules expressed on the surface of hiv are potential targets for neutralizing antibodies [ ] , as shown in figure . among the few broadly neutralizing hiv monoclonal antibodies, g is the only carbohydrate-directed that is able to recognize a cluster of high-mannose glycans on the viral envelope glycoprotein gp [ ] . this type of glycan is thus envisaged as a target to develop an hiv vaccine capable of eliciting g -like antibodies. gold nanoparticles coated with self-assembled monolayers of synthetic oligomannosides, which are present in gp , are able to bind g with high affinity and to interfere with g /gp binding [ ] . thiol-terminated oligosaccharides have been attached on gold nanoparticles and have been used in attempts to develop an hiv vaccine [ ] . two nanometer gold glyconanoparticles were coated with synthetic partial structures of several mannosides [ ] . the assembly of the antennas of the gp high-mannose type glycan on gold glyconanoparticles provided superior binding to the anti-hiv antibody g , which could help in the design of a carbohydrate-based vaccine against hiv. it was shown that conjugation to negatively charged gold glyconanoparticles could stabilize either the alpha-helix or beta-strand conformation of the third variable region (v peptide) of the hiv- gp [ ] . the peptide on the nanoparticles shows more stability toward peptidase degradation compared to the free peptide. v beta-glyconanoparticles produce antibodies in rabbits that recognize a recombinant gp , and the serum showed consistent neutralizing activity. these results potentially allow for the design of new fully synthetic hiv vaccine candidates [ ] . gold nanorods modified with poly(diallydimethylammonium chloride) or polyethyleneimine can significantly promote cellular and humoral immunity, as well as t-cells proliferation, through activating antigen-presenting cells, when compared to naked hiv envelope plasmid dna treatment in vivo [ ] . this makes gold nanorods promising dna vaccine adjuvants for hiv treatment. the use of gold nanoparticles in hiv vaccine development has been recently reviewed by several authors [ ] . after more than years since the discovery of hiv in , no effective vaccine is yet available [ , [ ] [ ] [ ] . some histocompatibility complex molecules expressed on the surface of hiv are potential targets for neutralizing antibodies [ ] , as shown in figure . among the few broadly neutralizing hiv monoclonal antibodies, g is the only carbohydrate-directed that is able to recognize a cluster of high-mannose glycans on the viral envelope glycoprotein gp [ ] . this type of glycan is thus envisaged as a target to develop an hiv vaccine capable of eliciting g -like antibodies. gold nanoparticles coated with self-assembled monolayers of synthetic oligomannosides, which are present in gp , are able to bind g with high affinity and to interfere with g /gp binding [ ] . thiol-terminated oligosaccharides have been attached on gold nanoparticles and have been used in attempts to develop an hiv vaccine [ ] . two nanometer gold glyconanoparticles were coated with synthetic partial structures of several mannosides [ ] . the assembly of the antennas of the gp high-mannose type glycan on gold glyconanoparticles provided superior binding to the anti-hiv antibody g , which could help in the design of a carbohydrate-based vaccine against hiv. it was shown that conjugation to negatively charged gold glyconanoparticles could stabilize either the alpha-helix or beta-strand conformation of the third variable region (v peptide) of the hiv- gp [ ] . the peptide on the nanoparticles shows more stability toward peptidase degradation the use of gold nanoparticles in designing vaccines against tick-borne encephalitis (tbe) was proposed for the first time by demenev et al. [ ] . the vaccine was prepared by conjugating colloid gold with a soluble tbe antigen. in animals immunized with the experimental vaccine, the protection coefficient and mean survival time were, respectively, . - . times and - % higher than in mice immunized with a commercial vaccine. moreover, the mean survival time was . - . times longer in animals injected with antibodies from mice immunized with the experimental vaccine, compared with the commercial one. in a more recent study, colloidal gold particles were used as carriers of protein antigen of the capsid of the tbe virus in the antiviral vaccine [ ] . after two inoculations, the mice in that study were found to resist challenge with , times the % lethal dose (ld ) of jev (beijing- strain), even when immunized with a relatively small dose of . mug of plasmid dna. japanese b encephalitis vaccine is an important vaccine to prevent this serious mosquito-borne disease caused by the virus [ , ] . there are some semiquantitative methods to determine this vaccine, such as plaque forming unit and the animal testing, but they have low sensitivity, are time-consuming, and have a high cost. a label-free amperometric immunosensor for fast and sensitive assay of japanese b encephalitis vaccine was reported with a specific response in the range . × − to . × − lg·pfu/ml (pfu/ml is plaque forming unit and lg is common logarithm) with a detection limit of × − lg·pfu/ml [ ] . an immunosensor based on the immobilization of antiserum of japanese b encephalitis on a gold electrode modified by [nano-au/co(bipyridine) + ] /nano-au/l-cysteine has also been reported [ ] . it exhibited fast potentiometric high sensitivity and long-term stability. these works are promising test methods for biological products. hepatitis is inflammation of the liver that may originate a yellow color in the skin and eyes and abdominal pain, among other symptoms that can be caused by viruses or from the abuse of alcohol and certain medications. there are several types of viral hepatitis: types a, b, c, d, and e. hepatitis a and e are often spread by contaminated food and water, while hepatitis b is mainly sexually transmitted but may pass from mother to child during pregnancy. types b and c are usually spread through figure shows a comparison between the viruses. hepatitis is inflammation of the liver that may originate a yellow color in the skin and eyes and abdominal pain, among other symptoms that can be caused by viruses or from the abuse of alcohol and certain medications. there are several types of viral hepatitis: types a, b, c, d, and e. hepatitis a and e are often spread by contaminated food and water, while hepatitis b is mainly sexually transmitted but may pass from mother to child during pregnancy. types b and c are usually spread through infected blood. hepatitis d can only infect people already infected with hepatitis b. figure shows a comparison between the viruses. concerning hepatitis b vaccine research, hbsag-dna can be introduced into the host by intramuscular or intradermal route using a needle with syringe. an alternative is using gene-gun, biolistic ® , powderjecttm, accell ® , or particle-mediated dna delivery, when dna-coated microscopic gold particles are transported using a needle-free device directly into the epidermic cells [ ] . micron-size gold particles were used as particulate adjuvants and coinjected intradermally with plasmid dna encoding the hbsag into mice [ , ] . the presence of gold particles accelerated the antibody response significantly, increased the percentage of responding animals, and shortened the time taken to reach maximal antibody titers by two weeks. these immunizations were effective in protecting mice against tumor challenge with cancer cells, expressing hbsag as a surrogate cancer antigen. good results were also obtained with minipigs [ ] . electrically activated plasmonic au nanoparticles can drive vibrational and dipole-like oscillations that are able to disrupt nearby cell membranes, allowing enhanced cell poration and facilitating the uptake of a model hepatitis c virus dna vaccine was recently reported [ ] . immunized mice showed up to -fold higher gene expression compared to control treatments (without nanoparticles) and exhibited significantly increased levels of both antibody and cellular immune responses. a new method of preparation of a vaccine for hepatitis e (heva) using in situ easy growth of gold clusters in the vaccine was recently reported [ ] . the prepared heva/au enhances its immune responses in vivo and reduces the potential toxicity of heva. the fluorescence of gold clusters enables the heva to be traceable, which may open a way to track the dynamic behavior of vaccines and further help to optimize an individual therapeutic regimen for immunotherapy [ ] . gold has been used in several other vaccines. some examples are reported below. gold nanoparticles were conjugated to a synthetic peptide of the vp capsid protein of the foot-and-mouth disease virus, which causes an acute, highly contagious infection of domestic and wild animals, transmittable to humans, for which the existing vaccines are not much effective [ ] . the resulting conjugate (with or without the use of complete freund's adjuvant) was compared to a commercial vaccine and to the native peptide, in immunization of guinea pigs. the titer and sensitivity of the antibodies were maximal for the combination comprising the nanoparticle-peptide conjugate and complete freund's adjuvant. gold nanoparticles were evaluated as vaccine carriers for enhancing the antibody response against a resembling foot-and-mouth disease virus peptide [ ] . particles with - nm in diameter stimulated the highest antibody levels and accumulated at the highest numbers in the spleen of mice. recently, the potential of chitosan functionalized gold nanoparticles (csaunps) for the transmucosal delivery of tetanus toxoid vaccine was shown [ , , ] . excellent stability was found for the formulation at recommended storage conditions. csaunps were also used as a carrier for the tetanus toxoid (tt) antigen along with the immunostimulant quillaja saponaria extract (qs) [ ] . the synthesized csaunps were spherical, around nm in size and conferred protection to tt against gastric hydrolysis. tt-qs-csaunps induced up to -fold immune responses compared to tt and tt-qs controls, after oral administration in mice. the delivery of tt and qs with functionalized csaunps might be a good approach for oral vaccine delivery [ ] . gold glyconanoparticles proved to be good carriers for a synthetic streptococcus pneumoniae type conjugate vaccine [ ] . a gold nanorod construct that displayed the major protective antigen of the respiratory syncytial virus (which causes pneumonia and wheezing in children and the elderly), the fusion protein (f) was reported, which can be a candidate vaccine preparation by the covalent attachment of viral protein using a layer-by-layer approach [ ] . gold nanoparticles of nm were conjugated to the matrix protein (m e) of influenza a virus m e through thiol-gold interactions [ ] . this virus ( figure ) is often responsible for seasonal epidemics and is also a high risk for pandemics [ ] . vaccination of mice with m e-au conjugates induced m e-specific igg serum antibodies, which significantly improved by addition of cpg as adjuvant, as mice immunized that way were fully protected against lethal pr [ ] . the same authors also showed that the inclusion of excess soluble m e antigen along with m e immobilized on au nanoparticles is vital for inducing high levels of antibody response, and in providing complete protection [ ] . also showed that the inclusion of excess soluble m e antigen along with m e immobilized on au nanoparticles is vital for inducing high levels of antibody response, and in providing complete protection [ ] . a recent study showed that animals immunized with a transmissible gastroenteritis virus, conjugated with colloidal gold nanoparticles, produced antibodies with a higher titer than those produced in response to the native virus [ ] . a gold nanoparticle antigen delivery approach was used together with a novel polysaccharide nanoparticulate adjuvant, and an effective t-cell vaccine was developed providing protection in animal models of listeriosis (a fatal infection for fetuses and newborns that causes meningitis and cutaneous lesions) [ ] . this antigen delivery approach against listeriosis, using gold nanoparticles and bacterial peptides, elicits protective cellular immunity, independent of the adjuvant used, either as a carbohydrate such as advax [ ] or a tlr- agonist such as dio- [ ] . gold glyconanoparticles loaded with listeriolysin peptide - (llo - ) or glyceraldehyde- -phosphate dehydrogenase - peptide (gapdh ) were successfully administrated to pregnant female mice as a vaccination for this disease [ ] . neonates from vaccinated mothers were free of bacteria and healthy, while non-vaccinated mice showed clear brain affections and cutaneous diminishment of melanocytes. amphiphilic surface ligand-coated au nanoparticles able to target myeloid dendritic cells in lymph nodes were used as a peptide antigen carrier, increasing the efficacy of a model vaccine in b -ova melanoma mouse models [ ] . gold nanoparticles were coated with the f -antigen of yersinia pestis (bacterium responsible for the plague), using n-hydroxysuccinimide and n-( -dimethylaminopropyl)-n′-ethylcarbodiimide a recent study showed that animals immunized with a transmissible gastroenteritis virus, conjugated with colloidal gold nanoparticles, produced antibodies with a higher titer than those produced in response to the native virus [ ] . a gold nanoparticle antigen delivery approach was used together with a novel polysaccharide nanoparticulate adjuvant, and an effective t-cell vaccine was developed providing protection in animal models of listeriosis (a fatal infection for fetuses and newborns that causes meningitis and cutaneous lesions) [ ] . this antigen delivery approach against listeriosis, using gold nanoparticles and bacterial peptides, elicits protective cellular immunity, independent of the adjuvant used, either as a carbohydrate such as advax [ ] or a tlr- agonist such as dio- [ ] . gold glyconanoparticles loaded with listeriolysin peptide - (llo - ) or glyceraldehyde- -phosphate dehydrogenase - peptide (gapdh( - )) were successfully administrated to pregnant female mice as a vaccination for this disease [ ] . neonates from vaccinated mothers were free of bacteria and healthy, while non-vaccinated mice showed clear brain affections and cutaneous diminishment of melanocytes. amphiphilic surface ligand-coated au nanoparticles able to target myeloid dendritic cells in lymph nodes were used as a peptide antigen carrier, increasing the efficacy of a model vaccine in b -ova melanoma mouse models [ ] . gold nanoparticles were coated with the f -antigen of yersinia pestis (bacterium responsible for the plague), using n-hydroxysuccinimide and n-( -dimethylaminopropyl)-n -ethylcarbodiimide hydrochloride coupling chemistry, and administrated to mice, triggering an igg antibody response to the f -antigen [ ] . the sera raised against f -antigen coupled to au nanoparticles were able to competitively bind to recombinant f -antigen, displacing protective macaque sera. target antigens identified in plasmodium falciparum, the parasite responsible for malaria, include surface proteins pfs and pfs / in male and female gametocytes and pfs expressed in zygotes and ookinetes. the codon-harmonized recombinant pfs in escherichia coil (chrpfs ) is able to produce malaria transmission-blocking antibodies in mice. chrpfs was also investigated, with gold nanoparticles of different shapes, size and physicochemical properties as adjuvants for induction of transmission blocking immunity, causing transmission blocking antibodies [ ] . these results show that gold nanoparticle-based formulations can be developed as nanovaccines to enhance the immunogenicity of vaccine antigens. gold nanoparticles were conjugated to n-terminal domains of pseudomonas aeruginosa flagellin recombinant protein through direct interaction of thiol molecules of the cysteines with gold and formation of au-s bond [ ] . mice that received aunp-flagellin(( - )) with freund's adjuvant produced high titers of anti-flagellin(( - )) antibodies that effectively recognized the native flagellin on the bacteria. synthetic virus-like particles (svlps) were prepared by incubating nm gold nanoparticles in a solution containing an avian coronavirus spike protein [ ] . after removing the free proteins, antigen-laden particles were recovered and showed morphological semblance to natural viral particles. vaccination with the svlps showed enhanced lymphatic antigen delivery, stronger antibody titers, increased splenic t-cell response, and reduced infection-associated symptoms, and provided superior antiviral protection when compared to a commercial whole inactivated virus vaccine [ ] . burkholderia mallei are bacteria responsible for the glanders disease, which is recently classified as a tier agent, since they can be weaponized for aerosol release, cause high mortality rates, and show multi-drug resistance, and for which there is so far no vaccine available [ ] . gold nanoparticles were covalently coupled with one of three different protein carriers (tethc, hcp , and flic) followed by conjugation to lipopolysaccharide (lps) generated significantly higher antibody titers compared with lps alone. in another study, a gold nanoparticle glycoconjugate composed of burkholderia thailandensis lps conjugated to flic was evaluated for the first time as a candidate vaccine for glanders on rhesus macaques (macaca mulatta) [ ] . it has recently been shown that the pichia pastoris yeast expression system was adequate for the production of recombinant-truncated proteins, and their apparent bioactivity suggests that torf , torf c, and torf d are potential candidate vaccines against cyprinid herpes virus infection. found in farmed gibel carp carassius gibelio, it is an infectious disease that recently emerged in china that has been troubling the aquaculture industry [ ] . colloidal gold-labelled immunoglobulin was used to confirm rv c protein localization on mycobacterium tuberculosis [ ] . these results, along with those obtained for other proteins, might lead to the discovery of select peptides that could have the potential to be included in a subunit-based vaccine for tuberculosis, an infectious disease that remains lethal around the world. biocompatible gold nanoparticles were complexed with anti-dengue virus small interfering rnas (sirna), and were able to enhance the sirna delivery and stability, becoming a novel strategy against a virus that causes flu-like symptoms, haemorrhagic fever, and death [ ] . escherichia coli as a model pathogen for the design of an antibacterial vaccine [ ] . the bacterial outer membrane vesicles were collected and successfully coated onto gold nanoparticles with a nm diameter. the resulting bacterial membrane-coated gold nanoparticles, when injected subcutaneously, induced rapid activation and maturation of dendritic cells in the lymph nodes of the vaccinated mice, generated durable antibody responses, and induced a high production of interferon gamma and interleukin- , but not interleukin- , indicating its capability of generating strong th -and th -biased cell responses against the used bacteria. a monoclonal antibody raised against human cytomegalovirus (cmv, a herpes virus) surface glycoprotein (gb) was chemically conjugated with gold nanoparticles [ ] . the gb-gold nanoparticles blocked viral replication, virus-induced cytopathogenic effects, and virus spread in cell culture without inducing cytotoxicity, and cells treated with them gained resistance to cmv infection. as discussed above, research on gold has indicated its potential for several applications in nanomedicine and its (bio)medical applications, including vaccines, and, as shown in this review, has been increasing. the overall conclusion is that the potential of gold for stimulating research in vaccines is considerable. the results will certainly lead to more practical and commercial applications, the full extent of which has yet to be envisaged. certainly, the use of gold carries added costs. as newer and more expensive vaccines are introduced and attempted to reach people of different ages and in new settings, the logistics systems must be strengthened and optimized, as recently highlighted and reviewed by zaffran et al. [ ] . catalysis by gold nanoparticles when gold is not noble: catalysis by nanoparticles gold catalysis catalysis by gold catalytic applications for gold nanotechnology anisotropic gold nanoparticles: preparation and applications in catalysis gold nanoparticles in biology and medicine: recent advances and prospects gold nanoparticles in biomedical applications: recent advances and perspectives biological applications of gold nanoparticles particulate inorganic adjuvants: recent developments and future outlook gold nanoparticles and their applications in biomedicine applications of nanotechnology in the agriculture, food, and pharmaceuticals use of nanoparticles to deliver immunomodulatory oligonucleotides current status of multiple antigen-presenting peptide vaccine systems: application of organic and inorganic nanoparticles vaccine delivery using nanoparticles applications of nanomaterials as vaccine adjuvants. hum. vaccines immunother oral delivery of nanoparticle-based vaccines synthetic nanoparticles for vaccines and immunotherapy gold nanoparticles and vaccine development gold nanocluster-based vaccines for dual-delivery of antigens and immunostimulatory oligonucleotides gold nanoparticles (aunps): a new frontier in vaccine delivery design of nanomaterial based systems for novel vaccine development recent advances in synthetic carbohydrate-based human immunodeficiency virus vaccines advanced nanobiomaterials: vaccines, diagnosis and treatment of infectious diseases recent advances in subunit vaccine carriers nanomaterial-based vaccine adjuvants understanding the immunogenicity and antigenicity of nanomaterials: past, present and future immunological properties of gold nanoparticles role of metallic nanoparticles in vaccinology: implications for infectious disease vaccine development vaccine technologies: from whole organisms to rationally designed protein assemblies review: cancer therapy and vaccination biomedicine: new insights from plant viruses nanovaccines and their mode of action glycodendrimers: versatile tools for nanotechnology. braz functional nanomaterials can optimize the efficacy of vaccines nanomedicine in the management of microbial infection-overview and perspectives nanomedicine scale-up technologies: feasibilities and challenges nanoparticle based tailoring of adjuvant function: the role in vaccine development the potential of nanoparticles for the immunization against viral infections learning from vaccines and progressing to antigen-specific immunotherapies advances in multifunctional glycosylated nanomaterials: preparation and applications in glycoscience nanoparticle-based modulation of the immune system real-time assessment of nanoparticle-mediated antigen delivery and cell response a study of the nucleation and growth processes in the synthesis of colloidal gold development of chitosan based gold nanomaterial as an efficient antifilarial agent: a mechanistic approach gold colloidal solution is used as an adjuvant, provides reducing toxicity of vaccines, to enhance their immunogenic activity and to provide stability of vaccine in storage. patent ru -c new adjuvant composition comprising colloidal gold, used for therapeutic vaccine or for inducing immune response gold nanoparticles as carriers for a synthetic streptococcus pneumoniae type conjugate vaccine gold nanoparticles as a vaccine platform: influence of size and shape on immunological responses in vitro and in vivo injectable nanomaterials for drug delivery: carriers, targeting moieties, and therapeutics high-density sub- -nm peptide-gold nanoparticle complexes improve vaccine presentation by dendritic cells in vitro gold nanoparticle conjugates: recent advances toward clinical applications polymeric nanoparticles potent vectors for vaccine delivery targeting cancer and infectious diseases tracking targeted bimodal nanovaccines: immune responses and routing in cells, tissue, and whole organism protection of non-human primates against glanders with a gold nanoparticle glycoconjugate vaccine synthesis of tumor-associated muc -glycopeptides and their multivalent presentation by functionalized gold colloids metal based frameworks for drug delivery systems gold nanoparticles: recent advances in the biomedical applications additives for vaccine storage to improve thermal stability of adenoviruses from hours to months assessment of gold nanoparticles as a size-dependent vaccine carrier for enhancing the antibody response against synthetic foot-and-mouth disease virus peptide structure function attributes of gold nanoparticle vaccine association: effect of particle size and association temperature intracellular accumulation and immunological properties of fluorescent gold nanoclusters in human dendritic cells different-sized gold nanoparticle activator/antigen increases dendritic cells accumulation in liver-draining lymph nodes and cd +t cell responses yeast-expressed bacteriophage-like particles for the packaging of nanomaterials polyelectrolyte multilayers assembled entirely from immune signals on gold nanoparticle templates promote antigen-specific t cell response impact of dose, route, and composition on the immunogenicity of immune polyelectrolyte multilayers delivered on gold templates construction and immunological evaluation of cpg-au@hbc virus-like nanoparticles as a potential vaccine ultrasmall graphene oxide supported gold nanoparticles as adjuvants improve humoral and cellular immunity in mice engineered cpg-antigen conjugates protected gold nanoclusters as smart self-vaccines for enhanced immune response and cell imaging endotoxin nanovesicles: hydrophilic gold nanodots control supramolecular lipopolysaccharide assembly for modulating immunological responses epidermal powder immunization induces both cytotoxic t-lymphocyte and antibody responses to protein antigens of influenza and hepatitis b viruses powder and particle-mediated approaches for delivery of dna and protein vaccines into the epidermis recent advances in hepatitis b vaccination nanoparticulate mediated transcutaneous immunization: myth or reality a solid-in-oil dispersion of gold nanorods can enhance transdermal protein delivery and skin vaccination a microarray mems device for biolistic delivery of vaccine and drug powders cancer: facts, causes, symptoms and research application of nanostructured drug delivery systems in immunotherapy of cancer: a review end results in hodgkin's disease and lymphosarcoma treated by the mixed toxins of erysipelas and bacillus prodigiosus, alone or combined with radiation in vitro administration of gold nanoparticles functionalized with muc- protein fragment generates anticancer vaccine response via macrophage activation and polarization mechanism glycopolymer-functionalized gold nanoparticles: a new strategy toward synthetic anticancer vaccines development of a novel cancer vaccine based on multivalent presentation of tumor-associated carbohydrate antigens on gold nanoparticle scaffolds design and synthesis of multifunctional gold nanoparticles bearing tumor-associated glycopeptide antigens as potential cancer vaccines imageable antigen-presenting gold nanoparticle vaccines for effective cancer immunotherapy in vivo gold nanoparticle mediated cancer immunotherapy gold nanoparticles displaying tumor-associated self-antigens as a potential vaccine for cancer immunotherapy current status and future directions of nanoparticulate strategy for cancer immunotherapy gold nanoparticles as an antigen and as an adjuvant multicopy multivalent' glycopolymer-stabilized gold nanoparticles as potential synthetic cancer vaccines glycopeptide-functionalized gold nanoparticles for antibody induction against the tumor associated mucin- glycoprotein in vivo gold nanoparticle delivery of peptide vaccine induces anti-tumor immune response in prophylactic and therapeutic tumor models role of nanotechnology in hiv/aids vaccine development gold nanoparticles coated with oligomannosides of hiv- glycoprotein gp mimic the carbohydrate epitope of antibody g gold manno-glyconanoparticles for intervening in hiv gp carbohydrate-mediated processes enhancing dendritic cell activation and hiv vaccine effectiveness through nanoparticle vaccination immunization with recombinant hla classes i and ii, hiv- gp , and siv p elicits protection against heterologous shiv infection in rhesus macaques design and synthesis of thiol-terminated oligosaccharides for attachment on gold nanoparticles: toward the development of an hiv vaccine assembling different antennas of the gp high mannose-type glycans on gold nanoparticles provides superior binding to the anti-hiv antibody g than the individual antennas negatively charged glyconanoparticles modulate and stabilize the secondary structures of a gp v loop peptide: toward fully synthetic hiv vaccine candidates surface-engineered gold nanorods: promising dna vaccine adjuvant for hiv- treatment perfection of methodical approaches to designing vaccines against tick-borne encephalitis inoculation of plasmids encoding japanese encephalitis virus prm-e proteins with colloidal gold elicits a protective immune response in balb/c mice a label-free amperometric immunosenor based on multi-layer assembly of polymerized o-phenylenediamine and gold nanoparticles for determination of japanese b encephalitis vaccine layer-by-layer self-assembly of films of nano-au and co(bpy)( )( +) for the determination of japanese b encephalitis vaccine accelerated immune response to dna vaccines enhancement of the effectiveness of electroporation-augmented cutaneous dna vaccination by a particulate adjuvant the assessment of local tolerance, acute toxicity, and dna biodistribution following particle-mediated delivery of a dna vaccine to minipigs electrically oscillating plasmonic nanoparticles for enhanced dna vaccination against hepatitis c virus assembly of hepatitis e vaccine by 'in situ' growth of gold clusters as nano-adjuvants: an efficient way to enhance the immune responses of vaccination use of a synthetic foot-and-mouth disease virus peptide conjugated to gold nanoparticles for enhancing immunological response gold nanoparticles as a potential carrier for transmucosal vaccine delivery enhanced mucosal immune responses against tetanus toxoid using novel delivery system comprised of chitosan-functionalized gold nanoparticles and botanical adjuvant: characterization, immunogenicity, and stability assessment quillaja saponaria extract as mucosal adjuvant with chitosan functionalized gold nanoparticles for mucosal vaccine delivery: stability and immunoefficiency studies gold nanorod vaccine for respiratory syncytial virus gold nanoparticle-m e conjugate coformulated with cpg induces protective immunity against influenza a virus m e-immobilized gold nanoparticles as influenza a vaccine: role of soluble m e and longevity of protection immunostimulatory effect of gold nanoparticles conjugated with transmissible gastroenteritis virus novel nanoparticle vaccines for listeriosis. hum. vaccines immunotherapeut a gold glyco-nanoparticle carrying a listeriolysin o peptide and formulated with advax™ delta inulin adjuvant induces robust t-cell protection against listeria infection pregnancy vaccination with gold glyco-nanoparticles carrying listeria monocytogenes peptides protects against listeriosis and brainand cutaneous-associated morbidities high-throughput quantitation of inorganic nanoparticle biodistribution at the single-cell level using mass cytometry conjugation of y. pestis f -antigen to gold nanoparticles improves immunogenicity nanovaccines for malaria using plasmodium falciparum antigen pfs attached gold nanoparticles induction of humoral immune response against pseudomonas aeruginosa flagellin( - ) using gold nanoparticles as an adjuvant synthetic virus-like particles prepared via protein corona formation enable effective vaccination in an avian model of coronavirus infection a gold nanoparticle-linked glycoconjugate vaccine against burkholderia mallei protective immunity in gibel carp, carassius gibelio of the truncated proteins of cyprinid herpesvirus expressed in pichia pastoris rv c protein peptide inhibiting mycobacterium tuberculosis h rv entry to target cells delivery of antiviral small interfering rna with gold nanoparticles inhibits dengue virus infection in vitro modulating antibacterial immunity via bacterial membrane-coated nanoparticles inhibition of cytomegalovirus infection and photothermolysis of infected cells using bioconjugated gold nanoparticles the imperative for stronger vaccine supply and logistics systems key: cord- -e m o c authors: lentini, giovanni; cavalluzzi, maria maddalena; habtemariam, solomon title: covid- , chloroquine repurposing, and cardiac safety concern: chirality might help date: - - journal: molecules doi: . /molecules sha: doc_id: cord_uid: e m o c the desperate need to find drugs for covid- has indicated repurposing strategies as our quickest way to obtain efficacious medicines. one of the options under investigation is the old antimalarial drug, chloroquine, and its analog, hydroxychloroquine. developed as synthetic succedanea of cinchona alkaloids, these chiral antimalarials are currently in use as the racemate. besides the ethical concern related to accelerated large-scale clinical trials of drugs with unproven efficacy, the known potential detrimental cardiac effects of these drugs should also be considered. in principle, the safety profile might be ameliorated by using chloroquine/hydroxychloroquine single enantiomers in place of the racemate. the current -ncov (sars-cov- ) causing covid- has demonstrated that our preparedness for emerging respiratory viral infections is far too inadequate. in the absence of any known efficacious therapeutic agent or vaccine, our effective measures against the disease are somehow similar to our ancestors a century ago: constant hand washing and social distancing. the desperate need to find drugs for covid- has brought the attention of numerous scholars to the repurposing of known drugs as tentative covid- therapeutics. it is logical that repurposing known drugs for covid- may be first considered from pharmacological agents with proven efficacy against other highly pathogenic viral infections. however, useful medicines could also be identified from non-etiotropic drugs. in the hunt for new antiviral agents, repurposing old drugs or repositioning experimental ones are not novel approaches, and they were proposed early to treat other viral infections such as the ebola outbreak [ ] . repurposing folk medicines was also suggested [ ] , while emphasis was given to cardiovascular drugs [ , ] . accordingly, antiviral agents, cardiovascular drugs [ ] , and traditional chinese remedies [ ] are being considered for repurposing against the sars-cov- infection. one of the options under investigation is the old antimalarial drug, chloroquine-a synthetic succedaneum of cinchona alkaloids [ ] (figure )-which gave promising results in a chinese clinical trial, where it was superior to the positive control treatment in more than patients [ ] . recently, wang et al. have revealed that remdesivir and chloroquine are highly effective in the control of the -ncov infection in vitro and suggested that they should be assessed in human patients [ ] . further clinical evidence is needed to support these successful preliminary reports, and the hazards related to the extension of chloroquine and its analog, hydroxychloroquine (figure ), to a wider set of patients should be carefully evaluated [ ] . the hazards related to the extension of chloroquine and its analog, hydroxychloroquine (figure ), to a wider set of patients should be carefully evaluated [ ] . besides the ethical concern related to accelerated large-scale clinical trials of drugs with unproven efficacy [ , ] , the known potential detrimental cardiac effects of chloroquine should also be taken into account [ ] . moreover, while being included in the essential medicine lists of several countries as an antimalarial [ ] , chloroquine may hamper cardiac function at clinically relevant doses and its safety margin is very narrow. the proarrhythmic activity of chloroquine is mainly due to its capacity to inhibit the cardiac inward rectifier potassium current and further, it can induce lethal ventricular arrhythmias. the off-target activity partially stems from the blockades of the human ether-à-go-go related gene (herg) [ ] and kir . potassium [ ] channels that may occur at low μm concentrations. additionally, while therapeutic doses of chloroquine typically result in plasma concentrations of - μm [ ] , peak levels up to μm could be achieved in the case of a clinical overdose [ ] . however, it should be noted that chloroquine is a chiral compound currently used as a racemate. it is generally accepted that stereochemistry may affect both the pharmacodynamics and admet of drugs [ ] . stereochemical aspects could thus be exploited to improve clinical safety and efficacy (risk-to-benefit ratio). the use of one of the separated enantiomerically pure chloroquine stereoisomers in lieu of its corresponding racemate-the so-called chiral switch approach [ ] -might found its rationale in the following considerations. chloroquine is a moderate inhibitor of herg with electrophysiology studies indicating ic values ranging from . to . μm, depending on the experimental setting used [ ] . site-directed mutagenesis studies revealed that chloroquine selectively blocks the above-mentioned potassium channels through structurally specific interactions. regarding the herg channel blockade, alanine-scanning mutagenesis and molecular docking studies indicated that chloroquine mainly interacts through specific cation-π and π-stacking interactions with tyr- and phe- of the proteic subunits lining the pore through a "foot in the door" type mechanism [ ] . a third possible point of interaction would be offered by ser- [ ] . thus, a three-point interaction binding might be envisaged and it may be anticipated that chloroquine off-target activity on cardiac functioning could display stereoselectivity. this means that one of the enantiomers could be less active as a herg blocker and, consequently, safer to humans. as stated above, chloroquine may prolong the qt interval of the electrocardiogram (ecg) and cause the potentially lethal long qt (lqt) syndrome (lqts), also interfering with the normal activity of kir . potassium channels with the binding site being located in the cytoplasmic pore of the channel [ ] . selective mutation studies demonstrated that at least three residues would be involved (glu- > met- > glu- ). thus, once again, d requirements might cause stereoselectivity of blocks and one of the chloroquine enantiomers might display weaker besides the ethical concern related to accelerated large-scale clinical trials of drugs with unproven efficacy [ , ] , the known potential detrimental cardiac effects of chloroquine should also be taken into account [ ] . moreover, while being included in the essential medicine lists of several countries as an antimalarial [ ] , chloroquine may hamper cardiac function at clinically relevant doses and its safety margin is very narrow. the proarrhythmic activity of chloroquine is mainly due to its capacity to inhibit the cardiac inward rectifier potassium current and further, it can induce lethal ventricular arrhythmias. the off-target activity partially stems from the blockades of the human ether-à-go-go related gene (herg) [ ] and kir . potassium [ ] channels that may occur at low µm concentrations. additionally, while therapeutic doses of chloroquine typically result in plasma concentrations of - µm [ ] , peak levels up to µm could be achieved in the case of a clinical overdose [ ] . however, it should be noted that chloroquine is a chiral compound currently used as a racemate. it is generally accepted that stereochemistry may affect both the pharmacodynamics and admet of drugs [ ] . stereochemical aspects could thus be exploited to improve clinical safety and efficacy (risk-to-benefit ratio). the use of one of the separated enantiomerically pure chloroquine stereoisomers in lieu of its corresponding racemate-the so-called chiral switch approach [ ] -might found its rationale in the following considerations. chloroquine is a moderate inhibitor of herg with electrophysiology studies indicating ic values ranging from . to . µm, depending on the experimental setting used [ ] . site-directed mutagenesis studies revealed that chloroquine selectively blocks the above-mentioned potassium channels through structurally specific interactions. regarding the herg channel blockade, alanine-scanning mutagenesis and molecular docking studies indicated that chloroquine mainly interacts through specific cation-π and π-stacking interactions with tyr- and phe- of the proteic subunits lining the pore through a "foot in the door" type mechanism [ ] . a third possible point of interaction would be offered by ser- [ ] . thus, a three-point interaction binding might be envisaged and it may be anticipated that chloroquine off-target activity on cardiac functioning could display stereoselectivity. this means that one of the enantiomers could be less active as a herg blocker and, consequently, safer to humans. as stated above, chloroquine may prolong the qt interval of the electrocardiogram (ecg) and cause the potentially lethal long qt (lqt) syndrome (lqts), also interfering with the normal activity of kir . potassium channels with the binding site being located in the cytoplasmic pore of the channel [ ] . selective mutation studies demonstrated that at least three residues would be involved (glu- > met- > glu- ). thus, once again, d requirements might cause stereoselectivity of blocks and one of the chloroquine enantiomers might display weaker interactions with respect to its optically active counterpart, thus resulting in a lesser detrimental effect on cardiac function. admet aspects might also contribute to the stereoselective clinically relevant outcomes. the binding of chloroquine with human plasma protein is enantioselective with the (s)-enantiomer displaying higher affinity than its enantiomer [ ] ; further, toxicity (ld ) is lower for the (s)-enantiomer in the mouse model [ ] . on the contrary, the desirable activity on covid- would mostly stem from structurally unspecific aspects such as basicity and lipophilicity. the formation of sars-cov particles occurs in the golgi apparatus [ ] . chloroquine is a basic tertiary amine that may enter the cell and increase the ph of acidic vesicles. due to its relatively high lipophilicity, the non-protonated fraction of chloroquine in the biophase may easily dissolve in the phospholipidic bilayer of cell membranes and enter the cell, where it becomes protonated and accumulates in low-ph organelles, including endosomes, golgi vesicles, and lysosomes [ ] . obviously, these processes are chirality-independent and even if both enantiomers share the same antiviral activity (regardless of their corresponding absolute configuration), the toxicologically safe enantiomer should be preferred. both enantiomers of chloroquine may be easily obtained from the corresponding racemate by optical resolution [ ] or through stereoselective synthesis [ ] . high enantiomeric purity (ep) values are mandatory for evaluating the stereoselectivity of drugs [ ] and convenient methods for assessing ep values of chloroquine enantiomers are now available [ ] . on the other hand, the studies on chloroquine enantiomers as antivirals might boost endeavours aimed at developing new chloroquine chiral analogs starting from this lead compound. medicinal chemists have developed easy rules of thumb to both accelerate optimization [ ] and reduce cardiac toxicity [ ] by facile chemical modifications without escaping from the "drug-like" space. given that useful drugs for treating new diseases might stem from relatively old drugs and that chloroquine has already shown some promise in pilot covid- trial studies, its full potential should be explored by testing enantiomerically pure analogs. beyond establishing the relative potency of the enantiomers against sars-cov- , their differential cardiotoxicity could lead to the identification of a more potent and safer lead. repurposed therapeutic agents targeting the ebola virus: a systematic review did ebola survivors use plant medicines, and if so, which ones? is there a hope from treatment with cardiovascular drugs? ebola therapy: developing new drugs or repurposing old ones? angiotensin receptor blockers as tentative sars-cov- therapeutics can chinese medicine be used for prevention of corona virus disease (covid- )? a review of historical classics, research evidence and current prevention programs pitfalls in a discovery: the chronicle of chloroquine breakthrough: chloroquine phosphate has shown apparent efficacy in treatment of covid- associated pneumonia in clinical studies remdesivir and chloroquine effectively inhibit the recently emerged novel coronavirus ( -ncov) in vitro of chloroquine and covid- the ebola clinical trials: a precedent for research ethics in disasters examining the ethics of clinical use of unproven interventions outside of clinical trials during the ebola epidemic chloroquine intoxication years of the who essential medicines lists: progress and challenges an evaluation of clinical drugs against the comprehensive in vitro proarrhythmia assay (cipa) proposed ion channel panel the molecular basis of chloroquine block of the inward rectifier kir . channel randomized dose-ranging controlled trial of aq- , a candidate antimalarial, and chloroquine in healthy volunteers treatment of severe chloroquine poisoning stereochemistry in drug action chiral switches collation, assessment and analysis of literature in vitro data on herg receptor blocking potency for subsequent modeling of drugs' cardiotoxic properties molecular determinants of voltage-dependent human ether-a-go-go related gene (herg) k+ channel block predictive models for herg channel blockers: ligand-based and structure-based approaches protein binding of chloroquine enantiomers and desethylchloroquine antimalarial activity of the optical isomers of chloroquine diphosphate proliferative growth of sars coronavirus in vero e cells chloroquine is a potent inhibitor of sars coronavirus infection and spread resolution of -chloro- ( -diethylamino- methylbutyl)aminoquinoline (chloroquine) into its enantiomers synthesis of chiral chloroquine and its analogues as antimalarial agents shouldn't enantiomeric purity be included in the "minimum information about a bioactive entity preparation of (−)-(r)- -( , , , , -pentafluorophenoxy)- -(phenyl-d )acetic acid: an efficient h nmr chiral solvating agent for direct enantiomeric purity evaluation of quinoline-containing antimalarial drugs ligand efficiency metrics in drug discovery: the pros and cons from a practical perspective human ether-à-go-go-related potassium channel: exploring sar to improve drug design funding: no research funding reported. no conflict of interest reported. key: cord- - nyje authors: piotrowska, dorota g.; głowacka, iwona e.; schols, dominique; snoeck, robert; andrei, graciela; gotkowska, joanna title: novel isoxazolidine and γ-lactam analogues of homonucleosides date: - - journal: molecules doi: . /molecules sha: doc_id: cord_uid: nyje homonucleoside analogues cis- and trans- having a ( -methoxycarbonyl)isoxazolidine framework were synthesized via the , -dipolar cycloaddition of nucleobase-derived nitrones with methyl acrylate. hydrogenolysis of the isoxazolidines containing thymine, dihydrouracil, theophylline and adenine moieties efficiently led to the formation of the respective γ-lactam analogues. γ-lactam analogues having -bromouracil and -chlorouracil fragments were synthesized by treatment of uracil-containing γ-lactams with nbs and ncs. isoxazolidine and γ-lactam analogues of homonucleosides obtained herein were evaluated for activity against a broad range of dna and rna viruses. none of the compounds that were tested exhibited antiviral or cytotoxic activity at concentrations up to µm. the cytostatic activities of all compounds toward nine cancerous cell lines was tested. γ-lactams trans- e (cl-ura) and cis- h (theo) appeared the most active toward pancreatic adenocarcinoma cells (capan- ), showing ic( ) values . and . µm, respectively. isoxazolidine cis- e (cl-ura) inhibited the proliferation of colorectal carcinoma (hct- ). nucleoside analogues belong to a group of important antiviral and antitumor drugs [ ] [ ] [ ] [ ] . however, resistance to drugs and their toxicity are considered major factors limiting the effectiveness of therapies. structural modifications of available drugs from a class of nucleoside analogues, including sugar and/or nucleobase residues, resulted in the discovery of a variety of therapeutically-useful antiviral [ ] (e.g., azidothymidine (azt) [ ] , carbovir [ ] , lobucavir [ ] , dioxolane t [ ] and lamivudine [ ] ) and anticancer [ , ] (e.g., cladribine [ ] , gemcitabine [ ] , azacitidine [ ] and cytarabine [ , ] , clofarabine [ ] ) agents ( figure ). on the other hand, the introduction of a methylene bridge between a nucleobase and the sugar partly led to the formation of homonucleosides, which are known for their resistance to hydrolytic or enzymatic cleavage, as well as for the rotational freedom when compared with the natural nucleosides. furthermore, it was shown that homonucleosides are substrates for cellular kinases and are able to pair with other nucleosides by watson-crick interactions without appreciable modifications of the structure of dna (or rna) molecules [ ] . -homonucleosides containing adenine or guanine were found to be active against herpes simplex virus (minimum inhibitory concentration (mic) = - µg/ml) and vaccinia virus [ ] [ ] [ ] , while compound (ic = . µg/ml) showed activity against the influenza type virus ah n ( figure ) [ ] . homonucleoside analogue of -deoxyuridine has been on the other hand, the introduction of a methylene bridge between a nucleobase and the sugar partly led to the formation of homonucleosides, which are known for their resistance to hydrolytic or enzymatic cleavage, as well as for the rotational freedom when compared with the natural nucleosides. furthermore, it was shown that homonucleosides are substrates for cellular kinases and are able to pair with other nucleosides by watson-crick interactions without appreciable modifications of the structure of dna (or rna) molecules [ ] . ′-homonucleosides containing adenine or guanine were found to be active against herpes simplex virus (minimum inhibitory concentration (mic) = - µg/ml) and vaccinia virus [ ] [ ] [ ] , while compound (ic = . µg/ml) showed activity against the influenza type virus ah n ( figure ) [ ] . homonucleoside analogue of ′-deoxyuridine has been described as a selective inhibitor of viral uracil-dna glycosylase (udg), while exerting only small effect on the human enzymes [ ] . incorporation of the isoxazolidine ring into a nucleoside framework as a sugar part replacer was first proposed by tronchet and co-workers [ , ] with the intention to study antiviral [ , [ ] [ ] [ ] [ ] [ ] [ ] , antibacterial [ ] and antifungal [ ] activities of the compounds. other research groups then intensively explored this field further. thus, a compound [ ] and phosphonylated derivatives [ ] and [ ] [ ] [ ] inhibited the reverse transcriptase activity of avian moloney virus (amv) and human immunodeficiency virus (hiv) at a level comparable with that of tenofovir ( nm) and fold higher than that of azt ( nm). moreover, their cytotoxicity was significantly lower ( % cytotoxic concentration (cc ) > µm) in comparison with that of azt (cc = . µm) ( figure ). in order to obtain compounds with a sufficient stability for hydrolytic or enzymatic cleavage, analogues of ′-homonucleosides - ( figure ) were also synthesized [ ] [ ] [ ] [ ] [ ] . on the other hand, the introduction of a methylene bridge between a nucleobase and the sugar partly led to the formation of homonucleosides, which are known for their resistance to hydrolytic or enzymatic cleavage, as well as for the rotational freedom when compared with the natural nucleosides. furthermore, it was shown that homonucleosides are substrates for cellular kinases and are able to pair with other nucleosides by watson-crick interactions without appreciable modifications of the structure of dna (or rna) molecules [ ] . ′-homonucleosides containing adenine or guanine were found to be active against herpes simplex virus (minimum inhibitory concentration (mic) = - µg/ml) and vaccinia virus [ ] [ ] [ ] , while compound (ic = . µg/ml) showed activity against the influenza type virus ah n ( figure ) [ ] . homonucleoside analogue of ′-deoxyuridine has been described as a selective inhibitor of viral uracil-dna glycosylase (udg), while exerting only small effect on the human enzymes [ ] . incorporation of the isoxazolidine ring into a nucleoside framework as a sugar part replacer was first proposed by tronchet and co-workers [ , ] with the intention to study antiviral [ , [ ] [ ] [ ] [ ] [ ] [ ] , antibacterial [ ] and antifungal [ ] activities of the compounds. other research groups then intensively explored this field further. thus, a compound [ ] and phosphonylated derivatives [ ] and [ ] [ ] [ ] inhibited the reverse transcriptase activity of avian moloney virus (amv) and human immunodeficiency virus (hiv) at a level comparable with that of tenofovir ( nm) and fold higher than that of azt ( nm). moreover, their cytotoxicity was significantly lower ( % cytotoxic concentration (cc ) > µm) in comparison with that of azt (cc = . µm) ( figure ). in order to obtain compounds with a sufficient stability for hydrolytic or enzymatic cleavage, analogues of ′-homonucleosides - ( figure ) were also synthesized [ ] [ ] [ ] [ ] [ ] . incorporation of the isoxazolidine ring into a nucleoside framework as a sugar part replacer was first proposed by tronchet and co-workers [ , ] with the intention to study antiviral [ , [ ] [ ] [ ] [ ] [ ] [ ] , antibacterial [ ] and antifungal [ ] activities of the compounds. other research groups then intensively explored this field further. thus, a compound [ ] and phosphonylated derivatives [ ] and [ ] [ ] [ ] inhibited the reverse transcriptase activity of avian moloney virus (amv) and human immunodeficiency virus (hiv) at a level comparable with that of tenofovir ( nm) and -fold higher than that of azt ( nm). moreover, their cytotoxicity was significantly lower ( % cytotoxic concentration (cc ) > µm) in comparison with that of azt (cc = . µm) ( figure ). in order to obtain compounds with a sufficient stability for hydrolytic or enzymatic cleavage, analogues of -homonucleosides - ( figure ) were also synthesized [ ] [ ] [ ] [ ] [ ] . in the isoxazolidine nucleoside/nucleotide analogues described so far, a nucleobase is attached to c ; therefore, the , -dipolar cycloaddition of nitrones and n-vinyl or n-allyl nucleobases has been found a convenient method for their preparation [ ] . recently, we designed isoxazolidine analogues of -homonucleos(t)ides (figure ), in which nucleobases were attached to c to underscore the fundamental difference to the already-described ones [ , ] . in the synthesis of homonucleosides , the , -dipolar cycloaddition was also employed, but in that case, nucleobase-derived nitrones and selected alkenes were applied [ , ] . since no significant activity of compounds toward the viruses tested was noticed, we wondered whether the replacement of an isoxazolidine moiety with a γ-lactam ring would lead to an improvement of antiviral properties of newly-designed compounds . the idea of incorporation of the γ-lactam moiety has been supported by the presence of this subunit in many natural products (figure ) , which showed antiviral [ , ] , cytotoxic [ ] [ ] [ ] [ ] [ ] , anti-inflammatory [ ] [ ] [ ] and antimicrobial [ , ] properties, among others. for this reason the assumption that homonucleoside analogues (scheme ) having the γ-lactam moiety could possess interesting biological properties as antiviral or cytotoxic is justified [ ] . in the isoxazolidine nucleoside/nucleotide analogues described so far, a nucleobase is attached to c ; therefore, the , -dipolar cycloaddition of nitrones and n-vinyl or n-allyl nucleobases has been found a convenient method for their preparation [ ] . recently, we designed isoxazolidine analogues of ′-homonucleos(t)ides (figure ), in which nucleobases were attached to c to underscore the fundamental difference to the already-described ones [ , ] . in the synthesis of homonucleosides , the , -dipolar cycloaddition was also employed, but in that case, nucleobase-derived nitrones and selected alkenes were applied [ , ] . since no significant activity of compounds toward the viruses tested was noticed, we wondered whether the replacement of an isoxazolidine moiety with a γ-lactam ring would lead to an improvement of antiviral properties of newly-designed compounds . the idea of incorporation of the γ-lactam moiety has been supported by the presence of this subunit in many natural products (figure ) , which showed antiviral [ , ] , cytotoxic [ ] [ ] [ ] [ ] [ ] , antiinflammatory [ ] [ ] [ ] and antimicrobial [ , ] properties, among others. for this reason the assumption that homonucleoside analogues (scheme ) having the γ-lactam moiety could possess interesting biological properties as antiviral or cytotoxic is justified [ ] . the synthetic strategy for γ-lactam homonucleosides relies on the , -dipolar cycloaddition of nucleobase-derived nitrones with methyl acrylate, followed by the hydrogenolysis of the n-o the synthetic strategy for γ-lactam homonucleosides relies on the , -dipolar cycloaddition of nucleobase-derived nitrones with methyl acrylate, followed by the hydrogenolysis of the n-o bond in an isoxazolidine scaffold, which was accompanied by the intramolecular cyclization to transform cycloadducts into compounds (scheme ). nucleobase-derived nitrones were synthesized from n-( -oxoethyl)nucleobases and nmethylhydroxylamine, as described previously [ , ] . the , -dipolar cycloadditions of the nucleobase-derived nitrones to methyl acrylate were carried out at °c in methanol for h (scheme , table ) and afforded mixtures of diastereoisomeric isoxazolidines cis- and trans- . the cis/trans ratios of the isoxazolidines were calculated from the h-nmr spectra of the crude reaction mixtures by the comparison of diagnostic resonances of the h c- protons in the isoxazolidine ring, as well as the signals of the respective protons of nucleobase moieties. in some cases, differences in resonances of the ch -n protons for diastereoisomeric isoxazolidines cis- and trans- were also diagnostic. the crude reaction mixtures of the diastereoisomers were subjected to column chromatography; however, only pure isomeric isoxazolidines cis- a and trans- a were isolated (table , entry a). in the other cases the respective fractions enriched in the isomers cis- b- i or scheme . retrosynthesis of γ-lactam analogues of -homonucleosides . the synthetic strategy for γ-lactam homonucleosides relies on the , -dipolar cycloaddition of nucleobase-derived nitrones with methyl acrylate, followed by the hydrogenolysis of the n-o bond in an isoxazolidine scaffold, which was accompanied by the intramolecular cyclization to transform cycloadducts into compounds (scheme ). nucleobase-derived nitrones were synthesized from n-( -oxoethyl)nucleobases and n-methylhydroxylamine, as described previously [ , ] . the , -dipolar cycloadditions of the nucleobase-derived nitrones to methyl acrylate were carried out at • c in methanol for h (scheme , table ) and afforded mixtures of diastereoisomeric isoxazolidines cis- and trans- . the cis/trans ratios of the isoxazolidines were calculated from the h-nmr spectra of the crude reaction mixtures by the comparison of diagnostic resonances of the h c- protons in the isoxazolidine ring, as well as the signals of the respective protons of nucleobase moieties. in some cases, differences in resonances of the ch -n protons for diastereoisomeric isoxazolidines cis- and trans- were also diagnostic. the crude reaction mixtures of the diastereoisomers were subjected to column chromatography; however, only pure isomeric isoxazolidines cis- a and trans- a were isolated (table , entry a). in the other cases the respective fractions enriched in the isomers cis- b- i or trans- b- i were collected. the subsequent separation of diastereoisomeric mixture of isoxazolidines cis- b- h or trans- b- h on an hplc column allowed us to obtain pure isomers cis- b- h and trans- b- h (table , entry b-h), and the amounts, however small, were sufficient to perform a full characterization of the newly-synthesized compounds and their further biological screening. unfortunately, the attempts at separating diastereoisomeric isoxazolidines cis- i and trans- i appeared fruitless, even with hplc. trans- b- i were collected. the subsequent separation of diastereoisomeric mixture of isoxazolidines cis- b- h or trans- b- h on an hplc column allowed us to obtain pure isomers cis- b- h and trans- b- h (table , entry b-h), and the amounts, however small, were sufficient to perform a full characterization of the newly-synthesized compounds and their further biological screening. unfortunately, the attempts at separating diastereoisomeric isoxazolidines cis- i and trans- i appeared fruitless, even with hplc. the relative configurations of isoxazolidines cis- a and trans- a have already been established [ ] . as we have recently described, ( -methoxycarbonyl)isoxazolidine cis- a was reduced with sodium borohydride in ethanol to the known ( -hydroxymethyl)isoxazolidine cis- [ ] (scheme ). during this transformation, the structure of the isoxazolidine skeleton remains unchanged, and the relative configurations of isoxazolidines cis- a and trans- a have already been established [ ] . as we have recently described, ( -methoxycarbonyl)isoxazolidine cis- a was reduced with sodium borohydride in ethanol to the known ( -hydroxymethyl)isoxazolidine cis- [ ] (scheme ). the relative configurations of isoxazolidines cis- a and trans- a have already been established [ ] . as we have recently described, ( -methoxycarbonyl)isoxazolidine cis- a was reduced with the relative configurations of isoxazolidines cis- a and trans- a have already been established [ ] . as we have recently described, ( -methoxycarbonyl)isoxazolidine cis- a was reduced with sodium borohydride in ethanol to the known ( -hydroxymethyl)isoxazolidine cis- [ ] (scheme ). during this transformation, the structure of the isoxazolidine skeleton remains unchanged, and therefore, a relative configuration of the isoxazolidine cis- a was unambiguously established. consequently, the isomeric ( -methoxycarbonyl)isoxazolidine was identified as trans- a. since similar spectral patterns for the respective isoxazolidine protons (h , h α, h β and h ) were observed in series of cis- b- i and trans- b- i when compared to those observed for cycloadducts cis- a and trans- a, their configurations were analogously assigned. the relative configurations of isoxazolidines cis- a and trans- a have already been established [ ] . as we have recently described, ( -methoxycarbonyl)isoxazolidine cis- a was reduced with sodium borohydride in ethanol to the known ( -hydroxymethyl)isoxazolidine cis- [ ] (scheme ). during this transformation, the structure of the isoxazolidine skeleton remains unchanged, and therefore, a relative configuration of the isoxazolidine cis- a was unambiguously established. consequently, the isomeric ( -methoxycarbonyl)isoxazolidine was identified as trans- a. since similar spectral patterns for the respective isoxazolidine protons (h , h ɑ, h β and h ) were observed in series of cis- b- i and trans- b- i when compared to those observed for cycloadducts cis- a and trans- a, their configurations were analogously assigned. figure , structure ) . surprisingly, the preferred conformations for dihydrouacil-containing isoxazolidines cis- g and trans- g could not be assigned, and sets of couplings indicate the existence of conformational equilibrium of isoxazolidine ring. figure , structure ) . surprisingly, the preferred conformations for dihydrouacil-containing isoxazolidines cis- g and trans- g could not be assigned, and sets of couplings indicate the existence of conformational equilibrium of isoxazolidine ring. for the either cis or trans isoxazolidines containing uracil or substituted uracil residues a- f, we also noticed that diastereotopic hydrogen atoms in ch -base units which resonate at a lower field ( . - . ppm) showed significantly smaller vicinal coupling constants to h-c ( . - . hz) than those associated with higher field signals ( . - . ppm) when values of . - . hz were observed. one may conclude that high-field hydrogens are antiperiplanar to h-c , while low-field ones are oriented gauche, as depicted by the newman projection ( figure ). although real reasons for the restricted rotation around c -ch base bond remain obscure to us at the moment, they may simply reflect repulsive interactions of sterically bulky ch -base groups with me-n and hβ-c -hα fragments. finally, attempts at transformation of isoxazolidine cycloadducts cis- /trans- into γ-lactam derivatives trans- /cis- were made, following the procedure applied previously for transformation for the either cis or trans isoxazolidines containing uracil or substituted uracil residues a- f, we also noticed that diastereotopic hydrogen atoms in ch -base units which resonate at a lower field ( . - . ppm) showed significantly smaller vicinal coupling constants to h-c ( . - . hz) than those associated with higher field signals ( . - . ppm) when values of . - . hz were observed. one may conclude that high-field hydrogens are antiperiplanar to h-c , while low-field ones are oriented gauche, as depicted by the newman projection ( figure ). although real reasons for the restricted rotation around c -ch base bond remain obscure to us at the moment, they may simply reflect repulsive interactions of sterically bulky ch -base groups with me-n and hβ-c -hα fragments. finally, attempts at transformation of isoxazolidine cycloadducts cis- /trans- into γ-lactam derivatives trans- /cis- were made, following the procedure applied previously for transformation of cis- a into trans- g [ ] . it was expected that since hydrogenation of the n-o bond in the isoxazolidine scaffold releases the amino group, the subsequent intramolecular cyclization to γ-lactams trans- /cis- should occur spontaneously (scheme ). consequently, the isoxazolidine cis- would provide trans- , while isoxazolidine trans- should be a substrate for the synthesis of cis-configured γ-lactam cis- . with that in mind, we concluded that not only can pure diastereoisomers cis- and trans- be used for the synthesis of trans- /cis- , but so can mixtures of the respective isomers cis- /trans- , although significantly enriched in one diastereoisomer to maintain control on the stereochemistry of the products formed. for the either cis or trans isoxazolidines containing uracil or substituted uracil residues a- f, we also noticed that diastereotopic hydrogen atoms in ch -base units which resonate at a lower field ( . - . ppm) showed significantly smaller vicinal coupling constants to h-c ( . - . hz) than those associated with higher field signals ( . - . ppm) when values of . - . hz were observed. one may conclude that high-field hydrogens are antiperiplanar to h-c , while low-field ones are oriented gauche, as depicted by the newman projection ( figure ). although real reasons for the restricted rotation around c -ch base bond remain obscure to us at the moment, they may simply reflect repulsive interactions of sterically bulky ch -base groups with me-n and hβ-c -hα fragments. finally, attempts at transformation of isoxazolidine cycloadducts cis- /trans- into γ-lactam derivatives trans- /cis- were made, following the procedure applied previously for transformation of cis- a into trans- g [ ] . it was expected that since hydrogenation of the n-o bond in the isoxazolidine scaffold releases the amino group, the subsequent intramolecular cyclization to γlactams trans- /cis- should occur spontaneously (scheme ). consequently, the isoxazolidine cis- would provide trans- , while isoxazolidine trans- should be a substrate for the synthesis of cisconfigured γ-lactam cis- . with that in mind, we concluded that not only can pure diastereoisomers cis- and trans- be used for the synthesis of trans- /cis- , but so can mixtures of the respective isomers cis- /trans- , although significantly enriched in one diastereoisomer to maintain control on the stereochemistry of the products formed. as we recently reported [ ] , the hydrogenation of uracil-containing isoxazolidine homonucleoside cis- a (b = ura) proceeded smoothly and the intramolecular cyclization of intermediary γ-aminocarboxylic ester to a γ-lactam was accompanied by hydrogenation of the uracil residue to produce the lactam trans- g (b = dih-ura) in a good yield ( %) instead of the expected compound trans- a (b = ura) ( table , entry a). similarly, cis- g (b = dih-ura) was obtained from trans- a (b = ura) ( %). as we recently reported [ ] , the hydrogenation of uracil-containing isoxazolidine homonucleoside cis- a (b = ura) proceeded smoothly and the intramolecular cyclization of intermediary γ-aminocarboxylic ester to a γ-lactam was accompanied by hydrogenation of the uracil residue to produce the lactam trans- g (b = dih-ura) in a good yield ( %) instead of the expected compound trans- a (b = ura) ( table , entry a). similarly, cis- g (b = dih-ura) was obtained from trans- a (b = ura) ( %). on the other hand, when a : mixture of thymine-containing isoxazolidines cis- b and trans- b (b = thy) was subjected to hydrogenation, the respective : mixture of γ-lactam trans- b and cis- b (b = thy) was obtained. from this mixture pure diastereoisomers were isolated after column chromatography followed by hplc ( table , entry b). during the hydrogenation of -halogenated uracil derivatives cis- /trans- (b = x-ura), we faced the same problem as in case of uracil derivatives. moreover, halogen atoms were removed from nucleobase skeletons. for example, hydrogenation of a : mixture of cis- d and trans- d isoxazolidines (b = br-ura) led to the formation of an inseparable mixture of trans- a, cis- a and trans- g and cis- g ( : : : ) ( table , entry d). analogously, a mixture of trans- a, cis- a, trans- g and cis- g ( : : : ) was obtained from the : mixture of isoxazolidines cis- e and trans- e (b = cl-ura) ( table , entry e). similarly, hydrogenation of a cis- c (b = f-ura) gave dihydrouracil-containing γ-lactam trans- g (b = dih-ura) ( table , entry c). furthermore, the respective mixtures of isoxazolidines cis- g/trans- g, cis- h/trans- h and cis- i/trans- i were successfully hydrogenated to provide γ-lactams containing dihydrouracil (trans- g/cis- g), theophylline (trans- h/cis- h) and adenine (trans- i/cis- i) as nucleobases ( table , entry g-i). to supplement a library of designed γ-lactam homonucleosides trans- /cis- with analogues having bromine (trans- d/cis- d) and chlorine (trans- e/cis- e) atoms, the halogenation of uracil derivatives was proposed [ ] [ ] [ ] . treatment of the above-mentioned (table , entry d), inseparable mixture of γ-lactams trans- a, cis- a, trans- g and trans- g ( : : : ) with n-bromosuccinimide (nbs) [ ] led to the formation of the mixture of γ-lactams trans- d, cis- d, together with unreacted trans- g and cis- g ( : : : ) (scheme ). the purification of this mixture on a silica gel column followed by hplc allowed us to isolate trans- d and cis- d in % and . % yields, respectively. similarly, the reaction of an analogous mixture of trans- a, cis- a, trans- g and cis- g with n-chlorosuccinimide (ncs) [ ] gave the respective mixture of compounds trans- e, cis- e, trans- g and cis- g ( : : : ), from which trans- e and cis- e were obtained in % and . % yields, respectively (scheme ). all isoxazolidines and γ-lactams obtained were evaluated for their activities against a wide variety of dna and rna viruses using the following cell-based assays: (a) human embryonic lung (hel) cell cultures: herpes simplex virus- (kos strain), herpes simplex virus- (g strain), vaccinia virus, thymidine kinase deficient (acyclovir-resistant) herpes simplex virus- (tk − kos acv r ), adenovirus- , human coronavirus ( e strain), cytomegalovirus (ad- and davis strains) and varicella-zoster virus (tk + vzv and tk − vzv strains); (b) hela cell cultures: vesicular stomatitis virus, coxsackie virus b and respiratory syncytial virus; (c) vero cell cultures: para-influenza- virus, reovirus- , sindbis virus, coxsackie virus b , punta toro virus and yellow fever virus; and (d) mdck cell cultures: influenza a virus (h n and h n subtypes) and influenza b virus. ganciclovir, cidofovir, acyclovir, brivudin, zalcitabine, zanamivir, alovudine, mycophenolic acid, amantadine, rimantadine, ribavirin, dextran sulfate (molecular weight , ds- ) and urtica dioica agglutinin (uda) were used as the reference compounds. the antiviral activity was expressed as the ec : the compound concentration required to reduce virus plaque formation (vzv) by % or to reduce virus-induced cytopathicity by % (other viruses). the cytotoxicity of the compounds tested toward the uninfected host cells was defined as the minimum cytotoxic concentration (mcc) that causes a microscopically detectable alteration of normal cell morphology. the % cytotoxic concentration (cc ), causing a % decrease in cell viability was determined using a colorimetric mts assay system. none of the tested compounds showed appreciable antiviral activity toward any of the tested dna and rna viruses at the concentration up to µm. at the same time, none of them affected the cell morphologies of used uninfected host cells. ganciclovir, cidofovir, acyclovir, brivudin, zalcitabine, zanamivir, alovudine, mycophenolic acid, amantadine, rimantadine, ribavirin, dextran sulfate (molecular weight , ds- ) and urtica dioica agglutinin (uda) were used as the reference compounds. the antiviral activity was expressed as the ec : the compound concentration required to reduce virus plaque formation (vzv) by % or to reduce virus-induced cytopathicity by % (other viruses). the cytotoxicity of the compounds scheme . synthesis of γ-lactams trans- d and cis- d (x = br), and trans- e and cis- e (x = cl). the % cytostatic inhibitory concentration (ic ) causing a % decrease in cell proliferation was determined for all isolated isoxazolidines and γ-lactams toward nine cancerous cell lines (capan- (pancreatic adenocarcinoma), hap (chronic myeloid leukemia), hct- (colorectal carcinoma), nci-h (lung carcinoma), dnd- (acute lymphoblastic leukemia), hl- (acute myeloid leukemia), k- (chronic myeloid leukemia), mm. s (multiple myeloma) and z- (non-hodgkin lymphoma)) and normal retina (non-cancerous) cells (htert rpe- ). docetaxel, etoposide and stauroporine were used as the reference compounds. all of the compounds tested were not toxic to non-cancerous retina cells (htert rpe- ) at concentrations up to µm. they were also not active against dnd- , hl- , k- , mm. s or z- cancer cells. all isoxazolidines and γ-lactams we tested, except cis- c (b = f-ura), exhibited moderate activity against pancreatic adenocarcinoma cells (capan- ) (ic = . to µm), and among them, γ-lactams trans- e (cl-ura) and cis- h (theo) were the most active, with ic values of . and . µm, respectively (table ) . in most cases, both cis-configured isoxazolidines and cis-configured γ-lactams were slightly less active when compared to the respective trans-isomers (e.g., cis- b-f versus trans- b-f, cis- h-i versus trans- h-i and cis- b versus trans- b, cis- d vs. trans- d); however, that correlation did to apply for uracil, dihydrouracil and theophiline-derivatives a, g and h. moreover, several isoxazolidines and γ-lactams inhibited the proliferation of hap (ic = . to . µm), hct- (ic = . to , µm) and nci-h cells (ic = . to . µm) ( table ) . to a solution of a nitrone ( . mmol) in methanol ( ml), methyl acrylate ( . ml) was added. the mixture was stirred at • c for h. the solvent was removed in vacuo and crude products were purified on silica gel columns using chloroform-methanol ( : , : , : , : , v/v) as eluents. the respective fractions were subjected to hplc on a x bridge prep, c , µm, obd, × mm column using water/methanol ( : , : , v/v) to afford pure isoxazolidines. a solution of the respective isoxazolidines cis- and trans- ( . mmol) in methanol ( ml) was stirred under atmospheric pressure of hydrogen over pd/c ( . mmol) with pd(oh) /c ( . mmol) at room temperature for - days. the suspension was filtered through a layer of celite. the solvent was removed in vacuo and crude products were chromatographed on silica gel columns with chloroform:methanol mixtures ( : , : and : , v/v) and then on hplc using a x bridge prep, c , µm, obd, × mm column and a water:methanol mixture ( : , v/v) as an eluent to give γ-lactams trans- and cis- . . (dd, h, j (hch) = . hz, j (hcc-h ) = . hz, hch-clura), . (s, h, ch n), . (ddd, h, j (h α-h β) = . hz, j = . hz, j = . hz, h α c ), . (ddd, h, j (h β-h α) = . hz, j = . hz, j = . hz, h β c ). the compounds were evaluated against different herpesviruses, including herpes simplex virus type (hsv- ) strain kos, thymidine kinase-deficient (tk − ) hsv- kos strain resistant to acv (acv r ), herpes simplex virus type (hsv- ) strain g, adeno virus- , human coronavirus, varicella-zoster virus (vzv) tk + strain oka, tk -vzv strains - , human cytomegalovirus (hcmv) strains ad- and davis; para-influenza- virus, reovirus- , sindbis virus, coxsackie virus b , punta toro virus, respiratory syncytial virus (rsv), vesicular stomatitis virus, yellow fever virus, influenza a virus subtypes h n (a/pr/ ) and h n (a/hk/ / ); and influenza b virus (b/hk/ / ). the antiviral assays were based on inhibition of virus-induced cytopathicity or plaque formation (for vzv) in human embryonic lung (hel) fibroblasts, african green monkey kidney cells (vero), human epithelial cervix carcinoma cells (hela), or madin darby canine kidney cells (mdck) . confluent cell cultures in microtiter -well plates were inoculated with ccid of virus ( ccid being the virus dose to infect % of the cell cultures) or with plaque forming units (pfu) (for vzv) and the cell cultures were incubated in the presence of varying concentrations of the test compounds. viral cytopathicity or plaque formation (vzv) was recorded as soon as it reached completion in the control virus-infected cell cultures that were not treated with the test compounds. antiviral activity was expressed as the ec or compound concentration required to reduce virus-induced cytopathicity or viral plaque formation by %. cytotoxicity of the test compounds was expressed as the minimum cytotoxic concentration (mcc) or the compound concentration that caused a microscopically detectable alteration of cell morphology. all assays were performed in -well microtiter plates. to each well was added ( − . ) × tumor cells and a given amount of the test compound. the cells were allowed to proliferate at • c in a humidified, co -controlled atmosphere. at the end of the incubation period, the cells were counted in a coulter counter. the ic ( % inhibitory concentration) was defined as the concentration of the compound that inhibited cell proliferation by %. a series of isoxazolidine analogues of homonucleosides cis- and trans- was synthesized by , -dipolar cycloaddition of nucleobase-derived nitrones and methyl acrylate. based on h-nmr vicinal coupling constants the preferred e conformations were established for the isoxazolidine ring in both cis- and trans- . hydrogenolytic transformation of isoxazolidines cis- and trans- into γ-lactams trans- and cis- was then performed to produce the respective γ-lactams containing thymine (trans- b/cis- b), dihydrouracil (trans- g/cis- g), theophylline (trans- h/cis- h) and adenine (trans- i/cis- i) as nucleobases by the application of the established protocol. since, during the hydrogenation of -halogenated uracil-containing isoxazolidine homonucleosides, the halogen atom was removed from a nucleobase skeleton, -bromouracil and -chlorouracil derivatives trans- d/cis- d (br-ura) and trans- e/cis- e (cl-ura) were obtained via the treatment of uracil-containing γ-lactams with nbs and ncs, respectively. all obtained isoxazolidine and γ-lactam homonucleosides were evaluated against a broad-spectrum of dna and rna viruses and appeared inactive at concentrations up to µm. antiproliferative properties of the obtained isoxazolidines and γ-lactams were evaluated on nine cancerous cell lines and several of them exhibited moderate inhibitory activity, and at the same time they were inactive toward normal retina cells. (c ), . (c ), . (c(o)och ), . (ch -brura) calcd for c h brn o : c, yield: %; a colorless amorphous solid; m.p. = - • c (crystallized from chloroform-methanol). ir (kbr, cm − ) ν max : (s, h, c(o)och ), . (dddd (s, h, ch n), . (ddd, h, j (h β-h α) = . hz, j (h β-h ) = calcd for c h brn o : c, yield: . %; a colorless amorphous solid; m.p. = - • c (crystallized from chloroform-methanol). ir (kbr, cm − ) ν max : (s, h, c(o)och ), . (dd, h, j (hch) = . hz (dddd, h, j (h -cch) = j (h -h β) = . hz, hc ), . (ddd, h, j (h α-h β) = . hz, j (h α-h ) = . hz, j (h α-h ) = (s, h, ch n) cdcl ) δ: . (c(o)och ), . (c ) yield: . %; a colorless amorphous solid; m.p. = - • c (crystallized from chloroform-methanol). ir (kbr, cm − ) ν max : hz, j (h -h α) = . hz, j (h -cch) = . hz, j (h -h β) hz, j (hcc-h ) = . hz, hch-clura), . (ddd, h, j (h α-h β) = . hz, j (h α-h ) = . hz (s, h cdcl ) δ: . (c(o)och ) yield: . %; a colorless amorphous solid; m.p. = - • c (crystallized from ethyl acetate-hexane). ir (kbr, cm − ) ν max : hz, j (h -h β) = . hz, hc ), . (dd, h, j (hch) = . hz, j (hcc-h ) = . hz, hch-iura) (ddd, h, j (h α-h β) = . hz, j (h α-h ) = . hz, j (h α-h ) = . hz, h α c ), . (s, h, ch n), . (ddd, h, j (h β-h α) = . hz, j (h β-h ) = . hz, j (h β-h ) = . hz, h β c ); c-nmr ( mhz, trans- -[( -hydroxy- -methyl- -oxopyrrolidin- -yl)methyl]- -methylpyrimidine- , ( h, h)-dione (trans- b) hz, j (h -h α) = . hz, hc ), . (dd, h, j (hch) = . hz, j (hcc-h ) = . hz, hch-thy), . (dddd, h, j (h -h β) = . hz, j (h -cch) = . hz, j (h -cch) j (h α-h β) = . hz, j (h α-h ) = . hz, j (h α-h ) = . hz, h α c ) hz, j (h β-h ) = . hz, j (h β-h ) = . hz, h β c ) (c ), . (c ), . (c ), . (c ) h)-dione (cis- b). yield: . %; a colorless amorphous solid; m.p. > • c (crystallized from methanol-chloroform). ir (kbr, cm − ) ν max : o) δ: . (q, h, j = . hz j (h -cch) = . hz, j (h -h β) = . hz, j (h -cch) (s, h hz, j (h α-h ) = . hz, h α c ) h β-h ) = . hz, j (h β-h ) = . hz, h β c ); c-nmr ( mhz, d o) δ: (c ) -hydroxy- -methyl- -oxopyrrolidin- -yl)metyl]dihydropyrimidine- , ( h, h)-dione (trans- g). yield: . %; a colorless amorphous solid; m.p. > • c with decomposition (crystallized from methanol-chloroform). ir (kbr, cm − ) ν max : j (hch) = . hz, j (hcc-h ) = . hz, hch-dihydroura), . - . (m, h), . (dd, h, j (hch) = . hz, j (hcc-h ) = . hz, hch-dihydroura (ddd, h, j (h β-h α) = . hz cd od) δ: . (c(o)), . (c ), . (c ), . (c ), . (ch -dihydroura), . (c ) yield: %; a colorless amorphous solid; m.p. > • c with decomposition (crystallized from methanol-chloroform). ir (kbr, cm − ) ν max : hch-dihydroura), . - . (m, h, hc ), . - . (m, h), . (dd, h, j (hch) = . hz, j (hcc-h ) = . hz, hch-dihydroura hz, j (h α-h ) = . hz, h α c ), . (ddd, h, j (h β-h α) = . hz, j (h β-h ) = . hz c(o)), . (c ), . (c ), . (c ), . (ch -dihydroura) yield: %; a colorless amorphous solid; m.p. > • c (crystallized from methanol-chloroform). ir (kbr, cm − ) ν max : , hch-the), . (dd, h, j (h -h α) = . hz, j (h -h β) = . hz, hc (ddd, h, j (h β-h α) = . hz, j (h β-h ) = . hz, j (h β-h ) = . hz cdcl ) δ: . (c(o)), . (c ), . (c ), . (c ), . (c ), . (c ), . (c ) yield: %; a colorless amorphous solid; m.p. > • c (crystallized from methanol-chloroform). ir (kbr, cm − ) ν max : j (hcc-h ) = . hz, hch-the), . (dd, h, j (h -h α) = . hz, j (h -h β) = . hz, hc ), . (dd, h, j (hch) = . hz, j (hcc-h ) = . hz, hch-the) (s, h, ch ), . (s, h, ch ), . (s, h (c ), . (c ), . (c ), . (c ), . (ch -the) yield: %; a colorless amorphous solid; m.p. > • c with decomposition (crystallized from water). ir (kbr, cm − ) ν max : (ddd, h, j (h β-h α) = . hz, j = . hz, j = . hz, h β c ) amino- h-purin- -yl)methyl]- -hydroxy- -methylpyrrolidin- -one (cis- i). a colorless oil. ir (kbr, cm − ) ν max : dmso-d ) δ: . (s, h, hc ), . (s, h, hc ) m, h), . (s, h, ch n), . - . (m, h, h α c ), . (dt, h j = . hz, j = . hz, h β c synthesis of γ-lactams cis- d and trans- d a solution of a : : : mixture of γ-lactams trans- a, cis- a mmol) in dmf ( ml) was stirred at room temperature for h. the solvent was removed in vacuo and the crude product was purified on a silica gel column using chloroform-methanol ( : , v/v) as an eluent. the respective fractions were subjected to chromatography on a x bridge prep, c , µm, obd, × mm column using water/methanol ( : , v/v) to give pure γ-lactams trans yield: %; a colorless amorphous solid; m.p. = - • c with decomposition (crystallized from methanol-chloroform). ir (kbr, cm − ) ν max : hz, j (h -h α) = . hz, hc ), . (dd, h, j (hch) = . hz, j (hcc-h ) = . hz, hch-brura hz, j (hcc-h ) = . hz, hch-brura) j (h α-h ) = . hz, j (h α-h ) = . hz, h α c ), . (ddd, h, j (h β-h α) = . hz, j (h β-h ) = hz, j (h β-h ) = . hz, h β c ); c-nmr ( mhz, cd od) δ: . (c(o)), . (c ), . (c ), . (c ), . (c ), . (c ) calcd for c h brn o : c, yl)methyl]pyrimidine- , ( h, h)-dione (cis- d). yield: . %; a colorless amorphous solid; m.p. = - • c with decomposition (crystallized from methanol-chloroform). ir (kbr, cm − ) ν max : -h α) = . hz, j (h -h β) = . hz, j (h -cch) = . hz, hc ), . (dd, h, j (hch) = . hz, j (hcc-h ) = . hz, hch-brura) hz, j (h α-h ) = . hz, j (h α-h ) = . hz, h α c ), . (ddd, h, j (h β-h α) = . hz h β c ). c-nmr signals of cis- d were extracted from the spectra of a : mixture of cis- d and trans- d c-nmr ( mhz cis- a, trans- g and trans- g ( . g, . mmol) and n-chlorosuccinimide ( . g, . mmol) in freshly distilled pyridine ( ml) was stirred at • c for h. the solvent was removed in vacuo and the crude product was purified on a silica gel column using chloroform-methanol ( : , v/v) as an eluent. the respective fractions were subjected to chromatography on an x bridge prep yield: . %; a colorless amorphous solid; m.p. > • c with decomposition (crystallized from methanol-chloroform). ir (kbr, cm − ) ν max : hch-clura), . (dddd, h, j (h -h β) = . hz, j (h -cch) = . hz, j (h -cch) j (h α-h β) = . hz, j (h α-h ) = . hz, j (h α-h ) = . hz, h α c ) hz, j (h β-h ) = . hz, j (h β-h ) = . hz, h β c ). c-nmr ( mhz, cd od) δ: . (c(o)), . (c ), . (c ), . (c ), . (c ), . (c ) -hydroxy- -methyl- -oxopyrrolidin- -yl)methyl]pyrimidine- , ( h, h)-dione (cis- e) antiviral nucleosides: chiral synthesis and chemotheraphy new developments in anti-hiv chemotherapy advances in the development of nucleoside and nucleotide analogues for cancer and viral diseases highlights in antiviral drug research: antivirals at the horizon a -year journey in search of selective antiviral chemotherapy hiv resistance to reverse transcriptase inhibitors potent and selective activity of a new carbocyclic nucleoside analog (carbovir: nsc ) against human immunodeficiency virus in vitro ( β, β)- -(hydroxymethyl)- -dioxolanyl]thymine): a new , -dideoxynucleoside prototype with in vitro activity against hiv new antivirals-mechanism of action and resistance development pyrimidine nucleoside analogs in cancer treatment nucleoside analogs: molecular mechanisms signaling cell death cladribine tablets: a review in relapsing ms a review of its pharmacology and clinical potential in non-small cell lung cancer and pancreatic cancer a review in myelodysplastic syndromes and acute myeloid leukaemia high-dose cytarabine (hd arac) in the treatment of leukemias: a review discovery and development of clofarabine: a nucleoside analogue for treating cancer -homonucleosides and their structural analogues: a review homo-n-nucleosides: incorporation into oligonucleotides and antiviral activity comparative efficacy of antiherpes drugs against different strains of herpes simplex virus a novel selective broad-spectrum anti-dna virus agent synthesis of -deoxy- -homo-n-nucleosides with anti-influenza activity by catalytic methyltrioxorhenium (mto)/h o oxyfunctionalization selective inhibition of herpes simplex virus type- uracil-dna glycosylase by designed substrate analogs dérivés de désoxy-hydroxylamino-sucres et radicaux libres diglycosylnitroxydes correspondants. communication préliminaire isoxazolidine analogs of nucleosides inhibition of hiv- replication by isoxazolidine and isoxazole sulfonamides pyrinodemins b-d, potent cytotoxic bis-pyridine alkaloids from marine sponge amphimedon sp synthesis and biological activity of isoxazolidinyl polycyclic aromatic hydrocarbons: potential dna intercalators inhibition of erbb (her ) expression with small molecule transcription factor mimics isoxazolidinyl polycyclic aromatic hydrocarbons as dna-intercalating antitumor agents design, synthesis and cytotoxicity of a new series of isoxazolidine based nucleoside analogues antibacterial activity, quantitative structure-activity relationship and diastereoselective synthesis of isoxazolidine derivatives via , -dipolar cycloaddition of d-glucose derived nitrone with olefin enhancement in antimicrobial activity of -(phenyl)- -( -butyl- -chloro- h-imidazolyl)- -butylate isoxazolidine enantioselective syntheses and cytotoxicity of n,o-nucleosides synthesis of c- truncated phosphonated carbocyclic -oxa- -azanucleosides as antiviral agents synthesis of phosphonated carbocyclic -oxa-aza-nucleosides: novel inhibitors of reverse transcriptase phosphonated carbocyclic -oxa- -azanucleosides as new antiretroviral agents effect of phosphonated carbocyclic -oxa- -aza-nucleoside on human t-cell leukemia virus type infection in vitro diastereo-and enantioselective synthesis of n,o-nucleosides enantioselective synthesis of homocarbocyclic- -oxo- -azanucleosides diastereoselective synthesis of homo-n,o-nucleosides diastereoselective synthesis of a collection of new homonucleoside mimetics containing pyrrolo[ , -b]isoxazoline and pyrrolidine rings synthesis of n,o-homonucleosides with high conformational freedom syntheses of isoxazolinyl and isoxazolidinyl nucleoside analogues synthesis of novel isoxazolidine analogues of homonucleosides novel isoxazolidine analogues of homonucleosides and homonucleotides structure of neooxazolomycin, an antitumor antibiotic specific inhibitors of plant transformation stereoselective synthesis of ( s, s)-and ( s, r)-aza-muricatacin from an l-glutamic acid derivative muricatacin: a simple biologically active acetogenin derivative from the seeds of annona muricata (annonaceae) synthesis of aza-muricatacin: an analogue of the bioactive muricatacin an acetogenin of annonaceae study of the structure-activity relationships of the acetogenin of annonaceae, muricatacin and analogues natural and synthetic α-methylenelactones and α-methylenelactams with anticancer potential crystal structure, absolute configuration, and phosphodiesterase inhibitory activity of (+)- -( -bromobenzyl)- evaluation of nigerian traditional medicines: . plants used for rheumatic (inflammatory) disorders fleming-tamao oxidation and masked hydroxyl functionality: total synthesis of (+)-pramanicin and structural elucidation of the antifungal natural product (-)-pramanicin biologically active γ-lactams: synthesis and natural sources antimycobacterial activities of -alkyl (or halo)- -substituted pyrimidine nucleoside analogs synthesis and anti-hiv activity of new modified , , -triazole acyclonucleosides synthesis of -c-alkylated- -iodo- -deoxypyrimidine nucleosides the authors declare no conflict of interest. key: cord- -xurmzmwj authors: lin, xiaoqian; li, xiu; lin, xubo title: a review on applications of computational methods in drug screening and design date: - - journal: molecules doi: . /molecules sha: doc_id: cord_uid: xurmzmwj drug development is one of the most significant processes in the pharmaceutical industry. various computational methods have dramatically reduced the time and cost of drug discovery. in this review, we firstly discussed roles of multiscale biomolecular simulations in identifying drug binding sites on the target macromolecule and elucidating drug action mechanisms. then, virtual screening methods (e.g., molecular docking, pharmacophore modeling, and qsar) as well as structure- and ligand-based classical/de novo drug design were introduced and discussed. last, we explored the development of machine learning methods and their applications in aforementioned computational methods to speed up the drug discovery process. also, several application examples of combining various methods was discussed. a combination of different methods to jointly solve the tough problem at different scales and dimensions will be an inevitable trend in drug screening and design. with the rapid development of both computer hardware, software, and algorithms, drug screening and design have benefited much from various computational methods which greatly reduce the time and cost of drug development. in general, bioinformatics can help reveal the key genes from a massive amount of genomic data [ , ] and thus provide possible target proteins for drug screening and design. as a supplement to experiments, protein structure prediction methods can provide protein structures with reasonable precision [ ] . biomolecular simulations with multiscale models allow for investigations of both structural and thermodynamic features of target proteins on different levels [ ] , which are useful for identifying drug binding sites and elucidating drug action mechanisms. virtual screening then searches chemical libraries to provide possible drug candidates based on drug binding sites on target proteins [ ] [ ] [ ] . with greatly reduced amount of possible drug candidates, in-vitro cell experiments can further evaluate the efficacy of these molecules. in addition to virtual screening, de novo drug design methods [ ] , which generate synthesizable small molecules with high binding affinity, provide another type of computer-aided drug design direction. artificial intelligence, e.g., machine learning and deep learning, is playing more and more important roles in the aforementioned computational methods and thus drug development [ ] [ ] [ ] . in this review, we will focus on developments of the last four computational methods as well as their applications in drug screening and design. predicted by the transition state theory. hence, they used isomorphism between probabilities obtained from the mc process and probability factors obtained from transition state theory [ ] , and converted the mc process to a time-dependent simulation with additional simplified modifications [ ] . the design, discovery, and development of drugs are complex processes involving many different fields of knowledge and are considered a time-consuming and laborious inter-disciplinary work [ ] [ ] [ ] [ ] . different drug design methods and virtual screening will be very useful to design and find rational drug molecules based on the target macromolecule that interacts with the drug and thus speed up the whole drug discovery process. here, we will discuss structure-based drug design, ligand-based drug design, and virtual screening. structure-based drug design must be performed with available structural models of the target proteins, which are provided by x-ray diffraction, nuclear magnetic resonance (nmr) or molecular simulation (homologous protein modeling, etc.) [ ] [ ] [ ] [ ] [ ] . keeping in mind the complexity of cancers which show diverse phenotypes and multiple etiologies, a one-size-fits-all drug design strategy for the development of cancer chemotherapeutics does not yield successful results. lately, arjmand et al. [ ] adopted a series of methods, such as the combination of x-ray crystal structures and molecular docking, to design, synthesize, and characterize novel chromone based-copper(ii) antitumor inhibitors. in general, after obtaining the structure of the receptor macromolecule by x-crystal single-crystal diffraction technique or multi-dimensional nmr, molecular modeling software can be used to analyze the physicochemical properties of drug binding sites on the receptor, especially including electrostatic field, hydrophobic field, hydrogen bond, and key residues. then, the small molecule database is searched, or the drug design technique is used to identify the suitable molecules whose molecular shapes match the binding sites of the receptor and binding affinity is high. then, these molecules are synthesized and their biological activities will be tested for further drug development. in short, structure-based drug design plays an extremely important role in drug design. unlike structure-based drug design, ligand-based drug design doesn't search small molecule libraries. instead, it relies on knowledge of known molecules binding to the target macromolecule of interest. using these known molecules, a pharmacophore model that defines the minimum necessary structural characteristics a molecule must possess in order to bind to the target can be derived [ , ] . then, this model can be further used to design new molecular entities that interact with the target. on the other hand, ligand-based drug design can also use quantitative structure-activity relationships (qsar) [ , ] in which a correlation between calculated properties of molecules and their experimentally determined biological activity is derived, to predict the activity of new analogs. both the pharmacophore model and qsar model will be discussed in detail in the following sessions. in recent years, the rapid development of computational resources and small molecule databases have led to major breakthroughs in the development of lead compounds. as the number of new drug targets increases exponentially, computational methods are increasingly being used to accelerate the drug discovery process. this has led to the increased use of computer-assisted drug design and chemical bioinformatics techniques such as high-throughput docking, homology search and pharmacophore search in databases for virtual screening (vs) technology [ ] . virtual screening is an important part of computer-aided drug design methods. it may be the cheapest way to identify potential lead compounds, and many successful cases have proven successful using this technology. the primary technique for identifying new lead compounds in drug discovery is to physically screen large chemical libraries for biological targets. in experiments, high-throughput screening identifies active molecules by performing separate biochemical analysis of more than one million compounds. however, this technology involves significant costs and time. therefore, a cheaper and more efficient calculation method came into being, namely, virtual high-throughput screening. the method has been widely used in the early development of new drug. the main purpose is to determine the novel active small molecule structure from the large compound libraries. it is consistent with the purpose of high-throughput screening. the difference is that virtual screening can save a lot of experimental costs by significantly reducing the number of compounds for the measurement of the pharmacological activity, while high-throughput screening needs to perform experiments with all compounds in the database. here, we will discuss common methods of virtual screening. molecular docking, which predicts interaction patterns between proteins and small molecules as wel as proteins and proteins, to evaluate the binding between two molecules [ ] , is widely used in the field of drug screening and design. the theoretical basis is that the process of ligand and receptor recognition relies on spatial shape matching and energy matching, which is the theory of "inducing fit". determining the correct binding conformation of small molecule ligands and protein receptors in the formation of complex structures is the basis for drug design and studying its action mechanism. molecular docking can be roughly divided into rigid docking, semi-flexible docking and flexible docking. in rigid docking, the structure of molecules does not change. the calculation method is relatively simple, and mainly studies the degree of conformation matching, so it is more suitable for studying macromolecular systems, such as protein-protein, protein-nucleic acid systems. in semi-flexible docking, the conformation of molecules can be varied within a certain range, so it is more suitable to deal with the interaction between proteins and small molecules [ ] . in general, the structure of small molecules can be freely changed, while macromolecules remain rigid or retain some of the rotatable amino acid residues to ensure computational efficiency. in flexible docking, the simulated system conformation is free to change, thus consuming more computing resources while improving accuracy. what's more, the establishment of binding sites in molecular docking methods is very important. for the first time, collins [ ] successfully determined the binding sites on the surface of proteins using a multi-scale algorithm and performed flexible docking of molecules, which greatly promoted the development of molecular docking. a pharmacophore is an abstract description of molecular features necessary for molecular recognition of a ligand by a biological macromolecule, which explains how structurally diverse ligands can bind to a common receptor site. when a drug molecule interacts with a target macromolecule, it produces a geometrically and energetically matched active conformation with the target. medicinal chemists found that different chemical groups in drug molecules have different effects on activity, and changes to some groups have a great influence on the interaction between drugs and targets, while others have little effect [ ] . moreover, it was found that molecules with the same activity tend to have some of the same characteristics. therefore, in , ehrlich proposed the concept of pharmacophores, which referred to the molecular framework of atoms with active essential characteristics [ ] . in , gund further clarified the concept of pharmacophores as a group of molecules that recognize receptors and form structural features of molecular biological activity [ ] . there are two main methods for the identification of pharmacophores. on one hand, if the target structure is available, the possible pharmacophore structure can be inferred by analyzing the action mode of receptor and drug molecule. on the other hand, when the structure of the target is unknown or the action mechanism is still unclear, a series of compounds will be studied for pharmacophores, and information on some groups that play a key role in the activity of compound will be summarized by means of conformational analysis and molecular folding [ ] . active compound that is suitable for constructing the model will be selected in the pharmacophore recognition process. then, conformation analysis is used to find the binding conformation of molecule, and to determine the pharmacophore [ ] . in recent years, with the development of compound databases and computer technology, the virtual screening of databases using the pharmacophore model has been widely used, and has become one of the important means to discover lead compounds. qsar is a quantitative study of the interactions between small organic molecules and biological macromolecules. it contains a correlation between calculated properties of molecules (e.g., absorption, distribution, metabolism of small organic molecules in living organisms) and their experimentally determined biological activity [ ] . in the case of unknown receptor structure, the qsar method is the most accurate and effective method for drug design. drug discovery often involves the use of qsar to identify chemical structures that could have good inhibitory effects on specific targets and have low toxicity (non-specific activity). with the further development of structure-activity relationship theory and statistical methods, in the s, d structural information was introduced into the qsar method, namely d-qsar. since s, with the improvement of computing power and the accurate determination of d structure of many biomacromolecules, structure-based drug design has gradually replaced the dominant position of quantitative structure-activity relationship in the field of drug design, but qsar with the advantages of small amount of calculation and good predictive ability [ ] still plays an important role in pharmaceutical researches. based on d structural characteristics of ligands and targets, d-qsar explores the d conception of bioactive molecules, accurately reflects the energy changes and patterns of interactions between bioactive molecules and receptors, and reveals the drug-receiving mechanism of body interactions. the physicochemical parameters and d structural parameters of a series of drugs are fitted to the quantitative relationship. then, the structures of new compounds are predicted and optimized. in short, d-qsar is actually a research method combining qsar with computational chemistry and molecular graphics. it is a powerful tool for studying the interactions between drugs and target macromolecules, speculating the image of simulated targets, establishing the relationship of drug structure activity, and designing drugs. computer-based de novo design methods of drug-like molecules are mainly for generating small molecule compounds with ideal physicochemical and pharmacological properties. in the past decades, fragment-based drug discovery had appeared as a novel concept that has proved a good prospect for improving lead optimization, in order to decrease the clinical attrition rates in drug design. it is an approach that uses small molecular fragments to deduce the biomolecular targets [ ] . fragment-based de novo design has obtained the long-term clinical success [ ] . despite the fact that modern drug discovery has made some successes in offering effective drugs, drug design has been affected by several factors, such as the tremendous chemical space for exploring drug molecules [ ] . further, as a large number of data increase in biological, chemical, and clinical medicine, it is obvious that the drug design should be solved with multiscale optimization methods, and concentrate on the data beyond molecular levels [ ] . thus, it is essential to discuss the function of multiscale models in drug discovery, and how they have predicted multiple biological properties in different biological targets. accordingly, we discuss the combined application of both the concept of fragment-based on de novo design and multiscale modeling. the fragment-based de novo design method starts with small building blocks. the initial molecular building blocks with desired properties are either elaborated upon (growing), directly connected (joining), or connected by a linker (linking). this process can be iterated until one or more molecules with the desired properties are obtained. there are two methods, namely structure-based and ligand-based methods [ ] . structure-based de novo design method searches novel ligands by using the d structural information of the protein target, which are usually constructed directly in the target protein binding site and evaluated by calculating the interaction energy of the target protein with ligands. nevertheless, in de novo ligand design method, the molecule structure of protein target is unknown, and the new molecule is suggested based structure analogous to the known ligand molecule. nowadays, proteochemometrics has emerged as a relatively new discipline for drug discovery. in this filed, qsar analysis is a powerful tool for the efficient virtual screening, which shows physicochemical properties of various compounds. compared to the classical qsar, the qm calculations use reactivity descriptors in ligand-based qsars, which provides an implicit model and calculate an exact enthalpy contributions of protein-ligand interactions. however, for the ab initio fragment, molecular orbital calculation in the structure-based qsars, which obtains an explicit model and a clear enthalpy, changes the binding energy in different additional conditions. moreover, it also calculates the free energy contribution of ligand-target complexes formation in structure-based and ligand-based qsar models. using a large number of ligand-target complexes to discuss the change of their binding affinity, more accurate optimization steps can be conducted based on good prediction and interpretation models [ ] . the key of any qsar model is how to accurately describe the molecule, and qm approaches provides a better understanding to the molecular and structural characteristics of ligands and drugs, so as to solve the problems existed in drug discovery. the qsar models of different scales are built according to the different computational precision, multiscale-qsar research object mainly refers to the structure description of the training set, and involves small molecules and macromolecules [ , ] . in micro-, meso-and macroscopic scales, different molecular approaches will be used. qm approaches are often used to perform precise calculations at microscales, such as atom-based qsar. molecular force field focuses on mesoscopic-scale simulation, such as fragment-based qsar. and coarse-grained study mainly performed in the macroscopic scales, such as macromolecule-based qsar and cell-based qsar. moreover, multi-scale can also be reflected by different dimensions used in different qsar models. comfa is a technique of d fragment-based qsar, which can complete skeleton transition and r-groups substitution, providing different structures for new drug design. besides, derived from proteochemometrics (pcm) [ ] , the . d kinochemometrics (kcm) approach using d descriptor for protein kinases and d fingerprints for ligands can greatly increase the efficiency as well as the precision compared the traditional d qsar methods [ ] . the multiscale qsar provides effective predictions for drug design, which integrates qsar more systematically and applies all existing qsar methods effectively. multiscale de novo drug design is a novel concept that combines qsar models, qm calculations [ ] and fragment-based drug discovery (fbdd) [ ] . here, the importance of explicit molecular descriptors is shown in a model from a molecular structural point of view through qm calculations. with the assembly of reasonable molecular fragments, the objective of drug design method is to produce a certain novel molecule that display highest biological activity, absorption, metabolism, elimination (adme) and lowest toxicity properties at different environments, which belong to the application range of qsar models. the multiscale de novo drug design methods can efficiently handle a large amount of biochemical/clinical data and obtain the chemical characteristics in order to improve the properties of the drug molecule. it is considered to be a more effective and safer method to discover new therapeutic agents. in the process of drug discovery, machine intelligence methods have mostly been used in the above-mentioned computational methods over the past few decades [ ] . with the booming era of "big" data, machine learning methods have developed into deep learning approaches, which are a more efficient way for drug designers to deal with important biological properties from large amount of compound databases. here, we introduce applications of machine learning methods in qsar analysis as well as the recent advances in deep learning methods. decision trees (dts) are a simple, interpretable and predictive machine learning method. ordinarily, there are two fundamental steps, that is, selecting properties and pruning for the decision trees building. the selected properties are considered as internal nodes, the branch representing the test result on the molecule, and the leaf node as a classification label. in order to avoid the complexity of the decision tree, the pruning program is used to prune the established tree. the dt is a typical classification algorithm, which is widely used in the prediction and auxiliary diagnosis of the disease, such as management decision-making, the classification mode for creating the metabolic disorder, and data mining of diabetes etc. [ ] . abdul et al. [ ] developed a task-based chemical toxicity prediction framework, and used a decision tree to obtain an optimum number of features from a collection of thousands of them, which effectively help chemists perform prescreening of toxic compounds effectively. the artificial neural network (ann) achieves problem-solving by mimicking brain function. just as the brain applies information obtained from past experience to solve new problems, a neural network can construct a system of "neurons" that reaches new decisions, classifications, and prediction based on previous experiences. the processing element is similar to a neuron, and a massive processing element is organized by the layers. they include three types: input, hidden, and output layers. ann benefits from high self-organization, robustness, and fault tolerance, and has been widely applied in prognosis evaluation and early prevention of diseases. lorenzo et al. [ ] used the interpretable ann to predict biophysical properties of therapeutic monoclonal antibodies, include melting temperature, aggregation onset temperature and interaction parameters as a function of ph and salt concentration from the amino acid composition. artificial neural networks had their first heyday in molecular informatics and drug discovery approximately two decades ago. currently, we are witnessing renewed interest in adapting advanced neural network architectures for pharmaceutical research by borrowing ideas from the deep learning field. compared with some other fields in life sciences, their applications in drug discovery is still limited. the support vector machine (svm) is one of the most promising machine learning methods that can use molecular descriptors to construct a predictive qsar models and deal with high-dimensional datasets. ann and multiple linear regression analysis were used to construct linear and nonlinear models, which were then compared with the results gained by svm. for linear models, the svm approaches use space mapping points to separate different classification for maximizing the range between different categories of points [ ] . further, for the nonlinear models, svms use nucleus mapping to transform into a high-dimension space for linear classification. at present, the svm approach has been widely used in modeling at different scales for drug discovery [ ] . the k nearest neighbor (knn) is one of the simplest and most intuitive algorithms among all machine learning methods, and is usually jointly used with other selection algorithms in the feature space. further, it is used for classification and regression based on example learning. normally, molecules are classified by votes of its closest neighbors, resulting in the most common class that molecules are distributed to its closest neighbors. here, the value of k is the number of closest neighbors. based on ligand-based virtual screening, knn can be viewed as a prolongation form chemical similarity searching to supervised learning, and the top search results predicted the best bioactivities. weber et al. [ ] tried two machine learning algorithms of classification (knn and rf) to analyze genotype-phenotype datasets of hiv protease and reverse transcriptase (rt). as a result, both algorithms had high accuracies for predicting the drug resistance for protease and rt inhibitors. the random forest (rf) is an ensemble learning approach involving the building of multiple dts based on the training examples. similar to knn algorithms, it is also used to for classification and to predict regression [ ] . compared to dts, it is impossible that rf over-fits the data, and the rf has been used for bioactivity data classification [ ] , toxicity modeling [ ] , and drug target prediction [ ] , etc. wang et al. [ ] used the rf approach to model the binding affinity of protein-ligand on hiv- proteases complexes, trypsin complexes, and carbonic anhydrase complexes, which demonstrated that individual representation and model construction for each protein family is a more reasonable way in predicting the affinity of one particular protein family. currently, the multiscale models can predict toxicity, activity, and adme properties of different proteins and microbial targets by integrating different genomes and proteomics. cheminformatics has played an important role in rationalize drug discovery. the qsar model has become the main auxiliary tool which can achieve virtual screening of various pharmacological characteristics. although the qsar model has been widely used in the search and design of new drug, classical qsar models can only predict the activity and toxicity of one biomolecule against one certain target. however, the multi-target qsar (mt-qsar) can be used to carry out rational drug design at multiple targets, which provides a better way to understand various pharmacological characteristic molecules including antibacterial activity and toxicity. furthermore, uniform multitasking models based on quantitative structure biological effect relationship (mtk-qsber) have been used in a lot of researches. these models were built by ann and the topological indices, which can predict the biological activity and toxicity correctly and classify the compounds in experimental conditions. meanwhile, these models used perturbation models to form structural-activity relationships between the site of infection and the drug, such as the ptml model [ ] and the chembl model [ ] , which has been applied in infectious diseases [ ] , immunology [ ] , and cancer [ ] widely. currently, the mtk-qsber model has been able to carry out the in-silico design and virtual screening of an antibacterial drug efficiently, and these antibacterial drugs have good biosafety. these methods have provided a powerful tool for in silico screening reasonable drugs. the deep learning network is a concept closely related to ann, which are learning of the concept of layering. in other words, it is a multiple learning approaches ranging from low to high levels. just when the molecular descriptors are not selected, the deep learning method will automatically select representations from original data and high-dimensional data [ ] . therefore, it allows deep learning to be applied to the model building of drug discovery [ ] . the convolutional neural networks (cnn) are most commonly used, which have made great progresses in the computer vision community [ ] , and been applied in the drug design fields including de novo drug molecule identification, protein engineering and gene expression analysis. with the rapid development of deep learning concepts such as cnn, the molecular modeler's tool box has been equipped with potentially game-changing methods. judging from the success of recent pioneering studies, we are convinced that modern deep learning architecture will be useful for the coming age of big data analysis in pharmaceutical research, toxicity prediction, genome mining and chemogenomic applications, to name only some of the immediate areas of application. kiminori et al. [ ] developed a fundamental technology that can predict the resistance of free cancer cells to fluorinated pyrimidine anticancer drugs by deep learning from the morphological image data taken from images. cai et al. [ ] developed a deep learning approach, termed deep human ether-a-go-go-related gene (herg), for the prediction of herg blockers of small molecules in drug discovery and post-marketing surveillance. the group found that deepherg models built by a multitask deep neural network (dnn) algorithm outperformed those built by single-task dnn, svm and rf. now, the drug development technologies usually include artificial intelligence-based (ai-based) techniques. most ai applications only concentrate on limited tasks. moreover, current ai can only direct patients' specific problems, it cannot make subjective inferences like doctors with the overall physical context of a patient. as a subfield of ai, ml can be successfully used for training in the quality of examples. however, this process is very time-consuming and costly. the development of ml techniques and the application of existing algorithms to process massive amounts of digital data resulted in higher requirements for computer hardware, which also increases the clinical cost. dl, which is also a subset of ml, and can process big data and create patterns by layers of neurons. however, it is difficult to understand how each decision is obtained by algorithm. ml methods have achieved great successes in the field of chemoinformatics to design and discover new drugs. an important innovation is the combination of ml methods and big data analysis to predict more extensive biological features. it is vital to discover more secure and efficient drugs by integrating structural, genetic information and pharmacological data from the scale of molecular to organism [ ] . in addition, dl approaches have proven to be a promising way for efficiently learning from a large variety of datasets for modern novel drug discovery. multiscale modeling of the drugs in an excitable system is critical because experiments on a single system scale cannot reveal the underlying effects of multiple drug interactions. a computationally based approach to predict the emergency effects of drugs on excitatory rhythms may form an interactive technology-driven process for the drug and disease screening industry, research and development academia, and patient-oriented medical clinic. there are potentially far-reaching implications because millions of people affected by arrhythmia each year will benefit from improved risk stratification of drug-based interventions. much progress has been made in developing multiscale computational modeling and simulation approaches for predicting effects of cardiac ion channel blocking drugs. structural modeling of ion channel interactions with drugs is a critical approach for current and future drug discovery efforts. modeling of drug receptor sites within an ion channel structure can be useful to identify key drug-channel interaction sites. drug interactions with cardiac ion channels have been modeled at the atomic scale in simulated docking and md simulations, as well as at the level of channel function to simulate drug effects on channel behavior [ , [ ] [ ] [ ] [ ] [ ] [ ] [ ] . structural modeling of drug-channel interactions at the atomic scale may ultimately allow for the design of novel high-affinity and subtype selective drugs for specific targeting of receptors for cardiac and neurological disorders. the world health organization (who) stated that cancer remains one of the most dangerous diseases today. considering that cancer is a multifactorial disease, there is increasing interest in multi-target compounds that can target multiple intracellular pathways. however, the study of large data sets for the analysis of anticancer compounds is difficult, with a large amount of data and high data complexity. for example, the chembl database [ ] compiles big datasets of very heterogeneous preclinical assays. bediaga et al. [ ] have reported a ptml-lda model of the chembl dataset for the preclinical determination of anticancer compounds. ptml is a model that combines perturbation theory (pt) ideas and ml methods to solve similar problems. they compared this model with other ptml models which was reported by speck-planche et al. [ , [ ] [ ] [ ] and then concluded that this is the only one that can predict activity against multiple cancers. speck-planche et al. also derived a multi-task (mtk) chemical information model combining broto moreau autocorrelation with ann from a dataset containing peptide cases. this model is used to virtually design and screen peptides with potential anti-cancer activity against different cancer cell lines and low cytotoxicity to a variety of healthy mammalian cells, and the model shows greater than % in both training and prediction (test)accuracy. in addition, due to the inherent complexity of tumors, it is necessary to analyze their growth at different scales. it includes many phenomena that occur at various spatial scales from tissue to molecular length. the complexity of cancer development is manifested in at least three scales that can be distinguished and described by mathematical models, namely microscale, mesoscale, and macroscale. wang et al. conducted a number of studies on how to use multiscale models for the identification and combination therapy of drug targets [ ] [ ] [ ] [ ] [ ] [ ] [ ] . this method is based on quantification of relationship between intracellular epidermal growth factor receptor (egfr) signaling kinetics, lung cancer extracellular epidermal growth factor (egf) stimulation and multicellular growth. the multiscale modeling of tumors combined with systemic pharmacology will contribute to the development of practical smart drugs. it will produce a comprehensive system-level approach to determine the dynamics and effects of existing and new drugs in preclinical trials, model organisms and individual patients. in addition, mathematical and computational studies will provide a better way to understand many factors that influence the effects of drugs, thus helping to uncover better ways to therapeutically interfere with disease. multiscale models can be also used to identify pathophysiological processes to allow disease staging. in many cases, like cancer, treatments vary depending on the stage of disease. the model can help determine prognosis, which is an important clinical determination that can help determine the right type of medication to be administered or discovered. several models focus on the neuronal network levels, including cutsuridis and moustafa for alzheimer's disease [ ] , and lytton for epilepsy [ ] . ann is a class of ml techniques that can be used for clinical analysis of big data including that related to drug testing, which is critical for drug discovery. in addition, anastasio [ ] introduced process algebra, a computer technology widely used to analyze complex computing systems, used here to calculate neurology. sirci et al. [ ] described how network (map) theory is used to identify similarities and differences between different pharmacological agents. in this type of study, each drug is a node, and the edges between drugs represent chemical and transcription-based interactions that characterize the drug. in addition, ferreira da costa et al. [ ] report the first ptml (pt + ml) study of a large number of chembl datasets for preclinical determinations of compounds for dopamine pathway proteins. molecular docking or ml models can be used to solve a specific protein, but these models cannot explain the large and complex large data sets of preclinical assays reported in public databases. pt models, on the other hand, allow us to predict the properties of a query compound or molecular system in an experimental analysis with multiple boundary conditions based on previously known reference cases. in their work, the best ptml model found in the training and external validation series has an accuracy of - %. hansch's model is a classic method for solving quantitative structural binding relationships (qsbr) in pharmacology and medicinal chemistry. abeijon et al. [ ] developed a new pt-qsbr hansch model based on pt and qsbr methods for a large number of drugs reported in chembl, focusing on a protein expressed in the hippocampus of the brain of alzheimer's disease (ad) patients. now, by decomposing how risks and causes are combined in complex systems to produce disease, and how to prevent or improve these diseases through multi-stage, multi-target, multi-drug techniques, multiscale modeling is gradually being grasped. from aids, hepatitis c, influenza, and other disease-related viruses to the current -ncov, we have been working hard to develop antiviral drugs targeting them. however, the unique structure and proliferation of the virus pose a natural challenge for drug development. viruses do not have their own cellular structure and metabolic system, and must replicate and proliferate in host cells. therefore, it is difficult to find compounds that target only viral targets without affecting the normal function of host cells. at present, the main way that some antiviral drugs work is to inhibit viral replication. however, many of the tools used for virus replication come from human cells, such as ribosomes, and the corresponding antiviral drugs will also bring great side effects to the human body. therefore, the discovery of drugs requires the introduction of a multi-scale model to screen out drugs that can inhibit viral replication while reducing the damage to the human body. so far, retroviral infections, such as hiv, are incurable diseases. chembl manages big data capabilities through complex datasets, which make the information difficult to analyze because these datasets describe numerous features for predicting new drugs for retroviral infections. without proper model, it is impossible to make full use of these features. hence, vásquez-domínguez et al. [ ] proposed a ptml model for the chembl dataset, which can be efficiently used for preclinical experimental analysis of antiretroviral compounds. the pt operator is based on a multi-conditional moving average, which combines different functions and simplifies the management of all data. the ptml model they proposed was the first to consider multiple features combined with preclinical experimental antiretroviral tests. in order to simultaneously explore antibacterial activity against gram-negative pathogens and in vitro safety related to absorption, distribution, metabolism, elimination, and toxicity (admet), the speck-planchee et al. [ ] further proposed the first mtk-qsber model. the accuracy of this model in both the training and prediction (test) sets is higher than %. they also have developed a chemoinformatic model for simultaneous prediction of anti-cocci activities and in vitro safety [ ] . the best model displayed accuracies around % in both training and prediction (test) sets. additionally, focusing on exploring anti-hepatitis c virus (hcv), the accuracy shown in the training and prediction (test) sets is higher than % using this model [ ] . cytotoxicity is one of the main concerns in the early development of peptide-based drugs. kleandrova et al. [ ] introduce the first multi-task processing (mtk) computational model focused on predicting both antibacterial activity and peptide cytotoxicity. gonzalez-diaz et al. [ ] developed a model called lnn-alma to generate complex networks of the aids prevalence with respect to the preclinical activity of anti-hiv drugs. multiscale models are also imperfect and have their limitations. models are expressions and simplifications of real life. no model can represent everything that can happen in the system. all models contain specific assumptions, and models vary widely in their comprehensiveness, quality, and utility. in other words, each model can only solve limited problems. hence, we need to integrate different computational models and data in order to make full use of these models. computational methods have come to play significant roles in drug screening and design. multiscale biomolecular simulations can help identify the drug binding sites on the target macromolecules and elucidate the drug action mechanisms. virtual screening can efficiently search massive chemical databases for lead compounds. de novo drug design provides alternative powerful way to design drug molecules from scratch using building blocks summarized and abstracted in previous successful drug discovery. ml is revolutionizing most computational methods in drug screening and design, which may greatly improve the efficiency and precision for the big data era. as we frequently emphasize, different models or efficient algorithms (e.g., dimensionality reduction) need to be integrated properly to achieve the comprehensive study of biological processes at multiple scales as well as accurate and effective drug screening and design. the integrated computational methods will accelerate drug development and help identify effective therapies with novel action mechanisms that can ultimately be applied to a variety of complex biological systems. the authors declare no conflict of interest. prediction of drug-target interaction networks from the integration of chemical and genomic spaces properties and identification of human protein drug targets critical assessment of methods of protein structure prediction (casp)-round xii multiscale modeling of biomolecular systems: in serial and in parallel virtual screening of chemical libraries computational protein-ligand docking and virtual drug screening with the autodock suite rapid virtual screening of enantioselective catalysts using catvs automated de novo drug design: are we nearly there yet? deep reinforcement learning for de novo drug design machine learning for molecular modelling in drug design. biomolecules machine learning based dimensionality reduction facilitates ligand diffusion paths assessment: a case of cytochrome p cam development of multiscale models for complex chemical systems: from h + h to biomolecules (nobel lecture) the many roles of computation in drug discovery role of molecular dynamics and related methods in drug discovery advancing drug discovery through enhanced free energy calculations excited state properties of non-doped thermally activated delayed fluorescence emitters with aggregation-induced emission: a qm/mm study exploring the dependence of qm/mm calculations of enzyme catalysis on the size of the qm region spectroscopy in complex environments from qm-mm simulations peptide folding kinetics from replica exchange molecular dynamics structural characterization of λ-repressor folding from all-atom molecular dynamics simulations macrolide antibiotics allosterically predispose the ribosome for translation arrest current tools and methods in molecular dynamics (md) simulations for drug design modeling structural dynamics of biomolecular complexes by coarse-grained molecular simulations the impact of molecular dynamics on drug design: applications for the characterization of ligand-macromolecule complexes molecular dynamics simulations and drug discovery the future of molecular dynamics simulations in drug discovery identification of drug binding sites and action mechanisms with molecular dynamics simulations assessing the performance of the mm/pbsa and mm/gbsa methods. . the accuracy of binding free energy calculations based on molecular dynamics simulations physical properties of the hiv- capsid from all-atom molecular dynamics simulations biomolecular interactions modulate macromolecular structure and dynamics in atomistic model of a bacterial cytoplasm online tools for protein ensemble pocket detection and tracking multiscale modeling in the clinic: drug design and development multiscale methods in drug design bridge chemical and biological complexity in the search for cures recent advances in fragment-based computational drug design: tackling simultaneous targets/biological effects monte carlo simulations of proton pumps: on the working principles of the biological valve that controls proton pumping in cytochrome c oxidase multiscale simulations of protein landscapes: using coarse-grained models as reference potentials to full explicit models realistic simulations of proton transport along the gramicidin channel: demonstrating the importance of solvation effects diverse strategies in drug discovery and development good practices in model-informed drug discovery and development: practice, application, and documentation advances in computational structure-based drug design and application in drug discovery expediting the design, discovery and development of anticancer drugs using computational approaches identification of potential crac channel inhibitors: pharmacophore mapping, d-qsar modelling, and molecular docking approach homology model versus x-ray structure in receptor-based drug design: a retrospective analysis with the dopamine d receptor new insights for drug design from the x-ray crystallographic structures of g-protein-coupled receptors an improved receptor-based pharmacophore generation algorithm guided by atomic chemical characteristics and hybridization types x-ray crystallographic structure of a teixobactin analogue reveals key interactions of the teixobactin pharmacophore design, synthesis and characterization of novel chromone based-copper (ii) antitumor agents with n, n-donor ligands: comparative dna/rna binding profile and cytotoxicity pharmacophore modeling and applications in drug discovery: challenges and recent advances searching for potential mtor inhibitors: ligand-based drug design, docking and molecular dynamics studies of rapamycin binding site best practices for qsar model development, validation, and exploitation rational drug design of antineoplastic agents using d-qsar, cheminformatic, and virtual screening approaches molecular docking and structure-based drug design strategies molecular docking as a popular tool in drug design, an in silico travel h-nmr of rh (nh ) phi + bound to d (tggcca) : classical intercalation by a nonclassical octahedral metallointercalator d pharmacophore modeling techniques in computer-aided molecular design using ligandscout Über den jetzigen stand der chemotherapie three-dimensional pharmacophoric pattern searching pharmacophore models and pharmacophore-based virtual screening: concepts and applications exemplified on hydroxysteroid dehydrogenases pharmacophore-based virtual screening identification of potential tumour-associated carbonic anhydrase isozyme ix inhibitors: atom-based d-qsar modelling, pharmacophore-based virtual screening and molecular docking studies revealing the macromolecular targets of complex natural products multi-objective molecular de novo design by adaptive fragment prioritization click chemistry for drug discovery legacy data sharing to improve drug safety assessment: the etox project multi-objective optimization methods in de novo drug design. mini rev using local models to improve (q) sar predictivity a multiscale simulation system for the prediction of drug-induced cardiotoxicity multiscale quantum chemical approaches to qsar modeling and drug design polypharmacology modelling using proteochemometrics (pcm): recent methodological developments, applications to target families, and future prospects prediction of protein kinase-ligand interactions through . d kinochemometrics chemoinformatics for medicinal chemistry: in silico model to enable the discovery of potent and safer anti-cocci agents fragment-based in silico modeling of multi-target inhibitors against breast cancer-related proteins quantitative structure-activity relationship: promising advances in drug discovery platforms machine learning method for knowledge discovery experimented with otoneurological data efficient toxicity prediction via simple features using shallow neural networks and decision trees application of interpretable artificial neural networks to early monoclonal antibodies development computational prediction of anti hiv- peptides and in vitro evaluation of anti hiv- activity of hiv- p -derived peptides in silico de novo design of novel nnrtis: a bio-molecular modelling approach automated prediction of hiv drug resistance from genotype data cct is a novel potent and selective chk inhibitor with oral efficacy alone and in combination with genotoxic anticancer drugs qsar based model for discriminating egfr inhibitors and non-inhibitors using random forest using random forest and decision tree models for a new vehicle prediction approach in computational toxicology identification of human drug targets using machine-learning algorithms a comparative study of family-specific protein-ligand complex affinity prediction based on random forest approach ptml model for proteome mining of b-cell epitopes and theoretical-experimental study of bm protein sequences from colima computational modeling in nanomedicine: prediction of multiple antibacterial profiles of nanoparticles using a quantitative structure-activity relationship perturbation model ptml combinatorial model of chembl compounds assays for multiple types of cancer big data deep learning: challenges and perspectives deep learning in neural networks: an overview in imagenet classification with deep convolutional neural networks deep learning recognizes ftd-resistant isolated cancer cells of colon cancer deep learning-based prediction of drug-induced cardiotoxicity data integration: challenges for drug discovery a computational model of induced pluripotent stem-cell derived cardiomyocytes incorporating experimental variability from multiple data sources multi-scale modeling of the cardiovascular system: disease development, progression, and clinical intervention binding pocket optimization by computational protein design molecular mechanism matters: benefits of mechanistic computational models for drug development parameterization for in-silico modeling of ion channel interactions with drugs a molecularly detailed nav . model reveals a new class i antiarrhythmic target a computational model to predict the effects of class i anti-arrhythmic drugs on ventricular rhythms cibrián-uhalte, e. the chembl database in chemoinformatics in anti-cancer chemotherapy: multi-target qsar model for the in silico discovery of anti-breast cancer agents rational drug design for anti-cancer chemotherapy: multi-target qsar models for the in silico discovery of anti-colorectal cancer agents unified multi-target approach for the rational in silico design of anti-bladder cancer agents. anti-cancer agent multiscale modeling of ductal carcinoma in situ a multiscale agent-based model of ductal carcinoma in situ mathematical modeling in cancer nanomedicine: a review mathematical modeling in cancer drug discovery mathematical modeling of tumor organoids: toward personalized medicine an in silico approach for assessing drug efficacy within a tumor tissue current advances in mathematical modeling of anti-cancer drug penetration into tumor tissues multiscale models of pharmacological, immunological and neurostimulation treatments in alzheimer's disease computer modeling of epilepsy: opportunities for drug discovery modeling neurological disease processes using process algebra computational drug networks: a computational approach to elucidate drug mode of action and to facilitate drug repositioning for neurodegenerative diseases gonzález-díaz, h. perturbation theory/machine learning model of chembl data for dopamine targets: docking, synthesis, and assay of new l-prolyl-l-leucyl-glycinamide peptidomimetics multi-target mining of alzheimer disease proteome with hansch's qsbr-perturbation theory and experimental-theoretic study of new thiophene isosters of rasagiline multioutput perturbation-theory machine learning (ptml) model of chembl data for antiretroviral compounds de novo computational design of compounds virtually displaying potent antibacterial activity and desirable in vitro admet profiles speeding up early drug discovery in antiviral research: a fragment-based in silico approach for the design of virtual anti-hepatitis c leads enabling the discovery and virtual screening of potent and safe antimicrobial peptides. simultaneous prediction of antibacterial activity and cytotoxicity ann multiscale model of anti-hiv drugs activity vs. aids prevalence in the us at county level based on information indices of molecular graphs and social networks key: cord- - umgvt authors: ma, li; yao, lei title: antiviral effects of plant-derived essential oils and their components: an updated review date: - - journal: molecules doi: . /molecules sha: doc_id: cord_uid: umgvt the presence of resistance to available antivirals calls for the development of novel therapeutic agents. plant-derived essential oils may serve as alternative sources of virus-induced disease therapy. previous studies have demonstrated essential oils to be excellent candidates to treat antiviral-resistant infection associated with their chemical complexity which confers broad-spectrum mechanisms of action and non-specific antiviral properties. however, almost no comprehensive reviews are updated to generalize knowledge in this regard and disclose the interplay between the components and their antiviral activities. this review provides an up-to-date overview of the antiviral efficacy of essential oils from a wide range of plant species and their characteristic components, as well as their overall mechanisms of action, focusing on the last decade. the roles of individual components relative to the overall antiviral efficacy of essential oils, together with the antiviral activity of essential oils in comparison with commercial drugs are also discussed. lastly, the inadequacies in current research and future research are put forward. this review will provide references in the design of new drug prototypes and improve our understanding of the proper applications of essential oils in the future. a virus is a small particle ( - nm) containing merely genetic substances wrapped with proteins and, sometimes, lipids [ ] . they infect host cells to fulfill self-replication. the viral diseases remain a significant worldwide concern. currently, few desirable antiviral drugs are available, and, in most cases, they exhibit either intracellular or extracellular antiviral properties. acyclovir, a nucleoside analogue against human herpes viruses (hsv), blocks viral replication via inhibition on viral dna polymerases [ ] . amantadine, an m ion channel inhibitor for influenza virus (ifv) a, blocks the influx of h + ion into the virus and prevents the virus from uncoating [ ] . zidovudine, a nucleoside analog reverse transcriptase inhibitor for the treatment of human immunodeficiency virus (hiv) infection, interferes with, through its azide moiety, the formation of phosphodiester linkages essential for viral dna amplification [ ] . however, the narrow spectrum of the mechanisms of action would lead to continuous drug-resistance [ ] , a phenomenon more prevalent in immunocompromised hosts, not to mention the side effects. therefore, discovery of new therapeutic alternatives with multiple viral targets to avoid resistance comes to the forefront. plant extracts play an important role in the folk medicine as therapeutic substitutes to relieve suffering. hitherto, natural products are still major resources for discovery of new therapeutic agents. plant derived-essential oils (eos) are complex volatiles composed of miscellaneous phytochemicals, such as monoterpenes, sesquiterpenes and phenylpropanoids, etc., the structures of which are frequently reviewed elsewhere [ , ] . essential oils as antibacterials, antifungals, antioxidants, etc., have been extensively investigated in numerous studies for decades [ ] [ ] [ ] . virucidal effects of eos extracted from numerous aromatic and herbal plants are also well documented on a variety of viruses, such as ifv, hsv, hiv, yellow fever virus, and avian influenza, etc. [ ] [ ] [ ] [ ] . in a recent study, cagno et al. [ ] reported that the eo from salvia desoleana atzei & v. picci significantly suppressed the acyclovir resistant hsv- strains with a % effective concentration value (ic ) of . µg/ml, far less than that of acyclovir ( . µg/ml). in another study, eos from five plant species (zataria multiflora, artemisia kermanensis, eucalyptus caesia, satureja hortensis, rosmarinus officinalis) also exhibited better anti-hsv activities than acyclovir [ ] . these results suggest that eos can be potential therapeutic agents for the treatment of virus infection as well as prototypes for new antiviral drug selection. although a number of studies investigated the antiviral activities of plant-derived eos in the past decades, they are fairly extensive and not interconnected to form an overall trend. the antimicrobial, antifungal, antioxidant and anti-inflammatory effects are extensively reviewed at times [ ] [ ] [ ] , while the up-to-date antiviral effects are seldom generalized. one related book chapter review published~ years ago summarized the inhibitory potency of common eos and eo-derived components and the overall mechanisms of action [ ] . however, the antiviral effects of eos relative to their components and commercial drugs, in particular, are inadequately reviewed and advances in eos as antivirals are not updated. another two reviews discussing biological activities of eos (antimicrobial, antifungal and antiviral activities) only allow limited space for antivirals [ , ] . therefore, in this review, we focus on in vitro antiviral studies published within years, with the purpose to provide up-to-date information on the antiviral properties of eos. animal-borne viruses are covered herein while phytoviruses are out the scope of the article. viruses attach, penetrate, and enter the host cell, where genetic substances are replicated, followed by the creation and release of new virions [ ] . adverse action on related targets involved in the infection phase will inhibit the viral infectivity ( figure ). mechanisms of action of eos (including their components) against viruses are usually determined via the manipulation of time-of-addition assays. cultured cells are pretreated with the eos for h prior to virus infection (pre-viral infection). a negative result indicates that eos do not affect viral attachment by blocking host cell receptors. alternatively, viruses are pretreated with eos for h followed by incubation simultaneously with host cells (simultaneous viral infection). a positive result indicates that eos interfere with free virions by modifying the virus envelope structure or masking the viral proteins, which are necessary for viral adsorption and entry into the host cells. otherwise, eos are added to the infected cells at different time intervals of the viral infection lifecycle (from penetration to progeny production) (post viral infection). in this case, the stage of viral infection cycle at which eos act against viruses can be accessed. so far, the time-of-addition assay is the most widely used procedure to investigate the overall intercellular and intracellular inhibitory properties of eos. collectively, eos and their components majorly act on free viruses directly (intercellular mode of action). multiple modes of action also exist, which may be eo-dependent. relying on one assay only nonspecific information on mechanisms of action is derived. for example, the time-of-addition experiment cannot tell whether the eos influence viral adsorption by destroying or masking the virus. fortunately, transmission electron microscopy (tem) imaging was used to visualize structural changes of the virus. with the aid of tem, gilling et al. [ ] found that murine norovirus (a non-enveloped virus) exposed to % oregano eo expanded in size from - nm to - nm in diameter but appeared intact in morphology. meanwhile, . % carvacrol treated relying on one assay only nonspecific information on mechanisms of action is derived. for example, the time-of-addition experiment cannot tell whether the eos influence viral adsorption by destroying or masking the virus. fortunately, transmission electron microscopy (tem) imaging was used to visualize structural changes of the virus. with the aid of tem, gilling et al. [ ] found that murine norovirus (a non-enveloped virus) exposed to % oregano eo expanded in size from - nm to - nm in diameter but appeared intact in morphology. meanwhile, . % carvacrol treated murine norovirus expanded from normal to~ nm in diameter, resulting in capsid disintegration. the capsid in the non-enveloped virus protects the viral rna from disintegration and triggers infection by adsorption to host cells [ ] . nevertheless, murine norovirus whose capsid was partially degraded may still be infectious since the rnase i protection assay revealed that capsid degradation did not lead to noticeable viral rna reduction. the results implied that the carvacrol interfered with virus adsorption to host cells via binding to or masking the capsid but not via structural damage to the virus. hemagglutinin (ha), an important membrane protein of the ifv, allows the virus to enter and exit the host cell [ ] . ha can cause the agglutination of red blood cells, so hemagglutination inhibition assay is usually used to test the effect of eos on viral adsorption to host cells. absence of agglutination indicates inhibition of ha activity. neuraminidase (na) is another important surface protein for initial ifv infection, which is also a target of the synthetic drugs, zanamivir and oseltamivir. summing up, eos tend to act on ha more than on na, which is dependent on types of the eo or compound. as an example, cedar leaf eo was found to strongly inhibit ha of ifv, while thujone and α-pinene, two major components of the eo, did not [ ] , for which specific underlying mechanisms are unclear. tat (trans-activator of transcription) is an essential protein for hiv transcription. tat interacts with tar (trans-activation-responsive region) rna, which is required for hiv- replication. therefore, tat/tar-rna complex could be a target of hiv- inhibitors. feriotto et al. [ ] examined the eo-treated tat/tar-rna complex using gel electrophoresis and found that eos of thymus vulgaris, cymbopogon citratus, and rosmarinus officinalis interacted directly with tat protein and destabilized tat/tar-rna complex. in an in silico study, pajaro-castro et al. [ ] calculated the affinity scores of eo-derived components to dengue virus proteins and then identified the interactions between the highly scored components and the viral proteins. the results showed that most of the components inhibited the dengue virus via binding to the nonpolar domain of the proteins through hydrophobic interactions. in silico studies conducted by sharma and kaur [ ] showed that , -cineole from eucalyptus eo effectively bound to covid- proteinase via hydrophobic interactions, hydrogen bond and ionic interactions. isothymol from the eo of ammoides verticillata (desf.) briq. was reported to be a good inhibitor against angiotensin converting enzyme , a receptor of covid- , via pi-h bonding [ ] . however, whether these interactions apply to cases with other viruses and eos involved and whether the polarity of the eo components affects the antiviral efficacy remain to be elucidated. during the infection process, the ifv uncoating step needs an acidic endosomal and lysosomal environment [ ] . an investigation of cellular endosomal/lysosomal ph alteration after eo application can track the effect of eos on early stages of the viral replication lifecycle. for example, garozzo et al. [ ] reported that tea tree eo and its major component terpinen- -ol inhibited viral replication by way of interrupting the acidification of intralysosomal compartment which is essential for virus uncoating. it is known that viral infection induces intracellular glutathione depletion in accompany with the redox change [ ] . eo-derived components, piperitenone oxide for instance, could inhibit the late stage of hsv- lifecycle by targeting on redox signaling pathway [ ] . also, eos may target genome related sites as revealed by genetic approaches [ ] . towards a certain type of virus, mechanisms of action may be eo dependent [ ] , possibly owing to the constitutional variations of the eos. summing up, the direct interaction of eos with free viruses tends to be the most common mode of action. antiviral activities are generally evaluated in vitro via cytopathic effect reduction assay, plaque reduction assay and viral yield reduction assay [ ] . generally, all antivirals inhibit the viruses in a dose-dependent manner. to ensure that the eos at the assayed concentrations do not exert toxicity on the host cells, we must test the cytotoxicity of eos, which is described in terms of cc ( % cytotoxic concentration), corresponding to the eo concentration that reduces the cell viability by %. viral activity is evaluated by the value of ic ( % infection concentration), i.e., the eo concentration required to reduce viral infection by %. the antiviral selectivity index (si), a measure of the therapeutic suitability of the eo, is calculated as the ratio of cc to ic [ ] . to define anti-infective potential in natural products, ic values should be below a threshold of µg/ml for mixtures and µm for pure compounds [ ] . an si value is > is deemed acceptable [ ] . the antiviral properties of eos from different aromatic plants and eo-derived components on different virus are summarized in tables and . human herpes viruses (e.g., hsv- and hsv- ), enveloped dna viruses, have received the most attention in the last decade. essential oils from star anise, australian tea tree, oregano, eucalyptus caesia, to name a few, have been demonstrated to exhibit high anti-hsv- activities in vitro (table ). table indicates that star anise eo is the most potent with an ic value of µg/ml and a si value of [ ] , far away from the recommended cutoff (ic < µg/ml; si > ). moreover, mentha suaveolens and australian tea tree are also potential efficient antivirals [ ] . antiviral screening studies using eos from umbelliferae, labiatae, myrtaceae, lamiaceae plants indicated that hsv- was susceptible to a wide range of aromatic plants [ , ] . with regard to the antiviral efficacy of eo-derived components, β-caryophyllene stands out with ic and si values of . µg/ml (equal to . µm) and (table ) , respectively. this indicates that β-caryophyllene-containing plants, such as black pepper, cannabis, cinnamon, oregano and cloves, may serve as potential sources for selection of hsv antivirals [ ] . in a study, a wide range of common components in eos have been screened for their anti-herpes activities and all exhibited high antiviral activities at the concentration range of . - . µg/ml with the exception of l-bornyl acetate and d-limonene [ ] . elsewhere, , -cinole [ ] , eugenol [ ] , and р-cymene [ ] were pointed out in a few studies to be inappropriate for the treatment of hsv- due to inefficacy (ic > µm) or high cytotoxicity (si < ). influenza viruses are enveloped rna viruses. essential oils and their constituents were frequently examined in vitro and reported to exhibit high therapeutic potential in a dose-dependent manner. choi [ ] screened the anti-influenza a/ws/ virus activities of eos and found that, among them, marjoram, clary sage, and anise oils exhibited higher efficacy (ic < µg/ml) than oseltamivir. elsewhere, eos from thymus vulgaris, cinnamomum zeylanicum, citrus bergamia, etc., were also reported to show high antiviral efficacy [ ] (table ) . as for eo-derived components, germacrone is among the most effective ones against ifv with a si value larger than , which may be due to its broad-spectrum of mechanisms of action by inhibiting multiple steps of viral replication [ ] . also, germacrone was shown to effectively inhibit multiple strains of feline caliciviruses, non-enveloped rna viruses [ ] . these results suggested that germacrone, a major component from rhizoma curcuma, a traditional chinese herb, could be used as a promising broad-spectrum therapeutic agent or a reference for novel antiviral drug development. interestingly, some components such as , -cinole and eugenol, which are ineffective against hsv- , was found to exhibit high anti-influenza activities [ , ] . additionally, other oxygen-bearing components such as β-santalol [ ] and terpinen- -ol [ ] were also reported to be major bioactive components against ifv. this contrasting result implies that the anti-hsv and anti-influenza effects of some components may be complementary. there seems to be a trend that non-oxygenated terpene hydrocarbons are more effective to hsv and oxygenated terpenes to ifv, a hypothesis which needs to be tested. antiviral studies in the past years focused on enveloped viruses, while non-enveloped viruses received relatively less attention. this is not surprising since a large portion of eos were reported to exert high antiviral activity prior to host cell infection via interaction with the envelope, while non-enveloped viruses have fewer active sites that the eos are able to target. the eo from osmunda regalis, a tunisian fern, demonstrated its superior anti-coxsackie activity with a ic value of . µg/ml and si value . [ ] , far more beyond the threshold. moreover, eucalyptus bicostata and dysphania ambrosioides eos also exhibited high anti-coxsackie activities [ , ] . the oregano eo and its primary component carvacrol were able to strongly inhibit human rotavirus and murine norovirus [ , ] . these studies indicated potential antiviral activities of eos against non-enveloped viruses. unfortunately, information was not available regarding detailed mechanisms of action and the bioactive compounds in eo mixture responsible for the antiviral efficacy, further hindering the exploration of their antiviral value. cymbopogon nardus eo was reported to inhibit hiv- reverse transcriptase activity with a ic value of . mg/ml (equal to µg/ml) and the author postulated that (s)-β-citronellol may be one of the bioactive component [ ] . thymus vulgaris, cymbopogon citratus, and rosmarinus officinalis eos were found to effectively inhibit hiv- tat/tar-rna interaction with an ic ranging from . to . µg/ml [ ] . however, the si values were all below the cutoff value of . nevertheless, these findings provide alternative choices for development of hiv antivirals. elsewhere, β-caryophyllene effectively inactivated dengue- virus targeting at intercellular and intracellular sites with ic and si value of . µm and . [ ] , respectively. more recently, eo-derived components, such as , -cineole [ ] and isothymol [ ] , were suggested to be potential inhibitors of covid- . antiviral effects of eos and their components were also observed on bovine viral diarrhoea virus [ ] , respiratory syncytial virus [ ] , yellow fever virus [ ] , caprine alphaherpesvirus [ ] , and zika virus [ ] , etc. the antiviral potential of eos and their components on non-enveloped viruses is worthy of further exploration. one thing worth noting is that, although the aromatic compound carvacrol showed a broad-spectrum antiviral effect (table ) , caution must be taken before application since carvacrol exhibits a high cytotoxicity, as reflected by the relatively low si values. venturi et al. [ ] found that exposure of glechon spathulata and glechon marifolia eos to light (for d) compromised their anti-hsv- efficacy, with a concomitant remarkable decrease in terpenes, especially non-oxygenated monoterpene hydrocarbons, which were postulated to be the major active antivirals. similarly, a bioassay-guided fractionation of the eo from salvia desoleana suggested that it is the terpene hydrocarbons (fraction ) other than the oxygenated monoterpenes (fraction ), with germacrene d ( %) and β-caryophyllene ( . %), the major component of the former fraction, and linalyl acetate ( . %), α-terpinyl acetate ( . %), , -cineole ( . %), and linalool ( . %), the later, accounting for the anti-hsv- activity [ ] . further antiviral test with individual linalyl acetate, α-terpinyl acetate, , -cineole, and linalool demonstrated that these oxygenated-monoterpenes had no antiviral effect on hsv- at all, while the non-oxygenated fraction (fraction ) showed higher antiviral efficacy (ic . µg/ml for fraction ) than the eo mixture (ic . µg/ml). elsewhere, astani et al. [ ] compared the anti-hsv- activities of six eo-derived phenylpropanoids and sesquiterpene and found that addition of an epoxide or hydroxyl function into the sesquiterpene hydrocarbons incurred decrease in their antiviral activity. these results suggest that increasing polarity (addition of oxygen) likely decreased the anti-hsv activity. on the contrary, lai et al. [ ] investigated the anti-hsv- effect of thymol-related monoterpenoids and found that the anti-hsv- effect decreased as the polarity of the substituents declines in the order: -qh > -nh > -ch > -h. the inconsistence remains unclear. ralambondrainy et al. [ ] reported that monoterpene-rich cymbopogon citratus and pelargonium graveolens eos exhibited higher antiviral effect on the ross river virus, while the sesquiterpene-rich vetiveria zizanioides eo showed an insignificant effect. however, it seems arbitrary for the author to draw an inference that the antiviral competency was ascribed to the monoterpenes without further experimental proofs. pajaro-castro et al. [ ] calculated and compared the binding affinities of a variety of eo-derived components to structural proteins of dengue viruses, including monoterpenes and sesquiterpenes, with or without oxygen-bearing functional groups, and found that the sequiterpenes hydrocarbons and some of their oxygenated counterparts, such as α-copaene, germacrene d, β-caryophyllene, and caryophyllene oxide, exhibited the highest affinity. according to pajaro-castro et al. [ ] , the eo components bound to dengue virus proteins majorly via hydrophobic force, from which it is extrapolated that nonpolar terpenes tend to show great interaction with viral proteins. here, we hypothesize that the binding force of the components to viral surface likely influences the anti-hsv effectiveness of the eo-derived components and the polarity of the components is one influencing factor of the binding affinity. however, limited information is available concerning the effect of polarity (or addition of oxygen) on antiviral efficacy. germacrone and β-caryophyllene, two characteristic components as we stated above, showed a relatively broad spectrum of antiviral activities. the antiviral capacities of their oxidized form, i.e., germacrene and caryophyllene oxide, however, are almost not addressed, making the evaluation of the roles of oxygen addition to the precursor (non-oxygenated terpene hydrocarbons) tough. therefore, extensive and systematic research with diverse viruses and eo-components needs to be attempted to make a relatively confirmatory inference. it is generally accepted that the eo chemistry determines its bioactivities. still, there are conflicts regarding the role that individual component plays in the overall eo antiviral activity. eucalyptus globulus and salvia officinalis both contain the major component , -cineole, however, the former was reported to have strong anti-h n activity (ic < . µg/ml) while the later was not [ ] , suggesting that other minor components may be more bioactive than the primary component. eugenol, a principle component of cinnamomum zeylanicum eo, was found to be as potent as the eo in treatment of h n [ ] , indicating that the antiviral efficacy of the eo could be ascribed to its principle component. pilau et al. [ ] found that mexican oregano eo was more efficient than its principle component, carvacrol, against hsv- , bovine viral diarrhoea virus, and respiratory syncytial virus, while the carvacrol was able to efficiently inhibit rotavirus on which the mexican oregano eo had no inhibitory effect at all at the tested concentration ( - µg/ml). these results suggest that individual terpene in eo may not contribute equally to the antiviral efficacy of the eo mixture. therefore, the antiviral efficacy of individual compounds relative to the eo mixture should be case dependent. indeed, in some cases, the eo mixture may provide higher si and a lower toxicity than their isolated single component [ , ] , making the eo blend stand out. but this does mean that the eo used as mixtures is always superior and preferable to its single isolate. there exist bioactive components, either minor or primary, that are responsible for eo bioactivity. from a new drug discovery point of view, isolation and in-detail studies of individual eo components that are far more bioactive than the eo mixture deserve our more attention. essential oils may be more potent and versatile than commercial drugs. for example, carvacrol and oregano eos were able to efficiently inhibit acyclovir-resistant hsv- [ ] . salvia desoleana eo efficiently suppressed acyclovir-sensitive and acyclovir-resistant hsv- strains. also, the author demonstrated, via chromatographic fractionation of the eo, that the primary isolate, germacrene d, likely accounted for the overall antiviral effect [ ] . similarly, liao et al. [ ] reported that germacrone showed more inhibitory efficacy than ribavirin against multiple ifv strains (including amantadine resistant strains) since it interferes with viral attachment via direct interaction with either the viruses or host cells as well as inhibits early phase of viral lifecycle by impairing viral protein expression and rna transcription. this suggests that the eo-derived components may avoid targets of m ion channel inhibitors (amantadine and rimantadine) and neuraminidase inhibitors (zanamivir and oseltamivir) or exhibit multiple mechanisms of action to override the drug-resistance. it is believed that the compositional complexity of eos confers co-occurrence of multiple bioactive components and synergism, which tends to render the antiviral competency of eos more diversified, in contrast to that of the synthetic drugs, which is virus specific. current studies demonstrated synergistic or additive activities when the commercial drugs are used in combination with eos. pourghanbari et al. [ ] found that the combination of oseltamivir and melissa officinalis eo enhanced the effect of oseltamivir on avian influenza a virus (h n ). in this study, . µg/ml of oseltamivir reduced the virus genome copy number to , /µl while the blend of . µg/ml of oseltamivir and . mg/ml of the eo reduced the number to near zero. additionally, mentha suaveolens eo and its principle component, piperitenone oxide, also exhibited synergistic activities against hsv- in combination with acyclovir [ ] . in the study, the number of viral titres in infected host cells was reduced by~ % when acyclovir ( . µg/ml) was administrated in combination with piperitenone oxide ( µg/ml) than when acyclovir was used alone. moreover, germacrone in combination with oseltamivir was reported to exhibit additive anti-influenza viral effect both in vitro and in vivo [ ] , namely, the antiviral capacity of the combination, in terms of inhibition concentration index [ ] or infected mice survival rate, was a sum of that of individual contributions. collectively, the synergistic activity of a combination of drugs and eos could be applied in future therapeutic treatment of infectious diseases due to such advantages as higher efficacy, broad-spectrum activity, lower cost, and less drug resistance. the existing references in the past ten years are rather limited and most of them investigated in vitro the antiviral effect of eos from various families of plants and eo-derived components. many studies showed the direct action against cell-free virions, the broad-spectrum antiviral activities and the antiviral versatility of eos. the antiviral potency was tested in clinical studies [ , ] and some achieved pleasant results. as an example, myrtle vaginal suppository containing . % myrtle eo was effective in patients with hpv infection with an hpv clearance rate of . % compared with that of placebo ( %) [ ] . taken together, eos hold promise as candidates for prophylactic and therapeutic treatment of virus-induced diseases in future. so far, mechanisms of action of the antiviral activity of eos are not yet fully illustrated, which is not conductive to a successful application of eos. currently, in most cases, investigators can only tell at which stage of viral infection that the eos action, while the specific sites and interaction mode involved in each step, for instance, are inadequately addressed. therefore, in-detail and in-depth investigation of mechanisms of action is the foremost thing in future. to construct a whole picture of modes of action, multiple experimental assays should be performed simultaneously. apart from tem imaging, cell-binding experiment, rnase i protection experiment, and other biochemical approaches, strategies such as in silico computational design, should be developed, if applicable. additionally, to our knowledge, the eo chemistry determines the bioactivity of the eos, efforts thus need to be made to further reveal the relationship between eo chemistry and the corresponding bioactivity. as discussed in the previous section, the antiviral effectiveness of eos can be contributed unequally to the active components, either minor or principle ones, and underlying synergism. in this case, fractionation of eo blend, identification of the bioactive individuals and evaluation of their antiviral effectiveness are fairly essential. existing studies evaluated the antiviral effect of commercially obtained eo-derived compounds, which may not be fully consistent with their fractionated counterparts from eos. the effects of chemical properties of stereoisomerism and polarity of the components on their antiviral effectiveness need to be figured out. the preparative high-performance chromatography in terms of thin-layer chromatography, gas chromatography, and liquid chromatography, as well as the state-of-the-art equipment for structural elucidation facilitates the fractionation of eo components and evaluation of their bioactivities. as an example, preparative chromatography-based fractionation combined with bioassay-guided test can be a feasible way for rapid screening and isolation of the bioactive individuals, which is essential for new antiviral drug discovery and development. last but not least, references regarding the antiviral effect of eos were not published in a large quantity in the past decade and the focus of the virus type is enveloped ones, especially hsv- and ifv. a few studies have already evidenced the great antiviral potency of some eos and their components on certain non-enveloped viruses. however, the mechanisms of action at the molecular level are inadequately elucidated and plant sources investigated are relatively limited. therefore, research in this regard deserves our further attention. antiviral and antifungal activity of essential oils: mechanisms and applications in vitro anti-herpes simplex virus- activity of salvia desoleana atzei & v. picci essential oil influenza a virus m ion channel activity is essential for efficient replication in tissue culture pharmgkb summary: zidovudine pathway influenza neuraminidase inhibitors: antiviral action and mechanisms of resistance. influenza other respir. viruses essential oils for the treatment of herpes simplex virus infections active essential oils and their components in use against neglected diseases and arboviruses the antibacterial, antifungal, and antioxidant activities of essential oil from different aromatic plants antioxidant, and immunomodulatory properties of essential oils: a systematic review chemical composition and antiviral activity of essential oils from citrus reshni hort. ex tanaka (cleopatra mandarin) cultivated in egypt chemical composition, antiviral against avian influenza (h n ) virus and antimicrobial activities of the essential oils of the leaves and fruits of fortunella margarita, lour. swingle, growing in egypt potential interaction of components from essential oils with dengue virus proteins susceptibility of herpes simplex virus type to monoterpenes thymol, carvacrol, p-cymene and essential oils of sinapis arvensis l., lallemantia royleana benth. and pulicaria vulgaris gaertn antiviral activity of some plant oils against herpes simplex virus type in vero cell culture antifungal investigations on plant essential oils. a review a status review on the medicinal properties of essential oils antioxidant and anti-inflammatory activities of essential oils: a short review antiviral effects of plant-derived essential oils and pure oil components essential oils of aromatic plants with antibacterial, antifungal, antiviral, and cytotoxic properties-an overview antiviral efficacy and mechanisms of action of oregano essential oil and its primary component carvacrol against murine norovirus capsid and infectivity in virus detection anti-influenza virus activity of essential oils and vapors the activity of cedar leaf oil vapor against respiratory viruses: practical applications chemical composition of essential oils from thymus vulgaris, cymbopogon citratus, and rosmarinus officinalis, and their effects on the hiv- tat protein function eucalyptol ( , cineole) from eucalyptus essential oil a potential inhibitor of covid corona virus infection by molecular docking studies in silico study the inhibition of angiotensin converting enzyme receptor of covid- by ammoides verticillata components harvested from western algeria requirement for vacuolar proton-atpase activity during entry of influenza virus into cells activity of melaleuca alternifolia (tea tree) oil on influenza virus a/pr/ : study on the mechanism of action in vitro inhibition of herpes simplex virus type replication by mentha suaveolens essential oil and its main component piperitenone oxide human immunodeficiency virus type (hiv- ) reverse transcriptase inhibitory effect of cymbopogon nardus essential oil in vitro comparison of three common essential oils mosquito repellents as inhibitors of the ross river virus antiviral activity of the lippia graveolens (mexican oregano) essential oil and its main compound carvacrol against human and animal viruses anti-infective potential of natural products: how to develop a stronger in vitro 'proof-of-concept survey of acyclovir-resistant herpes simplex virus in the netherlands: prevalence and characterization antimicrobial and antiviral effects of essential oils from selected umbelliferae and labiatae plants and individual essential oil components comparative study on the antiviral activity of selected monoterpenes derived from essential oils screening for antiviral activities of isolated compounds from essential oils inhibition of hsv- infection by pure compounds from thymus capitatus extract in vitro study on the effect of anti-respiratory viruses of patchouli oil in vitro the essential oil of tunisian dysphania ambrosioides and its antimicrobial and antiviral properties cytotoxic and antiviral activities of the essential oils from tunisian fern, osmunda regalis chemical composition of eucalyptus species' essential oils and the evaluation of their antibacterial, antifungal and antiviral activities in vitro inhibition of the bovine viral diarrhoea virus by the essential oil of ocimum basilicum (basil) and monoterpenes comparative study on in vitro activities of citral, limonene and essential oils from lippia citriodora and l. alba on yellow fever virus ayapana triplinervis essential oil and its main component thymohydroquinone dimethyl ether inhibit zika virus at doses devoid of toxicity in zebrafish essential oil composition, antioxidant, cytotoxic and antiviral activities of teucrium pseudochamaepitys growing spontaneously in tunisia inhibition of herpes simplex virus type by thymol-related monoterpenoids antiviral activity of monoterpenes beta-pinene and limonene against herpes simplex virus in vitro vimalanathan anti-influenza virus activities of commercial oregano oils and their carriers in vitro anti-viral effect of β-santalol against influenza viral replication germacrone inhibits early stages of influenza virus infection chemical constituents of essential oils possessing anti-influenza a/ws/ virus activity in vitro antiviral effect of germacrone on feline calicivirus xu, p. , -cineol protect against influenza-virus-induced pneumonia in mice virucidal activity of ginger essential oil against caprine alphaherpesvirus- chemical analysis and in vitro antiviral and antifungal activities of essential oils from glechon spathulata and glechon marifolia antiviral activity of the oseltamivir and melissa officinalis l. essential oil against avian influenza a virus (h n ) synergy, antagonism, and what the chequerboard puts between them reporting effectiveness of an extract of three traditional cretan herbs on upper respiratory tract infection: results from a double-blind randomized controlled trial the efficacy of vaginal suppository based on myrtle in patients with cervicovaginal human papillomavirus infection: a randomized, double-blind, placebo trial key: cord- -y vg frb authors: montané, xavier; kowalczyk, oliwia; reig-vano, belen; bajek, anna; roszkowski, krzysztof; tomczyk, remigiusz; pawliszak, wojciech; giamberini, marta; mocek-płóciniak, agnieszka; tylkowski, bartosz title: current perspectives of the applications of polyphenols and flavonoids in cancer therapy date: - - journal: molecules doi: . /molecules sha: doc_id: cord_uid: y vg frb the development of anticancer therapies that involve natural drugs has undergone exponential growth in recent years. among the natural compounds that produce beneficial effects on human health, polyphenols have shown potential therapeutic applications in cancer due to their protective functions in plants, their use as food additives, and their excellent antioxidant properties. the possibility of combining conventional drugs—which are usually more aggressive than natural compounds—with polyphenols offers very valuable advantages such as the building of more efficient anticancer therapies with less side effects on human health. this review shows a wide range of trials in which polyphenolic compounds play a crucial role as anticancer medicines alone or in combination with other drugs at different stages of cancer: cancer initiation, promotion, and growth or progression. moreover, the future directions in applications of various polyphenols in cancer therapy are emphasized. the appearance of the severe acute respiratory syndrome coronavirus (sars-cov- ) in december last year and its very rapid spread around the world in early , known to cause covid- disease, has evidenced, among other things, the importance of investing in research to improve the people's quality of life or eradicate diseases that still do not have an effective treatment. as observed in figure , there has been an exponential increase of research and publications related to the possible use of polyphenolic compounds in cancer therapy [ ] . the fact that polyphenols can be extracted using simple and green techniques-such as ultrasound-assisted extraction, and that after being sterilized, polyphenols preserve most of their properties intact-will contribute to the study of these compounds as potential anticancer drugs [ , ] . as observed in figure , there has been an exponential increase of research and publications related to the possible use of polyphenolic compounds in cancer therapy [ ] . the fact that polyphenols can be extracted using simple and green techniques-such as ultrasound-assisted extraction, and that after being sterilized, polyphenols preserve most of their properties intact-will contribute to the study of these compounds as potential anticancer drugs [ , ] . stilbenes or stilbenoids are hydroxylated derivatives of stilbene with a c -c -c chemical structure. these kinds of compounds are produced in various plants such as strawberries, grapes, peanuts, and cannabis [ ] . furthermore, various trees synthesize stilbenes as secondary products of heartwood that can act as antimicrobial and antioxidative substances. stilbenes share most of their biosynthesis pathway with chalcones, which is a class of flavonoids. the most representative compound of the stilbene family that has many health benefits is resveratrol [ ] . resveratrol ( , , ′-trihydroxy-trans-stilbene) is a natural polyphenol of the stilbene family. resveratrol is produced by several plants (grapes, almonds, beans, blueberries, raspberries, mulberries, peanuts, etc.) in response to infections and injuries or as a defense against different kinds of pathogens attacks, such as fungi or bacteria [ ] . furthermore, red wine also contains significant amounts of resveratrol. in , jang et al. were the first researchers that reported the inhibition of skin cancer development in mice by using resveratrol [ ] . since then, many investigations have suggested that resveratrol is able to prevent cancer or delay its onset [ ] . in point of fact, studies demonstrated that resveratrol has in vitro effects against a large range of human tumors: breast, skin, ovary, stomach, prostate, colon, liver, pancreas, cervix, thyroid carcinoma cells, lymphoid, and myeloid cancer cells [ ] . it has been proven that resveratrol shows beneficial effects at different stages of cancer (initiation, promotion, and progression of cancer). for example, resveratrol protects dna from reactive oxygen species (ros) and traps hydroxyls, superoxides, and free radicals produced in cellsevents that are usually related to the initiation of tumors [ ] . in another study, yin et al. demonstrated that the application of resveratrol inhibits the promotion and progression of a lung cancer cells in nude mice. however, the authors mentioned that further studies should be performed in order to evaluate other parameters, such as the applied dose of resveratrol [ ] . besides, clinical trials on humans have been performed with the use of resveratrol, obtaining satisfactory results [ ] [ ] [ ] . stilbenes or stilbenoids are hydroxylated derivatives of stilbene with a c -c -c chemical structure. these kinds of compounds are produced in various plants such as strawberries, grapes, peanuts, and cannabis [ ] . furthermore, various trees synthesize stilbenes as secondary products of heartwood that can act as antimicrobial and antioxidative substances. stilbenes share most of their biosynthesis pathway with chalcones, which is a class of flavonoids. the most representative compound of the stilbene family that has many health benefits is resveratrol [ ] . resveratrol ( , , -trihydroxy-trans-stilbene) is a natural polyphenol of the stilbene family. resveratrol is produced by several plants (grapes, almonds, beans, blueberries, raspberries, mulberries, peanuts, etc.) in response to infections and injuries or as a defense against different kinds of pathogens attacks, such as fungi or bacteria [ ] . furthermore, red wine also contains significant amounts of resveratrol. in , jang et al. were the first researchers that reported the inhibition of skin cancer development in mice by using resveratrol [ ] . since then, many investigations have suggested that resveratrol is able to prevent cancer or delay its onset [ ] . in point of fact, studies demonstrated that resveratrol has in vitro effects against a large range of human tumors: breast, skin, ovary, stomach, prostate, colon, liver, pancreas, cervix, thyroid carcinoma cells, lymphoid, and myeloid cancer cells [ ] . it has been proven that resveratrol shows beneficial effects at different stages of cancer (initiation, promotion, and progression of cancer). for example, resveratrol protects dna from reactive oxygen species (ros) and traps hydroxyls, superoxides, and free radicals produced in cells-events that are usually related to the initiation of tumors [ ] . in another study, yin et al. demonstrated that the application of resveratrol inhibits the promotion and progression of a lung cancer cells in nude mice. however, the authors mentioned that further studies should be performed in order to evaluate other parameters, such as the applied dose of resveratrol [ ] . besides, clinical trials on humans have been performed with the use of resveratrol, obtaining satisfactory results [ ] [ ] [ ] . curcuminoids are natural polyphenols that contain two phenol units joined through a linear diarylheptanoid. the presence of curcuminoids gives a yellow color to plants that contain these kinds of natural structures. the phenolic rings of curcuminoids are chemically modified with other chemical groups with the aim of overcoming some drawbacks of natural curcuminoids in clinical applications such as their poor solubility, low absorption, and bioavailability [ ] . among the curcuminoids, curcumin is one of the most known and studied structures with a high potential as medicine to treat different cancers, apart from also being useful in treating other types of diseases. nonetheless, the poor solubility of curcumin in water of acidic and physiological ph requires the use of diverse alternatives to avoid losing the effectiveness of curcumin as a medicine, such as the synthesis of other curcumin derivatives or the combination of curcuminoids with surfactants or co-surfactants. curcumin is a natural compound and the principal curcuminoid of turmeric plants, which is responsible for turmeric's yellow color [ ] . in addition to its applications in medicine, the use of curcumin has reached other fields. in the food industry, it has been used as a dietary supplement (it is sold as herbal supplement) or a food additive. additionally, it is used in cosmetics and other products. curcumin is commonly used in cancer therapies of different types of cancer: lung, cervix, prostate, breast, bone, and liver [ ] . nevertheless, the administration of free curcumin presents some drawbacks: poor solubility in water, instability in aqueous conditions, low bioavailability, and poor cellular uptake. to overcome these problems, two different solutions were attempted: - the synthesis of curcumin derivatives [ ] , and - the encapsulation of curcumin in different nanostructures ranging from liposomes to natural biopolymeric nanoparticles [ , ] . one of the curcumin derivatives used in breast and renal cancer therapies is dimethoxy curcumin. chen et al. recently proved that this curcumin derivative can be effective in the therapy of colon cancer cells due to causing the reduction of survivin expression and the enhancement of e-cadherin, a cell adhesion molecule, whose loss contributes to the formation of epithelial types of cancers such as carcinomas [ ] . recently, various research groups have reported that the combination of both curcumin and resveratrol can reduce the incidence of lung and prostate cancer [ , ] . lignans are diphenolic compounds found in a wide variety of plants including broccoli, beans, soybeans, rye, sesame seeds, pumpkin seeds, flax seeds, and some berries in very small amounts (µg of lignans per g of dry product) [ ] . their structure consists of two c -c units linked by β,β' bonds. lignans are one of the two main groups of phytoestrogens, which are well known for their good antioxidant properties. in fact, some antioxidant phytochemical compounds could be used as anticancer drugs as they are mimicking the functions of human hormones. some studies on rats showed that lignans prevent the growth of breast and prostate tumors [ , ] . numerous lignans could be considered as possible anticancer medicines due to their large pharmacologically valuable properties. among all of them, arctigenin, magnolol, and honokiol are the main lignans investigated in medicine. nonetheless, etoposide is a commercial lignin belonging to the podophilotoxin subfamily that is used in the treatment of different types of cancer such as lung cancer and breast cancer [ , ] . however, etoposide chemotherapy presents several side effects: low blood cell counts, vomiting, diarrhea, fever, loss of appetite, and alopecia. certain plants belonging to the family known as compositae produce arctigenin, especially the seeds of greater burdock (arctium lappa). some studies revealed that arctigenin inhibits the growth of various cancer cells: stomach, lung, liver, and colon, as well as leukocytes [ ] . at the same time, the addition of arctigenin intensifies the activity of caspase- , which is a protein that plays a crucial role in the death of carcinogenic cells. as a matter of fact, huang et al. demonstrated that the treatment of ovcar and skov ovarian cancers with arctigenin causes the apoptosis of cancer cells in vitro [ ] . one of the most used conventional anticancer drugs is doxorubicin, which is a medicine that belongs to the anthracycline family applied in the treatment of, among other cancers, bladder, stomach, ovaries, lung and thyroid cancers. however, doxorubicin exhibits side effects among which the most frequent are severe nauseas, vomiting, and alopecia [ ] . studies were conducted by lee et al. on adding natural products such as arctigenin to doxorubicin and determining the efficiency of both drugs in improving breast cancer treatment and reducing the side effects provoked by doxorubicin [ ] . the work concludes that the combination of arctigenin and doxorubicin induced the apoptosis of mda-mb- human breast cancer cells in vitro. the addition of arctigenin ameliorates the cellular uptake of doxorubicin, which causes the death of carcinogenic cells. another lignan that was tested in some studies on cancer therapy is magnolol. as its name indicates, magnolol is an isomer of honokiol found in magnolia bark [ ] . since ancient times, extracts from the bark of magnolia have been used in traditional chinese, korean, and japanese medicine. in the last decades, the research on the use of natural products in various cancer treatments has been focused on attempts of understanding mechanisms that induce the antitumor agents' response in the tumor cells [ ] . this year, su and co-workers elucidated the mechanism that reduces the endogen activity of nuclear factor kappa-light-chain-enhancer of activated b cells (nf-κb), which is a protein complex that controls dna transcription and cell survival. therefore, the cells that do not have regulated nf-κb can contribute to the onset and growth of various types of cancers. moreover, magnolol used in the treatment of colorectal cancer reduces the phosphorylation of protein kinase c delta type (pkcδ) and nf-κb, which are two proteins that are involved in tumour progression in vitro and in vivo [ ] . following the methodology used with other drugs, magnolol was co-encapsulated with trastuzumab, an anticancer drug commonly used in stomach or throat cancer therapies, and gold nanoparticles, building a nanocarrier cluster. the synthesized nanocarriers induced a specific photothermal near-ir response combined with targeted anticancer activity resulting in an improvement of magnolol cytotoxicity to breast cancer cells [ ] . as mentioned before, honokiol (also known as houpa or hnk) is a lignan isolated from the bark, seed cones, and leaves of trees belonging to the genus of magnolias, which includes around species. honokiol, which has been used in traditional eastern herbal medicines as an analgesic and together with magnolol, obovatol, and -o-methylhonokiol in the treatment of anxiety and mood disorders, has a spicy odor [ ] . honokiol is most frequently taken orally. in nature, honokiol and magnolol isomers are found together. usually, the separation and purification of both compounds had always been complexed, and it is commonly limited to hplc. in , amblard and co-workers developed a method in which the authors protect the near hydroxyl groups in magnolol to produce a magnolol acetonide that can be simply separated from honokiol via flash chromatography over silica [ ] . recent studies suggest that honokiol could be an effective agent in cancer treatment due to its physical properties-honokiol's ability to easily cross the blood-brain barrier and the bloodcerebrospinal fluid barrier-and its high bioavailability. many research studies have shown that honokiol can kill carcinogenic cells in melanoma, sarcoma, myeloma, and leukemia, as well as in bladder, lung, prostate, and colon cancers [ , ] . besides, honokiol enhances the apoptotic effects of some etoposides, such as doxorubicin. for instance, micelles with encapsulated doxorubicin and honokiol allow a controlled drug co-delivery that inhibits the progression of breast cancer tumors and reduces the doxorubicin side effects when compared with the micelles without honokiol [ ] . on the other hand, the effectiveness of honokiol in the fight with typically drug-resistant multiple myelomas and chronic b-cell leukemia has been proved by various authors [ , ] . ishitsuka and co-workers certified that honokiol presents the ability to kill drug-resistant multiple myeloma carcinogenic cells by varied mechanisms [ ] . another subgroup of polyphenols that can be found in several plants, especially in dried fruit, are phenolic acids. these compounds are characterized by containing a phenolic ring and an organic carboxylic acid function (c -c skeleton) [ ] . phenolic acids are divided in two classes: -derivatives of benzoic acid, and -derivatives of cinnamic acid. in general, derivatives of cinnamic acid are more common in plants than the derivatives of benzoic acid. despite that, some red fruit, onions, and black radish contain significant amounts of benzoic acid derivatives [ ] . to date, the phenolic acid that exhibited medicinal properties that turn it into a plausible candidate for cancer treatment is p-coumaric acid. p-coumaric acid p-coumaric acid (or -hydroxycinnamic acid) is an organic compound derived from cinnamic acid that can be found in a wide variety of edible plants (tomatoes, carrots, garlic, mushrooms, white beans, and others). moreover, p-coumaric acid found in pollen is a constituent of honey [ ] . additionally, p-coumaric can be synthesized from cinnamic acid or l-tyrosine by the action of -cinnamic acid hydroxylase (c h) or tyrosine ammonia lyase (tal) enzymes, respectively. during the last decade, few studies that evidenced the anticancer activity of p-coumaric acid in colon and gastric cancer cells have been published [ , ] . lately, jang et al. have shown that p-coumaric acid suppresses the growth of snu- gastric cancer cells [ ] . the most important group of polyphenols are flavonoids. the chemical structure of flavonoids is composed of carbon atoms comprising cycles of six carbon atoms linked by a -carbon chain (rings a and b, in figure ). the flavonoids family consists of over molecules that have been identified and isolated, but there are undoubtedly many more flavonoid structures to discover [ ] . honokiol can kill carcinogenic cells in melanoma, sarcoma, myeloma, and leukemia, as well as in bladder, lung, prostate, and colon cancers [ , ] . besides, honokiol enhances the apoptotic effects of some etoposides, such as doxorubicin. for instance, micelles with encapsulated doxorubicin and honokiol allow a controlled drug co-delivery that inhibits the progression of breast cancer tumors and reduces the doxorubicin side effects when compared with the micelles without honokiol [ ] . on the other hand, the effectiveness of honokiol in the fight with typically drug-resistant multiple myelomas and chronic b-cell leukemia has been proved by various authors [ , ] . ishitsuka and co-workers certified that honokiol presents the ability to kill drug-resistant multiple myeloma carcinogenic cells by varied mechanisms [ ] . another subgroup of polyphenols that can be found in several plants, especially in dried fruit, are phenolic acids. these compounds are characterized by containing a phenolic ring and an organic carboxylic acid function (c -c skeleton) [ ] . phenolic acids are divided in two classes: -derivatives of benzoic acid, and -derivatives of cinnamic acid. in general, derivatives of cinnamic acid are more common in plants than the derivatives of benzoic acid. despite that, some red fruit, onions, and black radish contain significant amounts of benzoic acid derivatives [ ] . to date, the phenolic acid that exhibited medicinal properties that turn it into a plausible candidate for cancer treatment is p-coumaric acid. p-coumaric acid p-coumaric acid (or -hydroxycinnamic acid) is an organic compound derived from cinnamic acid that can be found in a wide variety of edible plants (tomatoes, carrots, garlic, mushrooms, white beans, and others). moreover, p-coumaric acid found in pollen is a constituent of honey [ ] . additionally, p-coumaric can be synthesized from cinnamic acid or l-tyrosine by the action of -cinnamic acid hydroxylase (c h) or tyrosine ammonia lyase (tal) enzymes, respectively. during the last decade, few studies that evidenced the anticancer activity of p-coumaric acid in colon and gastric cancer cells have been published [ , ] . lately, jang et al. have shown that pcoumaric acid suppresses the growth of snu- gastric cancer cells [ ] . the most important group of polyphenols are flavonoids. the chemical structure of flavonoids is composed of carbon atoms comprising cycles of six carbon atoms linked by a -carbon chain (rings a and b, in figure ). the flavonoids family consists of over molecules that have been identified and isolated, but there are undoubtedly many more flavonoid structures to discover [ ] . flavonoids are found in abundance in colored vegetables (spinach) and fruit such as berries, blueberries, apples, grapes, oranges, strawberries, plums, and in some foods and beverages widely used in the human diet, including dark chocolate, nuts, red wine, tea, soy, and soy derivatives. flavonoids are found in abundance in colored vegetables (spinach) and fruit such as berries, blueberries, apples, grapes, oranges, strawberries, plums, and in some foods and beverages widely used in the human diet, including dark chocolate, nuts, red wine, tea, soy, and soy derivatives. flavonoids have a wide spectrum of functions in plants: -flavonoids attract pollinating insects through the color or smell that they give to the plant or its flowers, -filtration of uv light, -protection against herbivorous predators, -protection against fungi, -they are involved in the hormone auxin transport, -regulation of the cell cycle, -pigmented blue colors given by anthocyanins are responsible for the resistance of plants to the photooxidation of uv light from the sun, and - in carnivorous plants, they attract prey. usually, two criteria are used to classify flavonoids: -the chemical structure of the c heterocycle (if it is present), and -to which carbon of the c ring the b ring is attached (c and c in figure ). according to these two factors, seven groups of flavonoids can be distinguished: flavonols, flavones, flavanones, flavan- -ols, isoflavones, chalcones, and anthocyanidins ( figure ). the chemical structures of these groups are shown in figure . usually, two criteria are used to classify flavonoids: -the chemical structure of the c heterocycle (if it is present), and -to which carbon of the c ring the b ring is attached (c and c ′ in figure ). according to these two factors, seven groups of flavonoids can be distinguished: flavonols, flavones, flavanones, flavan- -ols, isoflavones, chalcones, and anthocyanidins ( figure ). the chemical structures of these groups are shown in figure . flavonols are a class of flavonoids based on the backbone -hydroxyflavone. there is a wide variety of flavonols, which depend on positions that can be hydroxylated ( figure ). many fruits (apples, peaches, oranges, blackberries, raspberries), vegetables (onions, broccoli, kale, brussels sprouts, cucumbers, lettuce, tomatoes, potatoes, spinach), leaves (aloe vera, rosemary, soybean, pinus sylvestris, holly, endive), seeds (grapes), and grains (several cereals including quinoa, buckwheat, barley, and oat) are rich sources of flavonols [ ] . flavonols are responsible for the color of flowers in some plants as well as protecting them from uv light and ros [ ] . furthermore, flavonols are bioactive polyphenols that are widely used due to their excellent antioxidant properties [ ] : -in medicine: antimicrobial, anti-inflammatory, antiaging, anticancer, or insecticidal agents. -in agriculture: as pesticides. kaempferol and quercetin are the main flavonols studied in medicine. nevertheless, other flavonols such as herbacetin, myricetin, and fisetin have also been investigated as anticancer drugs [ , ] . kaempferol is a flavonol that is found in plants, plant-derived foods, and traditional medicines, including in tea, kale, beans, spinach, and broccoli [ ] . once isolated, kaempferol is a yellow crystalline solid of poor solubility. one study reported by liu suggested that kaempferol intake contributes to approximately % of the total average intake of flavonols and flavones in a normal diet [ ] . during the last few years, numerous investigations provided new evidence of the anticancer mechanisms of kaempferol both in vitro and in vivo. discovering such mechanisms has enabled the analysis and understanding of kaempferol's role as an anticancer drug and afterwards may lead to an improvement of applied techniques and methods, such as the development of kaempferol-loaded targeted drug delivery systems [ ] . one of the cancers in which the effect of kaempferol has been studied the most is breast cancer [ ] . several research groups have proved the cytotoxicity of kaempferol against breast cancer cells both in vitro and in vivo: -by inhibiting the growth of cancer cells, flavonols are a class of flavonoids based on the backbone -hydroxyflavone. there is a wide variety of flavonols, which depend on positions that can be hydroxylated ( figure ). many fruits (apples, peaches, oranges, blackberries, raspberries), vegetables (onions, broccoli, kale, brussels sprouts, cucumbers, lettuce, tomatoes, potatoes, spinach), leaves (aloe vera, rosemary, soybean, pinus sylvestris, holly, endive), seeds (grapes), and grains (several cereals including quinoa, buckwheat, barley, and oat) are rich sources of flavonols [ ] . flavonols are responsible for the color of flowers in some plants as well as protecting them from uv light and ros [ ] . furthermore, flavonols are bioactive polyphenols that are widely used due to their excellent antioxidant properties [ ] : in medicine: antimicrobial, anti-inflammatory, antiaging, anticancer, or insecticidal agents. - in agriculture: as pesticides. kaempferol and quercetin are the main flavonols studied in medicine. nevertheless, other flavonols such as herbacetin, myricetin, and fisetin have also been investigated as anticancer drugs [ , ] . kaempferol is a flavonol that is found in plants, plant-derived foods, and traditional medicines, including in tea, kale, beans, spinach, and broccoli [ ] . once isolated, kaempferol is a yellow crystalline solid of poor solubility. one study reported by liu suggested that kaempferol intake contributes to approximately % of the total average intake of flavonols and flavones in a normal diet [ ] . during the last few years, numerous investigations provided new evidence of the anticancer mechanisms of kaempferol both in vitro and in vivo. discovering such mechanisms has enabled the analysis and understanding of kaempferol's role as an anticancer drug and afterwards may lead to an improvement of applied techniques and methods, such as the development of kaempferol-loaded targeted drug delivery systems [ ] . one of the cancers in which the effect of kaempferol has been studied the most is breast cancer [ ] . several research groups have proved the cytotoxicity of kaempferol against breast cancer cells both in vitro and in vivo: -by inhibiting the growth of cancer cells, -by stopping the progression and proliferation of cancer cells, and -by inducing cancer cells apoptosis. one of the latest investigations to clarify the mechanism of kaempferol as an anticancer drug against breast tumors was carried out by zhu et al. the authors mentioned that kaempferol induced apoptosis and dna damage in mda-mb- cancer cells by the upregulation of the phosphorylated form of the h a histone family member x (γh ax), caspase , caspase , and the protein serine/threonine kinase (p-atm) [ ] . da and co-workers tested kaempferol in prostate cancer cells [ ] . the authors concluded that the use of kaempferol against lncap prostate cancer cell lines led to cancer cells death and impeded cancer cell proliferation and invasion in a dose-dependent manner. quercetin is the most common flavonoid in human diet with an average daily consumption of - milligrams [ ] . quercetin is mainly found in red onions, kale, apples, grapes, broccoli, and tea. in red onions, quercetin represents around % of its dry weight. various in vitro and in vivo studies showed that quercetin is one of the most potent antioxidants of the flavonoid family [ ] , which makes it an ideal candidate for an anticancer drug. indeed, quercetin is the active ingredient of yang-yin-qing-fei-tang, which is a traditional chinese medicine. furthermore, quercetin exhibited cytotoxicity in various tumor cells, in breast, cervical, colon, liver, lung, gastric, prostate cancers, and in leukemia [ , ] . making use of the anticancer effects of quercetin, the most recent studies combined quercetin with other anticancer drugs with the aim of increasing the efficiency of cancer therapies. some examples are summarized below. one of the natural compounds that lately has been combined with quercetin in cancer therapy studies is curcumin. srivastavaa et al. showed that the mixture of quercetin and curcumin improved the inhibition of cancer cell proliferation by regulating the wnt/β-catenin signaling and promoting the carcinogenic cells death by distinct pathways [ ] . furthermore, sunoqrot and co-workers combined both curcumin and quercetin by preparing nanoparticles with encapsulated curcumin and a shell of quercetin covalently bonded with polyethylene glycol (peg) prepared in a one-pot procedure [ ] . once tested in vivo, these nanocarriers exhibited a controlled drug delivery of curcumin in physiological conditions, which makes it a potentially powerful tool in cancer therapy. it has also been observed that the addition of quercetin to docetaxel therapy in prostate cancer reduces the docetaxel resistance of carcinogenic cells. that increases the efficacy of cancer therapy resulting from an intensification of the apoptosis of cancer cells and the reduction of tumor proliferation and migration [ ] . flavones are a class of flavonoids with a chemical structure very similar to flavonols, from which they only differ in the non-hydroxyl substitution at the carbon -position of flavones ( figure ). flavones are basically found in herbs (parsley, thyme, chamomile, mint, chrysanthemum flowers) and red or purple plants and vegetables (apple skins, broccoli, cabbages, celery, onion leaves, carrots, and red peppers) [ ] . in plants, flavones usually act as defense mechanisms against diseases originated by pathogens. some of the flavones have been in use for many years. the most representative example is luteolin, which since ancient times has been used as yellow dye. apigenin has also been used to dye wool. moreover, wogonin is well known because it is one of the active ingredients of sho-saiko-to, which is a japanese herbal supplement [ ] . however, the interest in using this family of flavonoids in medicine has been growing because they demonstrate efficient antimicrobial, antioxidant, antifungal, anti-inflammatory, antimutagenic, and anticancer activity [ ] . inside the flavones family, the anticancer properties of apigenin and luteolin are widely investigated. apigenin, which is a yellow crystalline solid, is one of the flavones most commonly found in nature. many fruits and vegetables, such as parsley, celery, celeriac, carrot, oregano, and chamomile tea contain apigenin. in the particular case of chamomile tea, apigenin constitutes % of the total flavonoids content [ ] . for many centuries, apigenin has been widely used as a traditional medicine [ , ] . the excellent properties of this natural compound have prompted the study of its application as an anticancer drug [ , ] . in fact, various positive effects of apigenin administration, alone or in combination with other chemotherapeutic agents, in different types of cancer treatments were reported in the literature [ ] . the following aspects were mentioned: -inducing the death of cancer cell lines, -triggering both autophagy and apoptosis, -suppressing cancer cell migration and invasion, and -inducing the cancer cells cycle arrest. one of the recently carried out investigations mentions that apigenin promotes pancreatic cells death by increasing intracellular ros [ ] . in this work, montani et al. tried to understand the mechanism happening in cancer cells in which apigenin was applied. in fact, they suggested a biological mechanism occurring between heat shock protein (hsp ), a protein that stabilizes proteins involved in the growth of cancer cells, and tp gene mutations that reduce the cytotoxic effect of the chemotherapy with apigenin. the targeting of these molecules is an important anticancer strategy that has been extensively explored. on the other hand, liu et al. evaluated the synergistic effect in cancer therapy involving apigenin combined with metal ions [ ] . in this work, the authors examined the thermal stability of two flavones (apigenin and luteolin) when combined with ferrous or cupric ions, which negatively affects the anticancer activities of both flavones against human cervical cancer hela cells. luteolin is usually found in the leaves and bark of some plants. the major natural sources of luteolin are celery, thyme, dandelion, clover flower, ragweed pollen, chamomile, and perilla [ ] . due to its beneficial effects on the human body (antioxidative and anti-inflammatory properties, being a free radicals scavenger, promoting carbohydrate metabolism, and modulating the immune system), it is assumed that luteolin could perform an important role in cancer therapy [ , ] . some of the flavones have been in use for many years. the most representative example is luteolin, which since ancient times has been used as yellow dye. apigenin has also been used to dye wool. moreover, wogonin is well known because it is one of the active ingredients of sho-saiko-to, which is a japanese herbal supplement [ ] . however, the interest in using this family of flavonoids in medicine has been growing because they demonstrate efficient antimicrobial, antioxidant, antifungal, anti-inflammatory, antimutagenic, and anticancer activity [ ] . inside the flavones family, the anticancer properties of apigenin and luteolin are widely investigated. apigenin, which is a yellow crystalline solid, is one of the flavones most commonly found in nature. many fruits and vegetables, such as parsley, celery, celeriac, carrot, oregano, and chamomile tea contain apigenin. in the particular case of chamomile tea, apigenin constitutes % of the total flavonoids content [ ] . for many centuries, apigenin has been widely used as a traditional medicine [ , ] . the excellent properties of this natural compound have prompted the study of its application as an anticancer drug [ , ] . in fact, various positive effects of apigenin administration, alone or in combination with other chemotherapeutic agents, in different types of cancer treatments were reported in the literature [ ] . the following aspects were mentioned: -inducing the death of cancer cell lines, -triggering both autophagy and apoptosis, -suppressing cancer cell migration and invasion, and -inducing the cancer cells cycle arrest. one of the recently carried out investigations mentions that apigenin promotes pancreatic cells death by increasing intracellular ros [ ] . in this work, montani et al. tried to understand the mechanism happening in cancer cells in which apigenin was applied. in fact, they suggested a biological mechanism occurring between heat shock protein (hsp ), a protein that stabilizes proteins involved in the growth of cancer cells, and tp gene mutations that reduce the cytotoxic effect of the chemotherapy with apigenin. the targeting of these molecules is an important anticancer strategy that has been extensively explored. on the other hand, liu et al. evaluated the synergistic effect in cancer therapy involving apigenin combined with metal ions [ ] . in this work, the authors examined the thermal stability of two flavones (apigenin and luteolin) when combined with ferrous or cupric ions, which negatively affects the anticancer activities of both flavones against human cervical cancer hela cells. luteolin is usually found in the leaves and bark of some plants. the major natural sources of luteolin are celery, thyme, dandelion, clover flower, ragweed pollen, chamomile, and perilla [ ] . due to its beneficial effects on the human body (antioxidative and anti-inflammatory properties, being a free radicals scavenger, promoting carbohydrate metabolism, and modulating the immune system), it is assumed that luteolin could perform an important role in cancer therapy [ , ] . to enhance the anticancer effects of luteolin, the flavone is usually used together with other anticancer drugs. ren and co-workers demonstrated that the application of luteolin in combination with oxalipatlin, a conventional anticancer drug used to inhibit the development of cancer cells, stopped the proliferation of gastric cancer cells in vitro by the upregulation of the activity of caspase- and bax proteins [ ] . the construction of nanocarriers containing anticancer drugs allows obtaining controlled drug delivery systems. by the encapsulation of luteolin in polymeric micelles, hu et al. developed a thermosensitive nanocarrier that demonstrated an improved apoptosis of colorectal cancer cells compared to the administration of free luteolin [ ] . flavanones are colorless ketones derived from flavone. flavanones are found in a wide variety of foods included in our daily diet and in herbs [ , ] in citrus fruits, flavanones are usually glycosylated by a disaccharide in position ( figure ). they present different functions in plants: taste-modifying properties (eriodictyol, homoeriodictyol and sterubin), and -they are responsible for the bitter taste in citrus fruits (naringin). in the last decades, flavanones have gained a lot of importance in medicine for their antioxidant activity, radical scavenging, cardiovascular, anti-inflammatory, antiviral, and anticancer effects [ ] . naringenin and hesperetin are the most often investigated for being anticancer drugs. nevertheless, some tests were carried out using other flavanones such as didymin and alpinetin [ , ] . naringenin is a flavanone predominating in oranges and grapefruits. it is also found in bergamot, sour orange, tomatoes, cocoa, water mint, beans, etc. [ , ] . in some of these fruits, narigenin is present in its glycosidic form: naringin (which has attached a disaccharide neohesperidose via a glycosidic linkage at carbon ). as it has been proven in several studies, naringenin induces cytotoxicity in various carcinogenic cells of breast, stomach, liver, cervix, pancreas, colon cancers, and in leukemia [ ] . nevertheless, its poor solubility and instability in physiological medium limits the medical applications of naringenin. to solve these drawbacks, akhter et al. reported the encapsulation of naringenin in plga (poly(lactide-co-glycolid acid)) nanoparticles. moreover, they suggested that the encapsulated naringenin showed higher cytotoxicity when compared with free naringenin due to a more controlled drug release [ ] . another option that could enhance the anticancer properties of naringenin involves the synthesis of naringenin derivatives [ ] . an alternative recent study demonstrated naringenin's effectivity as an anticancer drug in breast cancer treatment is due to the activation of the caspase- protein and caspase- enzymes [ ] , while kumar and co-workers showed in vivo that naringenin showed antitumor effects on skin cancer [ ] . hesperetin and hesperetin's -o-glycoside (also known as hesperidin) are the main flavonoids found in lemons and sweet oranges [ ] . hesperetin's anticancer properties against specific tumors are well documented in numerous research publications: -it inhibits glucose uptake in various cancer cell lines [ , ] , -reduces the nf-κb activity, which leads to a decrease in tumor progression [ ] , and -upgrades the apoptosis via the induction of intracellular ros formation [ ] . in a more recent study, the addition of hesperetin improves the activity of cisplatin, which is an anticancer drug that is commonly used to treat lung cancer [ ] . it was observed that hesperetin inhibits mdr protein (multidrug resistance protein ), which is associated with the resistance to cisplatin developed in a great number of patients subjected to cancer therapy. curiously, the administration of both naringenin and hesperetin were tested in vitro and in vivo trials to analyze the anticancer effects in human pancreatic cancer [ ] . for the first time, the authors reported that the combination of both naringenin and hesperetin could be used as a potential non-toxic cancer therapy system that stops pancreatic cancer development. flavanols or flavan- -ols are another group of monomeric flavonoids. catechin and its derivatives are included in this group. natural sources of flavan- -ols are mainly the "tea plant" (camellia sinensis), and some cocoas. therefore, they are highly present in the human diet in both beverages (tea) and solid foods (chocolates) [ , ] . since studies of flavanols have started in the course of the last century, it has been found that these compounds provide resistance against dangerous trespassers, including microbes, fungi, insects, and herbivorous animals [ ] . thereby, the flavanols' health benefits have been broadly studied in humans. some investigations suggest that the intake of cocoa flavanols could help in the prevention of cardiovascular and metabolic diseases. indeed, the european food safety authority approved cocoa products containing mg of flavanols because they "help to maintain the elasticity of blood vessels, which contributes to normal blood flow" [ ] . epigallocatechin gallate (epigallocatechin- -gallate or egcg) is a catechin that is mostly found in tea and one of the polyphenolic compounds most commonly found in nature; it is also the ester of epigallocatechin and gallic acid [ ] . the objective of finding a correlation between green tea intake and the risk of cancer onset has been a well-studied topic [ ] . as an obvious example, the study presented by guo et al. [ ] validated that the consumption of green tea-and therefore catechins-up to seven cups a day provided a small reduction in the prostate cancer risk. moreover, egcg has been tested against certain cancer cell lines. in ht- colorectal cell lines, egcg upregulated the activity of tfr (transferrin receptor), which is a carrier protein for transferrin, and inhibited the activity of the ferritin-h protein via the iron chelation activity in ht- colorectal cancer cells [ ] . in another example, the synergistic effect of egcg and trail (tumor necrosis factor (tnf)-related apoptosis-inducing ligand), a protein that causes cell death, intensifies the activity of both caspase and the death receptor , causing the death of sw and hct colon cancer cells [ ] . despite the fact that egcg is commonly found in nature, this flavanol shows some drawbacks that limit its applications in cancer therapy (poor stability, low absorption, and hepatotoxicity) [ ] . so, the encapsulation of egcg can be a promising solution to minimize the limitations of the egcg use [ ] . the (−)-epicatechin molecule is a flavonoid of which large quantities are found in cocoa [ ] . the use of epicatechin in cancer therapy has been emerging over the last decade in the attempt to overcome some of the drawbacks of egcg [ , ] . pereyra-vergara and co-workers studied the effects and mechanism of (−)-epicatechin in breast cancer cells [ ] . it was shown that the addition of (−)-epicatechin to carcinogenic cells results in the apoptosis of the two tested breast cancer cell lines (mda-mb- and mcf- ). moreover, the authors proved that (−)-epicatechin increased the intracellular ros production and intensified the activity of bcl associated agonist of cell death (bad) and bcl- -like protein (bax), proteins that are associated with cell apoptosis. isoflavones are another type of biological active flavonoids. isoflavones are mostly found in plants of the leguminosea family. this family includes many species that are of great importance in the human diet (peas, lentils, licorice, beans, chickpeas, and carob), in animal fodder (alfalfa, clover, and carob) and as ornamental plants (mimosa and false acacia) [ , ] . since isoflavones present estrogenic properties, plants use these kinds of compounds as part of their natural defense system against the overpopulation of herbivores by controlling their male fertility [ ] . moreover, these properties make isoflavones good complementary therapeutic options in treating menopause and its symptoms such as osteoporosis, anxiety, emotional instability, and headaches. genistein and daidzein are the most studied compounds of this subgroup in terms of medical applications. nevertheless, other isoflavones such as glabridin and alpinumisoflavone have raised interest as potential cancer medicines in various types of cancer such as breast, liver, or thyroid cancers [ , ] . the isoflavone most reported in medicine is genistein, which is a phytoestrogen compound produced in soybeans. genistein was for the first time isolated in . however, it was not until the end of the last century that researchers started to explore its potential beneficial effects on human health and its possible applications as a medical compound in a wide range of diseases, including cardiovascular diseases, osteoporosis prevention, diabetes, and some types of cancers [ ] . it has been proven that genistein is involved in the regulation of different genes that are associated with the onset of cancers by various mechanisms [ ] . in a recent research, hsiao et al. studied the effects and mechanisms of genistein against leukemia cell lines. in fact, the application of genistein to hl- leukemia cells revealed that this natural medicine kills the carcinogenic cells via two different pathways (endoplasmatic reticulum stress and mitochondria-dependent pathway) in vitro and in mouse xenograft models in vivo [ ] . furthermore, different authors studied the effects of genistein when it is combined with other anticancer drugs [ ] . in a recent investigation, liu et al. tested mixtures of genistein and cisplatin in varied concentrations as a plausible anticancer agent in the treatment of cervical cancer cells [ ] . the authors proved that the addition of genistein improved the chemotherapeutic activity of cisplatin, requiring a lower dose of the drug in cancer treatment, which led to a reduction in the therapy side effects. the second isoflavone most commonly found in nature, which similar to genistein is also isolated from soybeans, is daidzein [ ] . the chemical structure of daidzein is very similar to genistein, without the hydroxyl group at position (table ) . rigalli et al. studied in vitro the effects of daidzein use in breast cancer therapy [ ] . in one of those studies, they proved that daidzein downregulated the expression of multidrug resistance-associated protein (mrp ) in both michigan cancer foundation- (mcf- ) and mda-mb- breast cancer cell lines. the reduction of this protein's activity is very important because mrp is involved in transporting many of the chemotherapeutic drugs out of the cells (for example, doxorubicin or mitoxantrone). in another study in vivo, mice were inoculated with t breast cancer cells and then treated with daidzein administered orally for days. in this case, the highest dose of daidzein ( mg/kg) was required to observe a considerable decrease in tumor size. at the same time, the authors reported that the combination of daidzein with regular exercise promotes the breast cancer cells apoptosis via the fas/fasl-mediated mechanism [ ] . chalcones are a class of polyphenolic compounds that are characterized by the presence of an aromatic ketone and an enone in their central core. many fruits such as citrus and apples, vegetables such as tomatoes, potatoes, shallots, and bean sprouts, and some edible plants such as licorice contain chalcones [ ] . besides, chalcones can be synthesized in the form of base-catalyzed aldol condensation of benzaldehydes with acetophenones (for example, sodium hydroxide) [ ] . the most studied chalcone in the field of medicine is ellagic acid, which has been investigated as a potential antitumor agent [ , ] . ellagic acid is an antioxidant that is found in various natural resources: in oak species such as white oak (quercus alba) and european red oak (quercus robur) or in medicinal fungi (phellinus linteus). peaches, pomegranates, grapes, strawberries, raspberries, pecans, walnuts, and raw chestnuts also contain a considerable amount of ellagic acid [ ] . the anti-proliferative and antioxidative properties of ellagic acid have encouraged researchers to study the health benefits of this natural compound. for years, the effects of treating tumors with ellagic acid have been studied by the evaluation of various alternatives (chemical modifications of ellagic acid or its encapsulation among other options) [ ] . one of the last studies that examined the breast cancer treatment with ellagic acid was published by yousuf et al. [ ] . this work evaluated the capacity of numerous phytochemicals in addition to ellagic acid (capsaicin, tocopherol, rosmarinic acid, ursolic acid, limonene, caffeic acid, and ferulic acid) to inhibit the activity of cyclin-dependent kinase (cdk ), which is an important gene associated with cancer progression. among all the tested natural compounds, ellagic acid showed the highest binding affinity for cdk , decreasing the tumor proliferation. however, the encapsulation of ellagic acid to enhance its poor solubility combined with an improvement of its controlled delivery was attempted by some research groups [ , ] . in a recent work, pirzadeh-naeeni et al. reported the nanoencapsulation of ellagic acid in two different biopolymers (schizophyllan and chitin), which were then tested against mcf- breast cancer cells [ ] . in this case, the controlled release of ellagic acid improved the cytotoxicity when compared with non-encapsulated ellagic acid. it also reduced the progression of tumor cells. anthocyanidins are water-soluble pigments found in plants. they are responsible for leaves, flowers, and fruit colors. some fruits included in the human diet are rich in anthocyanins: blueberries, raspberries, black rice, and black soybeans (normally known as dark fruit). the term anthocyanin was coined in by ludwig clamor marquart, a german pharmacist, to denote the blue pigment of red cabbage (brassica oleracea) [ ] . table . summary of various polyphenols, their chemical structures, and their anticancer effects. resveratrol dna protection against reactive oxygen species (ros), trap the hydroxyl and superoxide groups and the free radicals produced into the cells. inhibition of a lung cancer cells with the activation of caspase- . u. s. department of health and human services public health service food and drug administration status: bulk ingredient for human prescription compounding. [ , ] other colored fruits and vegetables, is one of the most common anthocyanidins [ , ] . the antitumour activity of delphinidin has been demonstrated by numerous researchers. in , jeong et al. studied the effect of delphinidin in prostate cancer treatment. they found that delphinidin increased the activity of caspase- , - , and - , in effect causing the death of cancer cells. moreover, they demonstrated that delphinidin intensified the roles of genes that induce the apoptosis of cancer cells and decreased the activity of some genes that dissuade killing the cancer cells [ ] . alternatively, delphinidin obstructs the progression of skov ovarian cancer cells in vitro by decreasing the akt pathway (a signal transduction pathway) activation, which can in result activate numerous factors that play a critical role in cancer migration [ ] . the chemical structure of the polyphenolic compounds mentioned in this review, their anticancer effects, and the corresponding references are summarized in table . as indicated in the table, the administration of resveratrol and quercetin has been approved by the food and drug administration (fda). [ [ ] [ ] [ ] , jeong et al. studied the effect of delphinidin in prostate cancer treatment. they found that delphinidin increased the activity of caspase- , - , and - , in effect causing the death of cancer cells. moreover, they demonstrated that delphinidin intensified the roles of genes that induce the apoptosis of cancer cells and decreased the activity of some genes that dissuade killing the cancer cells [ ] . alternatively, delphinidin obstructs the progression of skov ovarian cancer cells in vitro by decreasing the akt pathway (a signal transduction pathway) activation, which can in result activate numerous factors that play a critical role in cancer migration [ ] . the chemical structure of the polyphenolic compounds mentioned in this review, their anticancer effects, and the corresponding references are summarized in table . as indicated in the table, the administration of resveratrol and quercetin has been approved by the food and drug administration (fda). improves the cellular uptake of doxorubicin and reduces its side effects. apoptosis of mda-mb- breast cancer cells. [ , ] of cancer cells and decreased the activity of some genes that dissuade killing the cancer cells [ ] . alternatively, delphinidin obstructs the progression of skov ovarian cancer cells in vitro by decreasing the akt pathway (a signal transduction pathway) activation, which can in result activate numerous factors that play a critical role in cancer migration [ ] . the chemical structure of the polyphenolic compounds mentioned in this review, their anticancer effects, and the corresponding references are summarized in table . as indicated in the table, the administration of resveratrol and quercetin has been approved by the food and drug administration (fda). kappa-light-chain-enhancer of activated b cells (nf-κb) signaling through protein kinase c delta type (pkcδ) inactivation. upgrades the cytotoxicity of trastuzumab and increases the specificity to breast cancer cells. synergistic effects of honokiol and doxorubicin in breast cancer by suppressing the metastasis of carcinogenic cells and apoptosis induction. apoptosis of multiple myeloma cancer cells. [ , ] p-coumaric acid apoptosis of hct- colon cancer cells through ros mitochondrial pathway. inhibits the growth of snu- gastric cancer cells. [ [ ] [ ] [ ] kaempferol induces the apoptosis and dna damage in mda-mb- breast cancer cells by the upregulation of h a histone family member x (γh ax), caspase , caspase , and the protein serine/threonine kinase (p-atm). induces the apoptosis of lncap prostate cancer cells. impedes the proliferation of cancer cells. luteolin synergistic effects of luteolin and oxaliplatin: stops the proliferation of gastric cancer cell. promotes apoptosis and stops the proliferation of colorectal cancer cells. [ , ] u. s. department of health and human services public health service food and drug administration status: drug for further processing. promotes apoptosis of pancreatic cancer cells by increasing intracellular ros. damages dna of hela cervical cancer cells. inhibits the growth of cancer cells and induces its apoptosis. [ , ] luteolin synergistic effects of luteolin and oxaliplatin: stops the proliferation of gastric cancer cell. promotes apoptosis and stops the proliferation of colorectal cancer cells. [ , ] naringenin [ , ] naringenin apoptosis of breast cancer cells by the increase of the activity of caspase- and caspase- . suppression of skin cancer cells. [ , ] molecules , in conclusion, the exceptional antioxidative properties make polyphenols strong candidates for agents used in various types of cancer treatments. actually, the anticancer effects of several polyphenolic compounds have been mainly studied in in vitro cancer cells and in preclinical animal models. nevertheless, there are very few clinical data on many of the polyphenols application as anticancer medicines (clinical studies on cancer therapy involve only the most common polyphenols such as resveratrol, curcumin, and quercetin). nowadays, the vast majority of these clinical studies are still in progress. the research on cancer therapies involving varied polyphenol families, and particularly flavonoids, has contributed to the development of natural medicines that are less aggressive than conventional anticancer drugs. in fact, various research works proved that polyphenols could be used anthocyanidins have varied functions in plants: attracting pollinating insects, preventing the freezing of fruits such as grapes, and protecting plants against harmful uv radiation [ ] . moreover, these kinds of compounds are widely used in the food industry (preparation of food coloring, a parameter for determining wine quality) and in medical industry (decreased risk of contracting various diseases such as obesity, improved memory and age-related deficiencies, or improvement of the immunological system) due to their chemical and physical properties [ ] . some anticancer properties of anthocyanidins extracted from the plant cyanomorium coccineum have been recently described by rescigno et al., which demonstrated the antiproliferative effect of anthocyanidins against different leukemia cell lines [ ] . delphinidin, which can be found in red cabbage, grapes, berries, and sweet potatoes among other colored fruits and vegetables, is one of the most common anthocyanidins [ , ] . the antitumour activity of delphinidin has been demonstrated by numerous researchers. in , jeong et al. studied the effect of delphinidin in prostate cancer treatment. they found that delphinidin increased the activity of caspase- , - , and - , in effect causing the death of cancer cells. moreover, they demonstrated that delphinidin intensified the roles of genes that induce the apoptosis of cancer cells and decreased the activity of some genes that dissuade killing the cancer cells [ ] . alternatively, delphinidin obstructs the progression of skov ovarian cancer cells in vitro by decreasing the akt pathway (a signal transduction pathway) activation, which can in result activate numerous factors that play a critical role in cancer migration [ ] . the chemical structure of the polyphenolic compounds mentioned in this review, their anticancer effects, and the corresponding references are summarized in table . as indicated in the table, the administration of resveratrol and quercetin has been approved by the food and drug administration (fda). in conclusion, the exceptional antioxidative properties make polyphenols strong candidates for agents used in various types of cancer treatments. actually, the anticancer effects of several polyphenolic compounds have been mainly studied in in vitro cancer cells and in preclinical animal models. nevertheless, there are very few clinical data on many of the polyphenols application as anticancer medicines (clinical studies on cancer therapy involve only the most common polyphenols such as resveratrol, curcumin, and quercetin). nowadays, the vast majority of these clinical studies are still in progress. the research on cancer therapies involving varied polyphenol families, and particularly flavonoids, has contributed to the development of natural medicines that are less aggressive than conventional anticancer drugs. in fact, various research works proved that polyphenols could be used as chemotherapy adjuvant agents in cancer therapies. however, the process of discovering the polyphenols' mechanisms of action as anticancer drugs and their interactions with tumors requires further studies in order for those natural compounds to improve the actual therapeutic strategies. the authors declare no conflict of interest. cancer nanotherapy: concept for design of new drug cancer statistics molecular targeting therapy of cancer: drug resistance, apoptosis and survival signal advances in cancer therapy with plant based natural products new aspects of natural-killer-cell surveillance and therapy of cancer natural polymers: a source of inspiration discovery, development, and regulation of natural products. in using old solutions to new problems-natural drug discovery in the st century discovery, development, and regulation of natural products cancer chemoprevention with dietary phytochemicals dietary phytochemicals and human health phytochemicals modulate cancer aggressiveness: a review depicting the anticancer efficacy of dietary polyphenols and their combinations polyphenols as antimicrobial agents plant polyphenols: chemical properties, biological activities, and synthesis the pharmacology of avenanthramides: polyphenols ultrasound-assisted extraction of biologically active compounds and their successive concentration by using membrane processes stability and anti-proliferative properties of biologically active compounds extracted from cistus l. after sterilization treatments minireview-biological effects of resveratrol resveratrol and cancer: challenges for clinical translation resveratrol in cancer patients: from bench to bedside resveratrol scavenges reactive oxygen species and effects radical-induced cellular responses in vitro and in vivo evaluation of the antitumor efficiency of resveratrol against lung cancer. asian pac phase i randomized, double-blind pilot study of micronized resveratrol (srt ) in patients with hepatic metastases-safety, pharmacokinetics, and pharmacodynamics a phase study of srt (resveratrol) with bortezomib for patients with relapsed and or refractory multiple myeloma resveratrol reduces the levels of circulating androgen precursors but has no effect on, testosterone, dihydrotestosterone, psa levels or prostate volume. a -month randomised trial in middle-aged men biological activities of curcuminoids, other biomolecules from turmeric and their derivatives-a review compound from natural sources, a true scientific challenge-a review synthesis of curcumin derivatives and analysis of their antitumor effects in triple negative breast cancer (tnbc) cell lines photo-triggered capsules based on lanthanide-doped upconverting nanoparticles for medical applications encapsulation for cancer therapy dimethoxy curcumin induces apoptosis by suppressing survivin and inhibits invasion by enhancing e-cadherin in colon cancer cells potential of curcumin and resveratrol as biochemical and biophysical modulators during lung cancer in rats evaluation of biophysical as well as biochemical potential of curcumin and resveratrol during prostate cancer lignans in food and nutrition flaxseed and its lignan and oil components reduce mammary tumor growth at a late stage of carcinogenesis anticancer effects of a plant lignan -hydroxymatairesinol on a prostate cancer model in vivo irinotecan compared with etoposide in combination with platinum in previously untreated extensive stage small cell lung cancer: an updated systemic review a systematic review and pooled analysis of studies of oral etoposide in metastatic breast cancer molecular mechanisms of the action of arctigenin in cancer arctigenin promotes apoptosis in ovarian cancer cells via the inos/no/stat /survivin signalling nanoparticle delivery strategies to target doxorubicin to tumor cells and reduce side effects arctigenin enhances the cytotoxic effect of doxorubicin in mda-mb- breast cancer cells therapeutic applications of compounds in the magnolia family anticancer activity of natural compounds from plant and marine environment suppression of pkcdelta/nf-kappab signaling and apoptosis induction through extrinsic/intrinsic pathways are associated magnolol-inhibited tumor progression in colorectal cancer in vitro and in vivo near ir responsive targeted integrated lipid polymer nanoconstruct for enhanced magnolol cytotoxicity in breast cancer facile purification of honokiol and its antiviral and cytotoxic properties honokiol for cancer therapeutics: a traditional medicine that can modulate multiple oncogenic targets honokiol: a review of its anticancer potential and mechanisms synergistically enhanced antimetastasis effects by honokiol-loaded ph-sensitive polymer−doxorubicin conjugate micelles honokiol induces apoptosis in human lymphoid leukemia molt b cells the natural product honokiol induces caspase-dependent apoptosis in b-cell chronic lymphocytic leukemia (b-cll) cells honokiol overcomes conventional drug resistance in human multiple myeloma by induction of caspase-dependent and -independent apoptosis bioactivity of phenolic acids: metabolites versus parent compounds: a review dietary hydroxybenzoic acid derivatives-nature, occurrence and dietary burden in situ antioxidant and antimicrobial activities of naturally occurring caffeic acid, p-coumaric acid and rutin, using food systems events associated with apoptotic effect of p-coumaric acid in hct- colon cancer cells effects of p-coumaric acid on microrna expression profiles in snu- human gastric cancer cells anti-proliferative properties of p-coumaric acid in snu- gastric cancer cells anthocyanins and other flavonoids research trends in flavonoids and health phenol content in sprouted grains tautomerism of flavonol glucosides: relevance to plant uv protection and flower colour enhancement of oxidative and drought tolerance in arabidopsis by overaccumulation of antioxidant flavonoids dietary flavonoids: bioavailability, metabolic effects, and safety role of fisetin in chemosensitization health-promoting components of fruits and vegetables in the diet the mechanism of anticancer action and potential clinical use of kaempferol in the treatment of breast cancer kaempferol suppresses proliferation and induces cell cycle arrest, apoptosis, and dna damage in breast cancer cells kaempferol promotes apoptosis while inhibiting cell proliferation via androgen-dependent pathway and suppressing vasculogenic mimicry and invasion in prostate cancer review of the biology of quercetin and related bioflavonoids fruits and vegetables in the prevention of cellular oxidative damage quercetin is the active component of yang-yin-qing-fei-tang to induce apoptosis in non-small cell lung cancer pharmacological basis and new insights of quercetin action in respect to its anti-cancer effects curcumin and quercetin synergistically inhibit cancer cell proliferation in multiple cancer cells and modulate wnt/β-catenin signaling and apoptotic pathways in a cells bioinspired polymerization of quercetin to produce a curcumin-loaded nanomedicine with potent cytotoxicity and cancer-targeting potential in vivo quercetin reverses docetaxel resistance in prostate cancer via androgen receptor and pi k/akt signaling pathways the herbal medicine sho-saiko-to inhibits proliferation of cancer cell-lines by inducing apoptosis and arrest at the g( )-g( )-phase medicinal importance, pharmacological activities, and analytical aspects of hispidulin: a concise report curcumin and apigenin-novel and promising therapeutics against chronic neuroinflammation in alzheimer's disease in vitro anti-inflammatory effect of apigenin in the helicobacter pylori-infected gastric adenocarcinoma cells antal, i. study on the pulmonary delivery system of apigenin-loaded albumin nanocarriers with antioxidant activity apigenin and cancer chemoprevention a plant flavone playing noble roles in cancer prevention via modulation of key cell signaling networks. recent pat. anti-cancer drug discov apigenin in cancer therapy: anti-cancer effects and mechanisms of action mutant p , stabilized by its interplay with hsp , activates a positive feed-back loop between nrf and p that induces chemo-resistance to apigenin in pancreatic cancer cells the stability and activity changes of apigenin and luteolin in human cervical cancer hela cells in response to heat treatment and fe + /cu + addition intestinal absorption of luteolin and luteolin -o-beta-glucoside in rats and humans molecular targets of luteolin in cancer apoptosis induced by luteolin in breast cancer: mechanistic and therapeutic perspectives luteolin suppresses the proliferation of gastric cancer cells and acts in synergy with oxaliplatin thermosensitive in situ gel containing luteolin micelles is a promising efficient agent for colorectal cancer peritoneal metastasis treatment polyphenols: a concise overview on the chemistry, occurrence, and human health a comprehensive review on flavanones, the major citrus polyphenols didymin, a dietary flavonoid glycoside from citrus fruits, induces fas-mediated apoptotic pathway in human non-small-cell lung cancer cells in vitro and in vivo antiproliferative effect of alpinetin in bxpc- pancreatic cancer cells antiatherogenic properties of naringenin, a citrus flavonoid review of the flavonoids quercetin, hesperetin naringenin. dietary sources, bioactivities, and epidemiology inhibitory effects of naringenin on tumor growth in human cancer cell lines and sarcoma s- -implanted mice sonication tailored enhance cytotoxicity of naringenin nanoparticle in pancreatic cancer: design, optimization, and in vitro studies optimizing the flavanone core toward new selective nitrogen-containing modulators of abc transporters naringenin has a chemoprotective effect in mda-mb- breast cancer cells via inhibition of caspase- and - activities naringenin suppresses chemically induced skin cancer in two-stage skin carcinogenesis mouse model therapeutic potential of hesperidin and its aglycone hesperetin: cell cycle regulation and apoptosis induction in cancer models hesperetin impairs glucose uptake and inhibits proliferation of breast cancer cells natural compounds regulate glycolysis in hypoxic tumor microenvironment molecular mechanisms behind the biological effects of hesperidin and hesperetin for the prevention of cancer and cardiovascular diseases hesperidin: a promising anticancer agent from nature hesperetin reverses p-glycoprotein-mediated cisplatin resistance in ddp-resistant human lung cancer cells via modulation of the nuclear factor-κb signaling pathway combined administration of naringenin and hesperetin with optimal ratio maximizes the anti-cancer effect in human pancreatic cancer via down regulation of fak and p signaling pathway cocoa flavanols: natural agents with attenuating effects on metabolic syndrome risk factors scientific opinion on the modification of the authorisation of a health claim related to cocoa flavanols and maintenance of normal endotheliumdependent vasodilation pursuant to article ( ) of regulation (ec) no / following a request in accordance with article of regulation (ec) no epigallocatechin- -gallate (egcg): chemical and biomedical perspectives cancer prevention by tea: animal studies, molecular mechanisms and human relevance green tea and the risk of prostate cancer a systematic review and meta-analysis iron chelation properties of green tea epigallocatechin- -gallate (egcg) in colorectal cancer cells: analysis on tfr/fth regulations and molecular docking. evid-based complementary altern epigallocatechin- -gallate induces apoptosis as a trail sensitizer via activation of caspase and death receptor in human colon cancer cells the safety of green tea and green tea extract consumption in adults-results of a systematic review cancer therapeutics with epigallocatechin- -gallate encapsulated in biopolymeric nanoparticles flavor chemistry of cocoa and cocoa products-an overview antileukemic action of (−)-epicatechin in the spleen of rats with acute myeloid leukemia induction of apoptosis of sw human colon cancer cells by (−)-epicatechin isolated from bulnesia sarmienti apoptosis induced by (−)-epicatechin in human breast cancer cells is mediated by reactive oxygen species phenolic compounds as functional ingredients in beverages phytochemical mimicry of reproductive hormones and modulation of herbivore fertility by phytoestrogens. environ. health persp glabridin inhibits migration and invasion by transcriptional inhibition of matrix metalloproteinase through modulation of nf-kappa b and ap- activity in human liver cancer cells alpinumisoflavone inhibits tumor growth and metastasis in papillary thyroid cancer via upregulating mir- - p recent progress on biocompatible nanocarrierbased genistein delivery systems in cancer therapy the role of genistein and synthetic derivatives of isoflavone in cancer prevention and therapy genistein induces apoptosis in vitro and has antitumor activity against human leukemia hl- cancer cell xenograft growth in vivo anti-inflammatory and anticarcinogenic effect of genistein alone or in combination with capsaicin in tpa-treated rat mammary glands or mammary cancer cell line effects of genistein on anti-tumor activity of cisplatin in human cervical cancer cell lines nutritional epigenetic regulators in the field of cancer the phytoestrogens daidzein and equol inhibit the drug transporter bcrp/abcg in breast cancer cells: potential chemosensitizing effect synergetic inhibition of daidzein and regular exercise on breast cancer in bearing- t mice by regulating nk cells and apoptosis pathway a review on natural chalcones an update synthesis and anti-inflammatory activity of three nitro chalcones novel -amino chalcones: design, synthesis and biological evaluation antiproliferative and pro-apoptotic activities of -and -aminochalcones against tumor canine cells ellagic acid induces cell cycle arrest and apoptosis through tgf-β/smad signaling pathway in human breast cancer mcf- cells ellagic acid, sulforaphane, and ursolic acid in the prevention and therapy of breast cancer: current evidence and future perspectives ellagic acid controls cell proliferation and induces apoptosis in breast cancer cells via inhibition of cyclin-dependent kinase development of biodegradable nanoparticles for oral delivery of ellagic acid and evaluation of their antioxidant efficacy against cyclosporine a-induced nephrotoxicity in rats preparation of β-cd-ellagic acid microspheres and their effects on hepg cell proliferation a comparative study on schizophyllan and chitin nanoparticles for ellagic acid delivery in treating breast cancer anthocyanidins and anthocyanins: colored pigments as food, pharmaceutical ingredients, and the potential health benefits anthocyanin biosynthesis and degradation mechanisms in solanaceous vegetables: a review food applications and physiological effects of anthocyanins as functional food ingredients antiproliferative and antiviral extracts from sardinian maltese mushroom (cynomorium coccineum l.) updating the research on prodelphinidins from dietary sources influence of fruit juice processing on anthocyanin stability delphinidin induces apoptosis via cleaved hdac -mediated p acetylation and oligomerization in prostate cancer cells delphinidin inhibits bdnf-induced migration and invasion in skov ovarian cancer cells this article is an open access article distributed under the terms and conditions of the creative commons attribution (cc by) license key: cord- -knelqmzx authors: villas-boas, gustavo r.; rescia, vanessa c.; paes, marina m.; lavorato, stefânia n.; de magalhães-filho, manoel f.; cunha, mila s.; simões, rafael da c.; de lacerda, roseli b.; de freitas-júnior, renilson s.; ramos, bruno h. da s.; mapeli, ana m.; henriques, matheus da s. t.; de freitas, william r.; lopes, luiz a. f.; oliveira, luiz g. r.; da silva, jonatas g.; silva-filho, saulo e.; da silveira, ana p. s.; leão, katyuscya v.; matos, maria m. de s.; fernandes, jamille s.; cuman, roberto k. n.; silva-comar, francielli m. de s.; comar, jurandir f.; brasileiro, luana do a.; dos santos, jussileide n.; oesterreich, silvia a. title: the new coronavirus (sars-cov- ): a comprehensive review on immunity and the application of bioinformatics and molecular modeling to the discovery of potential anti-sars-cov- agents date: - - journal: molecules doi: . /molecules sha: doc_id: cord_uid: knelqmzx on march , , the world health organization (who) officially declared the outbreak caused by the new coronavirus (sars-cov- ) a pandemic. the rapid spread of the disease surprised the scientific and medical community. based on the latest reports, news, and scientific articles published, there is no doubt that the coronavirus has overloaded health systems globally. practical actions against the recent emergence and rapid expansion of the sars-cov- require the development and use of tools for discovering new molecular anti-sars-cov- targets. thus, this review presents bioinformatics and molecular modeling strategies that aim to assist in the discovery of potential anti-sars-cov- agents. besides, we reviewed the relationship between sars-cov- and innate immunity, since understanding the structures involved in this infection can contribute to the development of new therapeutic targets. bioinformatics is a technology that assists researchers in coping with diseases by investigating genetic sequencing and seeking structural models of potential molecular targets present in sars-cov . the details provided in this review provide future points of consideration in the field of virology and medical sciences that will contribute to clarifying potential therapeutic targets for anti-sars-cov- and for understanding the molecular mechanisms responsible for the pathogenesis and virulence of sars-cov- . . continental map of sars-cov- infection cases. data were collected on . . [ ] . designed by freepik. currently, in the absence of any efficient therapy known for the treatment of covid- infections and, also, to the process of developing new drugs is time-consuming and cumbersome, the use of bioinformatics as a tool can redirect old drugs against covid- , helping to identify treatments with known pharmacokinetic, pharmacodynamic and toxicity profiles [ ] . some recent studies have provided critical insights using bioinformatics and molecular modeling to help the rapid development of treatments that can be tested in clinical trials [ , ] . for example, using xml-like web effort (q-uel) systems to access relevant and emerging literature and interact with standard publicly available bioinformatics tools on the internet helped quickly identify sequences of amino acids that are well conserved across many coronaviruses, including sars-cov- . the theory behind q-uel has been described and developed in several essentially mathematical papers [ ] . q-uel is a biomedical and pharmaceutical data mining tool that comprises knowledge bearing tags as "probabilistic statements" from relatively structured data sources [ ] and specialist text [ ] , as well as "common sense" and general wisdom from thesauruses and encyclopedias, and automatic surfing of the internet [ ] . research using this type of tool can contribute to the proposition of specific synthetic vaccine epitope and peptidomimetic agents (see [ ] ). the role of bioinformatics in conjunction with molecular modeling in the search for methods of diagnosis, treatment and prevention of covid- is unquestionable. processes such as screening of bioactive compounds, modeling of biomacromolecule structures, primer selection and genetic sequencing compounds can be faster, more accurate and less expensive when aided by computer . . [ ] . designed by freepik. currently, in the absence of any efficient therapy known for the treatment of covid- infections and, also, to the process of developing new drugs is time-consuming and cumbersome, the use of bioinformatics as a tool can redirect old drugs against covid- , helping to identify treatments with known pharmacokinetic, pharmacodynamic and toxicity profiles [ ] . some recent studies have provided critical insights using bioinformatics and molecular modeling to help the rapid development of treatments that can be tested in clinical trials [ , ] . for example, using xml-like web effort (q-uel) systems to access relevant and emerging literature and interact with standard publicly available bioinformatics tools on the internet helped quickly identify sequences of amino acids that are well conserved across many coronaviruses, including sars-cov- . the theory behind q-uel has been described and developed in several essentially mathematical papers [ ] . q-uel is a biomedical and pharmaceutical data mining tool that comprises knowledge bearing tags as "probabilistic statements" from relatively structured data sources [ ] and specialist text [ ] , as well as "common sense" and general wisdom from thesauruses and encyclopedias, and automatic surfing of the internet [ ] . research using this type of tool can contribute to the proposition of specific synthetic vaccine epitope and peptidomimetic agents (see [ ] ). the role of bioinformatics in conjunction with molecular modeling in the search for methods of diagnosis, treatment and prevention of covid- is unquestionable. processes such as screening of bioactive compounds, modeling of biomacromolecule structures, primer selection and genetic sequencing compounds can be faster, more accurate and less expensive when aided by computer tools. experts from all over the world believe in the potential of bioinformatics in combating the covid- pandemic. bioinformatics contributes to understanding the variations in sars-cov- proteins and molecules , , of how the virulence of this pathogen can increase. based on this, this tool can also clarify how the virus subverts the immune system. our know-how about the molecular elements involved in triggering this type of response can bring to light essential points that may otherwise be neglected. in addition to issues related to bioinformatics and molecular modeling, understanding the participation of innate immunity in the infectious process is crucial for the screening of new target molecules for the treatment and diagnosis of covid- . it is well established in the current literature that innate immunity plays a central role in determining the outcome of viral infections in general [ ] . therefore, the present review explicitly focuses on this aspect of the immune system, demonstrating its role in the formation of the adaptive immune response downstream and its importance as a therapeutic target for the development of new drugs for the treatment of patients with severe covid- . researchers put much effort to understand the origin and pathophysiology of this novel coronavirus and have been testing multiple drugs to screen for therapeutic effective substances [ ] . despite little understanding about the pathophysiology and high pathogenicity of sars-cov- infection, early studies have shown that increased amounts of proinflammatory cytokines in serum (e.g., (interleukin il) il- β, il- , il- , interferon-γ (ifnγ), interferon-inducible protein (ip ), and monocytic chemotactic protein (mcp )) were associated with pulmonary inflammation and extensive lung damage in patients with severe acute respiratory syndrome (sars) [ ] . besides, research has shown that infection by mers-cov leads to a significant increase in serum levels of pro-inflammatory cytokines (ifnγ, tnf-α, il , and il ) [ ] . patients who required intensive care unit (icu) admission had higher concentrations of gcsf, ip , mcp , mip a, and tnf-α than those who did not require icu admission, suggesting that massive cytokine synthesis and secretion are associated with the severity of the disease [ ] . given the above, the present study aimed to develop a comprehensive review of aspects of bioinformatics and molecular modeling as auxiliary tools to pharmacology and immunology in the discovery of potential anti-sars-cov- agents. understanding these aspects streamlines the biomedical research process, without any operational costs. besides, we researched and gathered evidence of the interactions between sars-cov- and innate immunity. discovering new therapeutic targets and understanding pathophysiological processes are the key to the development of effective treatments and efficient management of patients who progress to severe sars-cov- infection, which can save lives worldwide. most coronavirus (cov) infections in humans are caused by low-pathogenicity species, causing common cold symptoms; however, they can eventually lead to serious infections in higher risk groups such as the elderly, children, patients with comorbidities (hypertension, diabetes mellitus, asthma, among others) and/or those suffering immunosuppression. prior to , two highly pathogenic and animal-derived coronavirus species (sars and mers) were responsible for outbreaks of severe acute respiratory syndromes. regarding human infection by sars-cov- , the clinical spectrum is not fully described, and the lethality, mortality, infectivity, and transmissibility pattern is not yet fully elucidated. in addition, there is no specific treatment with antivirals that are effective against sars-cov- or available vaccines and, currently, treatment is supportive and nonspecific for covid- in hospitalized patients with fever, accompanied by cough or sore throat and with dyspnea or o saturation below % or respiratory discomfort [ ] . cov, of the order nidovirales, coronavirin subfamily and coronaviridae family, is a single-stranded rna virus with diameter of - nm [ ] with appearance of crown under electron microscope ("coronam" is the latin term for "crown") due to the presence of glycoproteins in the viral envelope [ ] . it is a virus capable of infecting humans and a wide variety of other mammalian hosts (e.g., mice, swine, rats, dogs, cats, rabbits, horses, cattle, cetaceans and bats) and birds (chickens, pheasants and molecules , , of turkeys) and develop respiratory, enteric, liver and central nervous system (cns) diseases. based on its genotypic and serological characteristics, cov is classified into subfamilies, previously called groups , and . group and were composed of cov that has mammals as hosts and group was composed, until recently, only of avian cov [ ] [ ] [ ] [ ] [ ] . currently, the study group of the international committee for viral taxonomy (icvt) has proposed replacing the traditional groups by subfamilies alfacoronavirus (α-cov) (group ), betacoronavirus (β-cov) (group ) and gamacoronavirus (γ-cov) (group ). after that, the presence of a fourth cov subfamily was detected in birds and pigs and called deltacoronavirus (δ-cov) [ , ] . the most common human covs (hcov) are hcov-oc , hcov-hku , both β-covs of strain a, and hcov- e and hcov-nl , both α-covs. generally, they cause common colds and self-limited upper respiratory infections in immunocompetent individuals, that is, they are eliminated in a short period of time by the immune system without the need for intervention through specific pharmacotherapy. in immunocompromised and elderly individuals, lower respiratory tract infections may also occur. other hcov include sars-cov, sars-cov- (or sars-cov- ) and mers-cov (β-covs of lineage b and c, respectively). these cov categories can cause epidemics of varying clinical severity, with respiratory and extra-respiratory manifestations. regarding sars-cov, mers-cov, mortality rates are up to % and %, respectively [ ] and sars-cov- belongs to β-cov subfamily. an important feature of the sars-cov epidemic between and was the virus efficiency in transmitting from species such as masked palm civet (paguma larvata), raccoon dog (nyctereutes procyonoides) and the chinese ferret-badger (melogale moschata) and infect human populations (fig a) [ ] . it is postulated that sars-cov is the result of the recombination of covs transmitted from these animals to humans. however, research has shown that sars-cov has not been detected in domestic or wild masked palm civet [ ] , suggesting that these, and other animals marketed in wholesale seafood markets in china, were not the main reservoirs of the virus [ ] . cov similar to sars-cov was isolated from the chinese horseshoe bat (rhinolophus spp.) [ , ] , which were also marketed in live-animal markets, strongly suggesting that the virus may have recently been transmitted from bats to other mammals, such as masked palm civets, and later to humans (figure a ) [ ] . in addition to sars-cov, there are other situations of cross-transmission of covs among different animal species. bovine coronavirus (bcov) and hcov-oc have broad similarities. bcov is believed to have been transmitted from bovine hosts to humans a hundred years ago [ ] . previous studies have demonstrated the isolation of bcov from alpaca that showed signs of enteritis and in captive wild ruminants [ , ] , strongly suggesting that this cov has been transmitted to others species (figure b) . previous studies have shown that canine (ccov), feline (fcov) and swine coronaviruses exchanged genetic material at random, showing that they were infecting the same host. recombination processes among the first ccov and fcov (ccov-i and fcov-i) strains and an unknown cov resulted in two new groups of viruses, ccov-ii and fcov-ii. in addition, studies have shown that the sequence of the transmissible gastroenteritis virus (tgev) genetic material shows that this cov was originated through cross-transmission of ccov-ii species from an infected dog (figure c ) [ ] . molecules , , x for peer review of swine, rats, dogs, cats, rabbits, horses, cattle, cetaceans and bats) and birds (chickens, pheasants and turkeys) and develop respiratory, enteric, liver and central nervous system (cns) diseases. based on its genotypic and serological characteristics, cov is classified into subfamilies, previously called groups , and . group and were composed of cov that has mammals as hosts and group was composed, until recently, only of avian cov [ ] [ ] [ ] [ ] [ ] . currently, the study group of the international committee for viral taxonomy (icvt) has proposed replacing the traditional groups by subfamilies alfacoronavirus (α-cov) (group ), betacoronavirus (β-cov) (group ) and gamacoronavirus (γ-cov) (group ). after that, the presence of a fourth cov subfamily was detected in birds and pigs and called deltacoronavirus (δ-cov) [ , ] . the most common human covs (hcov) are hcov-oc , hcov-hku , both β-covs of strain a, and hcov- e and hcov-nl , both α-covs. generally, they cause common colds and selflimited upper respiratory infections in immunocompetent individuals, that is, they are eliminated in a short period of time by the immune system without the need for intervention through specific pharmacotherapy. in immunocompromised and elderly individuals, lower respiratory tract infections may also occur. other hcov include sars-cov, sars-cov- (or sars-cov- ) and mers-cov (β-covs of lineage b and c, respectively). these cov categories can cause epidemics of varying clinical severity, with respiratory and extra-respiratory manifestations. regarding sars-cov, mers-cov, mortality rates are up to % and %, respectively [ ] and sars-cov- belongs to β-cov subfamily. an important feature of the sars-cov epidemic between and was the virus efficiency in transmitting from species such as masked palm civet (paguma larvata), raccoon dog (nyctereutes procyonoides) and the chinese ferret-badger (melogale moschata) and infect human populations (fig a) [ ] . it is postulated that sars-cov is the result of the recombination of covs transmitted from these animals to humans. however, research has shown that sars-cov has not been detected in domestic or wild masked palm civet [ ] , suggesting that these, and other animals marketed in wholesale seafood markets in china, were not the main reservoirs of the virus [ ] . cov similar to sars-cov was isolated from the chinese horseshoe bat (rhinolophus spp.) [ , ] , which were also marketed in live-animal markets, strongly suggesting that the virus may have recently been transmitted from bats to other mammals, such as masked palm civets, and later to humans (figure a ) [ ] . . this virus has spread and adapted to wild animals, for example, masked palm civet, which is marketed for human consumption in wholesale seafood markets in china. the employees of these markets that manipulate these wild animals have been infected; however, they did not present important clinical signs, and symptoms were minimal. the process of adapting the virus to new hosts resulted in strains with efficient replication capacity in human hosts, which cause diseases with clinical conditions ranging from mild to severe and with great ability to spread from person to person; (b) oc coronavirus, whose natural reservoir are humans (hcov-oc ) and bovine coronavirus (bcov) are closely related. it is postulated that these coronaviruses originated in another animal species and subsequently have crossed their species. bcov has effectively spread among other animal species, for example, alpaca (south american mammal of the camelid family) and wild ruminants (such as deer); (c) currently, some canine viruses are believed to have common ancestors with feline species. this occurs with coronaviruses that infect these species. currently, feline coronavirus i (fcov-i) and canine coronavirus i (ccov-i) are believed to share a common ancestor. a recombination process (random exchange of genetic material) of ccov-i with an unknown coronavirus gave rise to a second type of canine coronavirus (ccov-ii). the recombination of ccov-ii with fcov-i in an unknown host gave rise to a second type of feline coronavirus (fcov-ii). there is evidence that ccov-ii was transmitted to pigs, originating the transmissible gastroenteritis virus (tgev) [ ] . note: this image was developed using the coreldraw software ( corel corporation id ). in addition to sars-cov, there are other situations of cross-transmission of covs among different animal species. bovine coronavirus (bcov) and hcov-oc have broad similarities. bcov is believed to have been transmitted from bovine hosts to humans a hundred years ago [ ] . previous this virus has spread and adapted to wild animals, for example, masked palm civet, which is marketed for human consumption in wholesale seafood markets in china. the employees of these markets that manipulate these wild animals have been infected; however, they did not present important clinical signs, and symptoms were minimal. the process of adapting the virus to new hosts resulted in strains with efficient replication capacity in human hosts, which cause diseases with clinical conditions ranging from mild to severe and with great ability to spread from person to person; (b) oc coronavirus, whose natural reservoir are humans (hcov-oc ) and bovine coronavirus (bcov) are closely related. it is postulated that these coronaviruses originated in another animal species and subsequently have crossed their species. bcov has effectively spread among other animal species, for example, alpaca (south american mammal of the camelid family) and wild ruminants (such as deer); (c) currently, some canine viruses are believed to have common ancestors with feline species. this occurs with coronaviruses that infect these species. currently, feline coronavirus i (fcov-i) and canine coronavirus i (ccov-i) are believed to share a common ancestor. a recombination process (random exchange of genetic material) of ccov-i with an unknown coronavirus gave rise to a second type of canine coronavirus (ccov-ii). the recombination of ccov-ii with fcov-i in an unknown host gave rise to a second type of feline coronavirus (fcov-ii). there is evidence that ccov-ii was transmitted to pigs, originating the transmissible gastroenteritis virus (tgev) [ ] . note: this image was developed using the coreldraw software ( corel corporation id ). in general, statistical data suggest that % of the population are asymptomatic cov carriers and that these viruses are responsible for about % to % of acute respiratory infections [ ] . as for transmission and infection, rarely animal covs infect people and spread among them, as occurred with sars-cov and mers-cov. at first, many of the patients with outbreaks of respiratory diseases caused by sars-cov- in wuhan, china, had some connection with a large seafood and live-animal market, strongly suggesting that the spread occurred from animal to people. however, after the outbreak began, an increasing number of patients supposedly did not have exposure to the animal market, also indicating the occurrence of spread from person to person [ ] . the possibility of sars-cov- transmission from seafood to humans is unlikely. as the wuhan seafood wholesale market also sells other animals, the sars-cov- natural host remains unknown [ ] . the sustained spread from people to people is occurring in the globe. this culminated in the statement that the now-pandemic sars-cov- outbreak constitutes a public health emergency of international concern (pheic) by who on january , in geneva, switzerland. person-to-person transmission cases have already been reported worldwide. transmission in health institutions, such as hospitals, has frequently occurred, having already been reported in almost all countries where sars-cov- is present. regarding the person-to-person spread that occurred with sars-cov or mers-cov, the main means is believed to have been through respiratory droplets produced when an infected person coughs or sneezes, similar to the way influenza and other respiratory pathogens are disseminated. in addition, aerosol transmission has been identified in patients undergoing airway procedures, such as endotracheal intubation or airway aspiration. in the population, sars-cov and mers-cov dissemination among people usually occurs after close contacts, and health professionals involved in assisting these patients are particularly vulnerable [ ] . a recent study conducted with women who were in the third trimester of pregnancy and who were confirmed with sars-cov- infection showed no evidence of vertical transmission, that is, from mother to child. despite that, all pregnant women underwent cesarean section and it remains unclear whether transmission can occur during normal delivery [ ] . further studies with pregnant women in other gestation periods and even in the third trimester should be carried out, since the sample used was small. studies like this are crucial for the care of pregnant women, since they are relatively more susceptible to respiratory pathogen infection and severe pneumonia. recent studies have shown that interpersonal sars-cov- transmission has occurred since mid-december in wuhan (china) and from there, considerable efforts to reduce transmission and control the current pandemic will be necessary, since the transmission dynamics is very similar in all places where the number of infected and dead people is high. furthermore, these results suggest that measures to prevent or reduce transmission should be urgently implemented in populations at risk [ ] . for this reason, all government spheres worldwide, together with regulatory agencies from the health area, recommended complete social distancing as an emergency measure to contain the spread of sars-cov- through interpersonal contact. it is important to highlight that, through the experience of chinese and italians, the possibility of sars-cov- transmission before the onset of symptoms cannot be excluded and although it seems uncommon, there is evidence that individuals who remain asymptomatic can transmit the virus. these data suggest that the use of isolation is the best way to contain the sars-cov- -associated pandemic [ ] . china's cdc and local cdcs used the chinese experience in the sars-cov- outbreak to conduct investigations that determined the viral incubation time. they demonstrated that this period can vary from to days to weeks, since the longest time from infection to symptoms was . days ( % ci, . - ) [ ] . in addition, these results showed that the new pandemic doubled every seven days, while the basic reproduction number (r -r zero) is . , that is, each patient transmits the infection to an additional . individuals, on average. it is important to note that the r estimates of the sars-cov epidemic in - were approximately [ ] . for viral infection to occur, the connection of the virus to a receptor expressed by host cells is the first step, followed by fusion with the cell membrane. specifically for sars-cov- , it is possible to deduce that the epithelial cells of lungs are its main target, since in part of infected patients, covid- progresses as an acute respiratory infection [ ] . previous studies have shown that human-to-human sars-cov transmission occurs by the association between the binding domain to the spike receptor located on the viral envelope and the cell receptor. recently, it was identified that cell receptor for sars-cov is the angiotensin-converting enzyme (ace ) [ , ] . it is important to note that the sequence of the binding domain to the spike receptor of the sars-cov- viral envelope is similar to that of sars-cov. these data strongly suggest that the entry into host cells occurs through the ace receptor ( figure ) [ ] . infection to an additional . individuals, on average. it is important to note that the r estimates of the sars-cov epidemic in - were approximately [ ] . for viral infection to occur, the connection of the virus to a receptor expressed by host cells is the first step, followed by fusion with the cell membrane. specifically for sars-cov- , it is possible to deduce that the epithelial cells of lungs are its main target, since in part of infected patients, covid- progresses as an acute respiratory infection [ ] . previous studies have shown that human-to-human sars-cov transmission occurs by the association between the binding domain to the spike receptor located on the viral envelope and the cell receptor. recently, it was identified that cell receptor for sars-cov is the angiotensin-converting enzyme (ace ) [ , ] . it is important to note that the sequence of the binding domain to the spike receptor of the sars-cov- viral envelope is similar to that of sars-cov. these data strongly suggest that the entry into host cells occurs through the ace receptor ( figure ) [ ] . it is assumed that covs that cause covid- in humans are related to bats (upper left corner). this selected subset of viruses has the necessary resources to infect the human respiratory tract (lower left corner), with a certain tropism for this system. infection (right panel) requires the interaction of spike proteins present in the sars-cov- viral envelope (s proteins) with host sites for type angiotensin-converting enzyme (ace ) present in the lung. subsequently, proteases present on the surface of pneumocytes cleave the s region of the s protein, the subunit responsible for the fusion of the s protein with the cell membrane. after cleavage, a series of conformational changes are triggered, resulting in the fusion between viral envelope and the target cell membrane. the structural features of sars-cov- that can facilitate infection in humans include: ( ) presence of reasons for binding to the s b receptor (rbms) (in purple) that bind to ace orthologous receptors. ace is believed to be orthologous because it exhibits homology to s b rbms (since they complement each other to the point of binding) and were probably duplicated from a common ancestor, shared by the two underlying sister species, where in the course of evolution, both receptors gradually differentiate but continue to have affinity for each other; ( ) an s a domain that provides additional interactions with the host and; ( ) a cleavage substrate for a furin protease (represented by the green starry shape bound to the protease at the bottom right of the figure . infection of pneumococytes during covid- . it is assumed that covs that cause covid- in humans are related to bats (upper left corner). this selected subset of viruses has the necessary resources to infect the human respiratory tract (lower left corner), with a certain tropism for this system. infection (right panel) requires the interaction of spike proteins present in the sars-cov- viral envelope (s proteins) with host sites for type angiotensin-converting enzyme (ace ) present in the lung. subsequently, proteases present on the surface of pneumocytes cleave the s region of the s protein, the subunit responsible for the fusion of the s protein with the cell membrane. after cleavage, a series of conformational changes are triggered, resulting in the fusion between viral envelope and the target cell membrane. the structural features of sars-cov- that can facilitate infection in humans include: ( ) presence of reasons for binding to the s b receptor (rbms) (in purple) that bind to ace orthologous receptors. ace is believed to be orthologous because it exhibits homology to s b rbms (since they complement each other to the point of binding) and were probably duplicated from a common ancestor, shared by the two underlying sister species, where in the course of evolution, both receptors gradually differentiate but continue to have affinity for each other; ( ) an s a domain that provides additional interactions with the host and; ( ) a cleavage substrate for a furin protease (represented by the green starry shape bound to the protease at the bottom right of the figure), which can provide greater sensitivity to cleavages by host proteases. anti-cov antibodies (shown at the bottom right of the figure) can prevent infection through the following mechanisms: (a) binding to s brbms of the virus, blocking access to ace receptor and consequently preventing the continuation of the process of virus fusion with the target cell; (b) distal connection in relation to rbms, generating steric impairment and, consequently, blocking the connection between the virus rbms and the ace receptor of the host cell; (c) binding in the s a region of the viral spike, blocking alternative connections to different receptors; and (d) binding to s , the region responsible for the fusion of the virus with the membrane of the target cell, consequently preventing fusion. as future perspectives, future research should aim at the development of protease inhibitor antiviral compounds, which play a crucial role in the fusion of the virus to the host cell membrane, suppressing the entry of the virus [ ] . note: this image was developed using the coreldraw software ( corel corporation id ). in a recent study, [ ] demonstrated the interactions of sars-cov- with the host organism, specifically interactions between spike proteins ("coronan") located in the viral envelope (s proteins), projected from the envelope, and the host ace receptors. covs derived from the most virulent bats are those in which s proteins have tropism by distinctly human receptors, as is the case with sars-cov- . after the first contact with humans, the virus migrates to lungs and s proteins interact with various proteins and receptors, which are factors of host susceptibility. such interactions cause substantial changes in the conformation of proteins involved in the infection process of host cells, triggering the fusion between virus and human cells and, consequently, the infection. it is noteworthy that specific antibodies against s proteins and antiviral agents that block the link between virus and human susceptibility factors can prevent sars-cov- infection [ ] (figure ). studies like these have shown that there is a wide spread of covs in their natural reservoirs and by demonstrating interactions between virus and human hosts and, consequently, how the infectious process occurs, they drive the development of vaccines and antiviral drugs. in addition, further studies should be aimed at the active surveillance of these viruses in geographic regions that go beyond the beginning of the sars-cov- pandemic outbreak. in the long term, broad-spectrum antiviral drugs and vaccines should be developed with the aim of controlling emerging infectious diseases caused by covs. in addition, regulatory agencies in the health sector should develop specific regulations to control domestication and consumption of wild animals. the emergence of the highly pathogenic sars-cov- led to the need for deeper knowledge on the biology and pathogenesis of cov. an emerging theme in the cov pathogenesis and covid- pathophysiology is the interaction between specific viral genes and the host's immune system, which acts as a key determinant in the regulation of disease virulence and development results. viral interactions with innate immune system play a key role in determining the course of the infection. the initial control of viral replication by type i interferons (ifn), complement system proteins and other innate immune mediators limit viral spread in the host during the early stages of the disease [ ] . in addition, the initial innate response also plays a key role in the development of the subsequent adaptive immune response [ ] . despite that, it is well established in current literature that the hyperactive innate immune response can also result in pathology and subsequent tissue damage. regarding innate immunity, there are two main pathways by which cells detect viruses that invade the organism and activate the ifn pathway [ ] . toll-like receptors (tlrs), which include tlr , tlr , tlr and tlr , are responsible for detecting viruses in endosomal compartments as they penetrate host cells. the cytoplasmic domain of tlrs (called card) contains two specific rna-helicases, rig-i and mda , which detect viral rna in the cell cytoplasm. for activation of both pathways, interactions between sensors with pathogen-associated molecular patterns (pamps) are required, such as, for example, single-stranded rnas associated with viral genomes or double-stranded rna, which is a by-product of viral replication, being common targets. different adapter proteins participate in signal transduction in the induction pathways of tlrs and cytoplasmic ifn. the tlr-dependent pathway uses tir-domain-containing adapter-inducing interferon-beta adapter proteins (trif) and/or the myeloid differentiation factor (myd ) and the ifn cytoplasmic induction pathway uses the mavs/ips- /visa/cardif mitochondrial adapter protein [ ] (figure ). the tlr-dependent pathway uses tir-domain-containing adapter-inducing interferon-beta adapter proteins (trif) and/or the myeloid differentiation factor (myd ) and the ifn cytoplasmic induction pathway uses the mavs/ips- /visa/cardif mitochondrial adapter protein [ ] ( figure ). after internalized, the virus exposes the genomic rna to the dsrna detection mechanism in the cell, that is, tlr , rigi and mda . these proteins are responsible for the irf- cascade signaling, leading to ifnb induction and, consequently, the production of ifnβ protein. the newly synthesized ifnβ can bind to ifn receptors on the surface of the same cell or surrounding cells and induce the synthesis of more ifn molecules. binding to ifn receptors activates the signal transducer and activator of transcription (stat ) signaling pathway to activate several distinct antiviral genes located in isre promoter elements [ , ] . note: this image was developed using the coreldraw software ( corel corporation id ). the complement system plays a crucial role in the innate immune response to viruses. it represents one of the main factors responsible for the development of pro-inflammatory reactions during these diseases [ , ] . research has shown that, just as in human infection, intranasal sars-cov infection in c bl/ j mice, results in virus replication with high rates in the lung, in addition to induction of inflammatory cytokines and chemokines and infiltration of immune cells in the pneumocytes [ ] . using c -deficient mice (c -/-), these researchers demonstrated that animals c -/-infected with sars-cov lost less weight and obtained a significant reduction in respiratory dysfunction when compared to control groups, despite viral loads equivalent in the lung. besides, a significantly lower rate of neutrophils and inflammatory monocytes were present in the lungs of c -/-mice than in the c bl/ j controls, and subsequent studies showed that lung injury was reduced, as were cytokine (il- , mainly) and chemokine levels in lungs and serum of c -/-mice than when purchased from animals in the control group [ ] . this suggests that c inhibition may significantly reduce the pulmonary inflammatory complications of sars-cov- infection. the decrease in neutrophilic infiltration in the lungs and the reduced levels of intrapulmonary and plasma il- that have been observed in mice with c -/-infected with sars-cov suggest a potential treatment that combines c inhibitors and anti-il- drugs [ ] . besides, as c is one of the first proteins synthesized by the complement system in the innate immune cascade, the anti-inflammatory potential for c blockage is broader. drugs like amy- are being investigated for this [ ] , which is currently being tested in patients with covid- [ ] . c blockade can simultaneously inhibit the synthesis of c a and c a. furthermore, this block reduces the intrapulmonary activation of c and the release of il- after internalized, the virus exposes the genomic rna to the dsrna detection mechanism in the cell, that is, tlr , rigi and mda . these proteins are responsible for the irf- cascade signaling, leading to ifnb induction and, consequently, the production of ifnβ protein. the newly synthesized ifnβ can bind to ifn receptors on the surface of the same cell or surrounding cells and induce the synthesis of more ifn molecules. binding to ifn receptors activates the signal transducer and activator of transcription (stat ) signaling pathway to activate several distinct antiviral genes located in isre promoter elements [ , ] . note: this image was developed using the coreldraw software ( corel corporation id ). the complement system plays a crucial role in the innate immune response to viruses. it represents one of the main factors responsible for the development of pro-inflammatory reactions during these diseases [ , ] . research has shown that, just as in human infection, intranasal sars-cov infection in c bl/ j mice, results in virus replication with high rates in the lung, in addition to induction of inflammatory cytokines and chemokines and infiltration of immune cells in the pneumocytes [ ] . using c -deficient mice (c -/-), these researchers demonstrated that animals c -/infected with sars-cov lost less weight and obtained a significant reduction in respiratory dysfunction when compared to control groups, despite viral loads equivalent in the lung. besides, a significantly lower rate of neutrophils and inflammatory monocytes were present in the lungs of c -/mice than in the c bl/ j controls, and subsequent studies showed that lung injury was reduced, as were cytokine (il- , mainly) and chemokine levels in lungs and serum of c -/mice than when purchased from animals in the control group [ ] . this suggests that c inhibition may significantly reduce the pulmonary inflammatory complications of sars-cov- infection. the decrease in neutrophilic infiltration in the lungs and the reduced levels of intrapulmonary and plasma il- that have been observed in mice with c -/infected with sars-cov suggest a potential treatment that combines c inhibitors and anti-il- drugs [ ] . besides, as c is one of the first proteins synthesized by the complement system in the innate immune cascade, the anti-inflammatory potential for c blockage is broader. drugs like amy- are being investigated for this [ ] , which is currently being tested in patients with covid- [ ] . c blockade can simultaneously inhibit the synthesis of c a and c a. furthermore, this block reduces the intrapulmonary activation of c and the release of il- from alveolar macrophages or other cells that express c a receptors (c ars) and/or c a receptors (c ars), causing the beneficial evolution and cure of lung injury [ ] . the role of complement system activation in the development of severe acute respiratory syndrome associated with sars-cov- infection is not yet fully understood, and clinical data are scarce. recent research has shown that the sars-cov, mers-cov, and sars-cov- "n" proteins bind to masp- , the essential serine protease in the complement activation lectin pathway, leading to over complement activation and lung injury severe inflammatory disease. thus, it is evident that the blockade between the interaction of protein n with masp- can significantly reduce the hyperactivation of complement induced by protein n and lung injury in vitro and in vivo. complement overactivation is present in patients with covid- , and a promising suppressive effect has been seen in patients with severe lung injury treated with an anti-c a monoclonal antibody [ ] . due to the cascade organization of the complement system, inhibitors that target c or its upstream activators may be more effective and, potentially, may prevent the initial stages of the infectious process that lead to severe lung inflammation. despite this, no substance that acts with these mechanisms of action has been approved for use in humans, although phase ii clinical studies are already underway [ ] . figure outlines the moment when complement system inhibitors can be used in lung injury associated with sars-cov- . molecules , , x for peer review of figure . a possible scheme for using complement system inhibitors in lung injury associated with sars-cov- : (a) sars-cov- penetrates the host's pneumocytes and uses the cellular machinery for protein synthesis and replication of the genetic material and causing activation of the complement system through different pathways; (b) complement activation contributes to the massive inflammatory response of pneumocytes observed in some patients with severe covid- . the inhibition of c or c can have significant therapeutic potential [ ] . note: this image was developed using the coreldraw software ( corel corporation id ). the mechanism of natural sars-cov- infection occurs very similar to previously studied sars-cov [ ] and produces adaptive immune responses against the structural antigens of these viruses in humans and animals [ ] . spike glycoproteins located in the viral envelope (s proteins) are figure . a possible scheme for using complement system inhibitors in lung injury associated with sars-cov- : (a) sars-cov- penetrates the host's pneumocytes and uses the cellular machinery for protein synthesis and replication of the genetic material and causing activation of the complement system through different pathways; (b) complement activation contributes to the massive inflammatory response of pneumocytes observed in some patients with severe covid- . the inhibition of c or c can have significant therapeutic potential [ ] . note: this image was developed using the coreldraw software ( corel corporation id ). the mechanism of natural sars-cov- infection occurs very similar to previously studied sars-cov [ ] and produces adaptive immune responses against the structural antigens of these viruses in humans and animals [ ] . spike glycoproteins located in the viral envelope (s proteins) are responsible for binding to the cell receptor and fusing the virus membrane with the host's cell membrane [ ] . these glycoproteins also act as antigens responsible for the activation of t cells and development of humoral and cellular immunity [ ] . literature data have demonstrated that the majority of antigenic peptides are located in structural proteins (mainly s protein) [ , ] . it is believed that hypervirulent cov variants, such as sars-cov- , have the capacity to develop viremia, producing systemic disease, being often fatal [ , [ ] [ ] [ ] [ ] [ ] [ ] . previous studies have shown that sars-cov infection of macrophages and dendritic cells leads to an aberrant cytokine/chemokine expression pattern [ ] , so that the ability to infect and replicate in such phagocytes appears to be a determinant factor for establishing the course of viral infection. in addition, it has recently been reported that sars-cov- causes lymphocyte depletion, resulting in high viral titers [ , , , ] . previous studies demonstrate the spread of sars-cov to lymphoid organs such as spleen, thymus, peyer plaques and mesenteric lymph nodes, developing intense lymphoid depletion [ , , ] . it is already well established in literature that lymphocytes and their subtypes are essential to maintain the immune system function in basal activity or during infectious processes. viral infections, immunodeficiency syndromes and other infectious diseases, such as tuberculosis, are known to determine abnormal plasma counts in lymphocyte levels and their subtypes [ ] [ ] [ ] ; and for such infections to evolve into more serious events, lymphopenia is crucial, since the reduction of this cell type is necessary for the survival and persistence of many microorganisms. in wuhan, where sars-cov- infection started, % of patients with covid- had significant lymphopenia induction [ ] . although the mechanisms that cause lymphopenia in cov infections are not fully understood, it is already known that lymphocytes can undergo lysis by direct or indirect viral action [ ] . previous studies have shown that indirect events, secondary to viral infection, are the main responsible for cell lysis through the synthesis of soluble factors such as ifn, cytokines and chemokines by host cells [ , ] . as already mentioned, very recent studies have identified that the sars-cov cell receptor is ace and that the sequence of the binding domain to the spike receptor of the sars-cov- viral envelope is similar to that of sars-cov, suggesting that the entry into host cells occurs through the ace receptor [ ] . however, the presence of ace receptors has been identified in the oral mucosa, in type ii pneumocytes, along the intestine and in the renal and cardiac endotheliums [ ] , with no evidence of the presence of these receptors in circulating mature lymphocytes. for this reason, it is believed that the cytolytic effect of sars-cov- that led infected patients to develop lymphopenia is not direct, that is, caused by the virus itself, since the receptor determines the tropism of the pathogen by the tissue. in addition, previous studies have demonstrated the presence of cov rna in lymphoid tissues, such as the thymus, in infected patients. this suggests that one of the lymphopenia mechanisms in these patients may be related to the reduction in the production of mature lymphocytes by the thymus, which is the target organ in several infectious diseases, including coronavirus [ ] . since these viruses infect the thymus and cause tissue damage, there is an important interference in the lymphocyte differentiation process, which can cause lymphopenia [ ] . it is well established in literature that t cells are crucial to produce immunoglobulins and, consequently, for the development of humoral and memory immunity. therefore, pharmacological treatments or the use of integrative and complementary practices that prevent the reduction or increase the levels of cells of the immune system can be essential tools in combination with treatment and/or prevention of sars-cov- infection. the use of bioinformatics and other computational tools in addition to molecular modeling has helped researchers from different areas in the search for strategies for diagnosing viral infection, in the development of vaccines for its prevention, as well as in the discovery of new anti-sars-cov- agents. the knowledge of the genome of a species based on the genetic sequencing technique is the starting point for the structure and function of its genes to be understood. in the context of diseases transmitted by microorganisms, such as sars-cov- , the mapping of the genome of microorganisms collected from infected patients in different regions of the world also allows tracing a transmission profile, including its dissemination in different regions and countries, contributing to the search for strategies to combat the disease and monitor mutations [ , ] . data from the ncbi genbank ® gene sequence database https://www.ncbi.nlm.nih.gov/genbank) accessed on / / indicated more than , nucleotide sequences inserted since december for sars-cov- , most of them coming from cities in china and the usa. in february , researchers from the university of são paulo and instituto adolf lutz, in brazil, together with researchers from the university of oxford, in the united kingdom, managed to elucidate and publish the complete gene sequence of the virus (genbank accession number mt ) obtained from of the first confirmed cases of the disease in brazil within just two days from the confirmation of the diagnosis [ ] . the speed in obtaining this information is only possible through the application of bioinformatics techniques. bioinformatics showed great advance in the s, mainly in view of the development of the genomics area, which generated large amount of biological data incompatible to be quickly analyzed in a manual way. the rapid and adequate manipulation of these data has been possible through the application of data comparison and analysis software and easy access to previously available data from the sharing and storage of information in virtual databases [ ] . in view of experimentally obtained sequences, it is possible to perform the alignment of these sequences to other sequences available in virtual databases, leading to the knowledge of the family to which the microorganism is related [ , , ] . in one of the first studies on the new virus that caused respiratory infections in china, the researchers were able to determine from the sequencing of rna obtained from bronchoalveolar fluid samples that the etiologic agent was rna virus of the coronaviridae family. in addition, using bioinformatics tools, it was possible to perform a phylogenetic analysis, revealing . % similarity of the nucleotide sequence of this virus with a group of coronaviruses of the genus betacoronavirus already identified in bats in china, which gave evidence of the virus origin [ ] (figure ). one of the algorithms most widely used for comparing biological sequences, whether from nucleotides to nucleic acids or from amino acids to proteins, is the blast (basic local alignment search tool) [ ] . the diversity of information on biological sequences from organisms of different degrees of complexity allows blast to be a starting point to understand the genetic relationship with other species, also tracing a possible origin. the use of such tool allowed [ ] to identify high similarity between sars-cov- genomic sequence extracted from ncbi genbank ® (genbank accession number mn ) and viral metagenomas found in the pangolin mammal present in the database. these studies, in turn, guided analyses that led to findings of high amino acid identity of s, e, m and n genes of pangolin coronavirus with those isolated from humans, suggesting that the virus capable of infecting humans may have emerged from a recombination of coronaviruses isolated from bat and pangolin. the large capacity for storing information in virtual databases made possible by advances in the field of information technology allows these databases to be continuously updated with new genomic sequences to be universally available. in the context of covid- , this characteristic was important for a better understanding of the origin of sars-cov- from the comparative analysis of genomic data of the new virus with others from the same family, suggesting its origin from natural selection, with modifications in its spike protein, more specifically in the host receptor binding domain, which may have enhanced its interaction and recognition by the human cell [ , ] . related [ , , ] . in one of the first studies on the new virus that caused respiratory infections in china, the researchers were able to determine from the sequencing of rna obtained from bronchoalveolar fluid samples that the etiologic agent was rna virus of the coronaviridae family. in addition, using bioinformatics tools, it was possible to perform a phylogenetic analysis, revealing . % similarity of the nucleotide sequence of this virus with a group of coronaviruses of the genus betacoronavirus already identified in bats in china, which gave evidence of the virus origin [ ] ( figure ). figure . bioinformatics as technologies applied to health as allies to coping with the disease: bioinformatics is a technology that assists researchers in coping with diseases by investigating genetic sequencing and seeking structural models of potential molecular targets present in sars-cov- . note: this image was developed using the coreldraw software ( corel corporation id ). one of the algorithms most widely used for comparing biological sequences, whether from nucleotides to nucleic acids or from amino acids to proteins, is the blast (basic local alignment figure . bioinformatics as technologies applied to health as allies to coping with the disease: bioinformatics is a technology that assists researchers in coping with diseases by investigating genetic sequencing and seeking structural models of potential molecular targets present in sars-cov- . note: this image was developed using the coreldraw software ( corel corporation id ). the rt-pcr test is considered the gold standard for diagnosing covid- [ ] . by this technique, the reverse transcriptase enzyme initially participates in transforming the viral rna into complementary dna (cdna). then, the genetic material is amplified. in this process, the regions of the genetic material to be amplified are recognized by polymerase by means of oligonucleotides complementary to the region of interest called primers [ ] . the step of primer selection is essential to ensure the quality of the analysis and requires a thorough analysis of the reference genetic sequence [ ] . it is also important to consider that recognition is specific, and the method can be applied worldwide, that is, genetic material conserved regions should be prioritized over regions of low similarity. in this context, bioinformatics can contribute by allowing the design of primers based on the comparison of different genetic sequences from different geographic regions, available in virtual databases, identifying regions conserved in the genome, which would enhance the method's specificity [ ] . in the work of [ ] , which is also recommended by who as a reference in primers to be used in rt-pcr tests, researchers used complete and partial genetic sequences of sars-related viruses present in the ncbi genbank ® database for the design of primers and, in view of the first publications of sars-cov- sequence on virological.org [ ] and gisaid [ ] , it was possible to align the primers proposed for these sequences and to select those with greater correspondence. several online servers and software have been used in order to optimize this process [ , , ] . one of the main strategies to control viral diseases has been vaccination prevention. the development of a vaccine involves the identification of either synthetic or natural antigens that can prevent or even treat a disease. for viral diseases, the most explored strategies involve the use of attenuated viruses in their composition, use of viral structures that can be neutralized by the immune system, or application of part of the viral genetic material related to the expression of proteins that can behave as antigens for the development of antibodies against the pathogen of origin [ ] . regardless of strategy to be used, knowledge about the viral genome and the structure and function of virus proteins is essential for a vaccine to be successfully developed. in this context, bioinformatics tools have also contributed to greater quality and speed in the process. the proposal of vaccines based on b and t-cell epitopes has been a possible strategy for obtaining vaccines for covid- prevention. such epitopes can be predicted from the analysis of amino acid sequences of proteins of the sars-cov- pathogen and comparison with virtual epitope databases [ , ] . even without studies on specific immune responses against sars-cov- , the genetic similarity between this virus and sars-cov obtained from sequential alignment and phylogenetic analysis of genomes indicates that possible common epitopes related to structural s and n proteins could be incorporated into vaccines to be developed, in view of the immunological response already observed against sars-cov structures [ ] . the search for regions conserved in genetic sequences of different coronavirus species aided by bioinformatics tools has also been a way to identify sequences of amino acids that can be models for the design of specific synthetic epitopes to compose vaccines for covid- prevention [ ] . the discovery and planning of new drugs that can be used in the treatment of covid- benefited from the several advances observed over the last decades in the field of bioinformatics. a potential antiviral agent should target processes or macromolecules essential for maintaining the cycle of viral replication and infection of new host cells [ ] . to do so, knowing the genetic sequence of the virus is not enough, but it is also important to determine the functions related to each of the genes that compose it. the sequential identity between genetic materials of different organisms may indicate equivalent genes in terms of function [ ] , which encode common proteins that can be exploited as molecular targets for the action of drugs aimed at the treatment of viral infections, for example [ ] . prior knowledge of potential molecular targets in other coronaviruses combined with the similarity of genetic sequences among viruses of this family opens several doors for the discovery of new bioactive compounds aided by computational tools [ ] . to modulate the action of a molecular target, it is important to know its three-dimensional structure. the determination of this structure is essential for the understanding of how its connection site is organized, how it interferes with its mechanism of action and, given this information, how agents that interfere with its functioning can be proposed. information on the structural organization of a protein is obtained by various techniques such as x-ray crystallography [ ] , cryogenic electron microscopy [ ] and nuclear magnetic resonance (nmr) [ ] . however, bioinformatics tools are those that enable the detailed analysis of data obtained by these techniques and, consequently, the proposition and refinement of three-dimensional models, also exploring comparative analyses with models already proposed [ ] . in the same way that there are virtual databases powered by genetic sequences, advances in bioinformatics have also enabled the creation of virtual databases of models of three-dimensional structures of biomacromolecules, which data are freely accessible and can be downloaded and viewed using visualization and molecular modeling software. the most popular three-dimensional structure database is the protein data bank (pdb-www.rcsb.org), with more than , structures inserted so far [ ] . so far, more than structural models of sars-cov- macromolecules were deposited in the pdb. most refer to structures proposed for the main protease complexed or not with possible inhibitors [ , ] , but structural models can also be found for the nucleocapsid protein, non-structural proteins nsp , nsp , nsp , nsp [ ] and nsp and spike glycoprotein [ , ] . the determination of structural models of the main sars-cov- protease, an important enzyme in the processing of polyproteins translated from viral rna in free forms and complexed with the α-ketoamide b inhibitor [ ] started from obtaining crystallographic data via x-ray diffraction. structural models were then determined by the molecular substitution method, using as reference a structural model from sars-cov already available in pdb, with % sequential identity. previously determined sars-cov spike glycoprotein models were also used as a reference in the construction of models of the same sars-cov- protein, whose structural data had been obtained from cryogenic electron microscopy [ ] . the construction of structural models using crystallographic data as a reference allows the knowledge of the real arrangement of the structure under given experimental condition. when determining the structure of the main protease complexed with b inhibitor, [ ] were able to "visualize" at molecular level, aided by computational tools, the main points of interaction between inhibitor and enzyme, which may contribute to direct structural modifications that lead to the optimization of the compound's inhibitory activity. likewise, the knowledge of interactions involved in the protein-protein complex between the rbd domain of the sars-cov- spike glycoprotein and the human cell ace receptor helps to better understand how viral recognition occurs and the consequent search for epitopes conserved in this viral protein that can be targets of neutralizing antibodies to enable the production of vaccines [ ] . obtaining crystals from biomolecules; however, can be a laborious or even unfeasible process [ ] . in these situations, we choose to build structural models using homology modeling. by this technique, a macromolecule of known three-dimensional structure with considerable degree of sequential identity to that to be determined is used as reference for the construction of this structural model [ ] . comparative studies between models determined with the aid of crystallography and models obtained by homology modeling indicate that they have high prediction quality and can be widely applied in studies of interaction between ligand and molecular target [ ] [ ] [ ] . considering the urgency of obtaining structural models of potential molecular targets present in sars-cov- and the availability of models of three-dimensional protein structure, mainly of other viruses of the coronaviridae family, with sequential similarity to those of the virus that causes covid- , homology modeling is the fastest, cheapest and most accessible way for structural determination. thus, many research groups have used this strategy to obtain structural models of different sars-cov- proteins, such as the nucleocapsid protein [ ] , envelope protein, orf a protein [ , ] , non-structural proteins (nsp) [ , , ] , clpro [ , ] and spike glycoprotein [ , , ] , which have been used for the discovery of antiviral agents and the development of vaccines against covid- . the use of molecular modeling at the early stages of searching for new bioactive compounds has contributed for the faster development of new drugs at reduced costs. in view of the urgency for effective treatments of covid- , virtual screening methods for bioactive compounds end up by optimizing this discovery pathway by allowing the initial selection of those that present steric and electronic similarities with compounds of known activity (drug planning based on the ligand) or that have the potential to strongly bind to the molecular target of interest (structure-based drug planning) [ ] . in view of the low number of compounds with known anti-sars-cov- activity and considering the availability of structural models of potential molecular targets of the virus, the strategy based on the structure of the molecular target has been further explored, in which molecular docking techniques play a key role in this planning method. molecular docking consists of anchoring a molecule at the binding site of a molecular target, evaluating its assumed conformations, always searching for the one that generates the lowest-energy complex or the most stable with the target [ ] . the binding energy of this complex can be related to its ability to interact and thus modulate the target's action. based on this information, it is possible to select, from virtual databases with a multitude of compounds, potential inhibitors, agonists, or antagonists, against a specific target, which will be hit compounds for more targeted structural changes. in this process of identifying new hit compounds via virtual screening, virtual libraries of chemical compounds, such as zinc databases (https://zinc.docking.org/) [ ] and pubchem (https: //pubchem.ncbi.nlm.nih.gov/) [ ] , offer a great diversity regarding structural patterns and origin (natural or synthetic), which can contribute to higher quality in the search process. these two databases were explored by [ ] to identify possible inhibitors of the sars-cov- main protease. from these chemical libraries, researchers selected compounds that already had the ability to inhibit proteases found in other organisms, totaling more than tested compounds, which were submitted to molecular docking studies against the main protease (pdb id lu ), thus determining the interaction energy of the target-ligand complex. in this study, the pubchem cid , compound (figure a) showed the greatest potential for inhibiting the enzyme, forming the most stable complex with the main protease. there are published works that have focused on the screening of compounds of natural origin, which have great historical importance in the process of discovering new drugs [ ] . libraries of phytochemical compounds with antiviral action from traditional chinese medicine plants have already been virtually screened against different sars-cov- molecular targets such as the clpro protein [ , ] , papain-like protease (plpro) and spike glycoprotein [ ] . among selected compounds, pubchem cid , , [ ] and quercetin [ ] showed strong interaction with clpro; dihydrotanshinone i with the spike glycoprotein; and cryptotanshinone, with both plpro there are published works that have focused on the screening of compounds of natural origin, which have great historical importance in the process of discovering new drugs [ ] . libraries of phytochemical compounds with antiviral action from traditional chinese medicine plants have already been virtually screened against different sars-cov- molecular targets such as the clpro protein [ , ] , papain-like protease (plpro) and spike glycoprotein [ ] . among selected compounds, pubchem cid , , [ ] and quercetin [ ] showed strong interaction with clpro; dihydrotanshinone i with the spike glycoprotein; and cryptotanshinone, with both plpro and clpro proteins [ ] (figure a ). in addition to phytochemicals, compounds of marine origin have also been in silico evaluated against the sars-cov- main protease [ ] . the virtual screening strategy adopted by the authors involved the initial construction of a pharmacophoric model based on the structure, which was created from information on the structure of the enzyme cocrystallized with n inhibitor (pdb id lu ). in this work, the virtual screening of structures from libraries of marine compounds was then carried out using the pharmacophoric model created as a molecular filter to select those that presented structural characteristics corresponding to the created model. subsequently, the selected compounds were submitted to anchoring at the active site of the enzyme as a way of assessing complementarity with the target. different compounds were identified as potential main protease inhibitors in this analysis, highlighting the compound with the greatest potential heptafuhalol a (figure a ). in addition to the virtual screening strategies adopted, another that has stood out among works searching for new anti-sars-cov- agents is the one that uses drug groups already approved, preferably those with antiviral action, such as search libraries, characterizing a drug repositioning strategy. the interest in studying compounds already in pharmaceutical use for other purposes has the advantages of reducing time and costs mainly related to the stage of drug discovery, but above all the fact of knowing the toxicological profile of the compound, previously attested in clinical trials [ ] . in two studies found in literature, the use of a set of drugs obtained from the zinc database [ ] was observed as a library of screening compounds. in the work of [ ] , the interaction between this set of drugs and the sars-cov- spike glycoprotein was evaluated, highlighting as a result the high potential for interaction between digitoxin and zorubicin (figure b ). in the work of [ ] , the set of drugs obtained from zinc was virtually evaluated regarding their interaction with different probable molecular targets of the virus, highlighting the potential of ribavirin against plpro; limecycline against clpro and valganciclovir against rna-dependent rna polymerase (rdrp) (figure b ). in the study by [ ] , the drugs analyzed were obtained from the sweetlead database [ ] and screened against the spike glycoprotein, with drug pemirolast (figure b ) being indicated as a promising inhibitor of the action of this glycoprotein. [ ] conducted in silico evaluation against sars-cov- rdrp only the potential inhibitor of drugs or compounds in clinical trials with anti-hcv action. among drugs tested, the potential of ribavirin is highlighted, which had also demonstrated potential antiviral action in the work of [ ] , but acting against the plpro target ( figure b ). currently, a comprehensive and standardized repository of knowledge of the interaction mechanisms between sars-cov- and the host has been created. the repository is called covid- disease map and was built with the contribution of experts using the results of published research, including bioinformatics data. this technology is a platform that contributes to the computational analysis of molecular processes involved in the -ncoc input and replication interactions in the host. besides, information about the immune system's performance, recovery of host cells, and repair mechanisms can be obtained through the covid- disease map [ ] . figure demonstrates the objective and the initial layout of the map and its operating cycle. a recent study involving the phylogenetic analysis of complete genomes of covid- , researchers revealed three central variants distinguished by changes in amino acids, which were called variants a, b, and c, with a being the ancestral type according to the bat outgroup coronavirus. viral strains a and c have been identified in many countries outside east asia, that is, in europe and the united states of america. in contrast, variant b is the most common type in east asia, and there is no evidence that its ancestral genome spread outside east asia without first undergoing mutations [ ] . this update is carried out according to the materials available in databases on the subject to support visual and computational exploration, as well as efforts to model diseases [ ] . note: this image was developed using the coreldraw software ( corel corporation id ). a recent study involving the phylogenetic analysis of complete genomes of covid- , researchers revealed three central variants distinguished by changes in amino acids, which were called variants a, b, and c, with a being the ancestral type according to the bat outgroup coronavirus. viral strains a and c have been identified in many countries outside east asia, that is, in europe and the united states of america. in contrast, variant b is the most common type in east asia, and there is no evidence that its ancestral genome spread outside east asia without first undergoing mutations [ ] . a phylogenetic analysis carried out in turkey, with the first genomes isolated from sars-cov- in this region, showed that the introduction of the virus in the country occurred before the first reported case of infection. the study demonstrated that the virus circulated in turkey from several independent international introductions and revealed a hub for inland transmission [ ] . another phylogenetic analysis carried out recently sequenced nine viral genomes of patients with covid- previously reported in connecticut, united states. the research placed most of these genomes with viruses sequenced from washington state. interestingly, when the researchers combined the genomic data obtained in the study with national and international travel patterns, they found that domestic introductions probably drove the initial transmission of sars-cov- in connecticut. this study provides evidence for the sustained and widespread transmission of sars-cov- in the usa and highlights the critical need for local surveillance [ ] . important research on the evolutionary and epidemiological dynamics of the current outbreak of covid- analyzed genomes of sars-cov- strains collected from china and other countries with sampling dates between december and february . the phylogenetic study results showed that the time to the most recent common ancestor was november , and the evolutionary rate of sars-cov- was . × − substitutions per site per year. these results highlight that the use of phylodynamic analyzes is crucial to provide insights into the possibilities of interventions to limit the spread of sars-cov- worldwide [ ] . disease map is selected and reviewed from databases and knowledge continuously. this update is carried out according to the materials available in databases on the subject to support visual and computational exploration, as well as efforts to model diseases [ ] . note: this image was developed using the coreldraw software ( corel corporation id ). a phylogenetic analysis carried out in turkey, with the first genomes isolated from sars-cov- in this region, showed that the introduction of the virus in the country occurred before the first reported case of infection. the study demonstrated that the virus circulated in turkey from several independent international introductions and revealed a hub for inland transmission [ ] . another phylogenetic analysis carried out recently sequenced nine viral genomes of patients with covid- previously reported in connecticut, united states. the research placed most of these genomes with viruses sequenced from washington state. interestingly, when the researchers combined the genomic data obtained in the study with national and international travel patterns, they found that domestic introductions probably drove the initial transmission of sars-cov- in connecticut. this study provides evidence for the sustained and widespread transmission of sars-cov- in the usa and highlights the critical need for local surveillance [ ] . important research on the evolutionary and epidemiological dynamics of the current outbreak of covid- analyzed genomes of sars-cov- strains collected from china and other countries with sampling dates between december and february . the phylogenetic study results showed that the time to the most recent common ancestor was november , and the evolutionary rate of sars-cov- was . × − substitutions per site per year. these results highlight that the use of phylodynamic analyzes is crucial to provide insights into the possibilities of interventions to limit the spread of sars-cov- worldwide [ ] . from studies like these, it is possible to suggest that phylogenetic networks developed with bioinformatics aid can also be used successfully to trace undocumented sources of covid- infection. after that, cases can be reported and quarantined to prevent the disease's recurrent spread worldwide [ ] . the contributions of bioinformatics and molecular modeling in elucidating essential targets for the planning and development of new drugs, and the analysis of already known compounds, support the search for safer and more effective treatments against sars-cov- infection. identifying targets, planning interactions, understanding the structure-activity relationship, coupling, and molecular dynamics allow a projection of the mechanism of antiviral action in silico. an example of this is the prospect of using molecules of natural origin tested in vitro or in vivo and which showed significant antiviral activity. therefore, the use of software to identify viral targets and the prediction of their interaction with the molecule under analysis is necessary so that that research can proceed safely and with a higher probability of success. the verification of the performance of these compounds through computational analysis can predict physical-chemical properties, which are related to pharmacodynamic and pharmacokinetic parameters, whose knowledge is crucial when it comes to the planning of new drugs [ ] . table presents studies where the researchers tested natural compounds from plants and fungi against sars-cov- . the table shows the results of these analyzes on viral replication. it provides an essential overview of the antiviral action of phytochemicals that can be studied in bioinformatics and molecular modeling for the design of new anti-sars-cov- therapies. in an attempt to mitigate the symptoms of covid- , accelerate recovery, and reduce the mortality rate, several known drugs are being tested against sars-cov- , aiming at an antiviral action, the development of effective treatments, and prevention of infections by opportunistic microorganisms. the interaction of these drugs with proteins and viral receptors can be clarified through bioinformatics, which can identify the target and, with the support of molecular modeling, test the structural overlap, chemical interactions, and molecular coupling. studies like these are fundamental to elucidate the antiviral action mechanism of the compounds, favoring the projection of molecular modifications that modulate the affinity and specificity of the drug and, consequently, its aspects of pharmacological potency and safety. proposals for the repurposing of drugs known are a more reliable option for the development of an efficient and developed therapy with the urgency that the moment requires, to reduce the number of deaths [ ] . table presents studies on the effects of using drugs already known, and therapies with combinations of drugs, and the results of these research on the clinical condition of that infected and viral replication. these studies serve as a basis for more accurate analyzes of the drug's mechanism of action and the test outcome. in the context of the covid- pandemic, some studies and clinical reports describe the clinical worsening of some patients. the condition became known as cytokine storm or hypercytokinemia and is associated with increased mortality in infected patients. among the proposed pharmacological strategies, is the use of anti-cytokine molecules, such as anti-interleukin drugs and inhibitors of ifn-γ and tnf-α (see figure ) , capable of reducing the inflammatory process. the elucidation of the mechanisms associated with the action of immunomodulatory drugs on hypercytokinaemia is essential for the knowledge of safety regarding the use of these drugs, the best pharmacotherapeutic management, and the effectiveness to reduce the risk of death. bioinformatics, when applied to the analysis of inflammatory mediators, is a tool capable of evaluating the virus's performance and its ability to trigger an intense inflammatory response and how anti-cytokine molecules can normalize this condition. in this sense, bioinformatics aims to plan therapies that prevent the disease from worsening without causing immunodeficiency [ , ] . table shows the research results that used anti-cytokine compounds on respiratory function and inflammatory indexes in patients with covid- . the table reveals the clinical outcomes of the use of these substances, which can be investigated with the aid of bioinformatics and molecular modeling as a potential therapy against hypercytokinaemia resulting from sars-cov- infection, significantly reducing patient mortality. humans mg of aztm once daily on day , followed by mg once daily for the next days no benefit was seen [ ] mg/kg intravenously days improvement in respiratory and laboratory parameters [ ] mg/kg once daily days the treatment associated with hemoadsorption, improved gas exchange and reduced levels of inflammatory mediators [ ] sarilumab humans mg intravenously days treatment was associated with faster recovery [ ] days reduction of inflammation and rapid recovery [ ] mg _ clinical improvement and lower mortality [ ] [ ] it is crucial to mention that several of the drugs mentioned in this review have already received temporary regulatory approval. for example, remdesivir, hydroxychloroquine, chloroquine, and azithromycin are being used with consent in treatment protocols, depending on the country. the combination of these substances has also been used. despite this, studies on its effectiveness in the different stages of sars-cov- infection are controversial, and additional research is needed to elucidate the efficacy of these substances in infected patients. the use of molecular modeling at the early stages of searching for new bioactive compounds contributes to the faster development of new drugs at significantly reduced costs. given the low number of compounds with known anti-sars-cov- activity and considering the availability of structural models of potential molecular targets of the virus, the strategy based on the structure of the molecular target has been further explored, in which molecular docking techniques play a crucial role in this planning method. the compound pubchem cid , showed the highest potential to inhibit sars-cov- proteases during studies in search of possible molecular targets against the virus. about phytochemicals, the virtual libraries that store information about these compounds have already been extensively explored. among the selected compounds, pubchem cid , , and quercetin showed a strong interaction with clpro from sars-cov- . also, dihydrotansinone i interact strongly with spike glycoprotein and cryptotanshinone, with proteins plpro and clpro. marine compounds were also evaluated in silico against the main sars-cov- protease. the results identified different compounds as potential inhibitors of this protease, highlighting heptafuhalol a as the most promising. another virtual screening strategy that stands out among the research is the one that uses groups of drugs with antiviral action already approved for use in humans, characterizing a drug repositioning strategy. it was shown that digitoxin and zorubicin have a high potential for interaction with the sars-cov- spike glycoprotein. another highlight was the potential for ribavirin to interact with the plpro, lymecycline against clpro, and valganciclovir against rna-dependent rna polymerase, essential proteins for the survival of sars-cov- . besides, the present review showed that the pemirolast is promising as an inhibitor of the action of spikes glycoproteins. in addition to issues related to bioinformatics and molecular modeling, the details provided in the present review envision future points of consideration in the field of virology and medical sciences that will contribute to understanding the molecular mechanisms responsible for the pathogenesis and virulence of sars-cov- well as the development of the human acute severe respiratory syndrome. it is well established that the pathophysiology and lung injury caused by sars-cov- is related to innate immunity and factors such as the release of pro-inflammatory cytokines and the complement system action. besides, we strongly emphasize that: ( ) the complement system is an essential element of protective immunity against pathogens, but its excessive or unregulated activation can result in critical tissue damage and; ( ) the viral s subunit can be an important target for future antiviral compounds. thus, anti-s antiviral compounds may be potential treatments for sars-cov- infection. during this unprecedented period, we encourage scientists to actively contribute to understanding the role of the complement system in the development of covid- . in addition to what was previously mentioned, all authors made due contributions to the conception or design of the work; or the acquisition, analysis, or interpretation of data; or the creation of new software used in the work; or have drafted the work or substantively revised it; has approved the submitted version and; agrees to be personally accountable for the author's own contributions and for ensuring that questions related to the accuracy or integrity of any part of the work, even ones in which the author was not personally involved, are appropriately investigated, resolved, and documented in the literature. all authors have read and agreed to the published version of the manuscript. funding: this research received no external funding. early transmission dynamics in wuhan, china, of novel coronavirus-infected pneumonia a novel coronavirus from patients with pneumonia in china severe acute respiratory syndrome related coronavirus: the species and its viruses-a statement of the coronavirus study group the impact of the covid- epidemic on the utilization of emergency dental services chloroquine and hydroxychloroquine in the treatment of covid- with or without diabetes: a systematic search and a narrative review with a special reference to india and other developing countries clinical features of patients infected with novel coronavirus in wuhan a pneumonia outbreak associated with a new coronavirus of probable bat origin discovery of a rich gene pool of bat sars-related coronaviruses provides new insights into the origin of sars coronavirus serological evidence of bat sars-related coronavirus infection in humans crystal structure of sars-cov- main protease provides a basis for design of improved α-ketoamide inhibitors who director-general's opening remarks at the media briefing on covid- . . available online situation report- world health organization declares global emergency: a review of the who novel coronavirus (covid- ) situation. available online evolutionary history, potential intermediate animal host, and cross-species analyses of sars-cov- preliminary bioinformatics studies on the design of a synthetic vaccine and a preventative peptidomimetic antagonist against the sars-cov- ( -ncov, covid- ) coronavirus data mining and clinical data repositories: insights from a , patient data set drug discovery using very large numbers of patents. general strategy with extensive use of match and edit operations suggestions for a web based universal exchange and inference language for medicine sars coronavirus and innate immunity the epidemic of -novel-coronavirus ( -ncov) pneumonia and insights for emerging infectious diseases in the future plasma inflammatory cytokines and chemokines in severe acute respiratory syndrome mers-cov infection in humans is associated with a pro-inflammatory th and th cytokine profile protocolo de tratamento do novo coronavírus ( -ncov) quantitative structure-activity relationship and molecular docking revealed a potency of anti-hepatitis c virus drugs against human corona viruses evaluation and treatment coronavirus fields virology molecular diagnosis of severe acute respiratory human coronavirus nl , a new respiratory virus navas-martin, s. coronavirus pathogenesis and the emerging pathogen severe acute respiratory syndrome coronavirus. microbiol molecular diversity of coronaviruses in bats remdesivir and chloroquine effectively inhibit the recently emerged novel coronavirus ( -ncov) in vitro interspecies transmission and emergence of novel viruses: lessons from bats and birds isolation and characterization of viruses related to the sars coronavirus from animals in southern china identification of a novel coronavirus in bats coronaviruses post-sars: update on replication and pathogenesis bats are natural reservoirs of sars-like coronaviruses severe acute respiratory syndrome coronavirus-like virus in chinese horseshoe bats van complete genomic sequence of human coronavirus oc : molecular clock analysis suggests a relatively recent zoonotic coronavirus transmission event bovine-like coronaviruses isolated from four species of captive wild ruminants are homologous to bovine coronaviruses, based on complete genomic sequences analysis of the genome sequence of an alpaca coronavirus preservation, and loss of a group a coronavirus accessory glycoprotein genome structure, replication, and pathogenesis clinical characteristics and intrauterine vertical transmission potential of covid- infection in nine pregnant women: a retrospective review of medical records dynamically modeling sars and other newly emerging respiratory illnesses: past, present, and future the epidemiology and pathogenesis of coronavirus disease (covid- ) outbreak receptor recognition by novel coronavirus from wuhan: an analysis based on decade-long structural studies of sars a tale of two viruses: the distinct spike glycoproteins of feline coronaviruses activation of the sars coronavirus spike protein via sequential proteolytic cleavage at two distinct sites spotlight sars coronavirus redux viruses and interferon: a fight for supremacy type interferons and the virus-host relationship: a lesson in détente viruses and interferons recent insights into emerging coronaviruses clinical characteristics of coronavirus disease in china coronavirus infections and immune responses complement activation contributes to severe acute respiratory syndrome coronavirus pathogenesis complement as a target in covid- ? clinical promise of next-generation complement therapeutics highly pathogenic coronavirus n protein aggravates lung injury by masp- -mediated complement over-activation function, and antigenicity of the sars-structure, function, and antigenicity of the sars-cov- spike glycoprotein dysregulated type i interferon and inflammatory monocyte-macrophage responses cause lethal pneumonia in sars-cov-infected mrice article dysregulated type i interferon and inflammatory monocyte-macrophage responses cause lethal pneumonia in sars-cov-infecte bat-to-human: spike features determining 'host jump' of coronaviruses sars-cov, mers-cov, and beyond the spike protein of sars-cov-a target for vaccine and therapeutic development cell responses to whole sars coronavirus in humans t-cell immunity of sars-cov: implications for vaccine development against mers-cov canine coronavirus highly pathogenic for dogs experimental infection of dogs with a novel strain of canine coronavirus causing systemic disease and lymphopenia prolonged depletion of circulating cd + t lymphocytes and acute monocytosis after pantropic canine coronavirus infection in dogs canine coronavirus: not only an enteric pathogen molecular characterization of hlj- , a recombinant canine coronavirus strain from china with an orf abc deletion genotypic characterization of canine coronaviruses associated with fatal canine neonatal enteritis in the united states natural history of a recurrent feline coronavirus infection and the role of cellular immunity in survival and disease acquisition of macrophage tropism during the pathogenesis of feline infectious peritonitis is determined by mutations in the feline coronavirus spike protein an update on canine coronaviruses: viral evolution and pathobiology epstein-barr virus-specific cd + t cells that re-express cd ra are apoptosis-resistant memory cells that retain replicative potential decreased ccr expression on cd + t cells of siv-infected sooty mangabeys expression of lymphocytes and lymphocyte subsets in patients with severe acute respiratory syndrome renin-angiotensin system: the unexpected flaw inside the human immune system revealed by sars-cov- immunity after natural exposure to enteric canine coronavirus does not provide complete protection against infection with the new pantropic cb/ strain the thymus is a common target organ articles genomic characterisation and epidemiology of novel coronavirus: implications for virus origins and receptor binding first cases of coronavirus disease (covid- ) in brazil, south america ( genomes the origins of bioinformatics multiple sequence alignment modeling: methods and applications genomic variance of the -ncov coronavirus analysis of therapeutic targets for sars-cov- and discovery of potential drugs by computational methods basic local alignment search tool isolation and characterization of -ncov-like coronavirus from malayan pangolins the proximal origin of sars-cov- detection of novel coronavirus ( -ncov) by real-time rt-pcr qpcr primer design revisited specific primer design for the polymerase chain reaction bioinformatic tools and guideline for pcr primer design primer-blast: a tool to design target-specific primers for polymerase chain reaction a bioinformatics workflow for the evaluation of rt-qpcr primer specificity: application for the assessment of gene expression data reliability in toxicological studies biotechnology applied to the development of vaccines a sequence homology and bioinformatic approach can predict candidate targets for immune responses to sars-cov- immunoinformatics-aided identification of t cell and b cell epitopes in the surface glycoprotein of -ncov preliminary identification of potential vaccine targets for the covid- coronavirus (sars-cov- ) based on sars-cov immunological studies accelerating drug development: antiviral therapies for emerging viruses as a model quantitative assessment of relationship between sequence similarity and function similarity structural elucidation of sars-cov- vital proteins: computational methods reveal potential drug candidates against main protease, nsp rna-dependent rna polymerase and nsp helicase the design of macromolecular crystallography diffraction experiments macromolecular structure determination by cryo-electron microscopy structural genomics: beyond the human genome project the protein data bank structure of mpro from covid- virus and discovery of its inhibitors crystal structure of nsp endoribonuclease nendou from sars-cov- crystal structure of the -ncov spike receptor-binding domain bound with the ace receptor cryo-em structure of the -ncov spike in the prefusion conformation making membrane proteins for structures: a trillion tiny tweaks homology modeling in drug discovery: overview, current applications, and future perspectives homology model versus x-ray structure in receptor-based drug design: a retrospective analysis with the dopamine d receptor comparison of a homology model and the crystallographic structure of human β-hydroxysteroid dehydrogenase type ( βhsd ) in a structure-based identification of inhibitors crystallographic structure versus homology model: a case study of molecular dynamics simulation of human and zebrafish histone deacetylase structural genomics of sars-cov- indicates evolutionary conserved functional regions of viral proteins whole genome sequence analysis and homology modelling of a c like peptidase and a non-structural protein of the sars-cov- shows protein ligand interaction with an aza-peptide and a noncovalent lead inhibitor with possible antiviral properties structural basis of sars-cov- cl pro and anti-covid- drug discovery from medicinal plants role of changes in sars-cov- spike protein in the interaction with the human ace receptor: an in silico analysis virtual screening strategies in drug discovery: a critical review progress in molecular docking zinc-a free database of commercially available compounds for virtual screening pubchem update: improved access to chemical data unrevealing sequence and structural features of novel coronavirus using in silico approaches: the main protease as molecular target the evolving role of natural products in drug discovery in silico screening of chinese herbal medicines with the potential to directly inhibit novel coronavirus putative inhibitors of sars-cov- main protease from a library of marine natural products: a virtual screening and molecular modeling study review of drug repositioning approaches and resources virtual screening of inhibitors against spike glycoprotein of novel corona virus: a drug repurposing approach repurposing therapeutics for covid- : supercomputer-based docking to the sars-cov- viral spike protein and viral spike protein-human ace interface an in silico database of approved drugs, regulated chemicals, and herbal isolates for computer-aided drug discovery anti-hcv, nucleotide inhibitors, repurposing against covid- covid- disease map, building a computational repository of sars-cov- virus-host interaction mechanisms phylogenetic network analysis of sars-cov- genomes phylogenetic analysis of sars-cov- genomes in turkey coast-to-coast spread of sars-cov- in the united states revealed by genomic epidemiology phylogenetic and phylodynamic analyses of sars-cov- a molecular modeling approach to identify effective antiviral phytochemicals against the main protease of sars-cov- structural and molecular modelling studies reveal a new mechanism of action of chloroquine and hydroxychloroquine against sars-cov- infection current and future developments in the treatment of virus-induced hypercytokinemia should we stimulate or suppress immune responses in covid- ? cytokine and anti-cytokine interventions prophylactic and therapeutic inhibition of in vitro sars-cov- replication by oleandrin anti-sars-cov- activities in vitro of shuanghuanglian preparations and bioactive ingredients effect of colchicine vs standard care on cardiac and inflammatory biomarkers and clinical outcomes in patients hospitalized with coronavirus disease association between treatment with colchicine and improved survival in a single-centre cohort of adult hospitalised patients with covid- pneumonia and acute respiratory distress syndrome remdesivir inhibits sars-cov- in human lung cells and chimeric sars-cov expressing the sars-cov- rna polymerase in mice remdesivir in adults with severe covid- : a randomised, double-blind, placebo-controlled, multicentre trial compassionate remdesivir treatment of severe covid- pneumonia in intensive care unit (icu) and non-icu patients: clinical outcome and differences in post-treatment hospitalisation status a trial of lopinavir-ritonavir in adults hospitalized with severe covid- lack of viral clearance by the combination of hydroxychloroquine and azithromycin or lopinavir and ritonavir in sars-cov- -related acute respiratory distress syndrome triple combination of interferon beta- b, lopinavir-ritonavir, and ribavirin in the treatment of patients admitted to hospital with covid- : an open-label, randomised, phase trial subcutaneous administration of interferon beta- a for covid- : a non-controlled prospective trial hydroxychloroquine in patients with mainly mild to moderate coronavirus disease : open label, randomised controlled trial hydroxychloroquine and azithromycin as a treatment of covid- : results of an open-label non-randomized clinical trial treatment with hydroxychloroquine, azithromycin, and combination in patients hospitalized with covid- in vitro antiviral activity and projection of optimized dosing design of hydroxychloroquine for the treatment of severe acute respiratory syndrome coronavirus (sars-cov- ) hydroxychloroquine, a less toxic derivative of chloroquine, is effective in inhibiting sars-cov- infection in vitro in vitro testing of combined hydroxychloroquine and azithromycin on sars-cov- shows synergistic effect early treatment of covid- patients with hydroxychloroquine and azithromycin: a retrospective analysis of cases in marseille, france safe use of hydroxychloroquine and its combination with azithromycin in the context of sars-cov- outbreak: clinical experience in a belgian tertiary center outcomes of , covid- patients treated with hydroxychloroquine/azithromycin and other regimens in marseille, france: a retrospective analysis effect of high vs low doses of chloroquine diphosphate as adjunctive therapy for patients hospitalized with severe acute respiratory syndrome coronavirus (sars-cov- ) infection the fda-approved drug ivermectin inhibits the replication of sars-cov- in vitro the anticoagulant nafamostat potently inhibits sars-cov- s protein-mediated fusion in a cell fusion assay system and viral infection in vitro in a cell-type-dependent manner the chemokine receptor antagonist cenicriviroc inhibits the replication of sars-cov- in vitro baricitinib as rescue therapy in a patient with covid- with no complete response to sarilumab tocilizumab treatment in covid- : a single center experience use of tocilizumab for covid- -induced cytokine release syndrome early administration of interleukin- inhibitors for patients with severe covid- disease is associated with decreased intubation, reduced mortality, and increased discharge effective treatment of severe covid- patients with tocilizumab tocilizumab for the treatment of severe covid- pneumonia with hyperinflammatory syndrome and acute respiratory failure: a single center study of patients in pilot prospective open, single-arm multicentre study on off-label use of tocilizumab in patients with severe covid- the combined use of tocilizumab and hemoadsorption in a patient with sars-cov- - -associated pneumonia: a case report interleukin- blockade with sarilumab in severe covid- pneumonia with systemic hyperinflammation: an open-label cohort study covid- pneumonia treated with sarilumab: a clinical series of eight patients use of anakinra in severe covid- : a case report favorable anakinra responses in severe covid- patients with secondary hemophagocytic lymphohistiocytosis interleukin- blockade with high-dose anakinra in patients with covid- , acute respiratory distress syndrome, and hyperinflammation: a retrospective cohort study pediatric crohn disease and multisystem inflammatory syndrome in children (mis-c) and covid- treated with infliximab eculizumab treatment in patients with covid- : preliminary results from real life asl napoli nord experience this article is an open access article distributed under the terms and conditions of the creative commons attribution (cc by) license we especially thank the research groups: research group on development of pharmaceutical products (p&dprofar); research group on biomolecules and catalyze; research group on biodiversity and health (biosa). the map for figure was "designed by freepik". we appreciate the opportunity to enrich our work with your art. we are grateful for the participation of everyone who contributed to the writing and revision of this manuscript. the authors declare no conflict of interest. key: cord- - dddrh e authors: el-faham, ayman; farooq, muhammad; khattab, sherine n.; abutaha, nael; wadaan, mohammad a.; ghabbour, hazem a.; fun, hoong-kun title: synthesis, characterization, and anti-cancer activity of some new n′-( -oxoindolin- -ylidene)- -propylpentane hydrazide-hydrazones derivatives date: - - journal: molecules doi: . /molecules sha: doc_id: cord_uid: dddrh e eight novel n′-( -oxoindolin- -ylidene)- -propylpentane hydrazide-hydrazone derivatives a–h were synthesized and fully characterized by ir, nmr (( )h-nmr and ( )c-nmr), elemental analysis, and x-ray crystallography. the cyto-toxicity and in vitro anti-cancer evaluation of the prepared compounds have been assessed against two different human tumour cell lines including human liver (hepg ) and leukaemia (jurkat), as well as in normal cell lines derived from human embryonic kidney (hek ) using mtt assay. the compounds e, f, a, c, and e revealed promising anti-cancer activities in tested human tumour cells lines (ic( ) values between and μm) as compared to the known anti-cancer drug -fluorouracil (ic( ) – μm). among the tested compounds, a showed specificity against leukaemia (jurkat) cells, with an ic( ) value of . μm, but this compound was inactive in liver cancer and normal cell lines. hydrazide-hydrazone derivatives are molecules containing a highly reactive group (co-nh-n=ch) and considered to be a good candidate for development of a new drug [ ] . recently, hydrazide-hydrazones have been considered to be of great interest in medicinal chemistry due to their diverse biological properties, including anti-microbial [ ] [ ] [ ] , anti-mycobacterial [ , ] , anti-convulsant [ ] , analgesic [ ] , anti-inflammatory [ ] , anti-platelet [ ] , anti-tubercular [ ] [ ] [ ] [ ] , and anti-tumoral activities [ ] [ ] [ ] [ ] [ ] [ ] . in addition, hydrazide-hydrazones have been reported to elicit anti-cancer [ ] [ ] [ ] [ ] and anti-hiv properties [ ] and they are therefore increasingly considered to be of great value in medicinal chemistry [ , [ ] [ ] [ ] [ ] [ ] . because of the remarkable biological value of isatin as an important constituent of bioactive compounds exhibiting caspase inhibitory [ , ] , antibacterial, and antiproliferative activity [ ] , schiff bases of isatin derivatives have antismallpox [ ] and gal receptor antagonist capabilities [ ] . in addition, the analogous of isatin derivatives displayed inhibitory activity against elf kinase activator [ ] , tnf-α, cdk [ ] and sars protease [ ] . isatin also displays antiviral [ ] , anti-inflammatory, analgesic [ ] , and anticonvulsant activities [ ] . valproic acid (vpa, ) has an important biological value as a potent antiepileptic molecule [ ] [ ] [ ] , as well as showing inhibition of angiogenesis both in vitro and in vivo [ ] [ ] [ ] [ ] . vpa also acts as a powerful histone deacetylase inhibitor [ , ] and induces differentiation and apoptosis in a variety of malignant cells in vitro [ ] . clinical trials with vpa have focused on acute myeloid leukaemia and the myelodysplastic syndromes. when it was used as mono therapy or in combination with all-trans retinoic acid, which synergizes in vitro, vpa achieved hematologic improvement in a subset of patients [ ] . as a continuation to our previously reported data [ ] [ ] [ ] , herein we designed eight isatin hydrazide-hydrazone derivatives, considering some of the factors responsible for such activity, including (i) the presence of isatin moiety; (ii) the presence of the hydrazide-hydrazone functionality; and (iii) valproic acid moiety ( figure ). all the prepared compounds were assessed against two different human tumour cell lines, including human liver (hepg ) and leukaemia (jurkat), as well as in normal cell lines derived from human embryonic kidney (hek ) using mtt assay. compound was prepared following the reported method [ ] and condensed with isatin derivatives a-h in the presence of - drops of glacial hoac and ethanol as the solvent to afford products a-h (scheme ). the structures of all synthesized compounds were in a good agreement with their spectral data. the ir spectra of the compounds a-g reveal absorption bands in the region - cm − corresponding to the (nh), a band at and cm − corresponding to the c=o group, and a peak around cm − related to the c=n bond. the nmr of all the products a-g are in good agreement with their structures (figures s -s ). as a prototype h-nmr of g showed multiple peaks at δ . - . , . - . , . - . , . - . ppm, and a broad singlet at δ . ppm corresponding to the valporic acid moiety ( ch , ch , and ch, respectively). also, two triplet peaks were observed at δ . and . ppm corresponding to the two methylene group (ch -ch br), respectively. the observed peaks in the aromatic region at δ . (t, h, ar-h), . (d, h, ar-h), . (t, h, ar-h), and . (brs, h, arh) are related to the isatin moiety, while the broad singlet peak at δ . ppm is related to the nh. the c-nmr of g showed peaks at δ . , . , . , . , . , . , and . ppm, corresponding to the valporic acid moiety and the two methylene groups, in addition to seven peaks related to the aromatic carbons and imino function group. the two peaks at δ . and . ppm corresponded to the carbonyl groups of the isatin moiety and the hydrazide group, respectively. it is expected that compound g could adopt two different geometrical isomers (z and e) as shown in figure a . therefore, it is considered worthwhile to model the compounds using molecular mechanics mm calculations. in addition, quantum chemical calculations were carried out with the gaussian suite of programs. geometry optimizations were carried out using the dft level (b lyp/ - g **) of theory to assess the relative stability of the z and e isomeric species. calculated relative energies of g z and e isomers are − . au and . au, respectively. computed energies indicate the stability of the z isomer over the e one by . au ( . kcal/mol) ( figure b ). the x-ray single crystal structure determination of compound g (ccdc: ) confirmed that the structure exists in the z-conformer rather than the e-conformer ( figure ). the details of data collection and structure refinement are listed in table , and the x-ray structures are shown in figure . compound g crystallized from ethanol in the triclinic space group p- . all bond lengths and angles are in normal ranges [ ] . in the crystal packing (figure ), the molecules are linked by intermolecular c a-h b···br b, c b-h d···o b and c a-h b···o a hydrogen bonds ( table ) into two-dimensional networks parallel to the bc plane ( figure and table ). the asymmetric unit contains two molecules of the compound with disorder in one of the ethylene arms. the molecular structure of compound g is composed of a -oxoindoline ring (c /n /c -c ) which is linked with two side chains at n and c . the two molecules in the asymmetric unit are with z configuration about the c =n double bond ( figure ). the z configuration of a is stabilized with intramolecular hydrogen n -h n···o ( table ). the single bond n -n is clearly characterized by the distances of . ( ) Å and . ( ) Å, respectively, for molecules a and b. the double bond of c =n is characterized by the distances of . ( ) Å and . ( ) Å, for molecules a and b, respectively. a b figure . ortep diagram of the asymmetric unit consisting of two symmetry-independent molecules. the ellipsoids are drawn at the % probability level with hydrogen atoms being shown as spheres of arbitrary radii. one of the molecules has disorder in the side chain c b-c b. ( ) . c b−h d···o b ii . . . ( ) . c a−h b···o a iii . . . ( ) . symmetry codes: reaction of valporic acid hydrazide with n-acetylisatin h proceeds in a different way, where the ring opening occurred to afford the product h. the ir and nmr data proved its structure in the open form, due to the attack of the hydrazide group on c instead of c [ ] . the ir spectrum of h reveals absorption bands in the region and cm − corresponding to the nh, a band at , , , and cm − corresponding to the c=o and c=n group, respectively. h-nmr of h showed multiple peaks at δ . - . , . - . , . - . , . - . , and a broad singlet peak at δ . ppm corresponding to the valporic acid moiety ( ch , ch , and ch , ch, respectively). the observed peak at δ . ppm is corresponding to the n-acetyl group. the observed peaks in the aromatic region at δ . (t, h), . (t, h), . (d, h), and . (d, h) ppm, are related to the phenyl ring, while the two singlet peaks at δ . and . ppm corresponded to the two nh. the c-nmr of h showed peaks at δ . , . , . , . , . , and . ppm corresponding to the valporic acid moiety group, while the acetyl group was observed at δ . ppm, in addition to six peaks related to the aromatic carbons at δ . , . , . , . , . , and . ppm. the three peaks observed at δ . , . , and . ppm related to the three conh groups, while the peak observed at δ . ppm is related to the α-ketoamide group. the cyto-toxicity activity was measured in vitro in hepg , jurkat, and hek cells using the mtt -( , -dimethylthiazol- -yl)- , -diphenylformazan colorimetric assay. for comparison, -fluorouracil ( -fu) was used as a standard anti-cancer drug. treatment with dimethylsulfoxide (dmso) was used as a control for the cancer cells. the anti-cancer activity of the new prepared compounds against the hepg cell line revealed that compounds a, h, a, b, f, g, and h showed no anti-cancer activity. similarly, the starting compounds and did not show promising anti-cancer activity in hepg cells. compound e exhibited higher potency against hepg cell line with ic = . ± . μm, which is much lower than the ic value of -fu ( . ± . μm). moreover, the results showed that compounds f, c, and e were also found to be potent and selective against hepg with ic values of . ± . , . ± . , and . ± . μm, respectively, which are also much lower than -fu ( figure and table ). the anti-cancer profile of newly synthesized compounds was also tested against leukaemia cells (jurkat cells, originated from human t lymphocytes). the compound c was most active, with an ic value of just . ± . μm, which was much lower than the positive control ( -fu ic = . ± . μm). similarly, the compounds f, a, and c showed strong activity and ic values were much lower as compared to -fu (table and figure ). the order of activity was f, a, e, and e in ascending order (table and figure ). the compound showed a weaker level of activity against jurkat cells (ic . ± . μm) and, indeed, this was much weaker than -fu (ic . ± . μm). finally the cyto-toxicities of these compounds were screened in the normal human embryonic kidney (hek ) cell line, in order to assess whether these compounds are active only in cancer cells or whether they possess toxicity against normal cells as well. the results revealed that the compound c turned out to be most toxic by disrupting the cell survival of normal cells (hek ) with ic . ± . μm. the compounds a, b, and g also possessed some level of cyto-toxicity against hek cells with ic values . ± . , . ± . , and . ± . μm, respectively. the ic values of these compounds in hek cells, however, are much higher compared to their activity in cancer cells (table and figure ). the compounds e, f, a, and e showed no activity in hek cells, but strong activity in liver (hepg ) and leukaemia (jurkat) cancer cells, suggesting that these compounds are potent and potentially viable anti-cancer molecules. the solvents used were of hplc reagent grade. melting points were determined with melting points were obtained in open capillary tubes using a mel-temp melting point apparatus (sigma-aldrich chemie gmbh, taufkirchen, germany) and are uncorrected and are uncorrected. infrared (ir) spectra were recorded on a perkin-elmer series fourier transform instrument (perkinelmer life and analytical sciences, shelton, ct, usa) as kbr pellets. nuclear magnetic resonance spectra ( h-nmr and c-nmr spectra) were recorded on mhz jeol spectrometer (jeol ltd., tokyo, japan) at room temperature. chemical shifts are reported in parts per million (ppm) and are referenced relative to residual solvent (e.g., chcl at δ . ppm for cdcl , dmso at δ . ppm for dmso-d ). spin multiplicities are represented by the following signals: singlet (s), broad singlet (br s), doublet (d), broad doublet (br d), doublet of doublets (dd), triplet (t), doublet of triplets (dt), and multiplet (m). elemental analyses were performed on a perkin-elmer elemental analyzer (perkinelmer inc., waltham, ma usa), and the values found were within ± . % of the theoretical values. follow-up of the reactions and checks of the purity of the compounds was done by tlc on silica gel-protected aluminum sheets (type gf , merck millipore, billerica, ma, usa) and the spots were detected by exposure to uv-lamp (company seven, montpelier, md, usa) at λ nm for a few seconds. the compounds were named using chem. draw ultra version , cambridge soft corporation. crystallographic data for the structure reported in this paper have been deposited at the cambridge crystallographic data center and allocated with the deposition numbers: ccdc for compound g. ccdc contains the supplementary crystallographic data for this paper. these data can be obtained free of charge via http://www.ccdc.cam.ac.uk/conts/retrieving.html (or from the ccdc, union road, cambridge cb ez, uk; fax: + ; e-mail: deposit@ccdc.cam.ac.uk). the product was prepared according to the reported procedure [ ] compounds e-h were prepared following the reported procedure [ ] . the entire prepared compounds were in a good agreement with the reported data. a solution of valproic hydrazide ( mg, mmol) in ethanol ( ml) was added to a solution of substituted isatin a-h ( mmol) in ethanol ( ml), and glacial acetic acid ( drops); the reaction mixture was refluxed for - h. the product was separated out on cooling, filtered, and recrystallized from ethanol or ethylacetate to afford n′-( -oxoindolin- -ylidene)- -propylpentanehydrazide derivatives a-h. the product was obtained as yellow solid, in % yield; single crystals were obtained by slow evaporation from ethanol. a good crystal with a suitable size was selected for x-ray diffraction analysis. data were collected on a d venture area diffractometer equipped with graphite monochromatic mok/α radiation (λ = . Å) at k. cell refinement and data reduction were done by bruker saint (bruker, madison, wi usa); the program used to solve structure and refine structure is shelxs- [ ] . the final refinement was performed by full-matrix least-squares techniques with anisotropic thermal data for non-hydrogen atoms on f . all the hydrogen atoms were placed in calculated positions and constrained to ride on their parent atoms. multi-scan absorption correction was applied by use of sadabs software [ ] . the stock concentration of the entire compound in dmso was mm and this concentration was used to prepare the working dilution. the final dmso concentration used in the experiments was ≤ . % as the working concentration. human liver cancer cell lines (hepg ), human leukemia cell line (jurkat), and human embryonic kidney cell line (hek ) cells were cultured in high glucose dulbecco's modified eagle medium (dmem: life technologies cat # ) supplemented with % fetal bovine serum (fbs: life technologies cat # ) in a humidified incubator with % co at °c. around × cells were seeded in each well of a -well cell culture plate and were allowed to adhere and grow for h. the jurkat cells grow in suspension so they were cultured and allowed to grow overnight before the addition of compounds. the compounds in serial dilutions ( , , , , , and μm) were added after h of culture and the cells were cultured for another h at °c. the cell viability was determined in each experiment using mtt -( , -dimethylthiazol- -yl)- , -diphenylformazan colorimetric assay. briefly, the treated or untreated cells were trypsenized and centrifuged, and the resulting pellet was re-suspended in μl of dmem serum-free medium and incubated at °c for h. after incubation, μl of mtt solution ( mg/ml in pbs: sigma aldrich cat #m ) was added to each well and further incubated for h. the plate was centrifuged at , rpm for min then the medium was removed from each well and -propanol containing . m hcl was added to dissolve the formazan produced in the cells. the optical density of the formazan product in solution was measured with a microplate reader at nm. the experiment was conducted in triplicate. data were calculated as percent of cell viability by the following formula: % cell viability = (mean absorbance in test wells/mean absorbance in control wells) × ( ) the ic values were calculated from the means of six different concentrations by bio data fit . using the software bio tool kit (version ; chang bioscience inc., castro valley, ca, usa) and online program http://ic .tk/index.html. all data were analyzed using origin (version . ; origin lab corp northampton, ma, usa). one-way anova analysis of variance and student t test was used to compare different experimental groups, and data were considered statistically significant for p values less than . . in conclusion, the screening results from human cancer and normal cell lines suggested that isatin derivatives were remarkably influenced by various substituents on the isatin ring and at the n-terminal of the hydrazide-hydrazone moiety. the data revealed that replacement of n-hydrogen at position of the isatin moiety a by methyl and benzyl of the targeted compound ( e and f) noticeably enhanced the activity against the selected two cell lines, hepg and jurkat, for compound f. the presence of the valproic acid moiety as hydrazide-hydrazone derivatives a make the compound more targeted towards jurakt cells in vitro. introducing the chlorine atom to the molecule c, meanwhile, increased the reactivity more significantly than with a and a towards the three cell lines hepg , jurkat, and hek . furthermore, the methyl group in the same analogous compound e increased the reactivity towards the two cell lines hepg and jurkat more than the benzyl group f. the rest of the compounds had no effect on the three cell lines. further studies to assess the effect of more derivatives on various cancer cell biomarkers are currently underway in our lab. supplementary materials can be accessed at: http://www.mdpi.com/ - / / / /s . microwave assisted synthesis and antimicrobial activity of -quinoxalinone- -hydrazone derivatives synthesis and antimicrobial activity of some new hydrazones of -fluorobenzoic acid hydrazide and -acetyl- , -disubstituted- , , -oxadiazolines synthesis, antibacterial and antifungal activity of some new thiazolylhydrazone derivatives containing -substituted cyclobutane ring efficacy of voriconazole in treatment of systemic scedosporiosis in neutropenic mice synthesis and antimycobacterial activity of ( , -diaryl- h-thiazol- -ylidene)-hydrazide derivatives synthesis, characterization and antibacterial activity of biologically important vanillin related hydrazone derivatives anticonvulsant properties of various acetylhydrazones, oxamoylhydrazones and semicarbazones derived from aromatic and unsaturated carbonyl compounds synthesis and analgesic activity of novel n-acylarylhydrazones and isosters, derived from natural safrole -acylthiose-micarbazides, , , -triazole- ( h)-thiones, , , -thia-diazoles and hydrazones containing -methyl- -benzoxazolinones: synthesis, analgesic-anti-inflammatory and antimicrobial activities new class of potent antinociceptive and antiplatelet h-phenothiazine- -acylhydrazone derivatives a new modification of anti-tubercular active molecules antituberculosis drugs: ten years of research tuberculosis chemotherapy: current drug delivery approaches new α-(n)-heterocyclichydrazones: evaluation of anti-cancer, anti-hiv and antimicrobial activity cyanoacetic acid hydrazones of -(and -) acetyl-pyridine and some derived ring systems as potential antitumor and anti-hcv agents synthesis and anti-tumor evaluation of novel hydrazide and hydrazide-hydrazone derivatives novel synthesis of hydrazide-hydrazone derivatives and their utilization in the synthesis of coumarin, pyridine, thiazole and thiophene derivatives with antitumor activity synthesis and anti-cancer evaluation of some new hydrazone derivatives of , -dimethylimidazo characterization of the conjugate of the ( -maleimidocaproyl) hydrazone derivative of doxorubicin with lactosaminated human albumin by c-nmr spectroscopy synthesis of new acridines and hydrazones derived from cyclic α-diketone for cytotoxic and antiviral evaluation discovery and sar of indole- -carboxylic acid benzylidene-hydrazides as a new series of potent apoptosis inducers using a cell-based hts assay anti hiv evaluation of benzo[d]isothiazole hydrazones synthesis, stereochemistry and biological activity of some novel long alkyl chain substituted thiazolidin- -ones and thiazan- -one from -undecenoic acid hydrazide -chloro- -methyl- -phenyl- h-pyrazol- -yl)methylene] / -substituted hydrazides: synthesis and anticonvulsant activity synthesis and structure-activity relationships of novel -arylmethyl- -aryl- h-pyrazole- -carbohydrazide hydrazone derivatives as potential agents against a lung cancer cells chemical synthesis and biological evaluation of triazole derivatives as inhibitors of inha and antituberculosis agents in silico screening, synthesis and in vitro evaluation of some quinazolinone and pyridine derivatives as dihydrofolate reductase inhibitors for anti-cancer activity n-benzylisation sulfonamide analogues as potent caspase- inhibitors: synthesis, in vitro activity, and molecular modeling studies synthesis and in vitro evaluation of sulfonamide isatin micheal acceptors as small molecule inhibitors of caspase- isatin-derived antibacterial and antifungal compounds and their transition metal complexes combinatorial optimization of isatin-β-thiosemicarbazones as anti-poxvirus agents arylimino- -indolones are potent and selective galanin gal receptor antagonists -diaryl- , -dihydroindol- -ones as antiproliferatives mediated by translation initiation inhibition isatins inhibit cyclooxygenase- -and inducible nitric oxide synthesis in a mouse macrophage cell line isatin compounds as noncovalent sars coronavirus c-like protease inhibitors synthesis, antibacterial, antifungal and antiviral activity evaluation of some new bis-schiff bases of isatin and their derivatives some pharmacological activities of new substituted pyrolloindoles, indolothiazepine and azole derivatives anticonvulsant activity of schiff bases of isatin derivatives valnoctamide as a valproate substitute with low teratogenic potential in mania: a double-blind, controlled, add-on clinical trial new antiepileptic drugs that are second generation to existing antiepileptic drugs valproic acid: second generation valproic acid inhibits angiogenesis in vitro and in vivo valproic acid inhibits angiogenesis in vitro and glioma angiogenesis in vivo in the brain modulation of angiogenesis-related protein synthesis by valproic acid valproic acid exerts anti-tumor as well as anti-angiogenic effects on myeloma histone deacetylase (hdac ) is specifically required for liver development in zebrafish valproic acid defines a novel class of hdac inhibitors inducing differentiation of transformed cells valproic acid for the treatment of myeloid malignancies teratogenic profile of valproic acid and newly synthesized derivatives in zebrafish embryos synthesis and biological activity of schiff base series of valproyl, n-valproyl glycinyl, and n-valproyl- -aminobenzoyl hydrazide derivatives biological screening of novel derivatives of valproic acid for anti-cancer and antiangiogenic properties. asian pac tables of bond lengths determined by x-ray and neutron diffraction. part . bond lengths in organic compounds microwave irradiation: synthesis and characterization of α-ketoamide and bis (α-ketoamide) derivatives via the ring opening of n-acetylisatin facile method for the synthesis of silver nanoparticles using -hydrazino-isatin derivatives in aqueous methanol and their antibacterial activity a short history of shelx the authors extend their sincere appreciation to the deanship of scientific research at king saud university for funding this work through research group project no. rgp- (saudi arabia). ayman el-faham and sherine khattab carried out the synthesis and designed the proposed methods and analyzed the data statistically together. hazem ghabbour and hoong-kun fun carried out x-ray method and the characterization; muhammad farooq, nael abutaha, and mohammad wadaan carried out all the biological activities studies. all authors read and approved the final manuscript. the authors declare no conflict of interest.molecules , key: cord- -ujvkcz s authors: papadakis, georgios; gerasi, maria; snoeck, robert; marakos, panagiotis; andrei, graciela; lougiakis, nikolaos; pouli, nicole title: synthesis of new imidazopyridine nucleoside derivatives designed as maribavir analogues date: - - journal: molecules doi: . /molecules sha: doc_id: cord_uid: ujvkcz s the strong inhibition of human cytomegalovirus (hcmv) replication by benzimidazole nucleosides, like triciribine and maribavir, has prompted us to expand the structure–activity relationships of the benzimidazole series, using as a central core the imidazo[ , -b]pyridine scaffold. we have thus synthesized a number of novel amino substituted imidazopyridine nucleoside derivatives, which can be considered as -(or )-aza-d-isosters of maribavir and have evaluated their potential antiviral activity. the target compounds were synthesized upon glycosylation of suitably substituted -aminoimidazopyridines, which were prepared in six steps starting from -amino- -chloropyridine. even if the new compounds possessed only a slight structural modification when compared to the original drug, they were not endowed with interesting antiviral activity. even so, three derivatives showed promising cytotoxic potential. human cytomegalovirus (hcmv) is a prevalent herpesvirus, with igg antibodies indicating past infection found in approximately % of adults in developed countries and almost % in developing ones [ ] . although hcmv infection rarely leads to clinical manifestations in immunocompetent hosts, there is an increasing amount of data associating lifelong viral persistence with vascular diseases (atherosclerosis [ ] , hypertension [ ] ) and the progression of some cancer types [ ] . in addition, hcmv is a major opportunistic pathogen in immunocompromised individuals, posing a serious threat to neonates, allograft recipients and aids patients [ ] . perinatal infection can cause irreversible hearing loss, blindness and mental retardation, while immunosuppressed adults may develop multi-organ failure syndrome, which is a life-threatening condition [ , ] . ganciclovir (gcv) and its orally bioavailable prodrug, valganciclovir, have served as gold standards for pre-emptive therapy and prophylaxis against hcmv in solid organ transplant patients [ ] as well as for the treatment of cmv retinitis [ ] for almost years. however, their myelosuppressive potential precludes their prophylactic use in stem cell transplant recipients, which has led to their administration, mostly upon engraftment [ ] . in addition, mutations mapping in the gene encoding for the ul kinase (responsible for the first phosphorylation of gcv towards its active triphosphate form) and in the ul gene encoding for the dna polymerase (target of ganciclovir triphosphate), have led to the emergence of drug resistant strains [ ] . furthermore, the dna polymerase inhibitors cidofovir and foscarnet, which are currently approved as second line agents, have several drawbacks that limit their clinical use, namely severe side effects and poor pharmacokinetic properties [ ] . it has become clear that there is an urgent need for better tolerated and more effective antiviral drugs, in order to fully address the health risks posed by hcmv. there are four compounds that have been considered or are at an advanced stage of clinical development for this purpose (figure ). brincidofovir is a per os administered hexadecyloxypropylester of cidofovir aimed at addressing the parent compound's dose-limiting renal toxicity [ ] . the development of brincidofovir for therapy of hcmv has been halted because of increased gastrointestinal toxicity of the oral formulation in adult hematopoietic cell transplant recipients. the non-nucleoside guanosine analogue cyclopropavir, which shares the same mechanism of action with gcv, has proven to be more potent in hcmv inhibition in vitro [ ] and phase i trials have been recently completed. furthermore, the search for novel molecular targets within the viral life cycle has led to the fast track approval of the terminase inhibitor letermovir in late , for the prophylaxis of hcmv infection and disease in adult hcmv-seropositive recipients of an allogeneic human stem cell transplant (hsct) [ ] . phase ii clinical trials are also about to be launched for the use of letermovir in paediatric patients who underwent an hsct. which has led to their administration, mostly upon engraftment [ ] . in addition, mutations mapping in the gene encoding for the ul kinase (responsible for the first phosphorylation of gcv towards its active triphosphate form) and in the ul gene encoding for the dna polymerase (target of ganciclovir triphosphate), have led to the emergence of drug resistant strains [ ] . furthermore, the dna polymerase inhibitors cidofovir and foscarnet, which are currently approved as second line agents, have several drawbacks that limit their clinical use, namely severe side effects and poor pharmacokinetic properties [ ] . it has become clear that there is an urgent need for better tolerated and more effective antiviral drugs, in order to fully address the health risks posed by hcmv. there are four compounds that have been considered or are at an advanced stage of clinical development for this purpose (figure ). brincidofovir is a per os administered hexadecyloxypropylester of cidofovir aimed at addressing the parent compound's dose-limiting renal toxicity [ ] . the development of brincidofovir for therapy of hcmv has been halted because of increased gastrointestinal toxicity of the oral formulation in adult hematopoietic cell transplant recipients. the non-nucleoside guanosine analogue cyclopropavir, which shares the same mechanism of action with gcv, has proven to be more potent in hcmv inhibition in vitro [ ] and phase i trials have been recently completed. furthermore, the search for novel molecular targets within the viral life cycle has led to the fast track approval of the terminase inhibitor letermovir in late , for the prophylaxis of hcmv infection and disease in adult hcmv-seropositive recipients of an allogeneic human stem cell transplant (hsct) [ ] . phase ii clinical trials are also about to be launched for the use of letermovir in paediatric patients who underwent an hsct. at the same time, the benzimidazole l-riboside maribavir is about to be evaluated in phase iii trials involving transplant recipients with hcmv infections that are refractory or resistant to the currently approved drugs as well as for its potential superiority over valganciclovir in hsct patients. maribavir was developed in the late s and has been proven to inhibit viral dna synthesis as well as nucleocapsid egress from the nucleus via the inhibition of the viral kinase ul [ ] . however, initial phase iii clinical trials failed to prove sufficient benefits for post-transplant at the same time, the benzimidazole l-riboside maribavir is about to be evaluated in phase iii trials involving transplant recipients with hcmv infections that are refractory or resistant to the currently approved drugs as well as for its potential superiority over valganciclovir in hsct patients. maribavir was developed in the late s and has been proven to inhibit viral dna synthesis as well as nucleocapsid egress from the nucleus via the inhibition of the viral kinase ul [ ] . however, initial phase iii clinical trials failed to prove sufficient benefits for post-transplant patients. this has later on been accredited to the pre-emptive therapy with gcv that several patients had received prior to surgery and to the low daily administered dose [ ] . as a continuation of our previous involvement with the synthesis and evaluation of purine isosteric bioactive nucleosides [ ] [ ] [ ] [ ] , we designed and synthesized a number of novel nucleoside derivatives, which can be considered as -(or )-aza-d-isosters of maribavir, having in mind that the d-enantiomer of maribavir possesses interesting anti-hcmv properties as well [ ] . our goal was to molecules , , of expand the structure-activity relationships of the benzimidazole series to the less-studied and more "purine-like" imidazo [ , -b] pyridine scaffold, retaining the pattern of the , -dichlorosubstitution in the new compounds. the diverse nature of the -amino groups in each of the final pair of isomeric nucleosides was at the core of our attempt to explore the spatial limitations of possible target enzymes as well as to gain some insight on potential interactions developed. within this context, we disclose herein the preparation and pharmacological evaluation of the -and -regioisomeric β-d-ribosides of , -dichloroimidazo [ , -b] pyridine, introducing various amino substituents at the vacant position of the imidazole ring. the synthetic route we envisaged in order to gain access to the target nucleosides involved the direct glycosylation of the suitably substituted imidazo [ , -b] pyridines a-d (scheme ). a common intermediate for the synthesis of these derivatives is , -dichloropyridine- , -diamine ( ), which was prepared following a five-step procedure, previously described by our group, starting from the pyridinamine [ ] . in the new compounds. the diverse nature of the -amino groups in each of the final pair of isomeric nucleosides was at the core of our attempt to explore the spatial limitations of possible target enzymes as well as to gain some insight on potential interactions developed. within this context, we disclose herein the preparation and pharmacological evaluation of the -and -regioisomeric β-d-ribosides of , -dichloroimidazo [ , -b] pyridine, introducing various amino substituents at the vacant position of the imidazole ring. the synthetic route we envisaged in order to gain access to the target nucleosides involved the direct glycosylation of the suitably substituted imidazo [ , -b] pyridines a-d (scheme ). a common intermediate for the synthesis of these derivatives is , -dichloropyridine- , -diamine ( ), which was prepared following a five-step procedure, previously described by our group, starting from the pyridinamine [ ] . amino substituted imidazopyridine derivatives a-c were prepared using a one-pot-two-stage procedure, which involves an initial treatment of with appropriately substituted n-alkylisothiocyanates in refluxing thf. the resulting mixture of isomeric thioureas undergoes rapid cyclodesulfurization in the presence of mercury oxide [ ] , yielding compounds a-c. concerning the preparation of the primary aminoderivative d, we implemented a different approach, which had been previously reported by townsend [ ] , for the synthesis of the corresponding benzimidazole analogue. thus, the addition of cyanogen bromide in a suspension of diamine in a : mixture of meoh and water provided d as the sole reaction product in very good yield. amino substituted imidazopyridine derivatives a-c were prepared using a one-pot-two-stage procedure, which involves an initial treatment of with appropriately substituted n-alkylisothiocyanates in refluxing thf. the resulting mixture of isomeric thioureas undergoes rapid cyclodesulfurization in the presence of mercury oxide [ ] , yielding compounds a-c. concerning the preparation of the primary aminoderivative d, we implemented a different approach, which had been previously reported by townsend [ ] , for the synthesis of the corresponding benzimidazole analogue. thus, the addition of cyanogen bromide in a suspension of diamine in a : mixture of meoh and water provided d as the sole reaction product in very good yield. the target n and n -ribonucleoside acetates were prepared under modified vorbrüggen conditions. the in situ formation of the persilylated heterocyclic bases by treatment of the corresponding heterocyclic compounds a-d with n,o-bistrimethylsilylacetamide (bsa) was followed by the addition of peracetylated β-d-ribofuranose and trimethylsilyltrifluoromethanesulfonate (tmsotf) (scheme ). thus, concerning the glycosylation reaction of compounds a-c, the major products isolated were the -β-d-ribosides a-c, as a mixture with a small amount of the corresponding α-anomers a-c. the n regioisomers were also obtained as mixtures of the -β-d ribosides a-c with their corresponding α-d anomers a-c. molecules , , x for peer review of reaction of compounds a-c, the major products isolated were the -β-d-ribosides a-c, as a mixture with a small amount of the corresponding α-anomers a-c. the n regioisomers were also obtained as mixtures of the -β-d ribosides a-c with their corresponding α-d anomers a-c. regarding the n regioisomers, we were able to isolate pure n -β-d nucleoside acetates a-c as well as their corresponding n -α-d anomers a-c, upon chromatographic purification. the site of ribosylation as well as the anomeric configuration were unambiguously determined on the basis of noe spectroscopy. taking into consideration the noe spectra of compounds a-c and a-c, we observed clear correlation peaks between the aromatic proton and protons of the furanose ring, determinant of the n -ribosylation pattern. in addition, we also noticed cross correlation peaks between ′-h and ′-h of the sugar moiety in the spectra of compounds a-c. such peaks were not observed on the noe spectra of a-c, thus clearly concluding that a-c were the n -α-d nucleoside products of the reaction, while a-c were their corresponding β-d anomers. deacetylation of a-c with methanolic ammonia provided the final compounds a-c. isolation of the pure -β-d nucleoside acetates proved to be difficult at this stage, so the mixtures of a-c with their corresponding α-anomers a-c were subjected to ammoniolysis, to provide the deprotected nucleosides. the ethylamino ( a) and isopropylamino ( b) derivatives were isolated in pure form by recrystallization, whereas an analytically pure sample of the benzylamino compound c was obtained upon purification with semi-preparative hplc. anomeric purity and configuration of compounds a-c were determined on the basis of h-nmr and noe spectra, respectively. in the latter ones, we observed clear correlation peaks between ′-h and ′-h of the ribofuranose moiety, while there was a profound absence of correlation peaks between the aromatic and sugar protons on the noe spectra of each of the aforementioned compounds. the close examination of d and d nmr spectra reveals that a simple differentiation between each pair of regio isomers can be easily made upon inspection of the chemical shift of the aromatic proton. this proton appears upfield in the case of the n -isomers a-d ( . - . ppm) whereas it shifts downfield ( . - . ppm) concerning the n -isomers a-d. applying the same reaction conditions for the glycosylation of the primary aminoderivative d, we isolated from the reaction mixture the -β-d nucleoside acetate ( d), as well as the -β-d isomer ( d), without detectable amounts of the corresponding α-anomers. deprotection of d and d with methanolic ammonia provided the respective final ribosides d and d. however, the major product of the glycosylation reaction of d turned out to be the di-ribosylated compound ( figure ), whose structure was elucidated on the basis of h-nmr, c-nmr, noe and mass spectra. two sets of sugar carbon peaks were observed on the c-nmr spectrum of , in the presence of only one set of aromatic carbon peaks. the sites of substitution as well as the anomeric configuration were assigned as exemplified in figure (namely n, -β-d) . upon careful examination of the noe scheme . reagents and conditions: (a) (i) n,o-bis(trimethylsilyl)acetamide, acn, reflux, h, regarding the n regioisomers, we were able to isolate pure n -β-d nucleoside acetates a-c as well as their corresponding n -α-d anomers a-c, upon chromatographic purification. the site of ribosylation as well as the anomeric configuration were unambiguously determined on the basis of noe spectroscopy. taking into consideration the noe spectra of compounds a-c and a-c, we observed clear correlation peaks between the aromatic proton and protons of the furanose ring, determinant of the n -ribosylation pattern. in addition, we also noticed cross correlation peaks between -h and -h of the sugar moiety in the spectra of compounds a-c. such peaks were not observed on the noe spectra of a-c, thus clearly concluding that a-c were the n -α-d nucleoside products of the reaction, while a-c were their corresponding β-d anomers. deacetylation of a-c with methanolic ammonia provided the final compounds a-c. isolation of the pure -β-d nucleoside acetates proved to be difficult at this stage, so the mixtures of a-c with their corresponding α-anomers a-c were subjected to ammoniolysis, to provide the deprotected nucleosides. the ethylamino ( a) and isopropylamino ( b) derivatives were isolated in pure form by recrystallization, whereas an analytically pure sample of the benzylamino compound c was obtained upon purification with semi-preparative hplc. anomeric purity and configuration of compounds a-c were determined on the basis of h-nmr and noe spectra, respectively. in the latter ones, we observed clear correlation peaks between -h and -h of the ribofuranose moiety, while there was a profound absence of correlation peaks between the aromatic and sugar protons on the noe spectra of each of the aforementioned compounds. the close examination of d and d nmr spectra reveals that a simple differentiation between each pair of regio isomers can be easily made upon inspection of the chemical shift of the aromatic proton. this proton appears upfield in the case of the n -isomers a-d ( . - . ppm) whereas it shifts downfield ( . - . ppm) concerning the n -isomers a-d. applying the same reaction conditions for the glycosylation of the primary aminoderivative d, we isolated from the reaction mixture the -β-d nucleoside acetate ( d), as well as the -β-d isomer ( d), without detectable amounts of the corresponding α-anomers. deprotection of d and d with methanolic ammonia provided the respective final ribosides d and d. however, the major product of the glycosylation reaction of d turned out to be the di-ribosylated compound (figure ) , whose structure was elucidated on the basis of h-nmr, c-nmr, noe and mass spectra. two sets of sugar carbon peaks were observed on the c-nmr spectrum of , in the presence of only one set of aromatic carbon peaks. the sites of substitution as well as the anomeric configuration were assigned as exemplified in figure (namely n, -β-d) . upon careful examination of the noe spectrum of , we observed a set of correlation peaks between the aromatic proton and protons of one sugar moiety as well as correlation peaks between the anomeric proton of each furanose with its corresponding -proton. molecules , , x for peer review of spectrum of , we observed a set of correlation peaks between the aromatic proton and protons of one sugar moiety as well as correlation peaks between the anomeric proton of each furanose with its corresponding ′-proton. compounds a-d and a-d were evaluated for their activity against vaccinia virus, adeno virus- , human coronavirus ( e), hsv- , hsv- , vzv, and hcmv (ad- and davis strains) in human embryonic lung cell cultures (hel) (tables s -s ). unfortunately, the new derivatives proved to lack antiviral activity against all viruses tested. in particular, the substitution of the benzimidazole pharmacophore present in maribavir by the imidazo [ , -b] pyridine scaffold, in combination with the replacement of the l-sugar configuration with its corresponding d-riboside, resulted in total loss of the anti-hcmv activity. nevertheless, upon determination of the cytotoxic properties of the new derivatives, compounds c, d and c, proved to possess moderate antiproliferative activity against the three cancer cell lines tested, namely human t-lymphocyte cells (cem), human cervix carcinoma cells (hela) and human dermal microvascular endothelial cells (hmec- ) (table s ) . among these compounds, both -benzylamino-substituted derivatives c and c showed antiproliferative activity against cem cell-line, with equipotent ic values ( ± and ± µ m) and at the same time, c proved active in inhibiting the growth of hela and hmec- cell lines, possessing ic values of ± µ m and ± µ m, respectively. the increased antiproliferative effects of c and c are worth investigating further and are currently under active search in our laboratories. tetrahydrofuran (thf) was distilled from sodium/benzophenone ketyl immediately prior to use. melting points were determined on a büchi apparatus and are uncorrected. h-nmr spectra and d (cosy, noesy, hsqc, hmbc) nmr spectra were recorded on a bruker avance iii or a bruker avance (bruker, karlsruhe, germany) drx instrument, whereas c-nmr spectra were recorded on a bruker avance iii in deuterated solvents and were referenced to tms (δ scale). the signals of h and c spectra were unambiguously assigned by using d nmr techniques: h h cosy, noesy, hsqc and hmbc. mass spectra were recorded with a ltq orbitrap discovery instrument, possessing an ionmax ionization source. column chromatography was performed on merck (merk, darmstadt, germany) silica gel ( . - . mm), unless specified otherwise. analytical thin layer chromatography (tlc) was carried out on precoated ( . mm) merck silica gel f- plates. preparative hplc was performed on a system equipped with two prep laballiance pumps (asi, richmond, ca, usa), a fortis c- ( μm) column (i.d. × mm) and a flash s dad detector (ecom, praha, czech republic). optical rotations were obtained on a perkin-elmer polarimeter (perkin elmer, shelton, ct, usa). compounds a-d and a-d were evaluated for their activity against vaccinia virus, adeno virus- , human coronavirus ( e), hsv- , hsv- , vzv, and hcmv (ad- and davis strains) in human embryonic lung cell cultures (hel) (tables s -s ). unfortunately, the new derivatives proved to lack antiviral activity against all viruses tested. in particular, the substitution of the benzimidazole pharmacophore present in maribavir by the imidazo[ , -b]pyridine scaffold, in combination with the replacement of the l-sugar configuration with its corresponding d-riboside, resulted in total loss of the anti-hcmv activity. nevertheless, upon determination of the cytotoxic properties of the new derivatives, compounds c, d and c, proved to possess moderate antiproliferative activity against the three cancer cell lines tested, namely human t-lymphocyte cells (cem), human cervix carcinoma cells (hela) and human dermal microvascular endothelial cells (hmec- ) (table s ) . among these compounds, both -benzylamino-substituted derivatives c and c showed antiproliferative activity against cem cell-line, with equipotent ic values ( ± and ± µm) and at the same time, c proved active in inhibiting the growth of hela and hmec- cell lines, possessing ic values of ± µm and ± µm, respectively. the increased antiproliferative effects of c and c are worth investigating further and are currently under active search in our laboratories. tetrahydrofuran (thf) was distilled from sodium/benzophenone ketyl immediately prior to use. melting points were determined on a büchi apparatus and are uncorrected. h-nmr spectra and d (cosy, noesy, hsqc, hmbc) nmr spectra were recorded on a bruker avance iii or a bruker avance (bruker, karlsruhe, germany) drx instrument, whereas c-nmr spectra were recorded on a bruker avance iii in deuterated solvents and were referenced to tms (δ scale). the signals of h and c spectra were unambiguously assigned by using d nmr techniques: h h cosy, noesy, hsqc and hmbc. mass spectra were recorded with a ltq orbitrap discovery instrument, possessing an ionmax ionization source. column chromatography was performed on merck (merk, darmstadt, germany) silica gel ( . - . mm), unless specified otherwise. analytical thin layer chromatography (tlc) was carried out on precoated ( . mm) merck silica gel f- plates. preparative hplc was performed on a system equipped with two prep laballiance pumps (asi, richmond, ca, usa), a fortis c- ( µm) column (i.d. × mm) and a flash s dad detector (ecom, praha, czech republic). optical rotations were obtained on a perkin-elmer polarimeter (perkin elmer, shelton, ct, usa). completed. the mixture was filtered warm through a celite pad, which was thoroughly washed with warm meoh. the solvent was then vacuum-evaporated and the residue was purified by column chromatography using a mixture of chcl /meoh ( / to / , v/v) as the eluent, to result in mg of a ( % overall yield). beige solid, mp > • c (dec) , (meoh). to a suspension of the imidazopyridine a ( mg, . mmol) in dry ch cn ( ml) was added n,o-bis-(trimethylsilyl)acetamide ( mg, . mmol) and the reaction mixture was refluxed for h. the suspension was then cooled to room temperature and , , , -tetra-o-acetyl-β-d-ribofuranose ( mg, . mmol) was added, followed by the dropwise addition of trimethylsilyltriflate ( . ml, . mmol) at • c. the mixture was refluxed for h, the solvent was vacuum evaporated, the residue was dissolved in etoac ( ml) and washed with a saturated nahco solution ( ml). the aqueous phase was extracted once more with etoac ( ml) and the combined organic layers were washed with brine ( ml), dried (na so ) and evaporated to dryness. the resulting oil was purified by column chromatography, using a mixture of chcl /meoh ( / to / , v/v) as the eluent, to afford a, as a mixture with its corresponding α-anomer a ( mg, total yield % for two anomers, a: a ( -β:α) ratio : , as estimated by [ , -b] pyridin- -amine ( b): these derivatives were prepared by a procedure analogous to that described for a, a, a, a, starting from imidazopyridine b ( mg, . mmol). purification was effected by column chromatography, using a mixture of chcl /meoh ( . / . to / , v/v) as the eluent, to afford b, as a mixture with its corresponding α-anomer b ( mg, % total yield for two anomers, b: b ( -β:α) ratio : , as estimated by h-nmr), b ( mg, % yield) and b ( mg, % yield). data the compounds were evaluated against different herpesviruses, including herpes simplex virus type (hsv- ) strain kos, a thymidine kinase-deficient (tk -) hsv- kos strain that is resistant to acv (acv r ), herpes simplex virus type (hsv- ) strain g, adeno virus- , human coronavirus, varicella-zoster virus (vzv) tk + strain oka, tk -vzv strain - , and human cytomegalovirus (hcmv) strains ad- and davis. the antiviral assays were based on inhibition of virus-induced cytopathicity or plaque formation (for vzv) in human embryonic lung (hel) fibroblasts. confluent cell cultures in microtiter -well plates were inoculated with ccid of virus ( ccid being the virus dose to infect % of the cell cultures) or with plaque forming units (pfu) (for vzv) and the cell cultures were incubated in the presence of varying concentrations of the test compounds. viral cytopathicity or plaque formation (vzv) was recorded as soon as it reached completion in the control virus-infected cell cultures that were not treated with the test compounds. antiviral activity was expressed as the ec or compound concentration required, to reduce virus-induced cytopathicity or viral plaque formation by %. cytotoxicity of the test compounds was expressed as the minimum cytotoxic concentration (mcc) or the compound concentration that caused a microscopically detectable alteration of cell morphology. alternatively, the cytostatic activity of the test compounds was measured based on inhibition of cell growth. hel cells were seeded at a rate of × cells/well into -well microtiter plates and allowed to proliferate for h. then, medium containing different concentrations of the test compounds was added. after days of incubation at • c, the cell number was determined with a coulter counter. the cytostatic concentration was calculated as the cc , or the compound concentration required to reduce cell proliferation by % relative to the number of cells in the untreated controls. the human cell lines used for the proliferation were hela (atcc #ccl- , cervical carcinoma), cem (t-lymphoblastoid cells) and hmec- (human microvascular endothelialcells). first, ( − . ) × cells were seeded onto standard -well microtiter plates and left to attach for h. on the next day, test compounds were added in five serial -fold dilutions. the cell growth rate was evaluated after h of incubation, using mtt assay. obtained results are expressed as an ic value, which stands for the concentration of the compound necessary for % growth inhibition. the ic values are calculated from concentration-response curve using linear regression analysis. each test was performed in quadruplicate in at least two individual experiments. supplementary materials: the following are available online: a figure indicating the numbering of the imidazopyridine nucleosides ( figure s ); h-and c-nmr spectra of the target nucleosides a-d and a-d, and of the di-ribosylated by-product ( figures s -s ) ; the noe spectra of target compounds a and a (figures s and s ); tables with the results of the antiviral (tables s -s ) and the cytotoxic (table s ) (co), . (co), . (co) pyridin- -amine ( c) and n-benzyl- , -dichloro- -( , , -tri-o-acetyl-α-d-ribofuranosyl)- h-imidazo[ , -b]pyridin- -amine ( c): these derivatives were prepared by a procedure analogous to that described for a, a, a, a, starting from imidazopyridine c ( mg, . mmol). purification was effected by column chromatography, using a mixture of cyclohexane/etoac ( / to / , v/v) as the eluent, to afford c, as a mixture with its corresponding α-anomer c ( mg, % total yield for two anomers, c: c ( -β:α) ratio : , as estimated by h-nmr), c ( mg, crude) and c ( mg, % yield). fractions containing c were pooled and subjected to column chromatography eluted with chcl /meoh ( . / . , v/v), yielding mg of pure c ( % yield). data for c: oil hr-ms (esi) m/z: calcd for c h cl n o na: [m + na] + = . , found . . data for c: oil , , -tri-o-acetyl-β-d-ribofuranosyl)- h-imidazo[ , -b]pyridin- -amine ( d) and , -dichloro- -( , , -tri-o-acetyl-β-d-ribofuranosyl)- h-imidazo[ , -b]pyridin- -amine ( d): these derivatives were prepared by a procedure analogous to that described for a and a, starting from d ( mg, . mmol). purification was effected by column chromatography chcl ). h nmr ( mhz, cdcl ) δ . (s, h, ch co), . (s, h, ch co), . (s, h (dd, h, h- , j , = . hz, j , = . hz), . (dd, h, h- , j , = . hz, j , = . hz), . (m, h, h- ), . (d, h, h- , j , = . hz), . (br s, h, d o exchangeable, nh ), . (s, h, h- ). c-nmr ( mhz, cdcl ) δ . (ch co), . (ch co) β-d-ribofuranosyl)- h-imidazo[ , -b]pyridin- -amine ( c): this compound was prepared by a procedure analogous to that described for a starting from c ( mg, . mmol, containing also the corresponding α-anomer c). purification was effected by column chromatography using a mixture of dcm/meoh: / to / (v/v) to afford an anomeric mixture β/α, in a / ratio ( mg). a second column chromatography was performed (dcm/meoh: . / . to / , v/v) and the fractions pooled ( mg) were enriched in the desired β-anomer c (β/αratio: / meoh). h nmr ( mhz, dmso-d ) δ . (br s (t, h, d o exchangeable, nh, j = . hz). c-nmr ( mhz, dmso-d ) δ purification was effected by column chromatography using a mixture of dcm/meoh: / to / (v/v) as the eluent, to result in d ( mg, % yield) (m, h, ch , overlapping with water of dmso-d ), . (dd, h, h- , j , = . hz, j , = -dichloro-n-isopropyl- -(β-d-ribofuranosyl)- h-imidazo[ , -b]pyridin- -amine ( b):this compound was prepared by a procedure analogous to that described for a starting from b ( mg, . mmol). purification was effected by column chromatography, using a mixture of dcm/meoh: / to / (v/v) as the eluent, to afford b ( mg, % yield) the pathogenesis of human cytomegalovirus detection of cytomegalovirus in atherosclerotic plaques and nonatherosclerotic arteries signature microrna expression profile of essential hypertension and its novel link to human cytomegalovirus infection the story of human cytomegalovirus and cancer: increasing evidence and open questions manifestations of human cytomegalovirus infection: proposed mechanisms of acute and chronic disease updated international consensus guidelines on the management of cytomegalovirus in solid-organ transplantation treatment outcomes of reduced-dose intravitreal ganciclovir for cytomegalovirus retinitis new vaccines and antiviral drugs for cytomegalovirus antiviral drug-and multidrug resistance in cytomegalovirus infected sct patients human cytomegalovirus dna replication: antiviral targets and drugs cmx to prevent cytomegalovirus disease in hematopoietic-cell transplantation potency and stereoselectivity of cyclopropavir triphosphate action on human cytomegalovirus dna polymerase first global approval function of human cytomegalovirus ul kinase in viral infection and its inhibition by maribavir maribavir and human cytomegalovirus-what happened in the clinical trials and why might the drug have failed? novel nucleoside analogues targeting hcv replication through an ns a-dependent inhibition mechanism synthesis of new nebularine analogues and their inhibitory activity against adenosine deaminase the application of mitsunobu cyclization for the synthesis of , -dideoxy-c-nucleosides designed as didanosine analogues the synthesis of the new c-nucleoside -deazaformycin b potent and selective inhibition of human cytomegalovirus replication by w , a benzimidazole l-riboside with a unique mode of action design, synthesis and anti-hbv activity evaluation of new substituted imidazo[ , -b]pyridines synthesis of -(alkylamino)benzimidazoles design, synthesis, and antiviral activity of certain , , -trihalo- -(beta-d-ribofuranosyl)benzimidazoles this article is an open access article distributed under the terms and conditions of the creative commons attribution the authors are grateful to leentje persoons and brecht dirix for excellent technical assistance in the antiviral and antiproliferative assays. the authors declare no conflict of interest. key: cord- -i l cq h authors: law, betty yuen kwan; mok, simon wing fai; wu, an guo; lam, christopher wai kei; yu, margaret xin yi; wong, vincent kam wai title: new potential pharmacological functions of chinese herbal medicines via regulation of autophagy date: - - journal: molecules doi: . /molecules sha: doc_id: cord_uid: i l cq h autophagy is a universal catabolic cellular process for quality control of cytoplasm and maintenance of cellular homeostasis upon nutrient deprivation and environmental stimulus. it involves the lysosomal degradation of cellular components such as misfolded proteins or damaged organelles. defects in autophagy are implicated in the pathogenesis of diseases including cancers, myopathy, neurodegenerations, infections and cardiovascular diseases. in the recent decade, traditional drugs with new clinical applications are not only commonly found in western medicines, but also highlighted in chinese herbal medicines (chm). for instance, pharmacological studies have revealed that active components or fractions from chaihu (radix bupleuri), hu zhang (rhizoma polygoni cuspidati), donglingcao (rabdosia rubesens), hou po (cortex magnoliae officinalis) and chuan xiong (rhizoma chuanxiong) modulate cancers, neurodegeneration and cardiovascular disease via autophagy. these findings shed light on the potential new applications and formulation of chm decoctions via regulation of autophagy. this article reviews the roles of autophagy in the pharmacological actions of chm and discusses their new potential clinical applications in various human diseases. autophagy is the catabolic process in which eukaryotic cells engulf and lysosomally digest intracellular contents. the process maintains metabolic balance and cellular quality by removing dysfunctional cytoplasmic constituents and recycling basic molecular building blocks. macroautophagy envelops and delivers the unfavoured intracellular contents to lytic compartment through the doubled-membraned autophagosome [ ] . autophagy-related proteins (atg) are the main underpinning mechanistic component of macroautophagy. about mammalian homologs of yeast atg have been discovered. these proteins are responsible for the nucleation and elongation of the isolation membrane through protein complex formation [ , ] . basal autophagy retains proper physiological functioning of cells through the control of cellular homeostasis, metabolic balance, and protein structural integrity. additionally, macroautophagy is known as type ii programmed cell death with its molecular regulation intricately interconnecting with the apoptotic mechanism [ ] [ ] [ ] [ ] . growing evidence also suggests involvement of macroautophagy in innate and adaptive immunity [ , ] . therefore hampered autophagy is closely associated with proteinopathies, metabolic and immunological disorders. in fact, macroautophagy failure is the molecular culprit of aberrant proteins accumulation in neurodegenerative diseases such as prion, alzheimer's and parkinson's [ ] [ ] [ ] . in metabolic diseases, altered macroautophagy is related to the mammalian tor performs its function by complexing with other proteins, and appears in two constitutively different forms which are the target of rapamycin complex and (mtorc and ) [ ] . canonically, mtorc masters the repression of autophagy responding to growth factor, hormone and amino acid signaling by direct interaction with the atg machinery and interference of autophagosome formation [ , ] . compared with mtorc , mtorc is better characterized which together with the tsc / (tuberous sclerosis complex / ) complex, an mtorc activities suppressor, underpinning the central molecular governance of mtor cascade. this pathway contributes mainly to the integration of growth factor and insulin signaling. the tsc / complexes collect the signals from cellular sensor such as insulin receptor at the plasma membrane. class i ptdlns k is then activated and phosphorylates phosphatidylinositol ( , )-bisphosphate (pip ) to phosphatidylinositol ( , , ) -bisphosphate (pip ). while class iii ptdins k product pi p is critical for autophagy, pip generated by class i ptdlns k are inhibitory. pip subsequently recruits and activates pkb [ ] . during cellular energy stress, the amp: atp ratio is increased because of intracellular atp depletion [ , ] triggering the upstream activator of ampk, liver kinase b (lkb kinase) [ ] . the activated ampk can interact directly with the regulatory-associated protein of mtor (raptor) subunit of mtorc inactivating the protein complex thereby upregulating autophagy [ ] . cytosolic calcium ion (ca + ) concentration is another positive stimulation of ampk-induced autophagy which is initiated by ca + /calmodulin-dependent kinase kinase β (camkkβ) and endoplasmic reticulum-localized bcl- [ ] . accumulation of misfolded proteins is the common feature of the different neurodegenerative diseases [ ] . the self-eating property of autophagy is cytoprotective which assists the clearance of the defective β-sheets enriched protein structure [ ] [ ] [ ] [ ] [ ] . clinically, the beclin level of ad's brain are found to be significantly depressed [ ] . transgenic mouse overexpressing beclin could improve parkinson's disease (pd) progression by reducing α-synuclein aggregation through the enhancement of autophagy [ ] . mutant huntingtin (htt) forms, the main source of neurotoxic activities of huntington disease (hd), are sensitive to beclin level as demonstrated by the increased accumulation of htt upon beclin deficiency [ ] . a mouse model of ad and in vitro studies showed that rapamycin effectively induced amyloid-β (aβ) clearance, and soothed the cognitive deficit [ ] . lithium inhibits inositol monophosphatase (impase) which reduces free inositol and ip levels promotes the removal of htt of hd and α-synuclein via autophagy [ , ] . repressing ip synthesis with the sodium salts of carbamazepine and valproate could induce similar therapeutic effects in the experimental models of hd [ ] [ ] [ ] . another mtor-independent autophagy inducer, trehalose, is also relevant to htt, α-synuclein and tau aggregations [ , ] . most tumor suppressors and oncogenes are actually cellular metabolism regulators responsible for metabolic pathways including aerobic glycolysis, glutaminolysis and one-carbon metabolism [ ] . the major cellular energy monitor ampk, and hence the downstream autophagy up-regulated by the kinase, are closely related to cancer progression in response to metabolic stresses. ampk activators such as -aminoimidazole- -carboxamide riboside (aicar) are strongly cytotoxic to the in vitro models of hepatic, gastric and prostate cancers [ ] [ ] [ ] . many other molecular messengers along the autophagy pathways are cancer-related. for example, the induction of the oncogenic mtorc attenuates autophagy and support cancer development [ ] [ ] [ ] [ ] . clinical trials with cytotoxic drugs rapamycin, temsirolimus and everolimus targeting mtorc to induce autophagy have been reported [ ] . therefore, cancer intervention with the use of autophagy inducers can trigger both autophagic cell death and physiological changes, such as cell cycle arrest, on cancer cells. this is of particular interest for most cancers and malignancies in regard to apoptotic resistance towards chemotherapies [ ] . the systemic balance of glucose and fatty acid is frequently lost in patients enduring metabolic syndromes such as obesity and glucose intolerance. it is evident that the corresponding complex regulatory networks are linked up by autophagy [ ] . chronic consumption of diets with high nutrient abnormally activates mtorc [ ] . the de novo hepatic lipogenesis can then be stimulated by mtorc and its substrate sterol regulatory element-binding protein c (srebp- c) during the obese state [ , ] , which further dampens the expressions of the atgs lc , beclin , atg and atg and thus the autophagic process [ ] . while activating autophagy with pharmacological inducers seems to be an ideal method for improving metabolic disorders, caloric restriction appears to be the best strategy for controlling these disorders [ ] . autophagy functions as an immune effector and directly delivers the intracellular microorganism or their components [ ] into the lysosome through a specific form of selective autophagy called xenophagy [ , ] . several medically important bacterial pathogens, such as mycobacterium tuberculosis (mtb), group a streptococcus, salmonella, shigella and listeria can be degraded by xenophagy. the sindbis virus, herpes simplex virus and the parasite toxoplasma gondii can also be eliminated by xenophagy [ , , ] . beyond the invading microbes, autophagy also targets the innate and adaptive immunity for preventing infections. autophagy negatively regulates il- β and il- expression through maintaining the quality of the intracellular milieu [ ] . the overexpression of il- β and il- of lipopolysaccharides (lps)-stimulated macrophages with the loss of atg l further clarified the role of autophagy in inflammatory immune responses [ ] . in vitro studies have verified the potential of some autophagic inducers in infection therapy. the hormonally active form of vitamin d , d , enhanced macrophage autophagy and prevented human immunodeficiency virus (hiv) replication [ ] . similarly, the small molecule pdk inhibitor ar cleared francisella tularensis [ ] and salmonella enterica serovar typhimurium [ ] . the therapeutic effects of the antibiotics cocktails containing isoniazid and pyrazinamide towards mtb-infected host cells also corresponded to their autophagy-inducing properties [ ] . autoimmunity is the aberrant activation of immune systems towards self-antigens due to central intolerance [ ] . autophagy is essential for loading the mhc ii compartment with intracellular antigens, a process involving the atg-lysosome interaction [ ] , implicating the importance of autophagy in the determination of cd + t lymphocytes receptor repertoire. also, the autoreactive thymocytes that have been programed in thymus can be eliminated by autophagy [ ] . besides, the autophagic activity is positively correlated with macrophagic expression of the proinflammatory cytokines tnf-α and il- which may also contribute to sle pathogenesis [ ] . glucocorticoids since autophagy dysregulation underlies a broad range of pathological conditions, the successful therapeutic outcomes of using autophagy modulators, along with the expanding discoveries regarding the molecular basis of autophagy, suggest the need of intensively investigating the pharmaceutical potential of compounds with autophagy-adjusting ability (table ) . while a number of synthetic autophagy regulators, such as tamoxifen, sorafenib and the water-soluble synthetic rapamycin temsirolimus, have been reported [ ] [ ] [ ] , natural autophagic compounds from chms are of interested because of their potential new therapeutic applications ( figure ). since autophagy dysregulation underlies a broad range of pathological conditions, the successful therapeutic outcomes of using autophagy modulators, along with the expanding discoveries regarding the molecular basis of autophagy, suggest the need of intensively investigating the pharmaceutical potential of compounds with autophagy-adjusting ability (table ) . while a number of synthetic autophagy regulators, such as tamoxifen, sorafenib and the water-soluble synthetic rapamycin temsirolimus, have been reported [ ] [ ] [ ] , natural autophagic compounds from chms are of interested because of their potential new therapeutic applications ( figure ). induction of autophagic cells death in glioblastoma, gastric adenocarcinoma [ ] ; inhibition of iav-induced autophagic cell death [ ] fructus piperis longi (bi bo) warms cold, expels cold, relieves pain piperlongumine promotion autophagic cell death of breast, kidney, prostate and lung cancer cells [ , ] blood regulating drugs rhizoma curcumae longae (jiang huang) regulates blood, moves blood, moves and regulates qi, descends the qi curcumin hinders α-synuclein accumulation in neural cells and suppression of the proliferation of glioma cells through induction of autophagy [ , ] radix salviae miltiorrhizae (dan shen) moves blood, breaks up blood stasis, cools heat, cools blood tanshinone iia induction of autophagic cell death of leukemia via activation of ampk/mtor, erk/mtor and p s k signaling [ ] ligusticum wallichii (chuan xiong) moves blood, moves and regulates qi, dispels wind ligustrazine induction of cytotoxic effects in hepatocellular carcinoma and protection of the kidney from neurotoxicity through autophagy [ , ] external using drugs venenum bufonis (chan su) opens the orifices, detoxifies, relieves pain bufalin induction of cell death in hepatoma cells and suppression of colon cancer cells proliferation through autophagy [ , ] gamboge (teng huang) detoxifies, disperses swelling, antiparasitic, alleviates itching gambogic acid amelioration of bladder cancer and induction of cytotoxic in leukemia cell through autophagy [ , ] spirit calming drugs radix polygalae (yuan zhi) anchors the yang, dislodges phlegm, opens the orifices onjisaponin b acceleration of the degradation of mutant α-synuclein and huntingtin in pc- cells through autophagy [ ] ganoderma lucidum (ling zhi) tonifies the heart and qi, calms and anchors the spirit ganoderic acid c reduction of accumulation of mutant huntingtins in pc- cells, induction of autophagic cell death in melanoma cells [ ] caulis polygoni multiflori (shou wu teng) calms and anchors the spirit, anchors the yang anthraquinones induction of autophagic cell death in c and u [ ] fructus schisandrae (wu wei zi) harmonizes and tonifies the yin and qi, secures the essence schisandra total lignin inhibition of d-galactose-induced brain tissue aging through autophagy [ ] semen ziziphi spinosae (suan zao ren) tonifies yin and blood, astringes and collects, anchors the yang jujuboside a, jujuboside b jujuboside b induces autophagic cell death in ags and hct human cancer cells and suppresses tumor growth [ ] succinum (ambrum) calms and anchors the spirit, sedates and cools the heart vitamin e succinate (ves) ves-induced autophagy participates in sgc- cell protection by inhibiting mtor axis phosphorylation [ ] . heat-clearing drugs are used to clear damp-heat, fire or heat in the blood and body fluids to maintain regular body temperature and normal hemostatis of body [ , ] . radix scutellariae (huang qin) has been shown to modulate inflammatory diseases like gastroenteritis and hepatitis, and is also effective in controlling tumorigeneses [ ] . the two main active components, baicalin and wogonin inhibit the release of proinflammatory mediators from different immunocellular components [ , ] . baicalin and wogonin might be effective for inducing cytotoxicity or inhibiting proliferation in various human hepatoma [ ] . the anti-cancer effects of radix scutellariae such as induction of cell death and cell cycle arrest could be mediated through autophagy [ ] . although the involvement of autophagy in the radix scutellariae-triggered anti-inflammatory property is still elusive, the progression of gastroenteritis and hepatitis are highly autophagic-related [ ] , suggesting the potential autophagic role of radix scutellariae in such diseases. all these observations suggest that a main part of the clinical functions of radix scutellariae as documented in chinese traditional medical references are manifested via autophagy. cortex phellodendri (huang bo) with berberine as its active ingredient after bark extraction has been traditionally prescribed for the treatment of pneumonia, tuberculosis, meningitis and liver cirrhosis [ , ] . cortex phellodendri is anti-inflammatory in nature, which helps to eliminate invading pathogens, ameliorates acetaldehyde-induced hepatic nf-κb activation during cirrhosis [ , ] , and inhibits glial proinflammatory inos (nitric oxide synthase) and tnf (tumor necrosis factor)-α activity [ ] . berberine modulates autophagic processes through the ampk/mtor signaling pathway [ , ] , therefore, the observed anti-inflammatory capability of cortex phellodendri was likely related to berberine-induced autophagy [ , ] . recently, the dietary supplement nexrutine ® which contains berberine, has been found to have therapeutic potential towards melanoma, multiple myeloma, prostate, pancreatic, breast and non-melanoma skin cancer [ ] [ ] [ ] [ ] [ ] . since berberine could induce both apoptosis and autophagy during tumorigenesis [ ] , autophagy may be responsible for the newly discovered anti-cancer properties of cortex phellodendri. rhizoma coptidis (huang lian), containing the active component berberine, has been traditionally used in alleviating inflammatory disorders, including type ii diabetes. pharmacological studies have reported the anti-inflammatory effects of rhizoma coptidis. for example, berberine was effective in repressing the adverse responses caused by atherosclerosis (as) [ ] . rhizoma coptidis was beneficial to the treatment of intestinal infections [ , ] , and gut-associated abnormalities such as irritable bowel syndrome [ ] . furthermore, berberine induced autophagy [ , ] and suppressed the pro-inflammatory phenotype of macrophages [ ] . in response to the anti-diabetic effect of rhizoma coptidis, autophagy was important for regulating the synthesis and secretion of insulin by pancreatic β-cells [ ] . recent studies have also reported the potential of rhizoma coptidis in neurodegeneration therapy [ , ] . with the protective role of autophagy in neurodegenative and inflammatory diseases, it is notable that the protective effect of rhizoma coptidis may be regulated through autophagy. radix sophorae flavescentis (ku shen) was used to alleviate toxicity, killed parasites and induced diuresis according to chinese medicinal theory [ ] . many traditional formulas contain radix sophorae flavescentis, for example, "xiaofeng san" was prescribed for treating cutaneous disorders [ ] , and "sanwu huangqin tang" has long been used for post-partum fevers arising from reproductive organ infections during childbirth [ ] . these therapeutic effects suggest the immunomodulatory and anti-inflammatory function of radix sophorae flavescentis may be attributed to the maintenance of systemic homeostasis by clearing off metabolic wastes through autophagy. the active component of radix sophorae flavescentis is matrine, which is pharmacologically related to the suppression of inflammation by inhibiting neutrophil infiltration and oxidative stress, as well as reducing the production of inflammatory mediators [ ] . also, matrine has been reported as t cell anergy inducer through regulating the expression of anergy-associated genes such as jumonji and cd [ ] . the autophagic effects of matrine have suggested radix sophorae flavescentis as a promising anti-cancer herb. for example, matrine was able to induce autophagic cell death against certain cancers [ , , ] , a pharmacological action which has not been reported in chinese medicinal documents. although no direct linkage between radix sophorae flavescentis-induced autophagy and its traditional immunity regulatory effects has been reported, owing to the significant role of autophagy in immunomodulation, the involvement of such a process cannot be neglected. herba rabdosiae (dong ling cao) has been extensively used in cancer therapy [ ] . this herbal drug was also effective in encountering inflammation, oxidative stress and pathogen invasion [ ] . oridonin, an active component of herba rabdosiae, attenuated neuroinflammation and associated oxidative stress by repressing the microglial production of nitric oxide and pro-inflammatory cytokines like il- b and il- [ ] . oridonin inhibited cancer growth by repressing the expression of proinflammatory mediators like il- and bone morphogenetic protein- (bmp- ) [ ] . therefore, the traditional anti-cancer property of herba rabdosiae is highly correlated to autophagy. radix isatidis (ban lan gen) is a popular herbal medicine, especially after the severe acute respiratory syndrome (sars) epidemic, for its clinical applications in upper respiratory tract infections, including the nose, throat, and sinuses [ ] . it is also prescribed for other viral infections like measles and hepatitis [ , ] , etc. the active components of radix isatidis are bis-benzylisoquinoline alkaloids-fangchinoline and tetrandrine. these active ingredients interact with invading pathogens and modulate the host responses. for example, fangchinoline could stop the replication of human immunodeficiency virus (hiv) type by disturbing the proteolytic process of viral gp [ ] ; tetrandrine suppressed hepatitis through the repression of nf-κb activation [ ] . in addition, fangchinoline activated autophagic cell death through the p /sestrin /amp pathway in hepatocellular carcinoma [ ], whereas tetrandrine inhibited leukemia cell proliferation and induced autophagy in vivo. to our knowledge, the use of radix isatidis in anti-cancer therapy has not been reported, therefore, these findings shed light to the development of the new usage of radix isatidis in oncology through induction of autophagy. however, the role of autophagy in mediating the traditional anti-inflammatory function of radic isatidis remains to be investigated. stephania japonica (qian jin teng) has been traditionally used for relieving fever, diarrhea, dyspepsia and urinary disease [ ] . cepharanthine and dauricine are the active components of the herb that have been commonly used for inflammatory and immunological disorders in chinese medicine [ , ] . animal and in vitro studies illustrated that cepharanthine suppressed cytokine synthesis, platelet aggregation, plasma membrane lipid peroxidation, and nuclear factor-κb (nf-κb) stimulation [ ] . the herb also facilitated the removal of free radicals and alleviated oxidative stress [ , ] . dauricine exhibited similar anti-inflammatory effects through repressing the expression of inflammatory indicators such as myeloperoxidase, il- bβ and tnf-α [ ] . recently, these two compounds have been postulated as novel anti-cancer drugs. for example, cepharanthine abolished the drug resistance property of cancer cells by modulating the activities of multidrug resistance factor abcc (mrp ) and atpase [ ] . dauricine had been reported to suppress cancer proliferation and invasion, and promote apoptosis through the nf-κb signaling pathway [ ] . although documentation associating autophagy with the immunomodulatory effects of cepharanthine, dauricine or stephania japonica is scarce, our laboratory discovered that both bioactive components induced autophagic cell death in apoptosis-resistant cancer cells [ ] . these findings suggest cepharanthine and dauricine repress cancer growth through autophagy induction, and stephania japonica may be a potential pharmaceutical agent for cancer. rhizoma polygoni cuspidati (hu zhang) has been used to relieve inflammation, coughing, fever, and provide diuretic, emmenagogue, emollient and stomachic actions [ , ] and against tumor activity [ ] . resveratrol, the active compound of rhizoma polygoni cuspidati, is a natural antibiotic [ ] which inhibits the growth of bacteria and fungi [ , ] . resveratrol exhibited its anti-inflammatory effect through inhibiting pro-inflammatory mediator synthesis [ ] . resveratrol also repressed tumorgenesis [ ] [ ] [ ] . increasing evidence has shown the autophagic role of resveratrol. for example, resveratrol attenuated the inflammatory phenotype of vascular endothelium through up-regulation of autophagy [ ] , and induced autophagic cell death in glioma and adenocarcinoma [ , ] . recently, resveratrol-induced cardioprotection was found to associate with mtorc -mediated autophagy and the up-regulation of antioxidant proteins [ , ] . therefore, autophagy could be the underlying molecular mechanism responsible for the protective effects of rhizoma polygoni cuspidati. herba scutellariae barbatae (ban zhi lian) has long been used for cancer therapy [ ] . it is also effective in treating inflammatory-related symptoms, for example, furunculosis, pyogenic infections, traumatic injury, edema, venomous snake bite, and jaundice [ ] . the active ingredient of the herb, pheophorbide, inhibited cytokines expression and monocyte activities, down-regulated macrophage expression of inos, and scavenged reactive oxygen species (ros) [ ] . in addition, pheophorbide was cytotoxic to hepatocellular carcinoma [ ] and breast adenocarcinoma [ ] . thus far, literatures correlating pheophorbide and autophagy were mostly about the photodynamic therapy of tumorigenesis involving the induction of autophagy and cancer cell death. in breast adenocarcinoma, pheophorbide induced autophagy through the erk signaling pathway [ ] . it also induced both apoptosis and autophagy via erk / and p in skin cancer [ ] . such autophagy-related therapeutic effect of pheophorbide has also been described in oral squamous carcinoma cells and hormone insensitive prostate cancer [ , ] . accordingly, the anti-cancer effect of herba scutellariae barbatae could be partly mediated by autophagy. nelumbo nucifera (lian hua) has been used for maintaining homeostasis, reducing anxiety, acting against bleeding, and repressing inflammation [ ] . neferine is one of the active components responsible for maintaining glucose and lipid balance during starvation [ ] . anti-inflammatory and anti-oxidative effects of neferine in neurodegenerative disease by suppressing nf-κb activation and lipid peroxidation have also been reported [ ] . protective effects of neferine towards inflammation and oxidative stress were observed in pulmonary fibrosis [ ] . emerging findings suggest that autophagy is one of the pharmaceutical targets of nelumbo nucifera. for example, neferine attenuated mutant huntingtin toxicity by inducing the ampk-mtor-dependent autophagic pathway [ ] . such findings correlated the psychopathological regulatory effects of neferine to autophagy. neferine induced autophagy via the ros mediated pathway in lung cancer [ ] , and was found effective in suppressing hepatocellular carcinoma [ ] . these findings confirmed the role of the compound in controlling inflammatory progression and its novel usage in tumorigenesis. therefore, triggering autophagy by nelumbo nucifera could be a new pharmaceutical strategy for encountering inflammatory, neurodegenerative and cancerous diseases. syzygium samarangense (lian wu) possessing the anti-free radical ability is indigenously used to manage inflammation-related conditions and removal of oxidative stress [ , ] . dimethyl cardamonin (dmc) is the active compound isolated from the leaves of syzygium samarangense contributing mainly to the anti-inflammatory and anti-oxidative properties. dmc protected the cells from ros damage by modulating the glutathione s-transferase and superoxide dismutase (sod) activities [ , ] . the compound also attenuated nf-κb activation and relieved cellular inflammatory phenotype with decreased serum level of proinflammatory cytokines [ , ] . recent studies have suggested autophagy induction by dmc was associated with proliferative arrest in colorectal carcinoma by stimulating the p /jnk-dependent signaling [ , ] . the therapeutic effects of dmc-induced autophagy have also been reported in cancers of the pancreas and prostate, myeloid leukemia and multiple myeloma [ ] . although the autophagic role of syzygium samarangense in anti-inflammation remains to be elucidated, the induction of autophagy is highly correlated to the syzygium samarangense-induced anti-cancer effects. trichosanthes kirilowii (cucumber) has been used as folk medicine for the treatment of inflammation and cancer [ ] . pharmacological research further proposed trichosanthes kirilowii as a potential remedy for suppressing hiv replication [ ] . cucurbitacin d, a major component of the herb, is an anti-inflammatory compound which inhibited pro-inflammatory mediator production via inos and nf-κb signaling [ , ] and cyclooxygenase- (cox- ) [ ] . cucurbitacin d was effective in the protection against hepatic and cardiovascular damages, the alleviation of diabetic condition, and the removal of invading microbes [ , ] . cucurbitacin d could be used for repressing tumorigenesis of colorectal carcinoma [ ] , breast adenocarcinoma [ ] , and leukemia [ ] . recent findings have demonstrated that structural analogues of cucurbitacin d, for example, cucurbitacins b, e and i, induced autophagy and are potentially beneficial to cancer intervention. cucurbitacin b triggered autophagy in breast cancer cells by manipulating the ros activities [ ] , and in melanoma cells via c-jun n-terminal kinase (jnk) activation [ ] . cucurbitacin e induced autophagy in cervical and breast cancer cells via activation of ampk signaling [ ] . similar therapeutic effects have also been reported in cucurbitacin i treatment of glioblastoma [ ] . all these observations point towards the likelihood of involvement of cucurbitacin d in autophagy-induced anti-tumor effects. mallotus philippensis (cu kang chai) has been used for alleviating inflammatory symptoms caused by bronchitis, rheumatism and infection. the herb is useful in eliminating parasite invasions such as tapeworm [ ] . rottlerin, one of the major active components of the herb, also manifested potent anti-inflammatory properties. rottlerin regulated inflammatory mediators including cox, protein kinase c δ, lipoxygenase, heme oxygenase and nf-κb [ ] . besides, it also suppressed the progression of malignant cancers by increasing the susceptibility of cancer cells towards apoptosis [ ] [ ] [ ] . in addition, the anti-tumor effects of rottlerin were associated with its autophagy activation property in certain cell types [ , , ] . rottlerin induced apoptosis and autophagic cell death in prostate [ ] and pancreatic cancers via the inhibition of pi k/akt/mtor signaling [ ] . rottlerin also triggered apoptosis and autophagic cell death in fibrosarcoma cells through a pi k/akt/mtor-independent pathway [ ]. these findings, together with the protective role of rottlerin in preventing the spreading of misfolded proteins including prion protein, amyloid aβ, and α-synuclein [ ], suggest that mallotus philippensis could be used as a novel therapeutic intervention for cancer-related and neurodegenerative disorders based on its autophagy-inducing ability. whether autophagy is involved in the traditional anti-inflammatory effects of rottlerin or mallotus philippensis is yet to be determined. rhizoma anemarrhenae (zhi mu) has been used for minor symptoms like cough, fever and constipation [ , ] . the herb was also effective in treating diabetes [ ] . one of the active components extracted from rhizoma anemarrhenae, timosaponin aiii, relieved inflammation and oxidative damages by regulating the cytosolic ca + concentration of endothelial cells [ ] , and neutrophilic superoxide generation stimulated by arachidonic acid [ ] . recent pharmacological studies demonstrated that the anti-tumor effect of timosaponin aiii was associated with autophagy. timosaponin aiii elicited autophagy and cytotoxicity in cervical cancer which was independent of apoptosis [ ] . it also repressed mtor activity, triggered er stress and autophagic cell death in breast cancer [ ] . as demonstrated in an insulin resistance rodent model, timosaponin aiii-induced autophagy may be responsible for its diabetes-ameliorating effect through activation of ampk [ ] . in neurodegeneration model, timosaponin aiii activated autophagy and facilitated the downstream sequestration of aggregation-prone ubiquitinated proteins [ ] . these findings implied the pharmaceutical potential of applying rhizoma anemarrhenae for the treatment of cancers and neurodegenerative diseases. these herbs are used when the zheng qi (normal body condition or upright qi) is weakened, for example, during recovery from illness, during childhood or in old age. the herbs have the ability to tonify (bu), nourish, supplement and strengthen human body [ , ] . radix glycyrrhizae (liquorice, gan cao) is traditionally used as adjuvant to modify the efficacy of other herbs in a single prescription of around % of chinese herbal formulas [ ] , which acted against inflammatory symptoms such as relieving cough, sore throat and phlegm production. it is important for maintaining a proper stomach function and is used for stomach ulcers. licochalcone a (la) and isoliquiritigenin (isl) are compounds extracted from radix glycyrrhizae. la exhibited anti-inflammatory effects which suppressed pro-inflammatory mediator expression [ , ] , and cleared cellular oxidative stress [ ] . isl demonstrated anti-oxidative [ ] and immunomodulatory effects [ ] . of note, emerging data suggest that the anti-cancer properties in both compounds are associated with autophagy. la induced autophagy via pi k/akt/mtor signaling which repressed cervical cancer growth [ ] . androgen-sensitive prostate adenocarcinoma and adenoid cystic carcinoma were sensitive to la-and isl-induced autophagic cell death mediated by mtor inhibition [ , ] . isl also suppressed breast cancer progression through autophagy induction [ ] . therefore, radix glycyrrhizae has the chemotherapeutic potential to function as an effective cancer therapeutic. further investigation is needed for clarifying the mediating role of autophagy in the traditional anti-inflammatory effects of the herb. radix dipsaci (xu duan) has been used for intervention in osteoporosis, strengthening of tendons and ligaments, and alleviating joint stiffness symptoms by promoting blood circulation in close proximity to the affected areas. the herb exhibits anti-inflammatory properties as reflected by its application in reducing abscesses, swellings, and sores [ ] . in addition, radix dipsaci helped to prevent abortion, which implies a regulatory role in the immune system [ ] . akebia saponin pa (as) is one of the bioactive components found in radix dipsaci, as induced autophagic and apoptotic cell death of gastric cancer cells through both the ampk/mtor and pi k/akt/mtor signaling and the downstream activation of p /jnk molecular pathway, which facilitated capase- -dependent apoptosis [ ] . this finding pointed towards the potential therapeutic role of radix dipsaci in cancers. also, the possibility that autophagy may participate in the immunomodulatory and anti-inflammatory functions of radix dipsaci should not be ignored. radix ginseng (ren shen) has been prescribed for maintaining bioenergetics balance as suggested by chinese herbalists for breathlessness, anorexia, hypodynamia, and diabetes [ ] . since, one of the main functions of autophagy is to retain energy homeostasis, it may be related to the traditional use of ginseng as mentioned. pharmacological studies revealed that the bioactive ginsenosides including rb , rg , rg , rh , re, and rd [ ] ameliorated inflammation, removed oxidative stress, stimulated immune system and regulated apoptosis. therefore, radix ginseng may cure more diseases than traditionally known. radix ginseng illustrated beneficial effects in neurodegenerations in part due to its anti-oxidative [ ] and anti-apoptotic properties [ ] . similar therapeutic uses of radix ginseng have also been reported in cardiovascular disease [ ] , and cancers [ ] . recent researches have focused on studying the autophagic mechanisms of radix ginseng. rb suppressed neurotoxicity through inhibiting autophagy by beclin- downregulation [ ] . the compound could also enhance cardiac muscle cell survival through autophagy [ ] . the minor rb -derived ginsenoside f [ ] and rg could regulate autophagy leading to the suppression of breast cancer stem cells [ ] and hepatocellular carcinoma [ ] , respectively, suggesting the role of autophagy in the potential new therapeutic action of ginseng. peschiera fuchsiaefolia (dao zhong sha ma cha) showed in vitro antimalarial activity against plasmodium falciparum [ ] . voacamine (voa), a bioactive alkaloid extracted from the herb [ , ] , has been reported to induce autophagic cell death of multidrug-resistant osteosarcoma, and inhibit the action of transporter p-glycoprotein (p-gp) [ ] . voa is the ligand of p-gp which expresses in kidney, gastrointestinal tract, brain, etc. [ ] . in fact, diabetes mellitus is associated with p-gp dysregulation [ ] . also, the blood brain barrier (bbb) p-gp was associated with abnormal protein aggregation in alzheimer's and parkinson's diseases, suggesting the potential use of the herb in neurodegenerative disorders [ ] . therefore, the well-known autophagic involvement in diabetes mellitus and neurodegenerations strongly advocated that peschiera fuchsiaefolia may act therapeutically as novel autophagy regulators under such pathological conditions. radix ophiopogonis (mai dong) has been used for treating inflammatory symptoms such as cough and phlegm production, and cardiovascular diseases [ ] . ophiopogonin (op)-b is one of the bioactive components, and was found to be an inducer of autophagy. in non-small cell lung cancer, op-b up-regulated autophagy of tumor cells through pi k/akt pathways, and induced apoptosis-independent cell death and silences [ ] . another active constituent, op-d exhibited anti-inflammatory effects through direct inhibition of mitochondrial ros synthesis [ ] . however, such an anti-inflammatory effect was not related to op-d-induced upregulation of autophagy, as the compound could in fact suppress autophagy per se [ ] . therefore, owing to the close relationship between inflammation progression and autophagy, together with the autophagy modulating role of op-b and op-d, it is predicted that the compounds may contribute to the anti-inflammatory activity of the herb by regulating autophagy. also, the findings of op-b in inducing autophagic cancer cell death suggest the alternative use of radix ophiopogonis in cancer therapy. these drugs help our body defenses against external stimulus, including invading pathogens, cold, heat, damp-wind or summer heat that may have a noxious effects on the human body, by maintaining a normal and healthy status of organ such as the stomach (wei qi) [ , ] . radix bupleuri (chai hu) has been used for counteracting different inflammatory conditions including pancreatitis, liver cirrhosis and fever, infections like malaria and common cold, modulating abnormal lipid metabolism and relieving depression [ ] . the bioactivities of the main components, the saikosaponins, are responsible for the mentioned clinical indications. for instance, saikosaponins modulated host immunity by manipulating pro-inflammatory mediator release and lymphocyte responses [ ] . saikosaponins also repressed viral replication [ ] and fever [ ] , reduced hepatotoxicity [ ] and acted as tranquilizers [ ] . direct evidence for the participation of autophagy in regulating such activities is however lacking. however, contemporary studies verified the anti-tumor effect of saikosaponins, in particular ssd (saikosaponin-d), through autophagy regulation. ssd was cytotoxic to different cancers, such as breast and cervical cancers by increasing autophagy-induced er stress via the camkkβ-ampk-mtor signaling [ , ] . in fact, formulated decoctions containing radix bupleuri have been prescribed for cancer therapy [ ] , which supports the idea that radix bupleuri is a novel autophagy enhancer exhibiting therapeutic effects towards cancers. rhizoma zingiberis recens (sheng jiang) has been used for centuries for the treatment of colds, arthritis, migraines, nausea and hypertension [ , ] . -gingerolis is the most abundant bioactive ingredient found in rhizoma zingiberis recens. it suppressed oxidative stress by inhibiting inos activity of activated macrophage [ ] . -gingerol affected the anti-inflammatory properties by regulating ca + , which may be related to the regulation of autophagy [ ] . the anti-mentic qualities of rhizoma zingiberis recens are also related to -gingerol through inhibition of the function of serotonin receptor [ ] . in addition, rhizoma zingiberis recens-mediated new anti-tumor functions via autophagy induction were further clarified. for instance, -gingerol induced cervical cancer cell death by upregulating caspase -mediated apoptosis, and autophagy partly via the repression of akt signaling [ ] . in pancreatic cancer, -gingerol induced cytotoxicity exclusively through autophagy by activating ampk-mtor signaling, which is a process independent of necroptosis and apoptosis [ ] . these herbs are responsible for treating painful obstruction (bi) syndromes due to wind (wind-bi syndrome), cold (cold-bi syndrome), dampness (dampness-bi syndrome) or heat (heat-bi syndrome), which are caused by poor qi or blood circulation [ , ] . radix tripterygii wilfordii (lei gong teng) has been used for the treatment of inflammation and overactive immune system, including rheumatoid arthritis, systemic lupus erythematosus, and dermatomyositis [ ] . celastrol is the bioactive component responsible for exhibiting the anti-inflammatory and anti-oxidative effects for alleviating autoimmune disorders such as chronic inflammation, neurodegenerative disease, and asthma [ ] . recent findings suggested the use of celastrol in cancer therapy as demonstrated by its inhibitory effects in different tumors [ ] . emerging data revealed that autophagy was one of the molecular machineries mediating these celastrol-induced therapeutic functions. by regulating the ros/jnk signaling pathway, celastrol triggered autophagy and apoptosis which further repressed the proliferation of osteosarcoma cells in both animal and cellular models [ ] . beside, autophagy induction by celastrol through the inhibition of pi k/akt/mtor signaling, have demonstrated therapeutic potential in inflammatory disorders such as crohn's disease, which implies that the traditional functions of radix tripterygii wilfordii are mediated through the regulation of autophagy [ ] . the compound also prevented neurodegeneration by inducing autophagy in affected neuronal cell death via the targeting jnk and pten-akt/mtor network [ ] . radix stephaniae tetrandrae (fang ji) is used for the treatment of edema, anti-hypertension and analgesic, and was usually used as decoction such as "fang ji huang qi tang" [ ] . the main bioactive constituents of the herb are fangchinoline and tetrandrine. both compounds are able to modulate cytokine expression and exhibit anti-inflammatory effects [ ] . fangchinoline reduced blood glucose levels, scavenged free radicals and reduced oxidative stress [ ] . tetrandrine exhibited anti-hypertensive action by disrupting ca + movement and no synthase activity [ ] . although the role of autophagy modulation in such alteration of glucose and ca + level remains elusive, both fangchinoline and tetrandrine have been reported to contribute their anti-tumor effect through autophagy. the proliferation and invasion of gastric cancer cells could be suppressed by fangchinoline mediated inhibition of pi k/akt signaling [ ] . the compound also triggered autophagic cell death by targeting the p /sestrin /ampk signaling in hepatocellular carcinoma [ ] . in leukemia cells, autophagy was induced by tetrandrine through the upregulation of notch and ros signaling [ ] . these autophagy-related anti-tumor activity stimulated by fangchinoline and tetrandrine encourage the further development of radix stephaniae tetrandrae as an effective drug for cancer therapy. radix plumbaginis zeylanicae (bai hua dan) has effects in relieving pain, activating blood circulation, and is used for menstrual disorders, detoxification, and elimination of intestinal worms. plumbagin, the bioactive component of the herb, is well known for its therapeutic safety and effectiveness [ ] . pharmacological studies demonstrated that plumbagin modulates the immune system and resolves inflammation [ , ] . the compound facilitates microbe clearance [ ] and modulates lipid metabolism [ ] . however, the role of autophagy in mediating these traditional functions of the herb remains to be elucidated. recently, the anti-tumor activities of plumbagin have been increasingly reported [ , ] . the anti-tumor properties of plumbagin were attributed to the autophagy induction ability of the compound. for instance, plumbagin induced autophagic cell death in breast cancer via the akt/mtor signaling pathway [ ] . the compound also triggered apoptosis and autophagic cell death in lung cancer and tongue squamous carcinoma cells through the mtor signaling pathway [ ] . these herbs are prescribed when there is an accumulation of dampness. this kind of disturbance in body fluid (water) metabolism is related to a dysfunction of the lung, spleen, kidney or bladder [ , ] . rhizoma alismatis (ze xie) is derived from the stem tuber of alisma orientale [ ] . the herb reduces the circulatory levels of cholesterol and blood sugar, and promotes urine production and perspiration, which helps to resolve symptoms like chronic nephritis and edema [ ] . alisol b is the active component of rhizoma alismatis which improved lipid metabolism by inhibiting the absorption and synthesis of cholesterol [ ] . alisol b -acetate, another important constituent of rhizoma alismatis, participates in inhibiting antibody-mediated allergic reactions, and lipopolysaccharide (lps)-induced inos and no production [ ] . up to now, it is still questionable if autophagy mediates the traditional functions of rhizoma alismatis or its bioactive constituents as mentioned. however, alisol b has been reported as a new autophagy inducer functioning through activation of camkk/ampk/mtor signaling, induction of apoptosis and triggering of cell death in breast cancer cells [ ] . the anti-tumor effects of alisol b -acetate have also been reported in hepatocellular carcinoma [ ] . therefore, rhizoma alismatis has high potential to be developed as an anti-cancer drug through regulation of autophagy. cortex magnoliae officinalis (hou po) helps remove chest stuffiness due to phlegm accumulation, and relieves distension, which suggest its regulatory role in the immune system [ ] . in consistence with the clinical indications of cortex magnoliae officinalis, the bioactive component, magnolol, alleviated acute pain and endothelial damage stimulated by inflammation [ ] . other pharmacological effects of the compound included the reduction of anxiety and irritability via the regulation of γ-aminobutyric acid (gaba) receptor expression [ ] . magnolol also possessed anti-fungal properties [ ] , and was applied for bone repair by regulating the activities of osteoclasts and osteoblasts [ ] . magnolol also interplayed with the autophagic process which was beneficial to cancer therapy, but seemed not to be related to the traditional immunomodulatory effects of the herb. it induced autophagic cell death of lung cancer by blocking the pi k/pten/akt pathway [ ] . ery , a compound derived from magnolol, activated autophagy and suppressed angiogenesis, causing apoptosis-independent cytotoxicity in prostate cancer cells [ ] . based on these observations, the effects of cortex magnoliae officinalis on autophagy appear to be a valuable research niche in the search of new cancer treatment modalities. these herbs are used to warm the interior organs, expel cold, tonify yang and rescue harmed yang qi and relieve pain. interior cold of the human body can be caused by exogenous cold, or kidney yang deficiency which finally results in spleen and heart yang deficiency [ , ] . fructus evodiae (wu zhu yu) was effective for the treatment of gastrointestinal and menstrual disorders, postpartum hemorrhage and headaches [ , ] . pharmacological studies of the bioactive component, evodiamine, showed that the compound is anti-inflammatory in nature by inhibiting cox- expression [ ] , and inducing blockage of preadipocytes differentiation [ ] . evodiamine also induced apoptosis and suppressed proliferation of cancer cells [ ] . in addition, evodiamine could induce autophagic cell death in glioblastoma by quenching calcium/jnk signaling and apoptosis [ ] . through the modulation of beclin- and bcl- expression, evodiamine induced autophagic cell death and apoptosis of gastric adenocarcinoma cells, respectively [ ] . in a drug screening test, evodiamine was found to inhibit autophagic cell death of infected cells upon viral inoculation of influenza a through ampk/tsc /mtor signaling [ ] . therefore, evodiamine regulated autophagy through a complex molecular network, further verification of such a circuit would help to discover and standardize the novel usage of evodiamine and fructus evodiae. further investigations correlating autophagy induced by evodiamine or fructus evodiae to their traditional use should also be undertaken. fructus piperis longi (bi bo) represses cough and fever, relieves allergic symptoms like asthma, helps cease pathogen invasions, reduces blood glucose level, induces coronary vasodilation and treats jaundice [ ] . the active components, piperlongumine and its derivatives, inhibit pro-inflammatory mediator synthesis [ ] . these compounds remove oxidative stress and prevent cardiac damage caused by ros [ ] . they also inhibit platelet aggregation and are used as crude drugs for promoting peripheral blood circulation [ ] . amongst the diverse pharmacological activities, the potential piperlongumine-induced cytotoxic effects towards the different cancer cells have aroused the most attention [ , ] . for example, autophagy induced by piperlongumine mediates the anti-tumor effects of the compound. piperlongumine attenuates akt/mtor signaling and promotes autophagic cell death of cancer cells originated from breast, kidney, prostate and lung [ , ] . piperlongumine induces autophagy by targeting the p signaling in osteosarcoma [ ] . apart from regulating tumorigenesis, the traditionally reported anti-inflammatory effect of the compound partly results from autophagy enhancement [ ] . these herbs are used to treat malfunctions in maintaining normal blood hemostasis, such as: ( ) mild forms of blood stagnation caused by slow blood flow which may lead to blood stasis; ( ) severe forms of stagnation due to congealment of phlegm, heat or cold, which lead to formation of solid masses and blood circulation stasis [ , ] . rhizoma curcumae longae (jiang huang) is a safe chm for alleviating intermittent fever and inflammation of the bronchi, kidney and gall bladder, counteracting infections such as leprosy and cold, and treating of edema, diarrhea and cancer [ , ] . curcumin, the main bioactive component of rhizoma curcumae longae, possesses a unique structure amongst other active constituents extracted from the herb. such characteristics conferred curcumin with pharmacological properties per se correlating with the clinical efficacy of rhizoma curcumae longae. the compound has been associated with the capability of preventing inflammation [ ] , tumor progression [ ] , and oxidative stress accumulation [ ] . of note, curcumin was pharmacologically beneficial to neurodegenerative disorders [ , ] . the compound was particularly suitable for neurodegeneration intervention since the compound could cross the bbb after oral administration, acting as an anti-inflammatory and antioxidant drug, and amyloid aggregation inhibitor [ , ] . in a parkinson's disease model, curcumin triggered autophagy in neural cells by suppressing the mtor/p s k signaling which hindered the downstream α-synuclein accumulation [ ] . in addition, curcumin-induced autophagy was correlated to its anti-cancer properties. by the up-regulation of erk / and akt/mtor/p s k signaling, curcumin induced autophagy and suppressed the proliferation of glioma cells [ ] , suggesting the possible autophagic role of curcumin in various disease models. radix salviae miltiorrhizae (dan shen) has been shown to prevent platelet aggregation and facilitate fibrinolysis [ ] . it is a traditional remedy for managing coronary heart disease [ ] . the herb was also used as ingredient in formulations for diabetes such as "tangzhiqing" [ ] , and was prescribed for hepatic and renal disorders [ ] . tanshinone iia is the main bioactive component abundantly found in radix salviae miltiorrhizae. it modulates inflammation and host immunity by acting on multiple targets depending on the cell types [ ] . for instance, tanshinone iia inhibited macrophagic nf-κb activation via the erk / , p and jnk pathways [ ] . in line with the clinical indications of radix salviae miltiorrhizae, tanshinone iia was cardioprotective through its action upon calcineurin/nfatc pathway [ ] . intriguingly, calcineurin regulated ampk-dependent autophagy of cardiomyocytes upon oxidative stress [ ] implying that the process may underlie the cardioprotective function of radix salviae miltiorrhizae. in terms of autophagy regulation, tanshinone iia has been reported to induce autophagic cell death of leukemia via activation of ampk/mtor and erk/mtor, as well as p s k signaling [ ] . such observations suggested new potential uses of radix salviae miltiorrhizae in the treatment of cancer via autophagy. rhizoma chuanxiong (chuan xiong) is well known for its efficacy in improving blood fluidity, coronary and systemic circulation [ ] . contemporary studies have demonstrated that the herb modulates the proliferation of vascular smooth muscle cells [ ] and prevents endothelial cell damage [ ] . ligustrazine (tetramethylpyrazine) is the active constituent which increases myocardial contractility and coronary circulation [ ] . in addition, this single molecule acts as an anti-oxidant to remove superoxide anion, hydroxyl and lipid peroxyl radical which induce oxidative damages in tissues [ ] . however, it is not known if autophagy is modulating the cardioprotective functions of the herb. ligustrazine exhibits neuroprotective and anti-inflammatory effects on brain disorders such as cerebral ischemia, through elevating nuclear factor e -related factor (nrf )/heme oxygenase- (ho- ) expression [ ] . recently, ligustrazine has been reported to induce autophagy which was associated with cytotoxic effects toward hepatocellular carcinoma [ ] . the ligustrazine-induced autophagic effect has also been demonstrated in protecting the kidney from neurotoxicity [ ] . therefore, the mechanisms underlying the rhizoma chuanxiong or ligustrazine-induced autophagic process are intricate. further investigations are needed to support the usage of the herb in pathological condition such as cancer and inflammation conditions. these kinds of compounds are applied to eliminate toxins, kill parasites, diminish swelling, relieve pain, expel pus and abscesses, improve wound healing, stop itching or bleeding [ , ] . venenum bufonis (chan su) is famous for its clinical application in resolving cardiovascular and inflammatory symptoms, including sore throat and tonsillitis, promoting urine production, and acting as an analgesic agent [ ] [ ] [ ] . in modern chinese medicine, venenum bufonis was used in liver cancer therapy [ ] . bufalin is one of the constituents of venenum bufonis exhibiting bioactivities related to the clinical indications described [ ] . recently, bufalin was found to regulate autophagy via cell type-dependent mechanisms to suppress tumorigenesis. in liver cancer, bufalin interacted with the atg , jnk, becn- and tnf signaling, and stimulated autophagic cell death of hepatoma cells like huh , hep b and ha t [ ] . on the other hand, bufalin stopped the proliferation of hepatocellular carcinoma by activating autophagy through the akt/mtor and ampk/mtor pathways respectively [ ] . the bufalin-induced autophagic cell death also effectively suppressed colon cancer cell proliferation via jnk activation [ ] , and the pten/akt pathways [ ] . through targeting ampk and the downstream p s k, bufalin modulated the apoptotic and autophagic activities of glioma cells [ ] . collectively, these findings support the traditional use of venenum bufonis in the treatment of cancerous diseases via autophagy regulation. garcinia hanburyi (teng huang) was used traditionally for treating inflammatory conditions and immunity dysregulation such as ulcerative gingivitis, skin infection, scald and burn, and chronic eczema [ ] . the herb was also applied to cease traumatic bleeding, attacking toxin and parasites [ ] . gambogic acid is the major active ingredient of garcinia hanburyi biologically alleviating pain, inflammation, and fever [ ] . recent investigations strongly suggest that gambogic acid is anti-tumor in nature by suppressing the proliferation of cancers of lung [ ] , liver [ ] , blood [ ] and stomach [ ] . further studies revealed that gambogic acid triggered autophagy and ameliorated bladder cancer by modulating the beclin- , p and nf-κb activities [ ] . by up-regulating the beclin- expression, gambogic acid induced cytotoxic in leukemia cells through the induction of autophagy and apoptosis [ ] . apparently, garcinia hanburyi and gambogic acid have the potential to be further developed as novel and effective anti-cancer therapeutic strategy. it is also important to verify if autophagy is mechanistically meditating the garcinia hanburyi-induced inflammatory and immunological regulations. these herbs can be used to treat mental syndromes related to heart (blood and yin) deficiency such as over-activity of the heart, nervousness, fright, restlessness, irritability, insomnia, palpitations or anxiety; or treat syndromes related to liver (yin and yang) deficiency such as dizziness and headaches. this group of drugs is also prescribed to pacify internal wind which can contribute to tremor, spasms, paraesthesias of the limbs, dizziness or difficulties in walking [ , ] . radix polygalae (rp) (yuan zhi) is commonly prescribed in many classical decoctions such as "kai xin san" [ ] , and "ding zhi wan" [ ] for the treatment of forgetfulness [ ] , anxiety [ ] , insomnia or depression [ ] . recent pharmacological studies have also reported the sedative-hypnotic [ ] , memory improving [ ] , cognitive recognition enhancing [ ] , antidepressant [ ] and neuroprotective effects [ ] of rp. rp was reported to inhibit the phosphatidylinositol -kinase (pi k)/akt or activate the n-methyl-d-aspartate (nmda) signaling pathways [ , ] . the active ingredients of rp such as onjisaponin b and other identified saponins, were proved to accelerate the clearance of neurodegenerative disease proteins such as huntingtin and α-synuclein, reduce aggregation and toxicity of mutant proteins through the induction of autophagy [ , ] . therefore, rp may play its traditional sedative effect through degradation of unwanted proteins or organelles by autophagy. ganoderma lucidum (ling zhi) is having efficacy in replenishing qi, stabilizing the nervous system and relieving cough and asthma, is commonly prescribed to treat insomnia, palpitation, cough and phlegm. it exerts tranquilizing effects through tonifying the heart, qi and blood. triterpenes are the major components of ganoderma lucidum. pharmacological studies have demonstrated that both ganoderma lucidum extract and ganoderic acid c could reduce accumulation of mutant huntingtins in pc- cells, alleviate neurotoxicity and behavioral deficits induced by -nitropropionic acid, and prevent or reverse memory loss resulting from sleep deprivation [ ] . additionally, it was also reported that ganoderma lucidum triterpene extract (glt) suppressed the proliferation of human colon cancer cells and inhibited tumor growth in a xenograft model, which were associated with the induction of autophagic cell death [ ] . furthermore, ganoderic acid activated autophagy, which facilitates immune recognition of cd + t cells; induced autophagic cell death and apoptosis of melanoma [ ] ganoderma lucidum triterpene extract induced autophagy which inhibited the development of colon cancer via p mapk signaling [ ] ; and suppressed gastric cancer cells through the repression of p [ ] . all these findings suggest that the traditional therapeutic role of ganoderma lucidum is in part related to autophagy regulation, which may also be responsible for the novel use of the herb in cancer treatment. caulis polygoni multiflori (shou wu teng) has been prescribed for nourishing blood, tranquilizing the mind and dispersing wind to treat insomnia, numbness of the skin and rheumatism in traditional chinese medicine. it was often combined with semen ziziphi spinosae (suan zao ren) and cortex albiziae (he huan pi) to treat insomnia, distraughtness and dizziness. modern pharmacological study has suggested an effect of caulis polygoni multiflori extract in protecting rats against ccl -induced hepatotoxicity through its antioxidant activities [ ] . the major active ingredients of shou wu teng, anthraquinones, possessed similar chemical structures but different bioactivities. for example, emodin, the most abundant anthraquinone in rhubarb, inhibits cellular proliferation and prevents metastasis of cancers through apoptosis. another major anthraquinone in rhubarb, rhein, inhibits the uptake of glucose and leads to cancer cell death caused by changes in membrane-associated functions [ ] . ananthraquinone-containing extract of shou wu teng possesses myocardial protective effects by maintaining antioxidant status under oxidative stress conditions [ ] . abnormal aggregation of tau protein is highly correlated with the pathogenesis of alzheimer's disease (ad), therefore, with the ability in mitigating aggregation and cytotoxicity of tau [ ] anthraquinones possesses high potential in modulating ad. together with the fact that ananthraquinones were able to induce autophagic cell death in cancer cells [ ] , shou wu teng may exert its traditional sedative function through the induction of autophagy, which was highly related to the modulation of neurodegenerative disease proteins, as well as cancers [ ] . fructus schisandrae (wu wei zi) has been prescribed to replenish qi, nourish the kidneys and tranquilize the mind to produce a sedative nephroprotective effect. besides, sc extract was co-administered with other medicine for reducing immunosuppressive drug (cyclosporine a)-induced side effects [ ] . schisandra total lignin (stl), the major active ingredient of fructus schisandrae, delayed mouse brain aging by attenuating apoptosis [ ] . in vivo experiments further demonstrated stl inhibited the d-galactose-induced brain tissue aging through regulating autophagy and inhibiting apoptosis in the mice. it has been reported that longevity-promoting regimens such as caloric restriction or inhibition of tor is associated with induction of autophagy [ ] . therefore, autophagy may be the mechanism responsible for the sedative and anti-ageing effect of fructus schisandrae. semen ziziphi spinosae (suan zao ren) is commonly prescribed as "suan zao ren tang" for sedation, nourishing the nerves, insomnia, palpitations, anxiety, dizziness, dry mouth and throat, red tongue, and clinical treatment of neurasthenia, heart neurosis and menopausal syndrome due to deficiencies of the heart and liver [ , ] . the active component of semen ziziphi spinosae, jujuboside b, was reported to inhibit platelet aggregation and target cardiovascular diseases associated with platelet hyperaggregation [ ] . furthermore, the anti-tumor activity of jujuboside b was reported to be associated with the induction of apoptosis and autophagy [ ] . another active neuroprotective component from semen ziziphi spinosae, jujuboside a, could mitigate learning and memory impairment in mice, by reducing the level of aβ , and inhibiting the activities of acetylcholinesterase (ache) and no in the hippocampus and cerebral cortex of mice [ ] . with its traditional effects in tranquilizing the mind and nourishing heart, blood and qi, current pharmacological studies have confirmed the neuroprotective and autophagic role of semen ziziphi spinosae. as autophagy was highly correlated to the maintenance of cellular homeostasis, which was important for normal function of brain, autophagy may be responsible for the pharmacological action and sedative effects of semen ziziphi spinosae. succinum (ambrum) has been prescribed for relieving convulsion, tranquilizing the mind, activating blood and removing stasis, inducing diuresis, treating irritability, epilepsy, algomenorrhea and amenorrhea in tcm [ ] . it was commonly prescribed with rhizoma acori graminei (shi chang pu) and radix polygalae (yuen zhi) in "hu po ding zhi wan" for treating palpitations, insomnia and forgetfulness [ ] . vitamin e succinate (ves), one of the active components of ambrum, was proved to induce autophagy via the inhibition of mtor [ ] . besides, ves worked as an anti-neoplastic agent through regulating apoptosis in cancer cells [ ] . as autophagy is also highly correlated with modulation of cancers, with the recently identified anti-cancer effect of jujuboside b and ves through induction of autophagy, both semen ziziphi spinosae and ambrum may possess new applications in anti-cancer therapy via its traditional tranquilizing effect, which was highly associated with the beneficial effect of autophagy. in addition, the chinese medicinal herbs including nelumbo nucifera, rhizoma acori graminei, radix salviae miltiorrhizae and radix ginseng also participate in tranquilization of the mind [ ] . nelumbo nucifera treats palpitations, insomnia and dreamful sleep [ ] in tcm. it is commonly prescribed as a formulation with radix polygalae, semen ziziphi spinosae and radix salviae miltiorrhizae. the active component of nelumbo nucifera, neferine, was identified as a novel autophagic enhancer which facilitates the degradation of mutant neurodegenerative disease proteins in vitro [ ] . with the beneficial effect of autophagy in maintaining normal homeostasis in cells, sedative tcms may play protective role in neurodegenerative diseases, through the removal of disease proteins by autophagy. preclinical or clinical models showed that pharmacological inhibition of autophagy can enhance the sensitivity of tumor cells towards multiple anti-cancer drugs. for example, inhibition of autophagy enhanced apoptosis induced by cetuximab [ ] or vorinostat [ ] . while cq enhanced the therapeutic efficacy of saracatinib [ ] in prostate cancer xenograft model, inhibition by -ma increased fluorouracil ( -fu) [ ] induced apoptosis with tumor regression in colon cancer xenografts. among these autophagy inhibitors, only cq or hcq were studied in humans as they cross the blood-brain barrier with hcq much preferred to human due to the less severe side effects [ ] . based on these preclinical data, phase i or ii trials were performed to evaluate the combinational use of autophagy inhibitors (hcq or cq) with various anti-cancer cytotoxic agents. however, there are limitations for their use in clinical practice due to the long half-life and high effective concentration of hcq. a phase i trial was performed to evaluate the combined use of hcq with temozolamide [ ] and radiation in glioblastoma patients. phase i or ii clinical trials evaluating the combination use of bortezomib and cq [ ] are ongoing in patients with recurrent carcinoma. a phase i trial of -deoxyglucose [ ] , an agent that blocks glucose metabolism, showed a reduction in autophagy, suggesting the role of autophagy in cancer therapy. similarly, the clinically used mood stabilizers lithium (valproate and carbamazepine) [ ] , induce mtor-independent autophagy and enhance the cellular degradation of aggregate-prone mutant huntingtin and α-synuclein. the hypertensive agent rilmenidine [ ] , a us food and drug administration-approved compound, showed protective effect in huntington's disease models and is now under further clinical trials. protein phosphatase a (pp a) agonists [ ] which favor induction of autophagy are currently under clinical trials for alzheimer's disease. the clinically used approved antidiabetic drug, metformin [ ] , attenuates disease development in some neurodegenerative diseases via ampk activation. -hydroxypropyl-β-cyclodextrin [ ] , effective in enhancing the clearance of lipids or autophagic substrate burden, can mitigate neurological deficits in an npc mouse model and is currently under early clinical trials for treating npc. another lipofuscinolytic agent, centrophenoxine [ ] , is an anti-aging antioxidant which possesses potential protective effect for late-stage dementia in small early clinical trials. however, factors such as blood-brain barrier penetration power of the compounds and cellular heterogeneity of brain tissue can also affect the clinical neuroprotective efficacy of autophagy drugs. for example, the different responses of neurons and glia to autophagy or drug must be considered in clinical trial evaluations [ ] . although the molecular mechanisms and functions of many autophagy modulators isolated from chm were intensively studied, we are still far away from translating these traditional herbs or compounds into clinical applications. knowledge concerning autophagy research is mainly related to non-selective autophagy describing the molecular responses upon starvation. however, increasing studies are evidencing the significance of selective autophagy which involves specific molecular mediators targeting particular kinds of unwanted intracellular materials [ , ] . therefore, it is important to clarify the potential discrepancies between selective and non-selective autophagy in terms of their response towards different therapeutic agents. on the other hand, the time of starting the autophagy modulatory treatment should also be considered. for example, early cancer development is likely to be prevented by autophagy induction, as cancer cells exploit nutrients generated by autophagy to survive under the stressful cellular environment at the later stages [ , ] . it is also important to take into account the cells type-specific property of autophagy which may otherwise minimize the efficacy of the applied herbs, and result in unfavourable side effects. the heterogeneous cell types as presented by hepatic lobules can well explain such a scenario. the liver consists mainly of parenchymal cells (hepatocytes) and around % of non-parenchymal cells such as hepatic stellate cells (hscs) [ ] . the functional consequences of autophagy induction on these cells vary under different diseased conditions. autophagy of hsc activated during the end stage of chronic liver disease and hepatic fibrosis could be alleviated by autophagy repression in vitro [ ] [ ] [ ] . in contrast, autophagy stimulation is beneficial upon most of the parenchymal cells-related hepatic malignancies. therefore special caution would need to be taken when applying the autophagic modulators in cancer therapy. an increasing number of chinese herbal medicines (chms) have been discovered as autophagy modulators. such autophagic-regulatory effects are therapeutically beneficial to a broad range of disorders which are consistent with their traditional usage. interestingly, many chm-induced autophagy findings described in this review also demonstrate novel applications in various pathological conditions. these potential therapeutic functions can be summarized into: ( ) anti-cancer; ( ) neuroprotective; ( ) cardiovascular-protective; and ( ) antiviral applications. for example, through the activation of autophagic cell death, tumorigeneses can be repressed by cortex phellodendri, radix sophorae lavescentis, radix isatidis, and stephania japonica which are not associated with cancer therapy according to chinese herbology. in fact, a plethora of chms having similar potential in preventing cancer progression have not been studied before. these herbs include nelumbo nucifera, syzygium samarangense, mallotus philippensis, rhizoma anemarrhenae, radix glehniae, radix ophiopogonis, radix glycyrrhizae, radix dipsaci, radix ginseng, radix codonopsis, rhizoma atractylodis macrocephalae, ganoderma lucidum, radix bupleuri, rhizoma zingiberis recens, radix tripterygii wilfordii, radix stephaniae tetrandrae, rhizoma alismatis, cortex magnoliae officinalis, fructus piperis longi, radix salviae miltiorrhizae, rhizoma chuanxiong, garcinia hanburyi, radix plumbaginis zeylanicae, and fructus evodiae. in contrast, fructus evodiae can inhibit autophagic cell death of influenza-infected cells which provides insight to the search of chms sharing such anti-viral properties. in the case of neurodegenerative disorders, nelumbo nucifera, mallotus philippensis, rhizoma anemarrhenae, radix ginseng, radix tripterygii wilfordii, and rhizoma curcumae longae can trigger previously unidentified autophagy-mediated neuronal survival. these groups of novel neuroprotective herbs may help remove misfolded protein aggregates via autophagy activation. also, rhizoma polygoni cuspidate can upregulate the cardiac myocytic autophagy to eliminate damaged proteins uncovering the innovative use of the herb in cardiovascular disorders. it should also be noted that a single chm usually contain more than one bioactive component. therefore, the downstream molecular networks modulated by these chms are complicated. precise investigations concerning how chms may influence the intricate autophagy machinery is needed, and will be a major challenge for further developing chm as practical autophagy regulators. when compared with western medicines, most of the reviewed autophagy modulators here are bioactive components constituting the chms, which have long been prescribed as decoctions or formulations in the chinese community with well-known pharmacological action, toxicity or side effects. therefore, clinical trials using these natural herbs may be more safe and reliable. also, the philosophy of chm emphasizes the comprehensive and persistent body balance, which tailors them to deal with the autophagy-related diseases, which are pathologically associated with the loss of overall cellular and physiological balance. in addition, the actions of chm are usually multi-targeting [ , ] , making them suitable medications for autophagy-related disorders which are usually polysymptomatic. therefore, detailed and systematic investigation concerning the interaction between chms and autophagy-related disorders in a comprehensive molecular approach is needed. positive findings in these areas could widen the scope of chm applications by suggesting novel intervention strategies, which have not been mentioned in the traditional chinese pharmacopeia. the authors declare no conflict of interest. the following abbreviations are used in this manuscript: (suppl. s ), s -s . the machinery of macroautophagy characterization of autophagosome formation site by a hierarchical analysis of mammalian atg proteins methods in mammalian autophagy research autophagic cell death: loch ness monster or endangered species? to die or not to die: that is the autophagic question self-eating and self-killing: crosstalk between autophagy and apoptosis autophagic cell death: the story of a misnomer autophagy and the immune system unveiling the roles of autophagy in innate and adaptive immunity role of galectin- in prion infections of the cns autophagy failure in alzheimer's disease and the role of defective lysosomal acidification the role of autophagy in parkinson's disease. cold spring harb atg deficiency exacerbates glucose intolerance in mice on high-fat diet the autophagy-related gene (atg ) is regulated by forkhead box o transcription factors and circadian rhythms and plays a critical role in hepatic autophagy and lipid metabolism defective regulation of adipose tissue autophagy in obesity extracellular nucleotides inhibit insulin receptor signaling, stimulate autophagy and control lipoprotein secretion macroautophagy in homeostasis of pancreatic β-cell autophagy in the pathogenesis of disease autophagy genes are essential for dauer development and life-span extension in c. elegans caloric restriction delays disease onset and mortality in rhesus monkeys genomic analyses reveal global functional alterations that promote tumor growth and novel tumor suppressor genes in natural killer-cell malignancies promotion of tumorigenesis by heterozygous disruption of the beclin autophagy gene reduced expression of lc b-ii and beclin in glioblastoma multiforme indicates a down-regulated autophagic capacity that relates to the progression of astrocytic tumors systemic treatment with the antidiabetic drug metformin selectively impairs p -deficient tumor cell growth saikosaponin-d, a novel serca inhibitor, induces autophagic cell death in apoptosis-defective cells natural small-molecule enhancers of autophagy induce autophagic cell death in apoptosis-defective cells z) , , , -trans-tetramethoxystilbene, a new analogue of resveratrol, inhibits gefitinb-resistant non-small cell lung cancer via selectively elevating intracellular calcium level autophagy in chronic obstructive pulmonary disease: homeostatic or pathogenic mechanism? autophagy in immunity: implications in etiology of autoimmune/autoinflammatory diseases autophagy in immunity and cell-autonomous defense against intracellular microbes hsc blockade by the therapeutic peptide p affects autophagic processes and endogenous mhcii presentation in murine lupus molecular therapies for systemic lupus erythematosus: clinical trials and future prospects fty increases cd expression and sensitizes mantle cell lymphoma cells to milatuzumab-mediated cell death pharmacological regulators of autophagy and their link with modulators of lupus disease effects of fty in mrl-lpr/lpr mice: therapeutic potential in systemic lupus erythematosus autophagic effects of chaihu (dried roots of bupleurum chinense dc or bupleurum scorzoneraefolium wild) tetramethylpyrazine (tmp) exerts antitumor effects by inducing apoptosis and autophagy in hepatocellular carcinoma oridonin up-regulates expression of p and induces autophagy and apoptosis in human prostate cancer cells spermidine and resveratrol induce autophagy by distinct pathways converging on the acetylproteome curcumin inhibits autophagy and apoptosis in hypoxia/reoxygenation-induced myocytes honokiol-induced apoptosis and autophagy in glioblastoma multiforme cells attenuation of aβ - -induced parallel autophagic and apoptotic cell death by gypenoside xvii through the estrogen receptor-dependent activation of nrf /are pathways erk / -dependent phosphorylation of gα-interacting protein stimulates its gtpase accelerating activity and autophagy in human colon cancer cells a heterotrimeric g-protein controls autophagic sequestration in the human colon cancer cell line ht- amino acids interfere with the erk / -dependent control of macroautophagy by controlling the activation of raf- in human colon cancer ht- cells synthesis and function of -phosphorylated inositol lipids beclin-phosphatidylinositol -kinase complex functions at the trans-golgi network regulation of membrane traffic by phosphoinositide -kinases induction of autophagy and inhibition of tumorigenesis by beclin jnk -mediated phosphorylation of bcl- regulates starvation-induced autophagy dap-kinase-mediated phosphorylation on the bh domain of beclin promotes dissociation of beclin from bcl-xl and induction of autophagy eaten alive: a history of macroautophagy mtor and cancer: insights into a complex relationship molecules and their functions in autophagy role of ampk-mtor-ulk / in the regulation of autophagy: cross talk, shortcuts, and feedbacks mammalian autophagy: core molecular machinery and signaling regulation amp-activated protein kinase: the energy charge hypothesis revisited amp-activated/snf protein kinases: conserved guardians of cellular energy the energy sensing lkb -ampk pathway regulates p kip phosphorylation mediating the decision to enter autophagy or apoptosis ampk phosphorylation of raptor mediates a metabolic checkpoint control of macroautophagy by calcium, calmodulin-dependent kinase kinase-β, and bcl- the roles of intracellular protein-degradation pathways in neurodegeneration examining the structure of the mature amyloid fibril structure of the cross-β spine of amyloid-like fibrils loss of autophagy in the central nervous system causes neurodegeneration in mice suppression of basal autophagy in neural cells causes neurodegenerative disease in mice molecular structure of amyloid fibrils: insights from solid-state nmr the autophagy-related protein beclin shows reduced expression in early alzheimer disease and regulates amyloid β accumulation in mice beclin gene transfer activates autophagy and ameliorates the neurodegenerative pathology in α-synuclein models of parkinson's and lewy body diseases regulation of intracellular accumulation of mutant huntingtin by beclin inhibition of mtor by rapamycin abolishes cognitive deficits and reduces amyloid-β levels in a mouse model of alzheimer's disease inositol and ip levels regulate autophagy: biology and therapeutic speculations lithium induces autophagy by inhibiting inositol monophosphatase small molecule enhancers of autophagy for neurodegenerative diseases novel targets for huntington's disease in an mtor-independent autophagy pathway stimulation of autophagy reduces neurodegeneration in a mouse model of human tauopathy trehalose, a novel mtor-independent autophagy enhancer, accelerates the clearance of mutant huntingtin and α-synuclein metabolic pathways promoting cancer cell survival and growth amp-activated protein kinase activators can inhibit the growth of prostate cancer cells by multiple mechanisms adenosine induces apoptosis in the human gastric cancer cells via an intrinsic pathway relevant to activation of amp-activated protein kinase sustained activation of amp-activated protein kinase induces c-jun n-terminal kinase activation and apoptosis in liver cells a mammalian protein targeted by g -arresting rapamycin-receptor complex target of rapamycin (tor): an integrator of nutrient and growth factor signals and coordinator of cell growth and cell cycle progression autophagy in tumor suppression and cancer therapy the hallmarks of cancer endoplasmic reticulum stress and the inflammatory basis of metabolic disease a branched-chain amino acid-related metabolic signature that differentiates obese and lean humans and contributes to insulin resistance effects of isoenergetic overfeeding of either carbohydrate or fat in young men mtorc activates srebp- c and uncouples lipogenesis from gluconeogenesis defective hepatic autophagy in obesity promotes er stress and causes insulin resistance circadian rhythms and metabolic syndrome: from experimental genetics to human disease pkr-dependent autophagic degradation of herpes simplex virus type autophagy fights disease through cellular self-digestion protection against fatal sindbis virus encephalitis by beclin, a novel bcl- -interacting protein cd induces macrophage anti-toxoplasma gondii activity by triggering autophagy-dependent fusion of pathogen-containing vacuoles and lysosomes autophagy proteins regulate innate immune responses by inhibiting the release of mitochondrial dna mediated by the nalp inflammasome loss of the autophagy protein atg l enhances endotoxin-induced il- β production vitamin d inhibits human immunodeficiency virus type and mycobacterium tuberculosis infection in macrophages through the induction of autophagy eradication of intracellular francisella tularensis in thp- human macrophages with a novel autophagy inducing agent host cell autophagy activated by antibiotics is required for their effective antimycobacterial drug action autophagy in immunity and inflammation autophagic compartments gain access to the mhc class ii compartments in thymic epithelium macroautophagy substrates are loaded onto mhc class ii of medullary thymic epithelial cells for central tolerance blockade of macrophage autophagy ameliorates activated lymphocytes-derived dna induced murine lupus possibly via inhibition of proinflammatory cytokine production ophiopogonin d attenuates doxorubicin-induced autophagic cell death by relieving mitochondrial damage in vitro and in vivo mammalian target of rapamycin regulates isoliquiritigenin-induced autophagic and apoptotic cell death in adenoid cystic carcinoma cells akebia saponin pa induces autophagic and apoptotic cell death in ags human gastric cancer cells neuroprotective effect of ginsenoside rb on glutamate-induced neurotoxicity: with emphasis on autophagy ginsenoside re enhances the survival of h c cardiac muscle cells through regulation of autophagy the plant alkaloid voacamine induces apoptosis-independent autophagic cell death on both sensitive and multidrug resistant human osteosarcoma cells anticancer effect of ginger extract against pancreatic cancer cells mainly through reactive oxygen species-mediated autotic cell death celastrol induces apoptosis and autophagy via the ros/jnk signaling pathway in human osteosarcoma cells: an in vitro and in vivo study celastrol prevents cadmium-induced neuronal cell death via targeting jnk and pten-akt/mtor network tetrandrine induces autophagy and differentiation by activating ros and notch signaling in leukemia cells plumbagin induces g -m arrest and autophagy by inhibiting the akt/mammalian target of rapamycin pathway in breast cancer cells alisol b, a novel inhibitor of the sarcoplasmic/endoplasmic reticulum ca + atpase pump, induces autophagy, endoplasmic reticulum stress, and apoptosis effect of magnolol on the function of osteoblastic mc t -e cells evodiamine: a novel anti-cancer alkaloid from evodia rutaecarpa a drug screening method based on the autophagy pathway and studies of the mechanism of evodiamine against influenza a virus piperlongumine promotes autophagy via inhibition of akt/mtor signalling and mediates cancer cell death piperlongumine induces apoptosis and autophagy in human lung cancer cells through inhibition of pi k/akt/mtor pathway curcumin ameliorates the neurodegenerative pathology in a t α-synuclein cell model of parkinson's disease through the downregulation of mtor/p s k signaling and the recovery of macroautophagy regulation of autophagy by polyphenolic compounds as a potential therapeutic strategy for cancer. cell death dis tanshinone iia induces autophagic cell death via activation of ampk and erk and inhibition of mtor and p s k in kbm- leukemia cells tetramethylpyrazine (tmp) protects against sodium arsenite-induced nephrotoxicity by suppressing ros production, mitochondrial dysfunction, pro-inflammatory signaling pathways and programed cell death anticancer effects of bufalin on human hepatocellular carcinoma hepg cells: roles of apoptosis and autophagy bufalin induces autophagy-mediated cell death in human colon cancer cells through reactive oxygen species generation and jnk activation. free radic gambogic acid induced oxidative stress dependent caspase activation regulates both apoptosis and autophagy by targeting various key molecules (nf-κb, beclin- , p and nbr ) in human bladder cancer cells gambogic acid induces death of k cells through autophagy and apoptosis mechanisms onjisaponin b derived from radix polygalae enhances autophagy and accelerates the degradation of mutant α-synuclein and huntingtin in pc- cells method and composition for inducing autophagy progress in phytochemicals' induction of autophagic cell death in cancer cells and regulation of nuclear receptors. chin can autophagy promote longevity? nat protective macroautophagy is involved in vitamin e succinate effects on human gastric carcinoma cell line sgc- by inhibiting mtor axis phosphorylation literature research of chinese medicine recipes for the treatment of psoriasis vulgaris with blood-heat syndrome type characterization of flavonoids in the traditional chinese herbal medicine-huangqin by liquid chromatography coupled with electrospray ionization mass spectrometry effects of wogonin, wogonoside, and , , , , -pentahydroxyflavone on chemical mediator production in peritoneal exduate cells and immunoglobulin e of rat mesenteric lymph node lymphocytes anti-inflammatory effects of scutellaria baicalensis extract via suppression of immune modulators and map kinase signaling molecules autophagy induced by baicalin involves downregulation of cd in smmc- cells in vitro autophagy: a primer for the gastroenterologist/hepatologist chinese traditional medicine chinese pharmaceutical science and technology publication co new protolimonoids from the fruits of phellodendron chinense acetaldehyde-induced interleukin- β and tumor necrosis factor-α production is inhibited by berberine through nuclear factor-κb signaling pathway in hepg cells extracts of bark from the traditional chinese herb phellodendron amurense inhibit contractility of the isolated rat prostate gland inhibition of gene expression and production of inos and tnf-α in lps-stimulated microglia by methanol extract of phellodendri cortex development of protein kinase activators: ampk as a target in metabolic disorders and cancer berberine attenuates autophagy in adipocytes by targeting becn phellodendron amurense bark extract prevents progression of prostate tumors in transgenic adenocarcinoma of mouse prostate: potential for prostate cancer management nexrutine ® inhibits tumorigenesis in mouse skin and induces apoptotic cell death in human squamous carcinoma a and human melanoma a cells regulation of cox- by cyclic amp response element binding protein in prostate cancer: potential role for nexrutine stat down regulates lc to inhibit autophagy and pancreatic cancer cell growth butanol fraction containing berberine or related compound from nexrutine inhibits nfκb signaling and induces apoptosis in prostate cancer cells berberine induces caspase-independent cell death in colon tumor cells through activation of apoptosis-inducing factor advance of studies on anti-atherosclerosis mechanism of berberine preventive effect of coptis chinensis and berberine on intestinal injury in rats challenged with lipopolysaccharides in vitro response of blastocystis hominis against traditional chinese medicine analgesic effect of coptis chinensis rhizomes (coptidis rhizoma) extract on rat model of irritable bowel syndrome berberine exerts neuroprotective actions against in vitro ischemia-induced neuronal cell damage in organotypic hippocampal slice cultures: involvement of b-cell lymphoma phosphorylation suppression berberine and total base from rhizoma coptis chinensis attenuate brain injury in an aluminum-induced rat model of neurodegenerative disease sophora flavescens ait.: traditional usage, phytochemistry and pharmacology of an important traditional chinese medicine the efficacy and safety of a chinese herbal product (xiao-feng-san) for the treatment of refractory atopic dermatitis: a randomized, double-blind, placebo-controlled trial simultaneous determination of baicalin, baicalein, wogonin, oxysophocarpine, oxymatrine and matrine in the chinese herbal preparation of sanwu-huangqin-tang by ion-paired hplc antiinflammatory effects of matrine in lps-induced acute lung injury in mice matrine induces cell anergy in human jurkat t cells through modulation of mitogen-activated protein kinases and nuclear factor of activated t-cells signaling with concomitant up-regulation of anergy-associated genes expression autophagy is involved in anticancer effects of matrine on sgc- human gastric cancer cells antitumor effect of matrine in human hepatoma g cells by inducing apoptosis and autophagy reversal effects of rabdosia rubescens extract on multidrug resistance of mcf- /adr cells in vitro oridonin ameliorates neuropathological changes and behavioural deficits in a mouse model of cerebral amyloidosis study on the inhibitory mechanism of oridonin in pancreatic cancers bxpc- cells by dna microarray. zhejiang zhongyiyao daxue xuebao efficacy and safety of ban-lan-gen granules in the treatment of seasonal influenza: study protocol for a randomized controlled trial ten lectures on the use of medicinals from the personal experience of jiao shu-de (jiao clinical chinese medicine) chinese medicinal teas: simple, proven, folk formulas for common diseases & promoting health fangchinoline inhibits human immunodeficiency virus type replication by interfering with gp proteolytic processing tetrandrine protects mice from concanavalin a-induced hepatitis through inhibiting nf-κb activation pharmacognostical studies on leaves of stephania japonica var dauricine induces apoptosis, inhibits proliferation and invasion through inhibiting nf-κb signaling pathway in colon cancer cells therapeutic potential of the biscoclaurine alkaloid, cepharanthine, for a range of clinical conditions potent antiperoxidation activity of the bisbenzylisoquinoline alkaloid cepharanthine: the amine moiety is responsible for its ph-dependent radical scavenge activity direct radical scavenging by the bisbenzylisoquinoline alkaloid cepharanthine inhibitory effect of dauricine on inflammatory process following focal cerebral ischemia/reperfusion in rats modulation of the atpase and transport activities of broad-acting multidrug resistance factor abcc (mrp ) a dictionary of plants used by man medicinal plants of china resveratrol as a chemopreventive agent: a promising molecule for fighting cancer fungicidal effect of resveratrol on human infectious fungi resveratrol antibacterial activity against escherichia coli is mediated by z-ring formation inhibition via suppression of ftsz expression anti-inflammatory responses of resveratrol resveratrol isolated from polygonum cuspidatum root prevents tumor growth and metastasis to lung and tumor-induced neovascularization in lewis lung carcinoma-bearing mice resveratrol interferes with akt activity and triggers apoptosis in human uterine cancer cells estrogen and resveratrol regulate rac and cdc signaling to the actin cytoskeleton of metastatic breast cancer cells resveratrol in cardiovascular health and disease cardioprotection by resveratrol: a novel mechanism via autophagy involving the mtorc pathway scutellaria barbata d don inhibits colorectal cancer growth via suppression of multiple signaling pathways research progress on anticancer effect of scuteiiaria barbata d.don and its mechanism anti-inflammatory activity of edible brown alga saccharina japonica and its constituents pheophorbide a and pheophytin a in lps-stimulated raw . macrophage cells pheophorbide a, a major antitumor component purified from scutellaria barbata, induces apoptosis in human hepatocellular carcinoma cells pheophorbide a-mediated photodynamic therapy induces autophagy and apoptosis via the activation of mapks in human skin cancer cells chinese drugs of plant origin: chemistry, pharmacology, and use in traditional and modern medicine neferine enhances insulin sensitivity in insulin resistant rats anti-amnesic activity of neferine with antioxidant and anti-inflammatory capacities, as well as inhibition of ches and bace neferine, a bisbenzylisoquinline alkaloid attenuates bleomycin-induced pulmonary fibrosis neferine isolated from nelumbo nucifera enhances anti-cancer activities in hep b cells: molecular mechanisms of cell cycle arrest, er stress induced apoptosis and anti-angiogenic response quantitative analysis of antiradical phenolic constituents from fourteen edible myrtaceae fruits antioxidant activities of some local bangladeshi fruits (artocarpus heterophyllus, annona squamosa, terminalia bellirica, syzygium samarangense, averrhoa carambola and olea europa). sheng wu gong cheng xue bao protective effects of , -dihydroxy- -methoxy- , -dimethylchalcone to pc cells against cytotoxicity induced by hydrogen peroxide hepatoprotective effects of , -dihydroxy- -methoxy- , -dimethylchalcone on ccl -induced acute liver injury in mice dimethyl cardamonin inhibits lipopolysaccharide-induced inflammatory factors through blocking nf-κb p activation blockade of nuclear factor-κb signaling pathway and anti-inflammatory activity of cardamomin, a chalcone analog from alpinia conchigera cardamonin induces autophagy and an antiproliferative effect through jnk activation in human colorectal carcinoma hct cells induction of autophagy by dimethyl cardamonin is associated with proliferative arrest in human colorectal carcinoma hct and lovo cells cucurbitacins-a promising target for cancer therapy anti-hiv agent trichosanthin enhances the capabilities of chemokines to stimulate chemotaxis and g protein activation, and this is mediated through interaction of trichosanthin and chemokine receptors inhibition of nitric oxide generation by , -dihydrocucurbitacin d in mouse peritoneal macrophages anti-inflammatory activity of two cucurbitacins isolated from cayaponia tayuya roots anticancer and antiinflammatory activities of cucurbitacins from cucurbitaandreana dihydrocucurbitacin b inhibits delayed type hypersensitivity reactions by suppressing lymphocyte proliferation cucurbitane triterpenoids from leucopaxillus gentianeus activated kras protects colon cancer cells from cucurbitacin-induced apoptosis: the role of p and p trichosanthes kirilowii ethanol extract and cucurbitacin d inhibit cell growth and induce apoptosis through inhibition of stat activity in breast cancer cells apoptosis induction through proteasome inhibitory activity of cucurbitacin d in human t-cell leukemia cucurbitacin e induces autophagy via downregulating mtorc signaling and upregulating ampk activity cucurbitacin i induces protective autophagy in glioblastoma in vitro and in vivo a helicobacter pylori treatment strategies and options: a review rottlerin sensitizes colon carcinoma cells to tumor necrosis factor-related apoptosis-inducing ligand-induced apoptosis via uncoupling of the mitochondria independent of protein kinase c rottlerin induces apoptosis via death receptor (dr ) upregulation through chop-dependent and pkc δ-independent mechanism in human malignant tumor cells pkcδ protects human breast tumor mcf- cells against tumor necrosis factor-related apoptosis-inducing ligand-mediated apoptosis tissue transglutaminase inhibits autophagy in pancreatic cancer cells screen for chemical modulators of autophagy reveals novel therapeutic inhibitors of mtorc signaling application of timosaponin aiii in anemarrhena to preparation of antitumor drugs the genus anemarrhena bunge: a review on ethnopharmacology, phytochemistry and pharmacology rhizoma anemarrhenae extract ameliorates hyperglycemia and insulin resistance via activation of amp-activated protein kinase in diabetic rodents effect of timosaponin a-iii, from anemarrhenae asphodeloides bunge (liliaceae), on calcium mobilization in vascular endothelial and smooth muscle cells and on vascular tension effect of steroidal saponins of anemarrhenae rhizoma on superoxide generation in human neutrophils timosaponin a-iii induces autophagy preceding mitochondria-mediated apoptosis in hela cancer cells timosaponin aiii is preferentially cytotoxic to tumor cells through inhibition of mtor and induction of er stress chinese medicinal herbs as source of antioxidant compounds-where tradition meets the future liquorice, a unique "guide drug" of traditional chinese medicine: a review of its role in drug interactions inhibition by licochalcone a, a novel flavonoid isolated from liquorice root, of il- β-induced pge production in human skin fibroblasts licochalcone a isolated from licorice suppresses lipopolysaccharide-stimulated inflammatory reactions in raw . cells and endotoxin shock in mice licochalcone a activates nrf in vitro and contributes to licorice extract-induced lowered cutaneous oxidative stress in vivo antioxidant constituents from licorice roots: isolation, structure elucidation and antioxidative capacity toward ldl oxidation. free radic inhibitory effects of some natural products on the activation of hyaluronidase and their antiallergic actions licochalcone a induces autophagy through pi k/akt/mtor inactivation and autophagy suppression enhances licochalcone a-induced apoptosis of human cervical cancer cells licorice and licochalcone-a induce autophagy in lncap prostate cancer cells by suppression of bcl- expression and the mtor pathway microrna- regulates chemoresistance-associated autophagy in breast cancer cells, a process modulated by the natural autophagy inducer isoliquiritigenin chinese materia medica: combinations and applications an illustrated chinese materia medica a comparison of the ancient use of ginseng in traditional chinese medicine with modern pharmacological experiments and clinical trials comparison of the pharmacological effects of panax ginseng and panax quinquefolium ginsenoside rg attenuates dopamine-induced apoptosis in pc cells by suppressing oxidative stress possible mechanisms of the protection of ginsenoside re against mptp-induced apoptosis in substantia nigra neurons of parkinson's disease mouse model cardiovascular diseases and panax ginseng: a review on molecular mechanisms and medical applications anti-cancer and potential chemopreventive actions of ginseng by activating nrf (nfe l ) anti-oxidative stress/anti-inflammatory pathways enzymatic biotransformation of ginsenoside rb and gypenoside xvii into ginsenosides rd and f by recombinant β-glucosidase from flavobacterium johnsoniae ginsenoside f induces apoptosis accompanied by protective autophagy in breast cancer stem cells (s)-ginsenoside rg is a novel inhibitor of autophagy and sensitizes hepatocellular carcinoma to doxorubicin antiplasmodial activity of the alkaloids of peschiera fuchsiaefolia elaborating the role of natural products-induced autophagy in cancer treatment: achievements and artifacts in the state of the art autophagy-mediated chemosensitizing effect of the plant alkaloid voacamine on multidrug resistant cells tissue distribution and chemical induction of multiple drug resistance genes in rats attenuated function and expression of p-glycoprotein at blood-brain barrier and increased brain distribution of phenobarbital in streptozotocin-induced diabetic mice blood-brain barrier p-glycoprotein function in neurodegenerative disease genetic distinction of radix adenophorae from its adulterants by the dna sequence of s-rrna spacer domains encyclopedia of medicinal plants in vivo and in vitro antiinflammatory activity of saikosaponins antiviral effects of saikosaponins on human coronavirus e in vitro antinociceptive and antipyretic properties of the pharmaceutical herbal preparation, radix bupleuri in rats discussion on the adverse drug reaction monitoring by community pharmacies evidence of nutriceutical effectiveness in the treatment of osteoarthritis zingiberis rhizoma: a comprehensive review on the ginger effect and efficacy profiles ]-gingerol inhibits nitric oxide synthesis in activated j . mouse macrophages and prevents peroxynitrite-induced oxidation and nitration reactions gingerols: a novel class of vanilloid receptor (vr ) agonists anti- -hydroxytryptamine effect of galanolactone, diterpenoid isolated from ginger [ ]-gingerol induces caspase dependent apoptosis and autophagy in cancer cells: drug-dna interaction and expression of certain signal genes in hela cells immunosuppressant discovery from tripterygium wilfordii hook f: the novel triptolide analog ( r)- -hydroxytriptolide (lldt- ) comparative bioavailability study of two cefixime formulations administered orally in healthy male volunteers celastrol inhibits tumor cell proliferation and promotes apoptosis through the activation of c-jun n-terminal kinase and suppression of pi k/akt signaling pathways celastrol ameliorates experimental colitis in il- deficient mice via the up-regulation of autophagy anti-inflammatory effects of the partially purified extract of radix stephaniae tetrandrae: comparative studies of its active principles tetrandrine and fangchinoline on human polymorphonuclear leukocyte functions anti-inflammatory effects of fangchinoline and tetrandrine anti-hyperglycemic effect of fangchinoline isolated from stephania tetrandra radix in streptozotocin-diabetic mice haemodynamic effects of chronic octreotide and tetrandrine administration in portal hypertensive rats fangchinoline targets pi k and suppresses pi k/akt signaling pathway in sgc cells perspectives on medicinal properties of plumbagin and its analogs plumbagin, a vitamin k analogue, abrogates lipopolysaccharide-induced oxidative stress, inflammation and endotoxic shock via nf-κb suppression plumbagin suppresses dendritic cell functions and alleviates experimental autoimmune encephalomyelitis antibacterial activity of some natural products against bacteria expressing a multidrug-resistant phenotype plumbagin inhibits cell growth and potentiates apoptosis in human gastric cancer cells in vitro through the nf-κb signaling pathway plumbagin ( -hydroxy- -methyl- , -naphthoquinone) suppresses nf-κb activation and nf-κb-regulated gene products through modulation of p and iκbα kinase activation, leading to potentiation of apoptosis induced by cytokine and chemotherapeutic agents plumbagin induces apoptotic and autophagic cell death through inhibition of the pi k/akt/mtor pathway in human non-small cell lung cancer cells ze xie diao xue zhi de yan jiu jin zhan chromatographic fingerprint analysis of herbal medicines alisol b acetate, a triterpene from alismatis rhizoma, induces bax nuclear translocation and apoptosis in human hormone-resistant prostate cancer pc- cells natural product agonists of peroxisome proliferator-activated receptor γ (pparγ): a review autophagy triggered by magnolol derivative negatively regulates angiogenesis magnolol, a major bioactive constituent of the bark of magnolia officinalis, exerts antiepileptic effects via the gaba/benzodiazepine receptor complex in mice antifungal activity of magnolol and honokiol magnolol-induced h cells death via autophagy but not apoptosis gastroprotective effect of fructus evodiae water extract on ethanol-induced gastric lesions in rats the protective effects of rutaecarpine on gastric mucosa injury in rats evodiamine abolishes constitutive and inducible nf-κb activation by inhibiting iκbα kinase activation, thereby suppressing nf-κb-regulated antiapoptotic and metastatic gene expression, up-regulating apoptosis, and inhibiting invasion evodiamine inhibits adipogenesis via the egfr-pkcα-erk signaling pathway cytotoxic effect of evodiamine in sgc- human gastric adenocarcinoma cells via simultaneous induction of apoptosis and autophagy inhibition of ccl ´i nduced liver fibrosis by piper longum linn anti-inflammatory and antiarthritic effects of piperine in human interleukin β-stimulated fibroblast-like synoviocytes and in rat arthritis models protective effect of piper longum l. on oxidative stress induced injury and cellular abnormality in adriamycin induced cardiotoxicity in rats piperlongumine, a constituent of piper longum l., inhibits rabbit platelet aggregation as a thromboxane a receptor antagonist piperlongumine selectively kills glioblastoma multiforme cells via reactive oxygen species accumulation dependent jnk and p activation antiproliferative effects of two amides, piperine and piplartine, from piper species piperlongumine induces autophagy by targeting p signaling development and mechanism investigation of a new piperlongumine derivative as a potent anti-inflammatory agent anticancer potential of curcumin: preclinical and clinical studies curcumin and cancer: an "old-age" disease with an "age-old" solution potential therapeutic effects of curcumin, the anti-inflammatory agent, against neurodegenerative, cardiovascular, pulmonary, metabolic, autoimmune and neoplastic diseases curcumin and cancer cells: how many ways can curry kill tumor cells selectively? anti-tumour and antioxidant activity of natural curcuminoids the chaperone-mediated autophagy receptor organizes in dynamic protein complexes at the lysosomal membrane dietary curcumin supplementation counteracts reduction in levels of molecules involved in energy homeostasis after brain trauma curcumin inhibits formation of amyloid β oligomers and fibrils, binds plaques, and reduces amyloid in vivo curcumin labels amyloid pathology in vivo, disrupts existing plaques, and partially restores distorted neurites in an alzheimer mouse model an overview of its chemistry, pharmacology, pharmacokinetics, and clinical use long-term treatment with danshen-gegen decoction protects the myocardium against ischemia/reperfusion injury via the redox-sensitive protein kinase c-ε/mkatp pathway in rats regulation effects on abnormal glucose and lipid metabolism of tzq-f, a new kind of traditional chinese medicine the general situation and progress of the modern research of red sage root (radix salviae miltiorrhizae) anti-inflammatory and immunomodulatory mechanism of tanshinone iia for atherosclerosis tanshinone iia inhibits lps-induced nf-κb activation in raw . cells: possible involvement of the nik-ikk, erk / , p and jnk pathways tanshinone iia protects against cardiac hypertrophy via inhibiting calcineurin/nfatc pathway calcineurin suppresses ampk-dependent cytoprotective autophagy in cardiomyocytes under oxidative stress advances of ligusticum chuanxiong inhibition of rat vascular smooth muscle cell proliferation by extract of ligusticum chuanxiong and angelica sinensis protective effect of ligusticum chuanxiong and angelica sinensis on endothelial cell damage induced by hydrogen peroxide ocular and cardiovascular pharmacology of tetramethylpyrazine isolated from ligusticum wallichii franch scavenging effects of tetramethylpyrazine on active oxygen free radicals tetramethylpyrazine reduces cellular inflammatory response following permanent focal cerebral ischemia in rats studies on cardiotonic steroids from the skin of japanese toad pharmacology and toxicology of toad venom bufotalin from venenum bufonis inhibits growth of multidrug resistant hepg cells through g /m cell cycle arrest and apoptosis cardiac glycosides are potent inhibitors of interferon-β gene expression bufalin induces g /m phase arrest and triggers autophagy via the tnf, jnk, becn- and atg pathway in human hepatoma cells bufalin inhibits hct colon cancer cells and its orthotopic xenograft tumor in mice model through genes related to apoptotic and pten/akt pathways bufalin induces the interplay between apoptosis and autophagy in glioma cells through endoplasmic reticulum stress anti-inflammatory, analgesic and antipyretic activities of the extract of gamboge from garcinia hanburyi hook f general gambogic acids inhibited growth of human hepatoma smmc- cells in vitro and in nude mice gambogic acid, a novel ligand for transferrin receptor, potentiates tnf-induced apoptosis through modulation of the nuclear factor-κb signaling pathway gambogic acid-induced g /m phase cell-cycle arrest via disturbing cdk -mediated phosphorylation of cdc /p in human gastric carcinoma bgc- cells characterization of multiple constituents in kai-xin-san prescription and rat plasma after oral administration by liquid chromatography with quadrupole time-of-flight tandem mass spectrometry a representative prescription for emotional disease, ding-zhi-xiao-wan restores -ht system deficit through interfering the synthesis and transshipment in chronic mild stress-induced depressive rats effects of tenuifolin extracted from radix polygalae on learning and memory: a behavioral and biochemical study on aged and amnesic mice anxiolytic and sedative-hypnotic activities of polygalasaponins from polygala tenuifolia in mice preclinical evidence of rapid-onset antidepressant-like effect in radix polygalae extract polygalasaponin xxxii from polygala tenuifolia root improves hippocampal-dependent learning and memory polygalasaponin f induces long-term potentiation in adult rat hippocampus via nmda receptor activation clionosterol and ethyl cholestan- -enol isolated from the rhizome of polygala tenuifolia inhibit phosphatidylinositol -kinase/akt pathway identification of novel autophagic radix polygalae fraction by cell membrane chromatography and uhplc-(q)tof-ms for degradation of neurodegenerative disease proteins triterpenes from ganoderma lucidum induce autophagy in colon cancer through the inhibition of p mitogen-activated kinase (p mapk) a possible cross-talk between autophagy and apoptosis in generating an immune response in melanoma methanolic extract of ganoderma lucidum induces autophagy of ags human gastric tumor cells antioxidant activities of polygonum multiflorum thunb., in vivo and in vitro anti-cancer properties of anthraquinones from rhubarb myocardial protective effect of an anthraquinone-containing extract of polygonum multiflorum ex vivo anthraquinones inhibit tau aggregation and dissolve alzheimer's paired helical filaments in vitro and in cells pharmacokinetic and nephroprotective benefits of using schisandra chinensis extracts in a cyclosporine a-based immune-suppressive regime schisandra total lignin attenuates apoptosis of endoplasmic reticulum pathway to delay mouse brain aging clinical efficacy of traditional chinese medicine, suan zao ren tang, for sleep disturbance during methadone maintenance: a randomized, double-blind, placebo-controlled trial suan zao ren tang as an original treatment for sleep difficulty in climacteric women: a prospective clinical observation zizyphus jujuba and its active component jujuboside b inhibit platelet aggregation antitumor activity of jujuboside b and the underlying mechanism via induction of apoptosis and autophagy jujuboside a, a neuroprotective agent from semen ziziphi spinosae ameliorates behavioral disorders of the dementia mouse model induced by aβ - identification and clinical application of amber analysis on characteristic of kaixin powder and its similar prescriptions vitamin e succinate is a potent novel antineoplastic agent with high selectivity and cooperativity with tumor necrosis factor-related apoptosis-inducing ligand (apo ligand) in vivo analysis of rule and clinicance of mind-calming medicine purgative genome of the long-living sacred lotus the epidermal growth factor receptor antibody cetuximab induces autophagy in cancer cells by downregulating hif- α and bcl- and activating the beclin /hvps complex autophagy inhibition enhances vorinostat-induced apoptosis via ubiquitinated protein accumulation autophagy blockade sensitizes prostate cancer cells towards src family kinase inhibitors inhibition of autophagy augments -fluorouracil chemotherapy in human colon cancer in vitro and in vivo model the role of autophagy in cancer: therapeutic implications pharmacokinetic analysis and pharmacodynamic evidence of autophagy inhibition in patients with newly diagnosed glioblastoma treated on a phase i trial of hydroxychloroquine in combination with adjuvant temozolomide and radination (abtc ) the antidepressants maprotiline and fluoxetine induce type ii autophagic cell death in drug-resistant burkitt's lymphoma targeting tumor metabolism with -deoxyglucose in patients with castrate-resistant prostate cancer and advanced malignancies autophagy modulation as a potential therapeutic target for diverse diseases pp a blockade inhibits autophagy and causes intraneuronal accumulation of ubiquitinated proteins amp-activated protein kinase: a potential player in alzheimer's disease chronic cyclodextrin treatment of murine niemann-pick c disease ameliorates neuronal cholesterol and glycosphingolipid storage and disease progression gerontopsychological studies using nai ("nürnberger alters-inventar") on patients with organic psychosyndrome (dsm iii, category ) treated with centrophenoxine in a double blind, comparative, randomized clinical trial the role of autophagy in neurodegenerative disease selective autophagy mediated by autophagic adapter proteins atg -family interacting motif crucial for selective autophagy deconvoluting the context-dependent role for autophagy in cancer autophagy as a target for anticancer therapy cooperation of liver cells in health and disease liver fibrosis a role for autophagy during hepatic stellate cell activation effects of fufang biejia ruangan pills on hepatic fibrosis in vivo and in vitro effect of bupleuri radix extracts on the toxicity of -fluorouracil in hepg hepatoma cells and normal human lymphocytes key: cord- -b y hxw authors: piotrowska, dorota g.; andrei, graciela; schols, dominique; snoeck, robert; grabkowska-drużyc, magdalena title: new isoxazolidine-conjugates of quinazolinones—synthesis, antiviral and cytostatic activity date: - - journal: molecules doi: . /molecules sha: doc_id: cord_uid: b y hxw a novel series of ( -diethoxyphosphoryl)isoxazolidines substituted at c with various quinazolinones have been synthesized by the , -dipolar cycloaddition of n-methyl-c-(diethoxyphosphoryl)nitrone with n -substitued -vinyl- h-quinazolin- -ones. all isoxazolidines were assessed for antiviral activity against a broad range of dna and rna viruses. isoxazolidines trans- f/cis- f ( : ), trans- h and trans- i/cis- i ( : ) showed weak activity (ec( ) = . , . and . μm) toward vzv (tk(+) strain) which was only one order of magnitude lower than that of acyclovir used as a reference drug. phosphonates trans- b/cis- b ( : ), trans- c, trans- e/cis- e ( : ) and trans- g appeared slightly active toward cytomegalovirus (ec( ) = – μm). compounds containing benzyl substituents at n in the quinazolinone skeleton exhibited slight antiproliferative activity towards the tested immortalized cells with ic( ) in the – μm range. nitrogen-containing heterocycles form the core of natural products (e.g., alkaloids) and they are also present in many pharmacophores as well as in numerous marketed drugs. among them, quinazolines and quinazolinones have drawn special attention due to the broad spectrum of biological activities of their derivatives, including sedative [ ] [ ] [ ] , anticancer [ ] [ ] [ ] [ ] , antiviral [ ] [ ] [ ] [ ] [ ] , antibacterial [ ] [ ] [ ] , antifungal [ , ] , anti-inflamatory [ , [ ] [ ] [ ] and antifibrotic [ , ] activities. several reviews focused on the synthetic strategies and biological activities of these compounds have been published [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] . the significant impact of various functional groups installed into quinazoline/quinazolinone frameworks on pharmacological properties have been proven. in the last decades several compounds containing the quinazolin- -one framework, which exhibited promising anticancer as well as antiviral properties, have been obtained ( figure ). furthermore, some biologically active substituted quinazolin- ( h)-ones were isolated from various fungi and bacteria species. for example, -( -hydroxybenzyl)quinazolin- ( h)-one ( ) was found in an entomopathogenic fungus isaria farinosa and its strong inhibitory properties on the replication of tobacco mosaic virus (tmv) [ ] were recognised, whereas its -( -hydroxybenzoyl) analogue present in fungus from penicillium genus appeared only slightly active toward tmv [ ] . moreover, compound exhibited significant cytotoxicity toward various cancer cell lines [ , ] . quinazolinone isolated from streptomyces sp. appeared cytotoxic against vero cells [ ] . very recently synthetic pyridine-containing analogue and its -substituted derivatives and have been obtained and their slight activity against influenza a virus was revealed [ ] . on the other hand, various , -disubstitued quinazolin- ( h)-ones, including compounds - , have been found to possess antitumor activity [ ] . slight activity against influenza a virus was revealed [ ] . on the other hand, various , -disubstitued quinazolin- ( h)-ones, including compounds - , have been found to possess antitumor activity [ ] . in continuation of our studies on antiviral and cytostatic activity of isoxazolidine analogues of c-nucleoside analogues, we designed a new series of compounds of the general formula containing a substituted quinazolinone moiety as a false nucleobase at c in the isoxazolidine ring and the diethoxyphosphoryl function attached at c . our synthetic strategy to compounds trans- /cis- relies on the , -dipolar cycloaddition of n-methyl-c-(diethoxyphosphoryl)nitrone [ ] with -vinyl- h-quinazolin- -ones substituted at n (scheme ). scheme . retrosynthesis of (isoxazolidinyl) phosphonates trans- /cis- . -vinyl- h-quinazolin- -ones modified at n with substituted benzyl groups were synthesized from commercially available -aminobenzamide ( ) by acylation with -chloropropionyl chloride followed by cyclization and dehydrohalogenation to prepare -vinyl- hquinazolin- -one ( a) as a key intermediate [ ] and a subsequent reaction with substituted benzyl bromides b-i [ ] (scheme ). moreover, compounds j (r = me) and k (r = et) were also obtained with intention to determine the influence of the benzyl substituent on biological activity of the designed isoxazolidines trans- /cis- . in the h-nmr spectra of compounds a-k characteristic signals for vinyl protons were observed in the . - . ppm (three doublets of doublets). in continuation of our studies on antiviral and cytostatic activity of isoxazolidine analogues of c-nucleoside analogues, we designed a new series of compounds of the general formula containing a substituted quinazolinone moiety as a false nucleobase at c in the isoxazolidine ring and the diethoxyphosphoryl function attached at c . our synthetic strategy to compounds trans- /cis- relies on the , -dipolar cycloaddition of n-methyl-c-(diethoxyphosphoryl)nitrone [ ] with -vinyl- h-quinazolin- -ones substituted at n (scheme ). slight activity against influenza a virus was revealed [ ] . on the other hand, various , -disubstitued quinazolin- ( h)-ones, including compounds - , have been found to possess antitumor activity [ ] . in continuation of our studies on antiviral and cytostatic activity of isoxazolidine analogues of c-nucleoside analogues, we designed a new series of compounds of the general formula containing a substituted quinazolinone moiety as a false nucleobase at c in the isoxazolidine ring and the diethoxyphosphoryl function attached at c . our synthetic strategy to compounds trans- /cis- relies on the , -dipolar cycloaddition of n-methyl-c-(diethoxyphosphoryl)nitrone [ ] with -vinyl- h-quinazolin- -ones substituted at n (scheme ). scheme . retrosynthesis of (isoxazolidinyl) phosphonates trans- /cis- . -vinyl- h-quinazolin- -ones modified at n with substituted benzyl groups were synthesized from commercially available -aminobenzamide ( ) by acylation with -chloropropionyl chloride followed by cyclization and dehydrohalogenation to prepare -vinyl- hquinazolin- -one ( a) as a key intermediate [ ] and a subsequent reaction with substituted benzyl bromides b-i [ ] (scheme ). moreover, compounds j (r = me) and k (r = et) were also obtained with intention to determine the influence of the benzyl substituent on biological activity of the designed isoxazolidines trans- /cis- . in the h-nmr spectra of compounds a-k characteristic signals for vinyl protons were observed in the . - . ppm (three doublets of doublets). scheme . retrosynthesis of (isoxazolidinyl) phosphonates trans- /cis- . -vinyl- h-quinazolin- -ones modified at n with substituted benzyl groups were synthesized from commercially available -aminobenzamide ( ) by acylation with -chloro-propionyl chloride followed by cyclization and dehydrohalogenation to prepare -vinyl- h-quinazolin- -one ( a) as a key intermediate [ ] and a subsequent reaction with substituted benzyl bromides b-i [ ] (scheme ). moreover, compounds j (r = me) and k (r = et) were also obtained with intention to determine the influence of the benzyl substituent on biological activity of the designed isoxazolidines trans- /cis- . in the h-nmr spectra of compounds a-k characteristic signals for vinyl protons were observed in the . - . ppm (three doublets of doublets). the , -dipolar cycloaddition of a nitrone with -vinylquinazolinones a-k led to the formation of diastereoisomeric mixtures of -substituted ( -diethoxyphosphoryl)isoxazolidines trans- and cis- with good ( %- %) diastereoselectivities (scheme , table ). ratios of cis/trans diastereoisomers were calculated from p-nmr spectra of crude reaction mixtures and confirmed by the analysis of h-nmr spectral data. crude mixtures of isoxazolidine cycloadducts were then subjected to purification on silica gel columns. however, attempts to isolate pure diastereoisomers were fruitful for trans- a the relative configurations of isoxazolidines trans- a and cis- a were established based on our previous studies on stereochemistry of cycloaddition of n-methyl-c-(diethoxyphosphoryl)nitrone ( ) with various vinyl aryls [ , ] since similar h-nmr spectral patters for the respective series of trans-and cis-isoxazolidines were observed. since for compound trans- a all necessary coupling constants were successfully extracted from the h-and c-nmr spectra, detailed conformational analysis was performed based on these data {j(h -h α) = . hz [ ] , j(h -h β) = . hz, j(h α-p) = . hz the , -dipolar cycloaddition of a nitrone with -vinylquinazolinones a-k led to the formation of diastereoisomeric mixtures of -substituted ( -diethoxyphosphoryl)isoxazolidines trans- and cis- with good ( %- %) diastereoselectivities (scheme , table ). ratios of cis/trans diastereoisomers were calculated from p-nmr spectra of crude reaction mixtures and confirmed by the analysis of h-nmr spectral data. crude mixtures of isoxazolidine cycloadducts were then subjected to purification on silica gel columns. however, attempts to isolate pure diastereoisomers were fruitful for trans- a (r = h), trans- c (r = -no -c h -ch ), trans- g (r = -f-c h -ch ), trans- h (r = -f-c h -ch ) and trans- j (r = me) only. table ). ratios of cis/trans diastereoisomers were calculated from p-nmr spectra of crude reaction mixtures and confirmed by the analysis of h-nmr spectral data. crude mixtures of isoxazolidine cycloadducts were then subjected to purification on silica gel columns. however, attempts to isolate pure diastereoisomers were fruitful for trans- a (r = h), trans- c (r = -no -c h -ch ), trans- g (r = -f-c h -ch ), trans- h (r = -f-c h -ch ) and trans- j (r = me) only. the relative configurations of isoxazolidines trans- a and cis- a were established based on our previous studies on stereochemistry of cycloaddition of n-methyl-c-(diethoxyphosphoryl)nitrone ( ) with various vinyl aryls [ , ] since similar h-nmr spectral patters for the respective series of trans-and cis-isoxazolidines were observed. since for compound trans- a all necessary coupling constants were successfully extracted from the h-and c-nmr spectra, detailed conformational analysis was performed based on these data {j(h -h α) = . hz [ ] , j(h -h β) = . hz, j(h α-p) = . hz scheme . synthesis of isoxazolidines cis- a-k and trans- a-k. reaction and conditions: a. toluene, ˝c, h. the relative configurations of isoxazolidines trans- a and cis- a were established based on our previous studies on stereochemistry of cycloaddition of n-methyl-c-(diethoxyphosphoryl)nitrone ( ) with various vinyl aryls [ , ] since similar h-nmr spectral patters for the respective series of transand cis-isoxazolidines were observed. since for compound trans- a all necessary coupling constants were successfully extracted from the h-and c-nmr spectra, detailed conformational analysis was performed based on these data {j (h -h α) = . hz [ ] , j (h -h β) = . hz, j (h α-p) = . hz [ , ] , j (h β-p) = . hz, j (h α-h ) = . hz, j (h β-h ) = . hz, j (cccp) = . hz [ , ] } and revealed that isoxazolidine ring in trans- a adopts a e conformation in which the diethoxyphosphoryl group resides in the equatorial position of the isoxazolidine ring while a quinazolinone substituent is located pseudoequatorially (figure ). on the other hand, cis configuration of the minor isomer was established from the corresponding couplings [j (h -h α) = . hz, j (h -h β) = . hz, j (h α-p) = . hz, j (h β-p) = . hz, j (h α-h ) = . hz, j (h β-h ) = . hz, j (cccp) = . hz] indicating the e conformation of the isoxazolidine ring ( figure ). the additional arguments to support our assignments follow from shielding of the ch ch op protons observed for the cis isomer (∆δ ca. . ppm) when compared with the trans- a. furthermore, it was found that on a h-nmr spectrum taken on the : mixture of cisand trans- a, the h-n proton in the quinazolinone ring of cis- a was significantly deshielded (∆δ = . ppm) when compared with the trans isomer, highly likely, as a result of the hydrogen bond formation with the phosphoryl oxygen amide, a phenomenon spatially achievable in the cis isomer only. since introduction of various substituents at n of quinazolinone moiety has no influence on the stereochemical outcome of the cycloaddition therefore configuration of the all major isoxazolidines were assigned as trans, thereby minor ones as cis. figure ). the additional arguments to support our assignments follow from shielding of the ch ch op protons observed for the cis isomer (Δδ ca. . ppm) when compared with the trans- a. furthermore, it was found that on a h-nmr spectrum taken on the : mixture of cis-and trans- a, the h-n proton in the quinazolinone ring of cis- a was significantly deshielded (Δδ = . ppm) when compared with the trans isomer, highly likely, as a result of the hydrogen bond formation with the phosphoryl oxygen amide, a phenomenon spatially achievable in the cis isomer only. since introduction of various substituents at n of quinazolinone moiety has no influence on the stereochemical outcome of the cycloaddition therefore configuration of the all major isoxazolidines were assigned as trans, thereby minor ones as cis. ganciclovir, cidofovir, acyclovir, brivudin, zalcitabine, zanamivir, alovudine, amantadine, rimantadine, ribavirin, dextran sulfate (molecular weight , , ds- ), mycophenolic acid, hippeastrum hybrid agglutinin (hha) and urtica dioica agglutinin (uda) were used as the reference compounds. the antiviral activity was expressed as the ec : the compound concentration required to reduce virus plaque formation (vzv) by % or to reduce virus-induced cytopathogenicity by % (other viruses). several isoxazolidines trans- /cis- were able to weakly inhibit the replication of tk + and tk − vzv strains with ec values in the range of . - μm ( table ). among them, phosphonates ganciclovir, cidofovir, acyclovir, brivudin, zalcitabine, zanamivir, alovudine, amantadine, rimantadine, ribavirin, dextran sulfate (molecular weight , , ds- ), mycophenolic acid, hippeastrum hybrid agglutinin (hha) and urtica dioica agglutinin (uda) were used as the reference compounds. the antiviral activity was expressed as the ec : the compound concentration required to reduce virus plaque formation (vzv) by % or to reduce virus-induced cytopathogenicity by % (other viruses). several isoxazolidines trans- /cis- were able to weakly inhibit the replication of tk + and tkv zv strains with ec values in the range of . - µm ( table ) . among them, phosphonates trans- f/cis- f ( : ) (r = -f-c h -ch ) (ec = . µm), trans- h (r = -f-c h -ch ) (ec = . µm), trans- i/cis- i ( : ) (r = , -dif-c h -ch ) (ec = . µm) were the most active toward tk + vzv oka strain, while exhibiting no activity toward tk´vzv strain. the activity of these isoxazolidines trans- /cis- against tk + vzv oka strain was -to -folds lower than that of the reference drug acyclovir. on the other hand, the ec values for the tk´vzv - strain (which is an acyclovir resistant strain) of the phosphonates trans- e/cis- e ( : ) (r = -no -c h -ch ) (ec = . µm) and trans- k/cis- k ( : ) (r = et) (ec = . µm) were comparable to that of acyclovir (ec = . µm). these derivatives showed similar ec 's for tk + and tk´vzv strains and therefore their potency against tk + vzv was approximately -fold lower compared to acyclovir. furthermore, compounds trans- b/cis- b ( : ) (r = c h -ch ), trans- c (r = -no -c h -ch ), trans- e/cis- e ( : ) (r = -no -c h -ch ) and trans- g (r = -f-c h -ch ) showed some activity against human cytomegalovirus (ec = - µm), although they were less active than ganciclovir and cidofovir used as the reference compounds ( table ) . none of the phosphonate derivatives here described showed activity against the other tested dna and rna viruses. the % cytostatic inhibitory concentration (ic ) causing a % decrease in cell proliferation was determined against murine leukemia l , human lymphocyte cem, human cervix carcinoma hela and immortalized human dermal microvacsular endothelial cells (hmec- ). isoxazolidines trans- a (r = h) and trans- j (r = me) did not inhibit cell proliferation at the highest tested concentration (i.e., µm), whereas trans- k/cis- k ( : ) (r = et) appeared slightly cytostatic towards the tested cell lines (ic = - µm). on the other hand (table , entries b to i), compounds having benzyl substituents at n in the quinazolinone moiety showed lower ic values (ic = - µm) thereby indicating that installation of functionalized benzyl groups was profitable for inhibitory properties. table . inhibitory effect of the tested compounds against the proliferation of murine leukemia (l ), human t-lymphocyte (cem), human cervix carcinoma (hela) and immortalized human dermal microvascular endothelial cells (hmec- ). to the solution of -vinyl- h-quinazolin- -one ( a, . mmol) in acetonitrile ( ml) potassium carbonate ( . mmol) was added. after min the respective benzyl bromide ( . mmol) was added and the reaction mixture was stirred under reflux for h. a solvent was removed and the residue was extracted with water ( ˆ ml). an organic layer was dried (mgso ), concentrated and the crude product was purified on a silica gel column with a methylene chloride: hexane mixture ( : , v/v) followed by crystallisation (chloroform-petroleum ether) to give pure quinazolinones b-e and g-i. . , . , . , . , . , . , . , . , . , . (s, n-ch ) . anal. calcd. for c to the solution of -vinyl- h-quinazolin- -one ( a, . mmol) in acetonitrile ( ml) potassium carbonate ( . mmol) was added. after min. iodomethane ( . mmol) or iodoethane ( . mmol) was added and the reaction mixture was stirred at ˝c for h. the solvent was removed and a residue was extracted with water ( ˆ ml). organic layer was dried (mgso ), concentrated and the crude product was purified on a silica gel column with methylene chloride:hexane mixture ( : , v/v) followed by crystallization (chloroform : petroleum ether) to give pure quinazolinones j [ ] or k. -methyl- -vinylquinazolin- ( h)-one ( j). amorphous solid, m.p. = ˝c- ˝c (reference [ ] m.p. = ˝c- ˝c). a solution of the nitrone ( . mmol) and the respective vinyl quinazolinone ( . mmol) in toluene ( ml) was stirred at ˝c until the disappearance (tlc) of the starting nitrone. all volatiles were removed in vacuo and crude products were subjected to chromatography on silica gel columns with a chloroform/methanol ( : , : , : , v/v) mixtures as eluents. diethyl trans-( -methyl- -( -oxo- , -dihydroquinazolin- -yl)isoxazolidin- -yl)phosphonate (trans- a). yellowish oil; ir (film, cm´ ) ν max : , , , , , , , , , , , diethyl trans-( -methyl- -( -( -nitrobenzyl)- -oxo- , -dihydroquinazolin- -yl)isoxazolidin- -yl)-phosphonate (trans- d). data noted below correspond to a : mixture of trans- d and cis- d. a yellowish oil; ir (film, cm´ ) ν max : , , , , , , , , , , , , (obtained on a : mixture of trans- f and cis- f) -fluorobenzyl)- -oxo- , -dihydroquinazolin- -yl)- -methylisoxazolidin- -yl)-phosphonate (trans- g) cdcl ): δ = . - . (m, h), . - . (m, h), . - . (m, h), . - . (m, h), . - . (m, h), . - . (m, h), . - . (m, h), . (s, h, n-ch ), . (dd, j (h -h β) = . hz, j (h -h α) = . hz, h, hc ) j (cf) = . hz, c '), . , . , . (d, j (cccf) = . hz, c '), . , . (d, j = . hz, c '), . , ch op), . (d, j (cop) = . hz, ch op) cdcl ): δ = . . anal. calcd. for c h fn o pˆ . h o: c, found: c, . -dihydroquinazolin- -yl)- -methylisoxazolidin- -yl)-phosphonate (trans- h) j (h α-h ) = . hz, j (h α-h ) = . hz, h, h α c ) (d, j (cccf) = . hz, c (s, c ), . (d, j (ccop) = . hz, ch ch op), . (d, j (ccop) = . hz, ch ch op). p-nmr ( mhz phosphonate (trans- i). data noted below correspond to a : mixture of trans- i and cis- i . (signals of trans- i were extracted from the spectra of a : mixture of trans- i and cis- i); h-nmr ( mhz m, h, h β c ), . (dddd, j (h α-p) = . hz, j (h α-h β) = . hz, j (h α-h ) = . hz, j (h α-h ) = . hz, h, h α c (dd, j (cf) = . hz, j (cccf) = . hz (dd, j (ccf) = . hz, j (ccccf) = . hz, c '), . (d, j (cp) = . hz, c ), . (d, j (cop) = . hz, ch op), . (d, j (cop) = . hz, ch op), . (d, j (cccf) = . hz, ch n) -yl)isoxazolidin- -yl)phosphonate (trans- j) j (h α-h ) = . hz, j (h α-h ) = . hz, h, h β c ) j (h -h β) = . hz, j (h -p) = . hz, h, hc ), . (dddd, j (h β-p) = . hz, j (h β-h α) = . hz j (h β-h ) = . hz, j (h β-h ) = . hz, h, h β c ) (d, j (cccp) = . hz, c ), . (d, j (cp) = . hz, c ), . (d, j (cop) = . hz, ch op), . (d, j (cop) = . hz, ch op) -ethyl- -oxo- , -dihydroquinazolin- -yl)- -methylisoxazolidin- -yl)phosphonate (trans- k). data noted below correspond to a : mixture of trans- k and cis- k (m, h, hc ), . (s, h, ch n), . - . (m, h, h α c , h β c ), . (t, j = . hz, h, ch ch ), . (t, j = . hz, h, ch ch op), . (t, j = . hz, h, ch ch op). c-nmr ( mhz after incubation at ˝c for two (l ), three (cem) or four (hela) days, the cell number was determined of isoxazolidine-containing quinazolinones trans- and cis- have been synthesised from n-methyl-c-diethoxyphosphorylnitrone ( ) and the respective n -substituted -vinyl-quinazolin-ones via the , -dipolar cycloaddition. the obtained isoxazolidine phosphonates trans- or the respective mixtures of trans- /cis- were evaluated against a variety of dna and rna viruses. among all tested compounds, isoxazolidines trans- f/cis- f ( : ), trans- h and trans- i/cis- i were slightly active toward tk + vzv strain (ec = . , . and . µm) without exhibiting cytotoxicity toward uninfected cells at concentration up to µm a multifaceted gabaa receptor modulator: functional properties and mechanism of action of the sedative-hypnotic and recreational drug methaqualone (quaalude) synthesis and anxiosedative and antidepressant properties of α-( -oxoquinazolin- ( h)-yl)carboxylic acid anilides anticonvulsant and sedative-hypnotic activity of some novel -( -( -substituted) phenyl- , , -oxadiazole- yl)- -styrylquinazoline- ( h)-ones synthesis and in vitro antitumor activities of novel -anilinoquinazoline derivatives new quinazolinone derivatives: synthesis, anti-inflammatory and antitumor activities synthesis and in vitro antitumor activity of substituted quinazoline and quinoxaline derivatives: search for anticancer agent synthesis and biological evaluation of a novel series of , -dibromo- ( h)quinazolinone derivatives as anticancer agents synthesis and antiviral activities of some , -disubstituted quinazoline derivatives synthesis, antiviral activity, d-qsar, and interaction mechanisms study of novel malonate derivatives containing quinazolin- ( h)-one moiety synthesis, anti-tobacco mosaic virus and cucumber mosaic virus activity, and d-qsar study of novel , -pentadien- -one derivatives containing -thioquinazoline moiety synthesis, antiviral and antimicrobial activities of quinazoline urea analogues development of (e)- -(( , -dimethylpiperazin- -ylidene)amino)- -nitro-nphenylbenzamide, ml : novel -amidinophenylbenzamides as potent inhibitors of venezuelan equine encephalitis virus antimicrobial study of newly synthesized -substituted indolo( , -c)quinazolines potential antimicrobial activities of quinazolinone derivatives quinazolinones linked amino acids derivatives as a new class of promising antimicrobial, antioxidant and anti-inflammatory agents synthetic and pharmacological evaluation of some -( -{((substitutedphenyl)methylene)amino}phenyl)- -bromo- -methylquinazolin- -one derivatives design, synthesis and biological evaluation of novel quinazoline-based anti-inflammatory agents acting as pde b inhibitors schiff's bases of quinazolinone derivatives: synthesis and sar studies of a novel series of potential anti-inflammatory and antioxidants synthesis of some new substituted azetidinonyl and thiazolidinonyl quinazolin- ( h)-ones as potential non-steroidal anti-inflammatory and analgesic agents halofuginone-the multifaceted molecule substituted quinazolinones as kinase inhibitors endowed with anti-fibroic properties deep eutectic solvent mediated synthesis of quinazolinones and dihydroquinazolinones: synthesis of natural products and drugs chemical characteristics, synthetic methods, and biological potential of quinazoline and quinazolinone derivatives recent advances of quinazolinone derivatives as marker for various biological activities quinazolinones as antimicrobial agents: a review quinazolinone: an overview quinazoline marketed drugs-a review synthetic approaches towards quinazolines, quinazolinones and quinazolinediones on solid phase a novel and other bioactive secondary metabolites from a marine fungus penicillium oxalicum f triazoles and other n-containing metabolites from the marine-derived endophytic fungus pencillinum chrysogenum en- triazole and dihydroimidazole alkaloids from the marine sediment-derived fungus penicillium paneum sd- quinazolinone alkaloids from actinomycete streptomyces sp. bcc -pyridinyl- ( h)-quinazolinone: a scaffold for anti-influenza a virus compounds design, synthesis, and molecular docking studies of -(furan- -yl)-quinazolin- -one derivatives as potential antiproliferative agents n-substituted c-diethoxyphosphorylated nitrones as useful synthons for the synthesis of α-aminophosphonates synthesis and reaction of some -vinyl- h-quinazolin- -ones a new mixed amino-amido n-heterocyclic carbene based on antranilin acid design, synthesis and cytotoxicity of a new series of isoxazolidine based nucleoside analogues design, synthesis, antiviral and cytostatic evaluation of novel isoxazolidine analogues of c-nucleotides vicinal proton coupling in nuclear magnetic resonance variation of vicinal phosphorus- -carbon-carbon-proton couplings with dihedral angle in phosphonates structural analysis of -c-phosphonates, -phosphinates, and -phosphine oxides of branched-chain sugars darstellung und kristallstruktur von endo- -dimethylphosphono-exo- -hydroxy-(-)-camphan zur bestimmung von j(cccp)-vicinalkopplungen c-and p-nmr spectra of -diethylphosphono- -hydroxycycloalkanes functionalized carbodiimide mediated synthesis of , -disubstituted quinazolin- ( h)-ones via the tandem strategy of c-nucleophilic addition and intramolecular nh-substitution cyclization this article is an open access article distributed under the terms and conditions of the creative commons attribution (cc-by) license the compounds were evaluated against different herpesviruses, including herpes simplex virus type (hsv- ) strain kos, thymidine kinase-deficient (tk´) hsv- kos strain resistant to acv (acv r ), herpes simplex virus type (hsv- ) strain g, varicella-zoster virus (vzv) strain oka, tkv zv strain - , human cytomegalovirus (hcmv) strains ad- and davis as well as feline herpes virus (fhv), the poxvirus vaccinia virus (lederle strain), para-influenza- virus, reovirus- , sindbis virus, coxsackie virus b , punta toro virus, respiratory syncytial virus (rsv), feline coronovirus the authors declare no conflict of interest. key: cord- - ljgkqy authors: završnik, davorka; muratović, samija; makuc, damjan; plavec, janez; cetina, mario; nagl, ante; de clercq, erik; balzarini, jan; mintas, mladen title: benzylidene-bis-( -hydroxycoumarin) and benzopyrano-coumarin derivatives: synthesis, ( )h/( )c-nmr conformational and x-ray crystal structure studies and in vitro antiviral activity evaluations date: - - journal: molecules doi: . /molecules sha: doc_id: cord_uid: ljgkqy we report on the synthesis of -hydroxycoumarin dimers – bearing an aryl substituent on the central linker and fused benzopyranocoumarin derivatives – and on their in vitro broad anti-dna and rna virus activity evaluations. the chemical identities and structure of compounds – were deduced from their homo- and heteronuclear nmr measurements whereas the conformational properties of , and were assessed by the use of d difference noe enhancements. unequivocal proof of the stereostructure of compounds , , and was obtained by single crystal x-ray diffraction method. the x-ray crystal structure analysis revealed that two -hydroxycoumarin moieties in the -trifluoromethylphenyl- and -nitrophenyl derivatives (compounds and , respectively) are intramolecularly hydrogen-bonded between hydroxyl and carbonyl oxygen atoms. consequently, the compounds and adopt conformations in which two -hydroxy-coumarin moieties are anti-disposed. antiviral activity evaluation results indicated that the -bromobenzylidene derivative of bis-( -hydroxycoumarin) (compound ) possesses inhibitory activity against hsv- (kos), hsv- (g), vaccinia virus and hsv- tk(-) kos (acv(r)) at a concentration of – μm and at a minimum cytotoxic concentration (mcc) greater than μm. compounds – , , and were active against feline herpes virus ( % effective concentration, ec( ) = – . μm), that is at a - -fold lower concentration than the mcc. numerous experimental studies indicate that natural and synthetic coumarins ( h- -benzopyran- ones) and their derivatives are endowed with excellent chemical reactivity and different bioactivity. thus, the natural coumarins play an important role in plant biochemistry and physiology. they act as antioxydants, enzyme inhibitors and precursors of toxic substances. they are also involved in the actions of plant growth hormones and growth regulators, the control over the respiration and photosynthesis, as well as in the defense against various infections [ ] . although most of the existing natural coumarins have been isolated from higher plants, some of them have been discovered in microorganisms, e.g., aminocoumarin antibiotics: novobiocin, coumermycin a and chlorobiocin (produced by the actinomycete streptomyces niveus) [ ] . synthetic coumarin derivatives have been obtained by chemical modification of the coumarin ring. as a substitution can conceptually occur at any of the six available sites of the basic molecule, these compounds are widely variable in structure and activity. the biological activities of coumarin derivatives, in particular their therapeutic application as anticoagulant and antibacterial agents [ ] , has stimulated further interest for the synthesis of this class of compounds. a variety of synthesized coumarin derivatives have been experimentally shown to exert pharmacological activities including inhibition of platelet aggregation, cytochrome p , and steroid -α-reductase. they have also been shown to exert efficient anti-proliferative, antifungal, anti-psoriasis, antiinflammatory, as well as antiviral activities [ ] [ ] [ ] [ ] [ ] . the interest in coumarins has recently increased significantly because it was found that they inhibit hiv (human immunodeficiency virus), by affecting integrase and reverse transcriptase, which play a critical role in the replicative cycle of hiv [ ] [ ] [ ] . the present study is focused on the antiviral activity evaluation of the benzylidene-bis- the benzylidene-bis-( -hydroxycoumarin) derivatives - and fused benzopyranocoumarin derivatives - were prepared by a sequence of reactions displayed in the scheme . in the first step of the synthesis the aldol condensation of -hydroxycoumarin ( -hc) with an appropriately substituted aldehyde linker followed by dehydration of aldol product (al) gave a chromone (cr). subsequent in situ reaction of the chromon with -hydroxycoumarin already present in excess in the reaction mixture gave dimeric coumarin derivatives - bearing an aryl substituent in the central methylene linker. in contrast, the chromone derivatives containing an ortho-substituted phenyl moiety (r = cl, f, oh, och ) gave under spontaneous cyclization the fused benzopyranocoumarin derivatives - . the compounds [ , , ] , [ , , ] , [ ] , [ ] , [ ] , [ ] , [ , ] , [ ] , [ ] , [ , ] and [ , ] that have been described previously were also synthesized for their antiviral activity evaluations. + r r r r h o (for r -r see figure a) i o o o oh -h o [r -r , c.f. figure a] [r -r , c.f. figure b] o oh o o oh o r r r r - (c.f. figure a) o oh o o o r r r - (c.f. figure b) o r cr r r r r al o o o r r r r the chemical identities and structures of - were confirmed by homo-and heteronuclear nmr measurements. h-, c-and f-nmr chemical shifts are reported in the experimental section. proton-decoupled c-nmr spectra showed c-f coupling constants that enabled straightforward identification of fluorinated carbon atoms and their neighbors. in the h spectrum of , , and , which were dissolved in non-polar solvents, well resolved hydroxyl protons were observed in the range from δ . to . ppm (see experimental section for details). the strongly deshielded signals suggest that oh protons are most probably involved in hydrogen bond formation. in addition, two set of h-nmr signals were observed for h and h " protons which indicates a slight difference in magnetic environment for otherwise symmetrical moieties. furthermore, hydroxyl protons, which were observed between δ . and . ppm for benzopyranocoumarin derivatives , and , are most likely involved in hydrogen bonds. hydrogen bonds described above partially determined conformational preferences of the studied compounds. conformational properties of , and were assessed with the use of d noe difference experiments. key noe enhancements are shown in figure . the saturation of h* in resulted in weak noe at h ' ( . %) and '-och ( . %, figure a ). likewise, the saturation of h* in gave moderate noe at h ' ( . %) and weak noe at '-och ( . %). these observations suggested nonrestricted rotation along c*-c ' bonds in and . no noes indicative of relative orientations of individual heterocyclic moieties were observed for . interestingly, benzopyranocoumarin derivatives , and showed two sets of signals in the h spectrum for coumarin protons. one set of h signals (h -h protons) exhibited broader line-widths with respect to multiplets attributed to h "-h " protons. this phenomenon was studied in more detail for by variable temperature experiments in the range from to k. as an example, broad multiplet at δ . ppm corresponding to h becomes sharper at higher temperatures ( figure ). this suggests that rotation along c*-c bond is restricted at lower temperatures, most likely due to formation of hydrogen bond between the c -oh and c " carbonyl group (for enumeration of atoms c.f. figure ). in ( figure a ) and (figure b) , two -hydroxycoumarin moieties are linked through a methylene bridge on which one hydrogen atom has been replaced with a phenyl ring bearing p-trifluoromethyl and o-nitro groups, respectively. in general, the geometry of the molecules agrees with closely related structures [ ] [ ] [ ] . this asymmetry, which is also found in similar structures, may be a consequence of steric crowding within the molecules. because of the same reason all principal bond angles about c are widened over normal tetrahedral values, ranging from . ( ) to . ( )° in and . ( ) to . ( )° in . the coumarin rings are slightly distorted from planarity, with two planes inclined at . ( )° and . ( )° to each other in and , respectively. the -hydroxycoumarin moieties are intramolecularly hydrogen bonded between hydroxyl and carbonyl oxygen atoms in both structures (see figures a and b and table s in esi), thus forming two eight-membered rings. thus, compounds and adopt a conformation in which two -hydroxycoumarin moieties are anti-disposed. benzylidene bis ( -hydroxycoumarin) derivatives and form supramolecular self-assemblies by c−h···o hydrogen bonds (figures s and s , see esi) in which infinite chains are extended by one f···f interaction between trifluoromethyl fluorine atoms in [f ···f i = . ( ) Å; (i): −x, −y, −z] and one π···π interaction in . the distance between the ring centroids of coplanar c '-c ' rings [α = o ] in is . ( ) Å, the planes are separated by . ( ) Å and centroids offset is ca. . Å. cyclized compound ( figure a ) crystallized with two independent molecules, two ethanol molecules and one water molecule in the asymmetric unit in monoclinic space group p /c. the bond lengths in two independent molecules of , denoted as a and b, are within σ values, and agree very well with the corresponding ones in ( figure b ). (table s , see esi). two independent molecules of , two ethanol molecules and water molecule are linked by six o−h···o and four c−h···o hydrogen bonds into three-dimensional network ( figure s , see esi). two c−h···π interactions participate also in supramolecular aggregation. on the contrary, only one hydrogen bond of c−h···o type links the molecules of , thus forming discrete centrosymmetric dimers via -membered rings ( figure s , see esi). of all evaluated compounds only the -bromobenzylidene derivative of bis( -hydroxycoumarin) showed potentially interesting inhibitory activity against hsv- (kos), hsv- (g), vaccinia virus and hsv- tk − kos (acv r ) in the range of - μm at a minimum cytotoxic concentration (mcc) greater than μm, whereas compound showed only slight activity against hsv- (kos), hsv- (g) and hsv- tk − kos (ec = - μm) in human embryonic lung hel cell cultures and no cytotostatic activity at µm (table , compounds - were also evaluated for their cytotoxicity and antiviral activities against coxsackie virus b and respiratory syncytial virus (rsv) in hel cell cultures; parainfluenza- virus, reovirus- , sindbis virus, coxsackie virus b and punta toro virus in vero cell cultures; and influenza virus subtypes a h n and h n , and influenza virus b. no specific antiviral effects (i.e., antivirally effective concentration > -fold lower than the minimal cytotoxic concentration) were noted for any of the evaluated compounds. melting points (uncorrected) were determined with büchi melting point b- . precoated merck silica gel f- plates were used for thin-layer chromatography (tlc) and the spots were detected under uv light ( nm). solvent system used for tlc was chloroform:methanol = : . mass spectra were recorded with an autospec: esi/q-tof premier instrument. h-nmr spectra were recorded at mhz, in deuterated dmso-d , on bruker (uxnmr/xwinnmr) spectrometer, using tetramethylsilane (tms) as internal reference. d and d nmr spectra were recorded at °c on varian unity inova spectrometers. h and c chemical shifts were referred to residual signal of cdcl , cd cl and dmso-d (δ tms . ppm). f chemical shifts were referenced externally with respect to ccl f (δ . ppm). h, c and f-nmr resonances were assigned on the basis of signal intensities and multiplicities in d spectra as well as correlation signals in d h- h cosy, h- c hsqc and h- c hmbc nmr spectra. elemental analyses were performed in the central analytic service, rudjer bošković institute zagreb, croatia, using a perkin elmer elemental analyser. the infrared spectra were obtained from potassium bromide triturate containing . % of the product on a perkin-elmer ft-ir spectrophotometer. all data were recorded at °c unless specified otherwise. . . . the compounds - were prepared by the following general procedure -hydroxycoumarin ( mmol) was dissolved in hot ethanol ( ml), the corresponding aldehyde ( mmol) was added, and the reaction mixture was refluxed for h. after cooling to room temperature, the solid was filtered off and crystallized to give the product benzylidene-bis-( hydroxycoumarin) derivatives - and -[ -oxo-( h)-benzopyrano[ , -b]benzopyran- -yl]- hydroxycoumarin derivatives - (figure ). ( ) [ , , ] . yield %; mp . ( ) [ , ] . ( ) [ , ] . ( ) [ , ] . program was used for data collection and processing. the intensities were corrected for absorption using the multi-scan absorption correction method [ ] . the crystal structures were solved by direct methods [ ] and all non-hydrogen atoms were refined anisotropically by full-matrix least-squares calculations [ ] based on f using the programs integrated in wingx [ ] program package. the hydrogen atoms attached to the o and o ' atoms in and , o a and o b atoms in and o atom in were found in a difference fourier map and are refined with o-h distance restraint of . Å. all other hydrogen atoms were treated using appropriate riding models, with shelxl defaults [ ] . fluorine atoms of trifluoromethyl group in are heavily disordered and restraints on anisotropic displacement parameters were therefore used in their refinement. the c and c atoms of ethanol molecules in are disordered with the site occupancy factors refined to . ( )/ . ( ) and . ( )/ . ( ) ratio, respectively. details of crystal data, data collection and refinement parameters are given in table . platon [ ] program was used for structure analysis and molecular and crystal structure drawings preparation. ccdc - contain the supplementary crystallographic data for this paper. these data can be obtained free of charge from the cambridge crystallographic data centre via www.ccdc.cam.ac.uk/data_request/cif. the antiviral assays, other than the anti-hiv assays, were based on inhibition of virus-induced cytopathic effect in human lung fibroblast [herpes simplex virus type (hsv- ) [strain kos], herpes simplex virus type (hsv- ) [strain g], vaccinia virus (vv) and vesicular stomatitis virus (vsv)], african green monkey kidney (vero, parainfluenza- , reovirus- , sindbis, coxsackie b , and punta toro virus), human cervix carcinoma (vesicular stomatitis virus, coxsackie virus b and respiratory syncytial virus (rsv)), feline crandell-rees kidney (crfk) (feline herpes virus, feline corona virus (fipv)) or madin-darby canine kidney (mdck) (influenza a [h n ; h n ] and influenza b) cell cultures. confluent cell cultures in microtiter -well plates were inoculated with ccid of virus ( ccid being the virus dose to infect % of the cell cultures) in the presence of varying concentrations ( , , , . and . µm) of the test compounds. viral cytopathicity was recorded as soon as it reached completion in the control virus-infected cell cultures that were not treated with the test compounds. the anti-hiv activity and cytotoxicity of the compounds were evaluated against wild-type hiv- strain iii b in mt- cell culture using the -( , -dimethylthiazol- -yl)- , diphenyltetrazolium bromide (mtt) method. mt- cells were suspended in culture medium at × cells/ml and infected with hiv at a multiplicity of infection of . . immediately after viral infection, µl of the cell suspension was placed in each well of a flat-bottomed microtiter tray containing various concentrations of the test compounds. after four days of incubation at °c, the number of viable cells was determined using the mtt method. compounds were tested in parallel for cytotoxic effects in uninfected mt- cells. a series of the benzylidene-bis-( -hydroxycoumarin) derivatives - and fused benzopyranocoumarin derivatives - were synthesized and evaluated for their antiviral activities on a broad panel of dna and rna viruses. x-ray crystal structure analysis of -trifluoromethylphenyl-and -nitrophenyl derivatives and revealed intramolecular hydrogen bonding between hydroxyl and carbonyl oxygen atoms of two -hydroxycoumarin moieties resulting in the formation of two eight-membered rings. accordingly, two -hydroxycoumarin moieties in compounds and are anti-disposed. the -bromobenzylidene derivative of bis( -hydroxycoumarin) ( ) exerted some inhibitory activity against hsv- (kos), hsv- (g), vaccinia virus and hsv- tk -kos (acv r ) in the range of - μm at minimum cytotoxic concentration (mcc) greater than μm, whereas the compounds - , , and exhibited rather pronounced anti-feline herpes virus activity (ec = - . μm) but with mcc at only - -fold higher than ec values. tables s with hydrogen-bonded geometries, figures s -s presenting hydrogen bonds for compounds , , and . (h , m, h), . (h ', d, j = . , h), . (h , m, h), . (h ", m, h), . (h ", m, h), . (h ', m, h), . (h , m, h), . (h ", m, h), . (h , b, h), . (h -methoxybenzopyrano dmso-d ): δ . (och , s, h), . (h*, s, h), . (h ', m, h), . (h ', m, h), . (h ', m, h), . (h , m, h), . (h , m, h), . (h ", d, j = . , h), . (h ", m, h), . (h , m, h), . (h ", dd, j = . , . , h), . (h ", dd, j = . , . , h), . (h , m, h), . ppm yield %; mp oh); h-nmr (dmso-d ): δ . (h*, b, h) elemental analysis. calc. for c h o n: c . , h . , n . , found: c . , h . . (h*, b, h), . (h ', d, j = . , h), . (h ', d, j = . , h), . (h , d, j = . , h), . (h , m, h), . (h ", m, h), . (h ", m, h), . (h , m, h), . (h ", dd, j = . , . , h), . (h , m, h) elemental analysis. calc. for c h o : c history of the development and applications of coumarin and coumarin-related compounds novobiocin and related coumarins and depletion of heat shock protein -dependent signaling proteins simple coumarins and analogues in medicinal chemistry: occurrence, synthesis and biological activity synthesis and antimicrobial evaluation of coumarin derivatives synthesis and antioxidant activity of selected -methylcoumarins synthetic approaches and biological activities of -hydroxycoumarin derivatives synthesis of hydroxycoumarins and hydroxybenzo[f]-or [h]coumarins as lipid peroxidation inhibitors studying plant-derived coumarins for their pharmacological and therapeutic properties as potential anticancer drugs. expert opin. drug disc synthesis and biological evaluation of coumarin-based inhibitors of nad(p)h: quinone oxidoreductase- (nqo ) synthesis and studies of new -(coumarin- -yloxy)- , -(substituted)-s-triazine derivatives as potential anti-hiv agents coumarin-based inhibitors of hiv integrase simian immunodeficiency virus is susceptible to inhibition by carbohydrate-binding agents in a manner similar to that of hiv: implications for further preclinical drug development synthesis and pharmacological investigations of some -hydroxycoumarin derivatives synthesis, toxicological and pharmacological assessment of some -hydroxycoumarin derivatives antiretroviral agents as inhibitors of both human immunodeficiency virus type integrase and protease synthesis, characterization, and cytotoxic activity of new lanthanum(iii) complexes of bis-coumarins structure and anticoagulant activity of bridge-substituted dicoumarols synthesis and anticoagulant activities of substituted , -diketochromans, biscoumarins, and chromanocoumarins synthesis and structure of structure of (phenyl)bis( -hydroxybenzo- h-pyran- -one- -yl)methane synthesis, structure and acid-base behaviour of some -hydroxycoumarin derivatives oxford diffraction ltd a short history of shelx wingx suite for small-molecule single-crystal crystallography single-crystal structure validation with the program platon this article is an open access article distributed under the terms and conditions of the creative commons attribution license support of this study by the ministry of science, education and sport republic of croatia (project nos. - - and - - ) is gratefully acknowledged. key: cord- -gh gaa authors: zannou, oscar; koca, ilkay; aldawoud, turki m. s.; galanakis, charis m. title: recovery and stabilization of anthocyanins and phenolic antioxidants of roselle (hibiscus sabdariffa l.) with hydrophilic deep eutectic solvents date: - - journal: molecules doi: . /molecules sha: doc_id: cord_uid: gh gaa deep eutectic solvents (dess) have got huge interest as new green and sustainable solvents for the extraction of bioactive compounds from plants in recent decades. in the present study, we aimed to investigate the effectiveness of hydrophilic des for the extraction of anthocyanin and polyphenol antioxidants from roselle. a natural hydrophilic des constituted of sodium acetate (hydrogen bond acceptor) and formic acid (hydrogen bond donor) designed to evaluate the total phenolic compound (tpc), total flavonoid (tfc), total anthocyanin (tacn), , -diphenyl- -picrylhydrazyl (dpph) radical scavenging and ferric reducing antioxidant power (frap) values of roselle. distilled water, % ethanol, and % methanol used as conventional solvents for comparison. the results indicated that the des prepared in molarity ratio (safa(m)) was the most efficient. subsequently, this prominent des selected for the optimization and the optimum extraction conditions were : . molarity ratio, % additional water, and ml solvent. tpc, tfc, tacn, frap, and dpph radical scavenging at the optimum point were . mg gae/g, . mg ece/g, . mg d s/g, . mmol ise/g, and . mmol te/g, respectively. the stability tests showed that anthocyanins were more stable in safa(m). these findings revealed that safa(m) is an effective green solvent for the extraction of polyphenols from various plants. roselle (hibiscus sabdariffa l.) is one of the most popular species belonging to the malvaceae family. they are highly desired by consumers, for containing several nutrients and phytochemical compounds that improve health. roselle is essential for the prevention and treatment of hypertension, hepatitis, cardiovascular diseases, atherosclerosis, and diabetes [ ] [ ] [ ] [ ] . the extracts had powerful effects on reducing high cholesterol, preventing cancer and hepatitis, and inhibiting pathogen microorganisms' growth [ , ] . roselle's red calyces are processed to juice and tea, or incorporated in confectionary (e.g., chocolate) and dairy products as natural flavoring, and coloring agents. the calyces remain as the primary by-product and provide natural antioxidants and odorants [ ] [ ] [ ] . food and plant bioactives are nowadays very important in supporting the immune system within the covid- pandemic era [ ] . roselle calyces contain bioactive compounds that are useful different letters (a, b, c) in the same column indicate statistical differences (p < . ). the ftir spectra of all the dess studied were shown in figure . the peaks at and cm − (figure a) were assigned to c-h stretching frequencies associated with formic acid and sodium acetate, respectively. the frequencies at and cm − were related to c=o of formic acid and sodium acetate, respectively. the stretching of c-o at cm − , ch bending at and cm − and stretching of c-c at cm − were used to recognize sodium acetate. from spectroscopy data, it can be assumed that the strong cooh...cooh bonds in sodium acetate and formic acid were broken down during the formation of sodium acetate and formic acid mixture to produce new strong intramolecular cooh bonds. this explains the low melting point and high viscosity of the pure safa [ ] . molecules , the dess prepared in molar and molarity ratios were encoded as safa and safam, respectively. each des was characterized by high viscosity ranged between . and . mpa.s ( table ). the presence of carboxyl groups and alkyl chains causes extremely high viscosity [ , ] . the pure safa ( % water content) was semi-solid at room temperature and displayed an extremely high viscosity of . mpa.s. this high viscosity is associated with the h bonding network generated by the combination of sodium acetate and formic acid in eutectic terms. the addition of water decreased des viscosity. the application of water in dess has been reported to weaken and disintegrate the dess nanostructure [ , ] . in the present study, the application of water decreased the viscosity to . mpa.s at % (v/v), . mpa.s at % (v/v), . mpa.s at % (v/v) and . mpa.s at % (v/v). the lowest viscosity was recorded in the des prepared in molarity ratio (safam), suggesting that it contained more water. therefore, the interaction between sodium acetate and formic acid might be the lowest since high water concentration in dess reduces the intermolecular and intramolecular reactions between the components constituting dess [ , , ] . different letters (a, b, c) in the same column indicate statistical differences (p < . ). the ftir spectra of all the dess studied were shown in figure . the peaks at and cm − (figure a) were assigned to c-h stretching frequencies associated with formic acid and sodium acetate, respectively. the frequencies at and cm − were related to c=o of formic acid and sodium acetate, respectively. the stretching of c-o at cm − , ch bending at and cm − and stretching of c-c at cm − were used to recognize sodium acetate. from spectroscopy data, it can be assumed that the strong cooh...cooh bonds in sodium acetate and formic acid were broken down during the formation of sodium acetate and formic acid mixture to produce new strong intramolecular cooh bonds. this explains the low melting point and high viscosity of the pure safa [ ] . after the addition of water, the stronger bonding between hbd and hba was affected and weakened with the appearance of o-h ( figure ). the absence of o-h in safa suggested that the mixture was too anhydrous. the stretching of o-h at cm − appeared progressively in dess, got higher as water content increased and became stronger in safam (figure b-e). similar phenomena have been reported in choline chloride and oxalic acid, as well as in choline chloride and glycol-based dess, and might be due to alternative possibilities for h bonding in des upon the introduction of water [ , ] . hammond et al. ( ) have revealed that the introduction of a small quantity of water affected immensely the nanostructure of des. however, ftir analysis showed that the shape of spectra was kept after addition of % water. this indicates the tolerance of des structure [ , ] . nonetheless, c-h stretching weakened upon addition of water and disappeared completely in safam. in the present study, sodium acetate:formic acid ( : molar ratio) was employed as a natural des and coded as safa to extract polyphenolic compounds, especially anthocyanins and to evaluate the antioxidant activity of roselle calyces. the major roadblock of dess which limits their applications and efficiency for phytochemicals is the high viscosity. this fact reduces consequently the mass transfer and solubility of phenolic compounds [ , , ] . to alleviate this constraint, des sodium acetate:formic acid was diluted with , , and % of water and encoded safa , safa , safa , and safa , respectively. furthermore, another sodium acetate:formic acid-based des was designed in molarity ratio : (safam), following the studies by the authors in [ ] , who have suggested the dess betaine:tartaric acid and betaine:citric acid prepared in molarity ratio. in addition, for the purposes of comparison, distilled water, % ethanol and % methanol were used after the addition of water, the stronger bonding between hbd and hba was affected and weakened with the appearance of o-h ( figure ). the absence of o-h in safa suggested that the mixture was too anhydrous. the stretching of o-h at cm − appeared progressively in dess, got higher as water content increased and became stronger in safa m (figure b-e). similar phenomena have been reported in choline chloride and oxalic acid, as well as in choline chloride and glycol-based dess, and might be due to alternative possibilities for h bonding in des upon the introduction of water [ , ] . hammond et al. ( ) have revealed that the introduction of a small quantity of water affected immensely the nanostructure of des. however, ftir analysis showed that the shape of spectra was kept after addition of % water. this indicates the tolerance of des structure [ , ] . nonetheless, c-h stretching weakened upon addition of water and disappeared completely in safa m . in the present study, sodium acetate:formic acid ( : molar ratio) was employed as a natural des and coded as safa to extract polyphenolic compounds, especially anthocyanins and to evaluate the antioxidant activity of roselle calyces. the major roadblock of dess which limits their applications and efficiency for phytochemicals is the high viscosity. this fact reduces consequently the mass transfer and solubility of phenolic compounds [ , , ] . to alleviate this constraint, des sodium acetate:formic acid was diluted with , , and % of water and encoded safa , safa , safa , and safa , respectively. furthermore, another sodium acetate:formic acid-based des was designed in molarity ratio : (safa m ), following the studies by the authors in [ ] , who have suggested the dess betaine:tartaric acid and betaine:citric acid prepared in molarity ratio. in addition, for the purposes of comparison, distilled water, % ethanol and % methanol were used as conventional solvents. roselle calyces were extracted with these solvents using ultrasound. the results of total phenolic table . globally, when compared the efficiency of the solvents used in this study, the dess were more efficient than the conventional solvents for the extraction of roselle polyphenolics, except safa and safa ( table ). the lowest results provided by safa and safa were due to their extremely viscosity ( table ). the viscosity of dess hinders the mass transfer in the extraction matrix, decreasing the extractability performance of dess [ , , , ] . the highest tpc was detected with safa m , following safa , safa , safa and safa , respectively. the tpc obtained with safa m was . , . and . % higher than the values obtained with distilled water, % ethanol and % methanol, respectively. similar results have been figured out in rosemary, where tpc obtained with acid-based dess was - % higher than ethanol [ ] . likewise, the highest tfc, tacn, dpph radical scavenging and frap were determined with safa m and safa . safa m yielded . , . and . -fold higher tfc than % ethanol, % methanol and distilled water, respectively. similarly, the tfc has been well-extracted from saffron with lactic acid-based des [ ] . however, sodium acetate:formic acid provided higher yields of tpc of roselle when compared to the yields of citric acid:glycerol ( . mg gae/g) and citric acid:ethylene glycol ( . mg gae/g) [ ] , except safa . in addition, tacn obtained with safa m was . , . and . -fold higher than % ethanol, distilled water, and % methanol, respectively. meanwhile, the values of tacn determined in this study were in accordance with the findings previously detected in roselle with citric acid:glycerol ( . mg c g/g) and citric acid:ethylene glycol ( . mg c g/g) [ ] . the high extraction yield of safa m is associated with the multiple hydrogen-bonding and low viscosity. the carboxyl group (-cooh) of formic acid presented more interactions between hydrogen-bonding [ ] . the high efficiency of acid-based dess for the extraction of polyphenolics have been proved and told to due to the h-bonding interactions among des components and the low viscosity [ ] [ ] [ ] [ ] [ ] . safa m was found to be an ideal medium for the extraction of roselle polyphenolics, allowing a high mass transfer and solubility of anthocyanins. the responses (total anthocyanin, total phenolic compound, dpph radical scavenging, frap and total flavonoid values) and the coded independent factors of experimental points were shown in table . a total of experimental points was investigated. tpc was ranged from . to . mg gae/g. the tfc values were found between . and . mg ece/g, while tacn values were ranged from . and . mg d s/g. the values of frap were found between . and . mmol ise/g, while dpph radical scavenging values were ranged from . and mmol te/g. the highest tpc, tfc, tacn, frap and dpph radical scavenging were detected at run (safa m : , % additional water and ml solvent). while, the lowest tpc and frap values were observed at run (safa m : . , % additional water and ml solvent), and the lowest dpph radical scavenging at run (safa m : . , % additional water and ml solvent). likewise, the lowest tfc and tacn values were detected in the run (safa m : . , % additional water and ml solvent) and run (safa m : . , % additional water and ml solvent). the lowest values of the responses were figured out at the runs where dess were prepared with additional water ranged between and %. at these experimental points, the viscosity was very low, indicating that the des might lose its intrinsic characteristics and become a simple aqueous solution. it has been reported that a very low viscosity induce less hydrogen-bonding and decrease the extraction yield of phytochemical compounds [ , ] . the highest values were detected in the runs where des did not require additional water. these findings revealed the importance of introduction of water in des and indicated that the optimum des will not have too low or too high viscosity. moreover, the highest responses were obtained with molarity ratio : . table . coded box-behnken design with the analytical responses. coded values analytical responses x (molarity ratio); x (additional water, %) and x (solvent ratio, ml). y (dpph radical scavenging, mmol te/g); y (tpc, mg gae/g); y (frap, mmol ise/g); y (tfc, mg ece/g) and y (tacn, mg d s/g). for the analysis of the optimization models, the regression coefficients were indicated at the least square for the second-order quadratic polynomial models. the stepwise option of response surface methodology was used to eliminate the non-significant parametric values [ ] . the reduced second-order models in terms of actual factors for the responses such as tpc, tfc, tacn, frap and dpph radical scavenging values of roselle calyces as a function of molarity ratio (x ), additional water (x ) and solvent to solid ratio (x ) were obtained as follows: these equations showed up the response patterns for individual measurement and intricacy of sceneries. the higher and positive parametric values translate the more significant the weight of the governing variable is [ ] . the results of the anova showing the effects of different parametric values on the responses along with r , adjusted r , predicted r , adequate precision and coefficient of variance (cv) were assigned in table . the model developed for tpc, which provided higher r ( . ) and adjusted r ( . ) was significant at p < . . the r and adjusted r values displayed a good closeness, indicating that there was strong conformity between the experimental findings and predicted values. the cv of . % revealed that the model was better reproducible for tpc, as cv expresses the standard deviation in percentage. the adequate precision ratio of . indicates that the model can be employed to explore the design because an adequate precision ratio higher than is desirable. the lack of fit which was non-significant indicated that the developed model was a good fit at f value . (p < . ). the predicted r of . for tpc is in reasonable agreement with the adjusted r of . ; i.e., the difference is less than . . the model developed for tfc was significant at f value . (p < . ) and had satisfactory r ( . ), adjusted r ( . ), cv ( . %) and adequate precision ( . ) . this model presented a non-significant lack of fit, conferring a good fitness to the model. furthermore, the r and adjusted r of the model developed for the tfc were very close and higher which implicated there was great conformity between the experimental and predicted values. the predicted r of . for tfc is in reasonable agreement with the adjusted r of . ; i.e., the difference is less than . . for tacn, the model was significant at f value . (p < . ) and had desirable r ( . ), adjusted r ( . ), cv ( . %) and adequate precision ( . ) . nonetheless, the predicted r of . is not as close to the adjusted r of . as one might normally expect. this may indicate a large block effect or a possible problem with the model and/or data. this model presented non-significant terms for lack of fit, indicating that the model was a good fit and adequate, since there was great conformity between the experimental and predicted values. in relation to the model developed for dpph radical scavenging, acceptable r ( . ), adjusted r ( . ), cv ( . %) and adequate precision ( . ) with significant terms at f value . (p < . ). the predicted r of . for dpph radical scavenging is in reasonable agreement with the adjusted r of . and a non-significant lack of fit approved the adequacy of the model. with respect to the model generated for frap, significant terms were found at f value . (p < . ) with higher r ( . ), adjusted r ( . ), cv ( . %) and adequate precision ( . ) . these results indicated that there was strong conformity between the experimental findings and predicted values, and the model adequate and reproducible for frap values of roselle calyces. in addition, the model generated for frap was good fit by providing a non-significant lack. these analytical findings proved that the parametric values for tpc, tfc, tacn, dpph radical scavenging and frap values by response surface methodology can be used for the prediction and optimization stages [ ] the linear, quadratic and interactions terms of the models on tpc, tfc, tacn, frap and dpph radical scavenging of roselle calyces were presented in table . the linear and quadratic terms had significant effects on tpc. however, the interaction terms of additional water and liquid to solid ratio had no significant effects on tpc with f values of . (p < . ). the parametric value having the most impactful effects on tfc was linear term of liquid to solid ratio with f value of . (p < . ), followed by the interaction terms of molarity ratio/liquid to ratio and additional water/liquid to solid ratio with f value of . (p < . ), and the quadratic terms of additional water with f value . (p < . ), and liquid to solid ratio with f value . (p < . ), respectively. all linear terms and quadratic effects on tacn. moreover, the interaction term of molarity ratio and additional water provided significant on tacn at f value . (p < . ). as shown in table , all the linear, interaction and quadratic units showed significant effects on dpph radical scavenging values of roselle calyces likewise, the linear units having the most impactful effects on frap were found to be additional water at f value . (p < . ), followed by liquid to solid ratio at f value . (p < . ), while the most impactful quadratic units were additional water f value . (p < . ), followed by molarity ratio f value . (p < . ) and liquid to solid ratio f value . (p < . ). in addition, the interaction of molarity ratio and additional water displayed significant effect on frap f value . (p < . ). the three-dimensional ( d) response surface plots of the models were used to interpret the effects of interactions between the variables on the responses (figure ). the d response surface plots showed that the tpc, tfc, tacn, frap and dpph radical scavenging values were strongly influenced by molarity ratio and additional water. from the figures, it can be observed that an increase in molarity ratio induced a rapid increase in tpc, tfc, tacn, frap and dpph radical scavenging values. this can be explained because the carboxyl group (-cooh) and sodium cation (-na + ) from sodium acetate would combine with the carboxyl group (-cooh) or the hydroxyl group (-oh) of formic acid. with the lower molarity ratio of formic acid, there are many specific groups from hba, which could not bond with the specific groups of hbd, leading to the precipitation of dess [ , ] . nonetheless, the effects of molarity ratio were moderate when compared to the effects of additional water. as demonstrated in figure , the addition of water led to a decrease in tpc, tfc, tacn, frap and dpph radical scavenging values. the highest results were recorded with % additional water. however, a slight increase in tacn was observed in - % of additional water. % additional water at the maximum response values might be explained because the dess used in the present study were prepared as molarity terms. the hydrophilic dess are greatly recommended for the isolation of bioactive compounds, facilitating their diffusion and solubility [ , ] . safa m was yet hydrophilic, implying that the further addition of water could be harmful to the des and decrease the extraction performance. the hydrophilic dess are easy to use, reduce the viscosity and preparation cost and increase polyphenolic extraction yields, nonetheless, a certain of amount of water could not be overpassed [ , , , ] . adding more water weakens the interactions between the hydrogen-bonding of different components, affecting the extraction yield [ ] . molecules , , x for peer review of the response surface methodology was performed to optimize tpc, tfc, tacn, frap and dpph radical scavenging values. the optimum conditions were : . molarity ratio, % additional water and ml solvent ratio. under these optimum conditions, the predicted tpc, tfc, tacn, frap and dpph radical scavenging values were . mg gae/g, . mg ece/g, . mg d s/g, . mmol ise/g and . mmol te/g, respectively. for confirmation, further analyses were performed under optimum conditions. the results were presented as . mg gae/g, . mg ece/g, . mg d s/g, . mmol ise/g and . mmol te/g for tpc, tfc, tacn, frap and dpph radical scavenging, respectively. the tacn of roselle calyces in safa m increased from . to . mg d s/g after the application of response surface methodology. these values were found to be higher than the total anthocyanin previously detected in roselle calyces which has been ranged between . and . mg/g [ , ] . this is due not only to the origin and variety of the plant material but mostly to the use of dess and uae for the extraction. the dess are natural and eco-friendly solvents which are prominent for the extraction of bioactive compounds including anthocyanins [ , , , , , , ] . the des (sodium acetate:formic acid) used in the present study extracted better anthocyanin from roselle calyces. furthermore, the application of uae combined with dess is well-adapted and accelerates the mass transfer of the analytes [ ] [ ] [ ] . the effects of molarity ratio, additional water and solvent ratio on the response anthocyanin was well-represented by perturbation graphic generated using response surface methodology ( figure ). as can be observed, the total anthocyanin of roselle calyces behaved differently with variables. the curvature observed indicated that tacn was sensitive to all the variables. however, the tacn was mostly affected by molarity ratio and additional water, as these variables showed great variations (figure ). the tacn of roselle calyces in safam increased from . to . mg d s/g after the application of response surface methodology. these values were found to be higher than the total anthocyanin previously detected in roselle calyces which has been ranged between . and . mg/g [ , ] . this is due not only to the origin and variety of the plant material but mostly to the use of dess and uae for the extraction. the dess are natural and eco-friendly solvents which are prominent for the extraction of bioactive compounds including anthocyanins [ , , , , , , ] . the des (sodium acetate:formic acid) used in the present study extracted better anthocyanin from roselle calyces. furthermore, the application of uae combined with dess is well-adapted and accelerates the mass transfer of the analytes [ ] [ ] [ ] . the effects of molarity ratio, additional water and solvent ratio on the response anthocyanin was well-represented by perturbation graphic generated using response surface methodology ( figure ). as can be observed, the total anthocyanin of roselle calyces behaved differently with variables. the curvature observed indicated that tacn was sensitive to all the variables. however, the tacn was mostly affected by molarity ratio and additional water, as these variables showed great variations (figure ) . the polyphenols recovered from different natural sources find applications in foods [ ] and cosmetics [ ] where stability is a critical factor for their successful implementation. the optimum experimental conditions figured out with response surface methodology were used to evaluate the heat and storage stabilities of tpc, tfc, tacn, dpph radical scavenging and frap of roselle. the polyphenols recovered from different natural sources find applications in foods [ ] and cosmetics [ ] where stability is a critical factor for their successful implementation. the optimum experimental conditions figured out with response surface methodology were used to evaluate the heat and storage stabilities of tpc, tfc, tacn, dpph radical scavenging and frap of roselle. [ ] [ ] [ ] . tacn was more stable at , and • c in safa m but decreased drastically at • c. similarly, the thermal degradation of anthocyanin in aqueous extracts of roselle has been reported to be crucial between and • c [ , ] . generally, the phytochemical compounds with antioxidant properties in roselle were found more stable in safa m . the strong thermal stability behavior of polyphenols in safa m could be associated with a strong hydrogen network between extracts and dess components [ ] . however, the thermal degradation rate increased by augmenting both temperature and heat time. the first-order reaction constant k and activation energy for isothermal kinetics of anthocyanin degradation were assigned in table . the values k figured out in this study confirmed the influence of temperature and time on anthocyanin. it was observed that k values increased with augmenting temperature from . × − at • c to . × − s − to • c for times varying between to min. other authors have also mentioned that k values increased with augmenting temperature and time [ , , [ ] [ ] [ ] . the activation energy (ea) was ranged from . to . kj/mol and was slightly less than ea values reported aqueous extracts of roselle [ , ] and in blackcurrant juice [ ] . order reaction constant k and activation energy for isothermal kinetics of anthocyanin degradation were assigned in table . the values k figured out in this study confirmed the influence of temperature and time on anthocyanin. it was observed that k values increased with augmenting temperature from . × − at °c to . × − s − to °c for times varying between to min. other authors have also mentioned that k values increased with augmenting temperature and time [ , , [ ] [ ] [ ] . the activation energy (ea) was ranged from . to . kj/mol and was slightly less than ea values reported aqueous extracts of roselle [ , ] and in blackcurrant juice [ ] . the degradation of anthocyanin in safa m was investigated during eighteen days at room temperature ( • c), • and − • c, and the results were represented in figure . the concentrations of anthocyanin decreased slowly in safa m with time at all temperatures. however, this decrease was steeper at higher temperature ( • c). the degradation rates were . , . and . % at • c, • c and − • c, respectively. the linear relation between the logarithm of total anthocyanins and time indicated a first-order kinetic for roselle anthocyanin degradation in safa m . this is in agreement with the previous investigations in aqueous extracts of roselle [ , ] . the anthocyanin degradation was too slow in safa m , especially after days, showing the strength of intramolecular and intermolecular reactions occurred between solute and safa m . nonetheless, the storage temperature had an influence on anthocyanin degradation ( table ). as can be seen, the values of k varied from . × − to . × − s − at − • c, from . × − to . × − s − at • c and from . × − to . × − s − at • c. during the storage, the values of the first-order rate constant (k) of the model increased . -fold. furthermore, the values of t / were ranged . × to . × s and the highest t / values were found at − • c and • c, respectively. k values detected in this study were low and t / values were higher when compared to k and t / values reported in previous studies [ , ] . this indicated that safa m was more anthocyanin-protective during the storage when compared to water. the degradation of anthocyanin in safam was investigated during eighteen days at room temperature ( °c), ° and − °c, and the results were represented in figure . the concentrations of anthocyanin decreased slowly in safam with time at all temperatures. however, this decrease was steeper at higher temperature ( °c). the degradation rates were . , . and . % at °c, °c and − °c, respectively. the linear relation between the logarithm of total anthocyanins and time indicated a first-order kinetic for roselle anthocyanin degradation in safam. this is in agreement with the previous investigations in aqueous extracts of roselle [ , ] . the anthocyanin degradation was too slow in safam, especially after days, showing the strength of intramolecular and intermolecular reactions occurred between solute and safam. nonetheless, the storage temperature had an influence on anthocyanin degradation ( table ). as can be seen, the values of k varied from . × − to . × − s − at − °c, from . × − to . × − s − at °c and from . × − to . × − s − at °c. during the storage, the values of the first-order rate constant (k) of the model increased . -fold. furthermore, the values of t / were ranged . × to . × s and the highest t / values were found at − °c and °c, respectively. k values detected in this study were low and t / values were higher when compared to k and t / values reported in previous studies [ , ] . this indicated that safam was more anthocyanin-protective during the storage when compared to water. roselle collected from the experimental farms of the university of agriculture of kétou, located in kétou province, benin republic. the fresh roselle calyces were sun-dried for days. the dried calyces were packed into cleaned and sterilized brown bottles and kept at • c for further use. prior analysis, the dried calyces were pulverized by a disintegrator (sinbo, coffee and spice grinder, scm ), sieved, and brown bottle at • c. distilled water purified by a millipore-q system (millipore billerica, massachusetts, usa). methanol (≥ , %), , -diphenyl- -picrylhydrazyl (dpph), , , -tris( -pyridyl)-s-triazine (tptz, ≥ . %), trolox ( %), sodium nitrite ( - . %), hydrochloric acid ( . - %), (-)-epicatechin, gallic acid (≥ . ) and sodium carbonate ( . - . %) were bought from sigma-aldrich. ethanol (≥ . %) purchased from isolab. folin-ciocalteu reagent, aluminum chloride, iron (iii) chloride, iron sulfate heptahydrate (≥ . %) and formic acid ( - %) brought from merck. sodium acetate anhydrous (≥ . %), glacial acetic acid ( . %), potassium chloride (≥ . %), and sodium hydroxide (≥ . %) obtained from carlo erba. the deep eutectic solvents (dess) were prepared based on previously reported methodologies with some changes [ , ] . the dess made in a molar ratio of two different components consisting of hydrogen bond donor (hbd) and hydrogen bond acceptor (hba), followed by the addition of , , , and % of distilled water (table ). sodium acetate used as hba [ ] and formic acid as hbd. another des prepared in the molarity ratio of sodium acetate and formic acid ( : ). the molarity ratio-based des formulated due to the fact that the anhydrous mixtures are very viscous, difficult to manipulate [ ] , and unsuitable for polyphenolics extraction. the molar ratio mixture prepared by mixing the molar mass of components as gram in appropriate ratios. the molarity ratio mixture prepared by mixing ingredients as molarity in the proper ratios. in this work, des components were placed in reaction flask at • c with constant stirring for h min to obtain a homogeneous liquid. the dess prepared in molar and molarity ratios were encoded as safa , and safa m , respectively. the viscosity of dess was measured using a rheometer (buchi, ch- flawil , switzerland) fitted with a parallel geometry with mm of diameter and gap mm. the measurements carried out as described in [ ] . ftir analysis of dess and extracts was carried out using a ftir spectrometer (perkin elmer, spectrum-two, usa, peservice ). diamond lens attenuated resistance was used. the spectrometer was adjusted in resolution and by selecting the norton-beer (n-b) strong apodization function. the range of all spectra was between the wavenumbers of and cm − . prior to every spectrum, a background reference was taken using an empty cell to ensure no interferences. then, spectrum intensity was transformed into relative transmittance, %t. ultrasound-assisted extractions (uae) performed in a sonication water bath (wuc-a h, daihan scientific co., ltd. seoul, korea). distilled water, % ethanol, and methanol were used as conventional solvents as they have exhibited high extraction performance of anthocyanins from roselle [ , , , ] . . g of comminuted roselle calyces added with ml of conventional solvents and dess. the mixture was ultrasonicated in an ultrasonic bath at room temperature ( • c) for min. the samples were left to cool and then filtered. the polyphenolic yield is affected by different operational factors such as temperature, time, liquid-solid ratio, the molar ratio of des, speed of agitator and particle size. herein, only three of them examined: liquid-to-solid ratio, molarity ratio, and additional water content. the des that provides the highest yield of anthocyanin was selected for the optimization process. the optimization parameters of the des examined systematically using response surface methodology based on the three-level box-behnken design (design expert software . ). the experimental design carried out with three independent variables of x (molarity ratio), x (additional water content), and x (solvent to solid ratio). the actual and coded values of the independent variables presented in table . the combinations of the molarity ratio of sodium acetate ( ) to formic acid ( , . and ), additional water ( %, % and %) and solvent to solid ratio ( : . , : . and : . ml/g) were independent variables chosen for uae. these variables regrouped in experimental points, including five replicates at the central point. total anthocyanin, total phenolic content, frap, dpph radical scavenging activity, and total flavonoid values investigated as the responses (y) for the design experiment. the experimental points, together with responses, were displayed in table . the experimental data fitted to the following quadratic polynomial model: the tpc of roselle extracts was determined using the folin-ciocalteu method [ , ] . a uv-spectrophotometer (thermo spectronic) used to read absorbance at nm, and the tpc in each extract was calculated from a calibration curve (y = . x − . ; r = . ), using gallic acid as a standard. the results were given as mg gallic acid equivalent (gae) g − dw. the total flavonoid was determined using a modified protocol [ , , ] . the absorbance was measured at nm after min in the dark at room temperature. the tfc was calculated from a calibration curve using epicatechin as standard (y = . x + . ; r = . ). the results estimated as mg epicatechin equivalents (ece) g − dw. a modified ph differential method [ ] was employed to quantify the total anthocyanin of roselle calyces. briefly, . ml aliquot of the extract diluted with . ml of buffer (consisting of . g of kcl, ml of distilled water and . ml hcl), ph . and another . ml aliquot of the extract diluted with . ml of buffer, (consisting of . g of sodium acetate, ml distilled water and ml hcl) solution ph . . the buffer solutions completed up to l with distilled water. afterward, the absorbance measured at nm, and the total anthocyanin content was calculated in function of mg delphinidin- -sambubioside equivalent (d s e) g − with the following equation, where a = (absorbance nm − absorbance nm ) ph . − (absorbance nm − absorbance nm ) ph . ; m w (molecular weight) = g·mol − for d s, df = dilution factor established in d, l = pathlength in cm and ε = , molar extinction coefficient, in l × mol − × cm − , for delphinidin- -sambubioside. frap assay performed according to the procedure of [ , ] . the frap values of the extracts were calculated from the calibration curve (y = . − . ; r = . ) using feso as a standard. the results were given as mmol feso equivalents (mmol ise g − dw) [ ] . dpph radical scavenging activity determined according to previous studies [ , , ]. an aliquot of µl of the sample added with ml dpph solution ( . mm in % methanol). the absorbance at nm was recorded. the dpph solution used as control. the reduction ratio of dpph determined with the following equation, where ac = absorbance of control and as = absorbance of extract. the dpph radical scavenging activity in each extract was calculated from a calibration curve (y = . x − . ; r = . ) considering the reduction ratio as y and using trolox as a standard. the results were given as mmol trolox equivalent (te) g − dw [ ] . for the thermal stability, ml of roselle extracts were put in the brown bottles with screw caps and placed in a preheated water bath at , , and • c. three bottles of each group were removed from the water bath after , , , and min and cooled to room temperature. to evaluate the kinetic modeling of tpc, tfc, tacn, dpph radical scavenging and frap values of des extracts of roselle, the first-order reaction rate constant (k) calculated [ , ] , where k is the constant rate (s − ), c is the initial concentration and c t is the concentration after the heating time (t) at a given temperature. the kinetics of thermal degradation of anthocyanin of roselle was evaluated with parameter activation energy (ea) which was determined as described in [ ] , where k is the constant rate (s − ), k ref is the constant rate (s − ) of a reference temperature t ref (k) . • c considered as reference temperature in the present study. ea is the activation energy (j mol − ) and r is the universal gas constant ( . j mol − k − ). the effect of storage time was investigated at − • c, • c, and ambient conditions in the dark. ml of roselle extracts were put in brown bottles and placed in the dark at − • c, • c and ambient temperature ( • c). three bottles of each group were removed and analyzed at days , , , and . the first-order rate constant (k) and half-life time (t / ) used to determine the kinetic modeling of anthocyanin degradation during the storage [ , ] : all studies were performed in triplicates and the mean values were determined. the software design expert . (trial version, stat-ease inc., mineapolis, mn usa) was used to design the experimentation along with data analysis. anova was used to determine the statistical relationship between factors. the adequacy of the models obtained was ascertained by screening the r , adjusted r , coefficient of variation (cv) and the value of fisher's test (f-value). the significance of the models and regression coefficients were measured at p < . . the relationship between independent variables and responses was checked by d graphics. the optimum conditions were determined according to the desirability function. one-way statistical analyses were carried out by anova with post-hoc duncan's test using spss (version ). the significance of the results was assessed at p ≤ . . the analysis of the effect of water addition revealed that the viscosity and des nanostructure decreased upon water addition. however, the extraction of phytochemical compounds and the antioxidant properties of roselle increased considerably after the water introduction. safa m with : molarity ratio was revealed to be the most efficient for the extraction of anthocyanins and polyphenol antioxidants from roselle calyces when compared to safa , safa , safa , safa , safa and conventional solvents. subsequently, this des was selected for the optimization using a box-behnken design paired with response surface methodology to determine the optimum point for the extraction of maximum anthocyanins from roselle. the optimum point was determined as : . molarity ratio, % additional water and ml solvent ratio. under these optimum conditions, tpc, tfc, tacn, frap and dpph radical scavenging were found to be . mg gae/g, . mg ece/g, . mg d s/g, . mmol ise/g, and . mmol te/g, respectively. the thermal and storage tests performed on roselle extracts showed that the phytochemical compounds mainly anthocyanins were more stable in safa m . this novel des represented a green and sustainable solvent for the extraction of bioactive compounds. the authors declare that there is no conflict of interest. phytochemical, pharmacological and toxicological aspects of hibiscus sabdariffa l.: a review pharmacological evidences for the extracts and secondary metabolites from plants of the genus hibiscus hibiscus tea (hibiscus sabdariffa l.) lowers blood pressure in pre-and mildly hypertensive adults antioxidant power quantification of decoction and cold infusions of hibiscus sabdariffa flowers hepatoprotective effects of hibiscus, rosmarinus and salvia on azathioprine-induced toxicity in rats antioxidant activity in different parts of roselle (hibiscus sabdariffa l.) extracts and potential exploitation of the seeds elucidation of key odorants in beninese roselle (hibiscus sabdariffa l.) infusions prepared by hot and cold brewing stability of hibiscus extract encapsulated by ionic gelation incorporated in yogurt the food systems in the era of the coronavirus (covid- ) pandemic crisis. foods phytochemistry, antioxidant capacity, total phenolic content and anti-inflammatory activity of hibiscus sabdariffa leaves identification of phenolic compounds in hibiscus sabdariffa polyphenolic rich extract (hpe) by chromatography techniques fernández-gutiérrez, a. selective extraction, separation, and identification of anthocyanins from hibiscus sabdariffa l. using solid phase extraction-capillary electrophoresis-mass spectrometry (time-of-flight/ion trap) isolation and characterization of anthocyanins from hibiscus sabdariffa flowers anthocyanins degradation during storage of hibiscus sabdariffa extract and evolution of its degradation products bioaccessibility of polyphenols released and associated to dietary fibre in calyces and decoction residues of roselle (hibiscus sabdariffa l.) anthocyanin-rich red dye of hibiscus sabdariffa calyx modulates cisplatin-induced nephrotoxicity and oxidative stress in rats anthocyanin-rich extract from hibiscus sabdariffa calyx counteracts uvc-caused impairments in rats recovery of high added-value components from food wastes: conventional, emerging technologies and commercialized applications emerging technologies for the production of nutraceuticals from agricultural by-products: a viewpoint of opportunities and challenges separation of functional macromolecules and micromolecules: from ultrafiltration to the border of nanofiltration techniques for extraction of bioactive compounds from plant materials: a review potential use of pulsed electric technologies and ultrasounds to improve the recovery of high-added value compounds from blackberries pressurized hot water extraction (phwe) for the green recovery of bioactive compounds and steviol glycosides from stevia rebaudiana bertoni leaves green extraction of natural products: concept and principles green extraction of polyphenols from grapefruit peels using high voltage electrical discharges, deep eutectic solvents and aqueous glycerol extraction of phytochemicals using hydrotropic solvents a facile water-induced complexation of lycopene and pectin from pink guava byproduct: extraction, characterization and kinetic studies characterization of xylitol or citric acid:choline chloride:water mixtures: structure, thermophysical properties, and quercetin solubility insights into the nature of eutectic and deep eutectic mixtures application of natural deep eutectic solvents for extraction and determination of phenolics in cajanus cajan leaves by ultra performance liquid chromatography extraction techniques with deep eutectic solvents an eco-friendly hydrophobic deep eutectic solvent-based dispersive liquid-liquid microextraction for the determination of neonicotinoid insecticide residues in water, soil and egg yolk samples enhanced-performance extraction of olive (olea europaea) leaf polyphenols using l-lactic acid/ammonium acetate deep eutectic solvent combined with β-cyclodextrin: screening, optimisation, temperature effects and stability deep eutectic solvents as extraction media for valuable flavonoids from natural sources effect of choline chloride-oxalic acid based deep eutectic solvent on the ultrasonic assisted extraction of polyphenols from aegle marmelos tailoring properties of natural deep eutectic solvents with water to facilitate their applications green tailoring with water of choline chloride deep eutectic solvents for the extraction of polyphenols from palm samples new low viscous hydrophobic deep eutectic solvents in vortex-assisted liquid-liquid microextraction for the determination of phthalate esters from food-contacted plastics the role of water in deep eutectic solvent-base extraction effect of water addition on choline chloride/glycol deep eutectic solvents: characterization of their structural and physicochemical properties extraction of polyphenolic antioxidants from orange peel waste using deep eutectic solvents deep eutectic solvents applied in the extraction and stabilization of rosemary (rosmarinus officinalis l.) phenolic compounds detoxification of olive mill wastewaters by liquid-liquid extraction with natural deep eutectic solvents highly efficient extraction of antioxidant polyphenols from olea europaea leaves using an eco-friendly glycerol/glycine deep eutectic solvent microwave-assisted extraction for recovery of polyphenolic antioxidants from ripe mango (mangifera indica l.) peel using lactic acid/sodium acetate deep eutectic mixtures thin-layer drying behaviour of vegetable wastes from wholesale market. dry optimization of heatand ultrasound-assisted extraction of anthocyanins from hibiscus sabdariffa calyces for natural food colorants effect of fixed bed drying on the retention of phenolic compounds, anthocyanins and antioxidant activity of roselle (hibiscus sabdariffa l.) citric acid-based deep eutectic solvent for the anthocyanin recovery from hibiscus sabdariffa through microwave-assisted extraction insights into the synthesis and properties of deep eutectic solvents based on cholinium chloride and carboxylic acids optimization and stabilization of the antioxidant properties from alkanet (alkanna tinctoria) with natural deep eutectic solvents the effect of water upon deep eutectic solvent nanostructure: an unusual transition from ionic mixture to aqueous solution. angew. chemie int natural deep eutectic solvents as new potential media for green technology application of natural deep eutectic solvents to the extraction of anthocyanins from catharanthus roseus with high extractability and stability replacing conventional organic solvents natural deep eutectic solvents from choline chloride and betainephysicochemical properties radojcic redovnikovic, i. green extraction of grape skin phenolics by using deep eutectic solvents saffron processing wastes as a bioresource of high-value added compounds: development of a green extraction process for polyphenol recovery using a natural deep eutectic solvent natural deep eutectic solvents providing enhanced stability of natural colorants from safflower (carthamus tinctorius) enhanced extraction of natural pigments from curcuma longa l. using natural deep eutectic solvents polyol-based deep eutectic solvents for extraction of natural polyphenolic antioxidants from chlorella vulgaris enhanced phenolic compounds extraction from morus alba l. leaves by deep eutectic solvents combined with ultrasonic-assisted extraction tailor-made natural deep eutectic solvents for green extraction of isoflavones from chickpea (cicer arietinum l.) sprouts application of response surface methodology for optimizing the recovery of phenolic compounds from hazelnut skin using different extraction methods enhanced extraction of phenolic compounds using choline chloride based deep eutectic solvents from juglans regia l natural deep eutectic solvents as alternatives for extracting phlorotannins from brown algae high selective purification of quercetin from peanut hull using protic deep eutectic mixture based liquid-liquid microextraction eco-friendly ultrasonic assisted liquid-liquid microextraction method based on hydrophobic deep eutectic solvent for the determination of sulfonamides in fruit juices optimization deep eutectic solvent-based ultrasound-assisted liquid-liquid microextraction by using the desirability function approach for extraction and preconcentration of organophosphorus pesticides from fruit ultrasound-assisted deep eutectic solvent as green and e fficient media combined with functionalized magnetic multi-walled carbon nanotubes as solid-phase extraction to determine pesticide residues in food products phenols recovered from olive mill wastewater as additives in meat products implementation of phenols recovered from olive mill wastewater as uv booster in cosmetics the kinetics of thermal degradation of polyphenolic compounds from elderberry (sambucus nigra l.) extract investigations on thermal degradation of phytochemicals from lavender extract. fascicle vi-food technol thermal stability of phytochemicals, hmf and antioxidant activity in cape gooseberry (physalis peruviana l.) aqueous extraction of anthocyanins from hibiscus sabdariffa: experimental kinetics and modeling thermal degradation kinetics of anthocyanins from blood orange, blackberry, and roselle using the arrhenius, eyring, and ball models thermal degradation kinetics of anthocyanin and visual colour of plum puree determination of the degradation kinetics of anthocyanins in a model juice system using isothermal and non-isothermal methods deep eutectic solvent-based extraction of polyphenolic antioxidants from onion (allium cepa l.) peel chemical and phytochemical properties and antioxidant activities of three pomegranate cultivars grown in south africa antioxidant properties of blackberry and blueberry fruits grown in the black sea region of turkey ultrasound-negative pressure cavitation extraction of phenolic compounds from blueberry leaves and evaluation of its dpph radical scavenging activity physico-chemical and antioxidant properties of cornelian cherry fruits (cornus mas l.) grown in turkey green synthesis of silver nanoparticles by bloom forming marine microalgae trichodesmium erythraeum and its applications in antioxidant, drug-resistant bacteria, and cytotoxicity activity kinetic modeling of anthocyanin extraction from grape marc this article is an open access article distributed under the terms and conditions of the creative commons attribution key: cord- -ljmkqdzx authors: parang, keykavous; el-sayed, naglaa salem; kazeminy, assad j.; tiwari, rakesh k. title: comparative antiviral activity of remdesivir and anti-hiv nucleoside analogs against human coronavirus e (hcov- e) date: - - journal: molecules doi: . /molecules sha: doc_id: cord_uid: ljmkqdzx remdesivir is a nucleotide prodrug that is currently undergoing extensive clinical trials for the treatment of covid- . the prodrug is metabolized to its active triphosphate form and interferes with the action of rna-dependent rna polymerase of sars-cov- . herein, we report the antiviral activity of remdesivir against human coronavirus e (hcov- e) compared to known anti-hiv agents. these agents included tenofovir (tfv), ′-ethynyl- -fluoro- ′-deoxyadenosine (efda), alovudine (flt), lamivudine ( tc), and emtricitabine (ftc), known as nucleoside reverse-transcriptase inhibitors (nrtis), and a number of ′-o-fatty acylated anti-hiv nucleoside conjugates. the anti-hiv nucleosides interfere with hiv rna-dependent dna polymerase and/or act as chain terminators. normal human fibroblast lung cells (mrc- ) were used to determine the cytotoxicity of the compounds. the study revealed that remdesivir exhibited an ec( ) value of . µm against hcov- e with tc( ) of > . µm against mrc- cells. parent nrtis were found to be inactive against (hcov- e) at tested concentrations. among all the nrtis and ′-o-fatty acyl conjugates of nrtis, ′-o-tetradecanoyl ester conjugate of ftc showed modest activity with ec( ) and tc( ) values of . µm and . µm, respectively. these data can be used for the design of potential compounds against other coronaviruses. human coronavirus e (hcov- e) is one of the seven known human coronaviruses, which include hcov-nl , hcov-oc , hcov-hku , mers-cov, sars-cov- , and sars-cov- . hcov- e is a member of the genus alphacoronavirus, which infects humans and bats [ ] . four of the human coronaviruses (hcov- e, hcov-hku , hcov-oc , and hcov-nl ) are associated with lower respiratory tract infections, including bronchiolitis and pneumonia [ , ] or upper respiratory tract infections characterized by rhinorrhea, nasal congestion, sore throat, sneezing, and cough that also may be associated with acute otitis media or asthma exacerbations [ ] . infection with hcov- e alone is most frequently associated with asymptomatic or mild disease and sometimes acute respiratory distress syndrome (ards) [ ] . the infection is also detected with other respiratory viruses, particularly with the human respiratory syncytial virus (hrsv) [ ] . these hcov related infections may also be molecules , , of accompanied by asthma exacerbations or acute otitis media. thus, exposure to hcov- e is low-risk for healthy adults. the hcov- e virus is easily accessible for the biosafety level (bsl ) laboratory. therefore, hcov- e may be a good initial model for the evaluation of antiviral compounds that could have potential applications against other coronaviruses, such as sars-cov- , the coronavirus that causes covid- . although the final selected compounds are still required to be evaluated against other coronaviruses to confirm their activity. as of may , , according to the johns hopkins coronavirus resource center, the human mortality of the covid- pandemic infection is , people, while , , people have been infected globally. currently, there are no approved drugs, monoclonal antibodies, or vaccines to treat or prevent human infections caused by sars-cov- . the discovery and approval of new compounds take several years. therefore, several existing drug and potential drug candidates such as remdesivir and other antiviral agents have been considered to be repurposed as covid- treatments. remdesivir (gs- , figure ) was developed by gilead and found to be effective against severe acute respiratory syndrome (sars) and middle east respiratory syndrome (mers) in animal models [ , ] . remdesivir was also evaluated by gilead for sars-cov- in early . it was then used by chinese medical researchers in patients for clinical testing in late january , suggesting a favorable inhibitory effect on sars-cov- (unpublished results). since then, several clinical trials of remdesivir have been initiated by china and the world health organization (who). furthermore, randomized clinical trials are ongoing or planned to determine the effect on improvements in patient recovery [ ] . remdesivir is still considered one of the most promising drug candidates for the treatment of covid- . take several years. therefore, several existing drug and potential drug candidates such as remdesivir and other antiviral agents have been considered to be repurposed as covid- treatments. remdesivir (gs- , figure ) was developed by gilead and found to be effective against severe acute respiratory syndrome (sars) and middle east respiratory syndrome (mers) in animal models [ , ] . remdesivir was also evaluated by gilead for sars-cov- in early . it was then used by chinese medical researchers in patients for clinical testing in late january , suggesting a favorable inhibitory effect on sars-cov- (unpublished results). since then, several clinical trials of remdesivir have been initiated by china and the world health organization (who). furthermore, randomized clinical trials are ongoing or planned to determine the effect on improvements in patient recovery [ ] . remdesivir is still considered one of the most promising drug candidates for the treatment of covid- . on may , , the us food and drug administration (fda stated that the potential benefits of remdesivir outweigh its known and potential risks for some patients with severe covid- , since a nih study demonstrated better recovery times than with placebo [ ] . the fda issued an emergency use authorization for remdesivir for the treatment of suspected or laboratory-confirmed covid- patients with severe disease. remdesivir is a phosphoramidite prodrug of an adenine c-nucleoside; it has a short plasma half-life ( . h) and is used in the iv dosage form. the compound is a broad-spectrum antiviral nucleotide prodrug that is metabolized first to the active triphosphate analog that inhibits rnadependent rna polymerase (rdrp), resulting in diminished viral rna replication [ ] . there is a sequence identity among the rdrp of coronaviruses. for example, the rdrp of sars-cov and sars-cov- share % sequence identity. therefore, drugs targeting viral rdrp proteins of sars-cov are likely to be effective for sars-cov- [ ] . remdesivir has been found to have 'broad-spectrum' anticoronavirus activity due to its potency against previously reported coronaviruses, such as mers-cov, hcov-nl , hcovoc , and hcov- e [ , , ] . therefore, we assumed that compounds that are active against hcov- e through interfering with rdrp could also exhibit promising antiviral activity against other coronaviruses such as sars-cov- . on may , , the us food and drug administration (fda stated that the potential benefits of remdesivir outweigh its known and potential risks for some patients with severe covid- , since a nih study demonstrated better recovery times than with placebo [ ] . the fda issued an emergency use authorization for remdesivir for the treatment of suspected or laboratory-confirmed covid- patients with severe disease. remdesivir is a phosphoramidite prodrug of an adenine c-nucleoside; it has a short plasma half-life ( . h) and is used in the iv dosage form. the compound is a broad-spectrum antiviral nucleotide prodrug that is metabolized first to the active triphosphate analog that inhibits rna-dependent rna polymerase (rdrp), resulting in diminished viral rna replication [ ] . there is a sequence identity among the rdrp of coronaviruses. for example, the rdrp of sars-cov and sars-cov- share % sequence identity. therefore, drugs targeting viral rdrp proteins of sars-cov are likely to be effective for sars-cov- [ ] . remdesivir has been found to have 'broad-spectrum' anti-coronavirus activity due to its potency against previously reported coronaviruses, such as mers-cov, hcov-nl , hcovoc , and hcov- e [ , , ] . therefore, we assumed that compounds that are active against hcov- e through interfering with rdrp could also exhibit promising antiviral activity against other coronaviruses such as sars-cov- . reverse transcriptase is an enzyme in the human immunodeficiency virus (hiv) and many retroviruses that convert the rna template to dna. the enzyme has three enzymatic functions at the same time: ( ) rna-dependent dna polymerase (rdrp), ( ) rnase h, and ( ) dna-dependent dna polymerase. the rna-dependent dna polymerase is used to synthesize a complementary dna strand to the rna template. after the removal of the rna strand from the rna-dna hybrid double helix by rnase h, the dna-dependent dna polymerase completes double-stranded dna synthesis [ ] . several nucleoside reverse transcriptase inhibitors (nrtis) have been shown to be anti-hiv agents. here, we selected five nrtis to be evaluated versue remdesivir against hcov- e. tenofovir (tfv, ) ( figure ) is a nucleotide analog of deoxyadenosine monophosphate, with activity against hiv- , hiv- , and hbv [ , ] . efda ( -ethynyl- -fluoro- -deoxyadenosine, ) is a highly promising potent nucleoside reverse transcriptase inhibitor (nrti) [ , ] . currently, efda is in clinical development by merck. -fluoro- -deoxythymidine (flt, alovudine, ) and its fatty acyl ester conjugates have been used as anti-hiv agents [ ] [ ] [ ] . , -dideoxy- -thiacytidine (lamivudine, tc, ) [ ] and , -dideoxy- -fluoro- -thiacytidine (emtricitabine, ftc, ) [ , ] are commercially available anti-hiv agents. we have previously shown that the conjugation of certain fatty acids to the anti-hiv nrtis, such as flt, tc and ftc, enhanced activity against x , r , cell-associated, and/or multi-drug resistant virus when compared with their parent nucleosides [ ] [ ] [ ] [ ] . thus, the antiviral activity of remdesivir against hcov- e was compared with selected nrtis and selected fatty acyl conjugates. the goal was to determine the antiviral activity and toxicity against hcov- e in an anti-coronavirus cytoprotection assay. the method could be used to select potential compounds rationally for evaluation against sars-cov- . nine compounds were evaluated against hcov- e in normal human fibroblast lung cells (mrc- ) at six test concentrations. the compounds included five nrtis (tfv ( ), efda ( ), flt ( ), tc ( ), and ftc ( )) and three fatty acyl conjugates of flt ( ), tc ( ), and ftc ( ) . we have previously reported the synthesis and evaluation of fatty acyl conjugates [ , , [ ] [ ] [ ] [ ] . fatty acylation of the parent nucleoside generated enhanced activity against cell-free, cell-associated, and/or multi-drug resistant virus. the antiviral efficacy and cellular toxicity data are summarized in table these data indicate that remdesivir acts as an antiviral agent against hcov- e, while anti-nrtis agents were found to be ineffective. this could be due to the unique interaction of remdesivir with rna-dependent rna polymerase in coronaviruses such as hcov- e, while nrtis inhibit reverse transcriptase. this enzyme has rna-dependent dna polymerase function. nrtis also act as dna synthesis chain terminators. the mode of interaction of remdesivir with rna polymerase and the crystal structure of protein-nucleotide have not been published yet. the structure of remdesivir is unique as a nucleotide prodrug, with the presence of a nitrile group at the position and both and -hydroxyl groups, leading to strong binding to rna polymerases that differentiates this compound from the other nucleoside analogs represented here. the structure of rna-dependent rna polymerase of sars-cov- was recently published [ ] . further structural modification of anti-hiv nucleosides could incorporate some functional groups for binding to rna polymerases, and be used for more rationale-based antiviral drug design against coronaviruses. furthermore, the determination of the crystal structure of remdesivir in terms of its binding with rdrp will provide insights into understanding the critical functional groups for the binding and design of the next generation of nucleoside-based inhibitors with higher binding affinities. a series of anti-hiv nucleosides and their fatty acyl derivatives were compared with remdesivir for antiviral activity against hcov- e in mrc- cells. among all the compounds, remdesivir was found to be potent, with an ec value of . µm and a therapeutic index of more than . µm. the -o-(tetradecanoul) ester derivative of ftc showed modest activity, with an ec value of µm. in general, nrtis did not show comparable activity against hcov- e, compared to remdesivir. this work advances scientific knowledge in the area of the testing of antiviral compounds and the activity of anti-hiv drugs against coronaviruses. this information could also be used to design compounds that are potentially effective against other coronaviruses, such as sars-cov- . the anti-hiv nucleosides were purchased from euro asia trans continental (bombay, india). the synthesis and evaluation of fatty acyl conjugates were conducted according to the previously reported procedures in our laboratory [ , , [ ] [ ] [ ] [ ] . the compounds were solubilized at mm in % dmso immediately before assay set up. the test materials were evaluated using a high test concentration of µm and five serial half-logarithmic dilutions in triplicate for the antiviral assay. the compounds were diluted to µm ( µl of mm stock) in a drug dilution tube containing µl of assay medium. three hundred twenty microliters ( µl) of the µm solution was transferred to µl of assay medium (half-log dilution) for a total of five serial dilutions. one hundred microliters of each concentration were added in triplicate wells for efficacy, duplicate wells for cytotoxicity, and a single well for colorimetric evaluation. remdesivir was purchased from medchem express (monmouth junction, nj) and evaluated as a positive control compound in the antiviral assay. the viral assay protocols were approved by the institutional biosafety committee (ibc) at imquest biosciences. mrc- cells were obtained from atcc (ccl- ) and passaged in the dmem medium supplemented with fbs ( %), penicillin ( u/ml), sodium pyruvate ( mm), l-glutamine ( mm), streptomycin ( µg/ml), and neaa ( . mm) using t- flasks before use in the antiviral assay. preceding the assay, the cells were divided into : to make sure they were in an exponential growth coronavirus e (hcov- e) was obtained from atcc (vr- ) and grown in mrc- cells (atcc# ccl- ) for the production of a stock virus pool. a pretitered aliquot of the virus was removed from the freezer (− • c). the aliquot was allowed to thaw slowly to room temperature in a biological safety cabinet. the virus was resuspended and diluted into assay medium (dmem supplemented with % heat-inactivated fbs, penicillin ( u/ml), l-glutamine ( mm), streptomycin ( µg/ml)), in such a way that µl of virus was added to each well. this amount was determined to result in to % cell death at days postinfection. each plate contained virus control wells (cells plus virus), triplicate cell control wells (cells only), drug toxicity wells according to the compound (cells plus drug only), as well as triplicate experimental wells (drug plus cells plus virus). a cell viability of more than % for the cells was utilized in the assay. the cells were resuspended at × cells per well in the tissue culture medium. then, the cells (a volume of µl) were added to flat-bottom microtiter plates. the microtiter plates were incubated at • c/ % co overnight to allow cell adherence to occur. after incubation of the test compounds at • c in a % co incubator for six days, the plates were stained with the tetrazolium dye , -bis( -methoxy- nitro- -sulfophenyl)- -[(phenylamino)carbonyl]- h-tetrazolium hydroxide (xtt). xtt tetrazolium is known to be metabolized by the mitochondrial enzymes of metabolically active cells into a soluble formazan product. this reaction makes it possible to promptly perform quantitative analyses of the inhibition of virus-induced cell killing by antiviral test substances. xtt was prepared daily as a stock solution of mg/ml in rpml . phenazine methosulfate (pms) solution was prepared in pbs ( . mg/ml) and stored in the dark at − • c. xtt/pms stock was prepared immediately before each use by adding a volume of µl of pms per ml of xtt solution. xtt/pms ( µl) was added to each well of the plate, and the plate was reincubated for h at • c. plates were sealed with adhesive plate sealers. then, they were shaken gently or inverted several times to mix the soluble formazan product. the plates were read spectrophotometrically at / nm with a molecular devices vmax plate reader (molecular devices, llc., san jose, ca, usa). the raw data were collected from softmax pro (version , molecular devices, llc, san jose, ca, usa) and imported into a microsoft excel xlfit (version , from idbs lab, boston, ma, united states) spreadsheet for analysis using four-parameter curve fit calculations. the antiviral activity and toxicity with graphic representation of the data were provided in a plate analysis report (par) summarizing the individual compound activity and a summary table of calculated tc , ec , and tl values. the graphical presentation shows the percentages of cell viability and of reduced viral viral cytopathic effect (cpe) at each test concentration. human coronaviruses: a review of virus-host interactions coronavirus e-related pneumonia in immunocompromised patients clinical and molecular epidemiological features of coronavirus hku -associated community-acquired pneumonia human coronaviruses a rare case of human coronavirus e associated with acute respiratory distress syndrome in a healthy adult prophylactic and therapeutic remdesivir (gs- ) treatment in the rhesus macaque model of mers-cov infection broad-spectrum antiviral gs- inhibits both epidemic and zoonotic coronaviruses compassionate use of remdesivir for patients with severe covid- coronavirus susceptibility to the antiviral remdesivir (gs- ) is mediated by the viral polymerase and the proofreading exoribonuclease learning from the past: possible urgent prevention and treatment options for severe acute respiratory infections caused by -ncov arguments in favour of remdesivir for treating sars-cov- infections broad spectrum antiviral remdesivir inhibits human endemic and zoonotic deltacoronaviruses with a highly divergent rna dependent rna polymerase rna-dependent dna polymerases anti-hiv activity of adefovir (pmea) and pmpa in combination with antiretroviral compounds: in vitro analyses anti-human immunodeficiency virus activity and cellular metabolism of a potential prodrug of the acyclic nucleoside phosphonate -r-( -phosphonomethoxypropyl)adenine (pmpa) -deoxy- -c-ethynyl- -fluoroadenosine: a nucleoside reverse transcriptase inhibitor with highly potent activity against wide spectrum of hiv- strains, favorable toxic profiles, and stability in plasma concise synthesis of the anti-hiv nucleoside efda -substituted , -dideoxynucleoside analogues as potential anti-hiv (htlv-iii/lav) agents synthesis, in vitro anti-hiv activity, and biological stability of -o-myristoyl analogue derivatives of -fluoro- , -dideoxythymidine (flt) as potential bifunctional prodrugs of flt in vitro anti-hepatitis b virus activities of "-o-myristoyl analogue derivatives of "-fluoro- ", "-dideoxythymidine (flt) and "-azido- ", "-dideoxythymidine (azt) treatment of chronic hepatitis b in hiv co-infected patients emtricitabine, a new antiretroviral agent with activity against hiv and hepatitis b virus emtricitabine (ftc) for the treatment of hiv infection synthesis and anti-hiv activities of phosphate triester derivatives of -fluoro- , -dideoxythymidine and -azido- , -dideoxythymidine synthesis and biological evaluation of fatty acyl ester derivatives of , -didehydro- , -dideoxythymidine synthesis and biological evaluation of fatty acyl ester derivatives of (-)- , -dideoxy- -thiacytidine emtricitabine prodrugs with improved anti-hiv activity and cellular uptake structure of the rna-dependent rna polymerase from covid- virus we acknowledge imquest biosciences for assistance in conducting the antiviral assays. the authors declare no conflict of interest.molecules , , key: cord- -gu cact authors: li, miao; liu, qin; cui, yajuan; li, dong; wang, hexiang; ng, tzi bun title: isolation and characterization of a phaseolus vulgaris trypsin inhibitor with antiproliferative activity on leukemia and lymphoma cells date: - - journal: molecules doi: . /molecules sha: doc_id: cord_uid: gu cact a . -kda trypsin inhibitor was purified from phaseolus vulgaris cv. “gold bean” with an isolation protocol including ion exchange chromatography on deae-cellulose (diethylaminoethyl-cellulose), affinity chromatography on affi-gel blue gel, ion exchange chromatography on sp-sepharose (sulfopropyl-sepharose), and gel filtration by fplc (fast protein liquid chromatography) on superdex . it dose-dependently inhibited trypsin with an ic( ) value of . μm, and this activity was reduced in the presence of dithiothreitol in a dose- and time-dependent manner, signifying the importance of the disulfide linkage to the activity. it inhibited [methyl-( )h] thymidine incorporation by leukemia l cells and lymphoma mbl cells with an ic( ) value of . μm and . μm, respectively. the inhibitor had no effect on fungal growth and the activities of various viral enzymes when tested up to μm. protease inhibitors have been isolated from the seeds of different monocots and dicots, including maize [ ] , wheat [ ] , wampee [ ] , bitter gourd [ ] , momordica cochinchinensis [ ] , and legumes [ ] [ ] [ ] . some of them display a variety of biological activities including antifungal [ ] , immunomodulatory [ ] and antitumor/antiproliferative [ , ] activities. thus, they have drawn the attention of many researchers. one class of protease inhibitors is trypsin inhibitors. trypsin inhibitors are divided into kunitz type [ ] , bowman-birk type [ , ] , and squash type [ ] . the three types have a molecular mass of about kda, kda, and kda, respectively. the soybean produces both kunitz and bowman-birk inhibitors [ ] , while melons belonging to family cucurbitaceal produce squash-type inhibitors [ ] . leguminous seeds are a rich source of proteins, including protease inhibitors, lectins [ ] , antifungal proteins [ ] , ribosome inactivating proteins [ ] , and arcelins [ ] . some of the proteins have interesting biological activities, such as insecticidal [ ] , immunomodulatory [ ] , antifungal [ ] , and antitumor [ , ] activities. the intent of the present study was to isolate a trypsin inhibitor from the gold bean and to test it for inhibitory action on tumor cells, viral enzymes, and fungal growth. upon ion exchange chromatography on deae-cellulose, the bean extract was separated into three fractions of approximately equal size, an unadsorbed fraction d and two adsorbed fractions d and d . trypsin inhibitory activity was located in fraction d . when fraction d was subjected to affinity chromatography on affi-gel blue gel, the activity was recovered in the larger unadsorbed fraction b (data not shown). upon ion exchange chromatography on sp-sepharose, fraction b was resolved into a small unadsorbed fraction s and two large adsorbed fractions (s and s ) of approximately equal size. activity resided in fraction s . further purification of s on superdex yielded two fractions, su and su . only fraction su exhibited trypsin inhibitory activity. su was the purified trypsin inhibitor (gbti). the yields of the various chromatographic fractions are presented in table . the n-terminal sequence of the trypsin inhibitor is shown in table . gbti exhibited a molecular mass of . kda in both sodium dodecyl sulfate-polyacrylamide gel electrophoresis (sds-page) and gel filtration ( figure ). its trypsin inhibitor activity remained unchanged in the temperature range - • c, was reduced by % at • c, and, was totally abolished after exposure to • c. upon ion exchange chromatography on deae-cellulose, the bean extract was separated into three fractions of approximately equal size, an unadsorbed fraction d and two adsorbed fractions d and d . trypsin inhibitory activity was located in fraction d . when fraction d was subjected to affinity chromatography on affi-gel blue gel, the activity was recovered in the larger unadsorbed fraction b (data not shown). upon ion exchange chromatography on sp-sepharose, fraction b was resolved into a small unadsorbed fraction s and two large adsorbed fractions (s and s ) of approximately equal size. activity resided in fraction s . further purification of s on superdex yielded two fractions, su and su . only fraction su exhibited trypsin inhibitory activity. su was the purified trypsin inhibitor (gbti). the yields of the various chromatographic fractions are presented in table . the n-terminal sequence of the trypsin inhibitor is shown in table . gbti exhibited a molecular mass of . kda in both sodium dodecyl sulfate-polyacrylamide gel electrophoresis (sds-page) and gel filtration ( figure ). its trypsin inhibitor activity remained unchanged in the temperature range - °c, was reduced by % at °c, and, was totally abolished after exposure to °c. trypsin inhibitor-enriched fractions are highlighted in boldface. results represent mean ± sd, n = . trypsin inhibitor-enriched fractions are highlighted in boldface. results represent mean ± sd, n = . gbti inhibited trypsin with an ic of . µm ( figure ). dithiothreitol (dtt) treatment lowered the trypsin inhibitory activity in a dose-and time-dependent manner ( table ). the ic values of the inhibitory effects of gbti on l cells and mbl cells were . µm and . µm, respectively (table ) . there was no inhibition on hiv- reverse transcriptase when it was tested at various concentrations up to µm ( table ). the inhibitor had no effect on the activities of hiv- integrase (table ) and sars coronavirus proteinase at µm. there was no inhibitory action on fungal growth at µm (table ) . table . inhibition rate (%) of dithiothreitol (dtt) on the activity of gbti and soybean trypsin inhibitor after incubation at • c for different durations. results are presented as mean ± sd (n = ). different letters (e.g., a,b and c ) indicate statistically significant differences (p < . ) when (i) data at the same time point and different dtt concentrations or (ii) data at the same dtt concentration, but different time points, were analyzed by analysis of variance followed by duncan's multiple range test. inhibition rate (%) of purified or soybean trypsin inhibitor at x mm dtt = (trypsin inhibitory activity of purified or soybean trypsin inhibitor−trypsin inhibitory activity of purified or soybean trypsin inhibitor in presence of x mm dtt)/trypsin inhibitory activity of purified or soybean trypsin inhibitor × %. results are presented as mean ± sd (n = ). different letters (e.g., a,b,c and d ) indicate statistically-significant differences (p < . ) when data were analyzed by analysis of variance followed by duncan's multiple range test. gold bean trypsin inhibitor (gbti) is unadsorbed on affi-gel blue gel, but adsorbed on deae-cellulose. this chromatographic behavior is distinctly different from that of other leguminous antifungal proteins which are unadsorbed on the ion exchanger and adsorbed on the affinity chromatography media [ , ] . hence, the purification procedure described herein provides a convenient means to separate trypsin inhibitors from antifungal proteins which may be present in the same legume. the trypsin inhibitor from gold beans demonstrate some sequence resemblance to other leguminous trypsin inhibitors including those of cowpea, mung bean, and garden pea. it is noteworthy that it exerts a potent antiproliferative action on both l cells and mbl cells. some of the legume trypsin inhibitors, for instance, field bean trypsin inhibitors, inhibit in vitro proliferation of cancer cells and metastasis in vivo [ , ] . gold bean trypsin inhibitor is dissimilar from broad bean trypsin inhibitor [ ] in that the former lacks hiv- reverse transcriptase inhibitory activity. the lack of hiv- reverse transcriptase inhibitory activity in gold bean trypsin inhibitor is similar to the findings on lily bulb trypsin inhibitor [ ] . gold bean trypsin inhibitor is also devoid of any inhibitory effect on hiv- integrase and sars coronavirus proteinase. the absence of antifungal activity in gold bean trypsin inhibitor is also in agreement with the observation on some trypsin inhibitors, such as lily bulb trypsin inhibitor [ ] . the results demonstrate that hiv- reverse transcriptase inhibitory and antifungal activities of trypsin inhibitors have structural requirements different from those of trypsin inhibitory activity. the importance of the disulfide linkage in gold bean trypsin inhibitor to its trypsin inhibitory activity is revealed by the ability of the reducing agent dithiothreitol to reduce the activity. this is reminiscent of the results on trypsin-chymotrypsin inhibitor from vigna mungo seeds [ ] . gold bean trypsin inhibitor (gbti) is unadsorbed on affi-gel blue gel, but adsorbed on deae-cellulose. this chromatographic behavior is distinctly different from that of other leguminous antifungal proteins which are unadsorbed on the ion exchanger and adsorbed on the affinity chromatography media [ , ] . hence, the purification procedure described herein provides a convenient means to separate trypsin inhibitors from antifungal proteins which may be present in the same legume. the trypsin inhibitor from gold beans demonstrate some sequence resemblance to other leguminous trypsin inhibitors including those of cowpea, mung bean, and garden pea. it is noteworthy that it exerts a potent antiproliferative action on both l cells and mbl cells. some of the legume trypsin inhibitors, for instance, field bean trypsin inhibitors, inhibit in vitro proliferation of cancer cells and metastasis in vivo [ , ] . gold bean trypsin inhibitor is dissimilar from broad bean trypsin inhibitor [ ] in that the former lacks hiv- reverse transcriptase inhibitory activity. the lack of hiv- reverse transcriptase inhibitory activity in gold bean trypsin inhibitor is similar to the findings on lily bulb trypsin inhibitor [ ] . gold bean trypsin inhibitor is also devoid of any inhibitory effect on hiv- integrase and sars coronavirus proteinase. the absence of antifungal activity in gold bean trypsin inhibitor is also in agreement with the observation on some trypsin inhibitors, such as lily bulb trypsin inhibitor [ ] . the results demonstrate that hiv- reverse transcriptase inhibitory and antifungal activities of trypsin inhibitors have structural requirements different from those of trypsin inhibitory activity. the importance of the disulfide linkage in gold bean trypsin inhibitor to its trypsin inhibitory activity is revealed by the ability of the reducing agent dithiothreitol to reduce the activity. this is reminiscent of the results on trypsin-chymotrypsin inhibitor from vigna mungo seeds [ ] . a lectin, an antifungal protein and a trypsin inhibitor can be isolated from the gold bean [ , ] . all three proteins can be regarded as defense proteins that protect the plant from pathogenic and predatory organisms. previously, a trypsin inhibitors have been isolated from phaseolus vulgaris. however, they have not been examined for biological activities other than trypsin inhibitory activity [ ] [ ] [ ] [ ] [ ] [ ] [ ] . in conclusion, a new trypsin inhibitor was purified from gold bean. it dose-dependently inhibited trypsin, and this activity was reduced in the presence of dithiothreitol in a dose-and time-dependent manner, signifying the importance of the disulfide linkage to the activity. it inhibited [methyl- h] thymidine incorporation by leukemia l cells and lymphoma mbl cells. a water extract of gold beans phaseolus vulgaris cv. "gold bean" from mainland china ( g) was made by homogenizing them in distilled water ( ml/g). the homogenate was then centrifuged ) . the unadsorbed proteins (fraction b ) were dialyzed against mm nh ac buffer (ph ) and applied on a . × cm column of sp-sepharose (ge healthcare, uppsala, sweden). after elution of unadsorbed proteins (fraction s ), the column was eluted with a - m nacl concentration gradient in the nh ac buffer. the first adsorbed fraction (s ) was then subjected to gel filtration on a superdex hr / column (ge healthcare) in . m nh hco buffer (ph . ). the second absorbance peak (su ) represented purified trypsin inhibitor (gbti). the assay for trypsin inhibitory activity was carried out by addition of test sample ( µl) to µl of a % casein solution in . m tris-hcl buffer (ph . ). trypsin ( µl of a . mg/ml solution) was then added and the mixture was incubated at • c for min before . ml % trichloroacetic acid was added to terminate the reaction. after centrifugation, the absorbance of the supernatant, which reflects the amount of casein fragments, was measured at nm [ ] . the purified trypsin inhibitor was subjected to sodium dodecyl sulfate-polyacrylamide gel electrophoresis (sds-page, woodinville, wa, usa) for molecular mass determination in accordance with the procedure of laemmli and favre [ ] . gel filtration on an fplc-superdex column (ge healthcare, uppsala, sweden), which had been calibrated with molecular mass markers including phosphorylase b ( kda), bovine serum albumin ( kda), ovalbumin ( kda), carbonic anhydrase ( kda), soybean trypsin inhibitor ( kda) and α-lactalbumin ( . kda) (ge healthcare), was conducted to determine the molecular mass of the protein. the n-terminal sequence of the trypsin inhibitor was determined by using a hewlett-packard hp g a edman degradation unit (hewlett packard company, palo alto, ca, usa) and an hp hplc system (hewlett packard company, palo alto, ca, usa). the purified trypsin inhibitor was incubated in a water bath at different temperatures from • c to • c for min, then immediately chilled on ice. the reaction mixture was then subjected to the assay of trypsin inhibitory activity. the purified trypsin inhibitor ( . µm) was incubated with dithiothreitol (dtt) at the final concentration of . , , and mm for , , and min at • c, respectively. for comparison, soybean trypsin inhibitor purchased from sigma chemical co., (st. louis, mi, usa) ( . µm) was similarly treated. the reaction was terminated by adding iodoacetamide at twice the amount of thiol functions at each dtt concentration. the remaining trypsin inhibitor activity was measured at ph . , as described above. the highest iodoacetamide concentration used in the test was devoid of any effect on the activity of trypsin and the trypsin inhibitory activity of purified trypsin inhibitor and soybean trypsin inhibitor [ ] . the antiproliferative activity of the purified protein was determined as follows. the cell lines l (leukemia) and mbl (lymphoma) were purchased from american type culture collection. the cell line was maintained in dulbecco modified eagles' medium (dmem) supplemented with % fetal bovine serum (fbs) and mg/l streptomycin and iu/ml penicillin at • c in a humidified atmosphere of % co . cells ( × ) in their exponential growth phase were seeded into each well of a -well culture plate (nunc, roskilde, denmark) and incubated for h before addition of the trypsin inhibitor. incubation was carried out for another h. radioactive precursor, µci ([ h-methyl]-thymidine, from ge healthcare), was then added to each well and incubated for hrs. the cultures were then harvested by a cell harvester. the incorporated radioactivity was determined by liquid scintillation counting [ ] . the assay for hiv reverse transcriptase inhibitory activity was carried out in view of the report that trypsin inhibitors manifest this activity [ , ] . it was conducted according to instructions supplied with the assay kit from boehringer mannheim (basel, switzerland). the assay takes advantage of the ability of reverse transcriptase to synthesize dna, starting from the template/primer hybrid poly (a) oligo (dt) . the digoxigenin-and biotin-labeled nucleotides in an optimized ratio are incorporated into one of the same dna molecule, which is freshly synthesized by the reverse transcriptase (rt). the detection and quantification of synthesized dna as a parameter for rt activity follows a sandwich elisa protocol. biotin-labeled dna binds to the surface of microtiter plate modules that have been pre-coated with strepatavidin. in the next step, an antibody to digoxigenin, conjugated to peroxidase enzyme, binds to the digoxigenin-labeled dna. in the final step, the peroxidase substrate is added. the peroxidase enzyme catalyzes the cleavage of the substrate, producing a colored reaction product. the absorbance of the sample at nm can be determined using a microtiter plate (elisa) reader and is directly correlated to the level of rt activity. a fixed amount ( - ng) of recombinant hiv- reverse transcriptase was used. the inhibitory activity of the protein was calculated as percent inhibition compared to a control without the protein [ ] . the positive control used was kale antifungal peptide. the plasmid that expressed his-tagged wild-type hiv- integrase, pt - -his (y|tx)-hiv- -in, was a generous gift from professor s.a. chow (school of medicine, ucla). to express the protein, a -liter culture of e. coli bl (de ) cells containing the expression plasmid was grown at • c until od reached . - . . cells were induced by addition of . mm iptg (isopropyl-β-d-thiogalactopyranoside) and harvested, after h of incubation, by centrifugation at × g for min at • c cells were suspended at a concentration of . g/ml wet cell paste in mm tris-hcl buffer (ph . ) containing . mm edta (ethylenediamine tetraacetic acid), mm β-mercaptoethanol, . m nacl and mm imidazole. lysozyme was added to a concentration of . mg/ml. after incubation at • c for h, the lysate was sonicated and centrifuged at , × g at • c for min. the pellet was homogenized in ml buffer a ( mm tris-hcl, ph . , m nacl, mm β-mercaptoethanol) containing mm imidazole. the suspension was rotated at • c for h, and cleared by centrifugation at , × g at • c for min. the supernatant was loaded onto a -ml chelating sepharose column (ge healthcare) charged with mm niso and was equilibrated with buffer a containing mm imidazole. the column was washed with five column volumes of buffer a containing mm imidazole, and the protein was eluted with three column volumes of buffer a containing and mm imidazole, respectively. protein containing fractions were pooled, and edta was added to a final concentration of mm. the protein was dialyzed against buffer b ( mm hepes, ph . , mm edta, m nacl, % glycerol) containing mm β-mercaptoethanol, and then against buffer b containing mm dithiothreitol. aliquots of the protein were stored at − • c [ ] . a non-radioactive elisa-based hiv- integrase assay was performed according to the dna-coated plate method. in this study, µg of smal-linearized pbluescript sk was coated onto each well in the presence of m nacl as target dna. the donor dna was prepared by annealing vu br ( '-biotin-gtgtggaaaatctctagcagt- ') and vu ( '-actgctagagattttccacac- ') in mm tris-hc , ph . , mm edta and . m nacl at • c, followed by min at room temperature. integrase reaction was performed in mm hepes ( -( -hydroxyethyl)- -piperazineethanesulfonic acid) (ph . ) containing mm mncl , mm nacl, mm dithiothreitol and . % nonidet-p (sigma). after the integrase reaction, the biotinylated dna immobilized on the wells was detected by incubation with streptavidin-conjugated alkaline phosphatase (boehringer-mannheim, mannheim, germany), followed by colorimetric detection with mg/ml p-nitrophenyl phosphate in % diethanolamine buffer (ph . ) containing . mm mgcl . the absorbance due to the alkaline phosphatase reaction was measured at nm. the ribosome inactivating protein luffin was used as a positive control [ ] . the activity of sars coronavirus (cov) protease was indicated by a cleavage of designed substrate which was composed of two proteins linked by a cleavage site for sars cov protease. the reaction was performed in a mixture containing µm sars cov protease, µm sample, µm substrate, and buffer [ mm tris-hcl (ph . ), mm nacl and mm beta-mercaptoethanol] for min at • c. after min, the reaction was stopped by heating at • c for min. then the reaction mixture was analysed by sds-page. if sars cov protease is inhibited by the test sample, there is only one band, which is the intact substrate, shown in sds-page [ ] . this assay was conducted in view of the report on antifungal activity of some trypsin inhibitors [ ] . the assay for antifungal activity toward mycosphaerella arachidicola and fusarium oxysporum was carried out in mm × mm petri dishes containing ml of potato dextrose agar. after the mycelial colony had developed, sterile blank paper disks ( . cm in diameter) were placed at a distance of . cm away from the rim of the mycelial colony. an aliquot ( µl) of the purified trypsin inhibitor was added to a disk. the plates were incubated at • c for h until mycelial growth had enveloped the disks containing the control and had formed crescents of inhibition around the disks containing samples with antifungal activity [ ] . the positive control used was kale antifungal peptide. a . -kda trypsin inhibitor was purified from phaseolus vulgaris cv. "gold bean". it was adsorbed on deae-cellulose, unadsorbed on affi-gel blue gel, and adsorbed on sp-sepharose. it dose-dependently inhibited trypsin with an ic value of . µm. about % and all of the trypsin inhibitory activity were abolished after treatment with . mm dithiothrietol for min and min of incubation, respectively. [methyl- h] thymidine incorporation by leukemia l cells and lymphoma mbl cells was inhibited with an ic value of about µm. mycelial growth in fusarium oxysporum and mycosphaerella arachidicola were unaffected. the activities of hiv- reverse transcriptase, hiv- integrase and sars coronavirus proteinase were unaltered after exposure to the trypsin inhibitor. amino acid sequence and secondary structural analysis of the corn inhibitor of trypsin and activated hageman factor primary structure and reactive site of a novel wheat proteinase inhibitor of subtilisin and chymotrypsin a homodimeric sporamin-type trypsin inhibitor with antiproliferative, hiv reverse transcriptase-inhibitory and antifungal activities from wampee (clausena lansium) seeds bitter gourd proteinase inhibitors: potential growth inhibitors of helicoverpa armigera and spodoptera litura isolation of a trypsin inhibitor with deletion of n-terminal pentapeptide from the seeds of momordica cochinchinensis, the chinese drug mubiezhi protein proteinase inhibitors in legume seed-overview trypsin inhibitor from poecilanthe parviflora seeds: purification, characterization, and activity against pest proteases effects of the medicago scutellata trypsin inhibitor (msti) on cisplatin-induced cytotoxicity in human breast and cervical cancer cells a bowman-birk-type trypsin-chymotrypsin inhibitor from broad beans wolfenstein-todel, c. a novel trypsin inhibitor from peltophorum dubium seeds, with lectin-like properties, triggers rat lymphoma cell apoptosis characterization and pharmacological properties of a novel multifunctional kunitz inhibitor from erythrina velutina seeds formation of bowman-birk inhibitors during the germination of horsegram (dolichos biflorus) reductive unfolding and oxidative refolding of a bowman-birk inhibitor from horsegram seeds (dolichos biflorus): evidence for "hyperreactive" disulfide bonds and rate-limiting nature of disulfide isomerization in folding a comparison of the short and long term effects of insecticidal lectins on the activities of soluble and brush border enzymes of tomato moth larvae (lacanobia oleracea) a mitogenic defensin from white cloud beans (phaseolus vulgaris) purification and characterization of novel ribosome inactivating proteins, alpha-and beta-pisavins, from seeds of the garden pea pisum sativum molecular characterization of a new arcelin- gene distribution of proteinases and carbohydrates in the midgut of the larvae of the sweet potato weevil cyclas formicarius and response of proteinase to inhibitors from sweet potato concurrent purification of two defense proteins from french bean seeds: a defensin-like antifungal peptide and a hemagglutinin treatment with field bean protease inhibitor can effectively repress ethylnitrosourea (enu)-induced neoplasms of the nervous system in sprague-dawley rats the field bean protease inhibitor has the potential to suppress b f melanoma cell lung metastasis in mice isolation and characterization of a novel trypsin inhibitor from fresh lily bulbs trypsin-chymotrypsin inhibitors from vigna mungo seeds an antifungal peptide from phaseolus vulgaris cv. brown kidney bean purification and characterization of a lectin from phaseolus vulgaris cv. (anasazi beans) characterization of trypsin inhibitors from tora-mame seeds, one of the japanese cultivars of phaseolus vulgaris trypsin isoinhibitors from garden beans (phaseolus vulgaris) isolation and characterization of some proteinase inhibitors from phaseolus vulgaris var. nanus natural plant enzyme inhibitors: isolation and properties of a trypsin/chymotrypsin inhibitor from kidney bean (phaseolus vulgaris) the isolation and characterization of a trypsin inhibitor from kintoki bean (phaseolus vulgaris) the isolation of two proteins, glycoprotein i and a trypsin inhibitor, from the seeds of kidney bean (phaseolus vulgaris) enzyme inhibitory activities of two leguminosae: phaseolus vulgaris l. pods and vicia faba l. hulls. trypsin inhibitory activity maturation of the head of bacteriophage t . i. dna packaging events baeyens-volant, d. the papaya kunitz-type trypsin inhibitor is a highly stable beta-sheet glycoprotein a novel lectin from pseudostellaria heterophylla roots with sequence simularity to kunitz-type soybean trypsin inhibitor marmorin, a new ribosome inactivating protein with antiproliferative and hiv- reverse transcriptase inhibitory activities from the mushroom hypsizigus marmoreus the authors declare no conflict of interest.molecules , , key: cord- -qboon uv authors: zheljazkov, valtcho d.; sikora, vladimir; dincheva, ivayla; kačániová, miroslava; astatkie, tess; semerdjieva, ivanka b.; latkovic, dragana title: industrial, cbd, and wild hemp: how different are their essential oil profile and antimicrobial activity? date: - - journal: molecules doi: . /molecules sha: doc_id: cord_uid: qboon uv hemp (cannabis sativa l.) is currently one of the most controversial and promising crops. this study compared nine wild hemp (c. sativa spp. spontanea v.) accessions with registered cultivars, eight breeding lines, and one cannabidiol (cbd) hemp strain belonging to c. sativa l. the first three groups had similar main essential oil (eo) constituents, but in different concentrations; the cbd hemp had a different eo profile. the concentration of the four major constituents in the industrial hemp lines and wild hemp accessions varied as follows: β-caryophyllene – % and . – . %; α-humulene . – . % and . – . %; caryophyllene oxide . – . % and . – . %; and humulene epoxide , . – . % and . – . %, respectively. the concentration of cbd in the eo of wild hemp varied from . to . % of the total oil while cbd in the eo of the registered cultivars varied from . to %; cbd in the eo of the breeding lines and in the cbd strain varied from . to % and . to . %, respectively. the concentrations of δ -tetrahydrocannabinol (thc) in the eo of the three groups of hemp were significantly different, with the highest concentration being . %. the eo of wild hemp had greater antimicrobial activity compared with the eo of registered cultivars. this is the first report to show that significant amounts of cbd could be accumulated in the eo of wild and registered cultivars of hemp following hydro-distillation. the amount of cbd in the eo can be greater than that in the eo of the usa strain used for commercial production of cbd. furthermore, this is among the first reports that show greater antimicrobial activity of the eo of wild hemp vs. the eo of registered cultivars. the results suggest that wild hemp may offer an excellent opportunity for future breeding and the selection of cultivars with a desirable composition of the eo and possibly cbd-rich eo production. hemp (cannabis sativa l.) is a new-old crop, one of the most controversial and promising crops due to its multiple utilizations, and contains a wide array of biologically active substances synthesized and accumulated in different plant parts [ ] . industrial hemp has been grown for grain and fiber for many decades in europe, asia, and north america. in addition, there is wild hemp (c. sativa spp. spontanea vavilov, also known as spontaneous), which is native to both central and eastern europe as well as parts of asia, and is found as a weed in agricultural fields. hemp essential oil (eo) can be extracted using various extraction methods, the simplest and most natural using either steam or hydro-distillation, as is the case with many other eo crops. hydro-distilled or steam distilled hemp eo are generally preferred by consumers and can be incorporated into a number of certified organic products; the market for organic food and non-food products reached us$ billion in in the usa alone [ ] . currently, cannabidiol (cbd) production and markets in many countries are depressed due to the overproduction and the covid- situation. for example, hemp production in the usa has increased rapidly and by mid- , there were around , licensed acres to grow hemp [ ] . that acreage in would have generated an estimated us$ . billion income, or around % of the total value of all cash crops in the usa [ ] . however, due to the depressed markets and expensive feminized seed (us$ /seed), there is now significant interest in hemp eo from industrial hemp cultivars and even from wild hemp. indeed, a number of producers and processors in the usa are developing new products based on naturally extracted hemp eo or cannabinoids. overall, research suggests that the hemp eo has medical significance and may also be utilized as an ingredient in commercial insect repellents and biopesticides [ , ] . some cultivars such as finola have been employed for commercial production of eo, as hemp eo has commanded high prices in recent years. hemp terpenes in the eo contribute to the aroma of various cannabis genotypes, and so far, around different terpenes have been reported in this plant [ , [ ] [ ] [ ] . current thinking is that terpenes have played a key role in the selection of medical/recreational and cbd type cannabis because their concentration is positively correlated to some of the cannabinoids [ ] . the hemp eo profile depends on genotype, growth conditions, and extraction method (steam, hydro, co , or solvent extraction) [ ] . of the various groups of terpenes, monoterpenes such as limonene, β-myrcene, α-pinene, β-pinene, and linalool (containing carbon units) comprise the major portion of the volatile oil fraction [ ] . these eo constituents are widely found in the eo of other plant species such as spices, eo crops, and medicinal herbs; they are not specific to cannabis. sesquiterpenes (with carbons) such as β-caryophyllene, α-humulene, caryophyllene oxide, and β-phellandrene are also present in higher concentrations in hemp extracts [ , ] . monoterpene composition can distinguish between monoecious and dioecious hemp cultivars [ ] . furthermore, certain terpenoids are highly correlated to the concentration of cbd and ∆ -tetrahydrocannabinolic acid (thca); consequently, they have been proposed as a chemotaxonomic classification tool and to distinguish drug-type cannabis in nevada [ ] . overall, hemp varieties (cultivars) with a higher concentration of monoterpenes have a more pleasant aroma compared with the varieties with higher amounts of sesquiterpenes. the monoterpenes pinene and limonene are the determinants of cannabis aroma in the immediate vicinity of the plant [ ] . hydro-distilled eo from industrial hemp cultivars may contain cbd [ ] ; the interaction of environment and genetics plays a role in the hemp eo profile. hemp eo (distilled from leaves, inflorescences, and thinner stems) has shown biological activity against several targets of pharmaceutical interests s. aureus, h. pylori, candida, and malassezia spp., enzymes, and cancer cell lines [ ] . the eos (collected from inflorescences after blooming) of cvs. carmagnola, fibranova, and futura have shown significant antimicrobial activity against g + and gbacteria and yeast, and the effect depended on the cultivar and seeding date [ ] . this study addresses a knowledge gap and current industry interest towards hemp eo with different origins and profile. the hypothesis was that wild hemp would have a different eo content, composition, and antimicrobial activity compared with the eos of registered industrial hemp cultivars, new hemp breeding lines, and a hemp strain (unregistered cultivar) that is currently used for the commercial production of cbd. the objective of this study was to compare nine wild hemp accessions (c. sativa spp. spontanea) sampled from agricultural fields in northeastern serbia with eu registered cultivars, eight breeding lines, and one cbd hemp strain (belonging to c. sativa) with respect to their eo profile and antimicrobial activity. the eo yield of the wild hemp accessions varied from . to . ml/ g for air-dried material and the yield of the breeding lines was . to . , while the eo yield of the registered cultivars was . to . ml/ g of dried material. however, the overall differences in oil yield between the three groups of hemp were not significantly different, with an overall mean of . ml/ dried material (table ) . table . analysis of variance (anova) p-values that show the significance of the effect of cultivar on constituents, square root of mean squares error (root mse) that represents the common standard deviation, and the overall mean oil content (v/w, volume oil per dry weight) and concentration (%) of the five constituents with no significant difference between the cultivars and cbd. the p-values that show significant (p < . ) and marginally significant ( . ≤ p < . ) effect and need multiple means comparison are shown in bold. overall, the eos of the wild hemps and registered cultivars in this study were similar to those reported previously: . to . % in fresh inflorescences [ ] , . to . % depending on the collection time with higher eo yield from plants sampled earlier (in september than in october) [ ] , and . % in stems and . % in the leaves of wild hemp from austria [ ] , respectively. however, the eo content of the usa hemp strain, utilized for cbd production in the usa and grown near the field trials was . to . %. for the wild hemps, two locations (kovacica and susara) were randomly selected among the nine locations (slavka, kovacica, buro, daleka zemlia, susara, saykaj, perez, titelski breg, and paluka) to be used as two replications to represent wild hemp in the statistical analyses. as indicated in the materials and methods section, one-way anova was completed to determine the significance of differences between the mean constituents obtained from nine hemp cultivars from northeast serbia. the constituents were: α-pinene, β-pinene, isocaryophyllene (γ-caryophyllene), β-caryophyllene, α-(e)-bergamotene, (z)-β-farnesene, caryophyllene oxide, humulene epoxide , selina- -en- -ol, caryophylla- ( ), ( )-dien- α-ol, caryophylla- ( ), ( )-dien- β-ol, -hydroxy-(z)-caryophyllene, β-bisabolol, α-bisabolol, cbd, and δ -tetrahydrocannabinol. overall, the eo profile of the wild hemp was different from that of one of the registered cultivars and the new breeding lines (supplementary tables s -s ). the eo constituents whose concentrations were significantly different are shown in bold in table . the mean concentrations of the five constituents and cbd from the above list that did not have significant differences between the wild and the registered cultivars are shown in table . the constituents whose means were significantly different are shown in table . the comparative concentrations of the eo constituents in this and previous reports cited here are summarized in table . table . mean concentration (%) of isocaryophyllene (γ-caryophyllene) [ ] , β-caryophyllene [ ] , α-(e)-bergamotene [ ] , caryophyllene oxide [ ] , humulene epoxide [ ] , selina- -en- -ol [ ] , caryophylla- ( ), ( )-dien- β-ol [ ] , β-bisabolol [ ] , α-bisabolol [ ] , and δ -tetrahydrocannabinol (dronabinol) [ ] obtained from the cultivars. the concentration range of α-pinene (bicyclic monoterpene) in the eo was from non-detected (n.d.) in three accessions of wild hemp to . % in wild hemp slavka; from n.d. to . % in the registered cultivars; and . to . % in the new breeding lines (supplementary tables s -s ). however, when grouped together, the concentration of α-pinene in the eo was not statistically different between the three groups of hemp. the concentration of α-pinene in the eo of the usa hemp strain (grown in the vicinity of the registered cultivars in this study and extracted the same way for the same time duration) was . to . % of the total eo. the monoterpenes pinene and limonene are the determinants of cannabis aroma in the immediate vicinity of the plant [ ] . α-pinene concentration in the eo in this study was comparable to previous reports from studies that used either steam or hydro-distillation [ ] [ ] [ ] [ ] , but was lower than that in other reports [ , , ] (table ). the eo of wild hemp from austria contained % of this compound in stems, but a much lower concentration in the leaves [ ] , while the eo of spontaneous (wild) hemp from hungary contained . , . , and . % of this compound in the leaves, male, and female flowers, respectively. these differences may be due to the environment (growing conditions including latitude and altitude), the plant parts analyzed, and/or the genetics (cultivar). the concentration range of γ-caryophyllene in the eo was . to . % in wild, . to . % in the registered cultivars, and . to . % in the eo of the breeding lines (supplementary tables s -s ) . γ-caryophyllene concentration in the eo of the usa strain was . %. the concentration of β-caryophyllene was to % in the eo of wild hemp, to % in the registered cultivars, and to % in the eo of the breeding lines (supplementary tables s -s ) . overall, statistically, the highest concentration of β-caryophyllene was found in the eo of cv. spic and the lowest in the eos of cvs. simba and dioica (table ) . β-caryophyllene in the usa strain eo was . to . %. β-caryophyllene in the eo of this study was similar to those reported in previous studies [ , , , , , ] (table ) . the data from this study and previous reports suggest that the concentration of β-caryophyllene could vary significantly depending on the cultivar. (e)-β-caryophyllene is known as the major eo constituent in hemp [ ] . this is one of the c. sativa eo caryophyllane-and humulane-type sesquiterpenes that include sesquiterpenes (e)-β-caryophyllene, (z)-β-caryophyllene, caryophyllene oxide, and the ring-opened isomer α-humulene (α-caryophyllene) [ ] . this eo compound has been shown to function in vivo as a non-psychoactive cb receptor ligand in foodstuff [ ] . the concentration of α-(e)-bergamotene (bicyclic sesquiterpene) ranged from . to . % in the wild hemp, . to . % in the registered cultivars, and . to . % in the breeding lines (supplementary tables s -s ) . overall, the concentration of α-(e)-bergamotene was statistically higher in cvs bacalmas and helena, and the lowest in cv. cs (carmagnola selezionata) ( table ). the concentration of this compound in the usa strain eo was . %. overall, the concentration of this eo compound in wild hemp from this study was similar to that in the literature reports on wild hemp from austria and hungary [ , ] . the concentration of caryophyllene oxide (bicyclic sesquiterpenoid) was . to % of the total eo in wild hemp, . to . % in registered cultivars, and . to % in the eo of the breeding lines (supplementary tables s -s ). the concentration of caryophyllene oxide was statistically higher in the eos of cvs. bacalmas, helena, and sequieni, and lowest in the eo of cv. carmagnola ( table ). the concentration of this compound in the usa strain eo was . to . %. caryophyllene oxide in the eo of wild hemp from austria was . % [ ] , while its concentration in the eo of spontaneous hemp from hungary was . , . , and . % in the leaves, male, and female flowers, respectively [ ] . the concentration of humulene epoxide (bicyclic sesquiterpenoid) was . to . % in wild hemp, . to . % in registered hemp cultivars, and . to . % in the eo of the breeding lines (supplementary tables s -s ) . overall, the concentration of humulene epoxide was statistically higher in the cvs. bacalmas and sequieni, and the lowest in cv. spic ( table ). the concentration of this compound in the usa hemp strain eo was . %. the concentration of this compound in the eo of wild hemp from austria was < . % [ ] , while in the eo of spontaneous hemp from hungary, it was . , . , and . % in the leaves, male, and female flowers, respectively [ ] . selina- -en- -ol (bicyclic sesquiterpenoid) was . to . % in the eo of wild hemp, n.d. to . % in the eo of the registered cultivars, and . to . % in the eo of the breeding lines (supplementary tables s -s ). its concentration was statistically highest in the eo of cv. dioica and lower in the eos of cvs. bacalmas and spic (table ) . hydro-distillation (hd); steam-distillation (sd); headspace solid-phase microextraction (hs-spme); solvent extraction (se); microwave-assisted extraction (mae). the concentration of caryophylla- ( ), ( )-dien- β-ol (bicyclic sesquiterpenoid) was . to . % in the eo of wild hemp; from n.d. to . % in the eo of the registered hemp cultivars; and . to . % in the eo of the breeding lines (supplementary tables s -s ). its concentration was statistically the highest in the eo of the wild hemp and the lowest in the eo of cv. spic ( table ). the concentration of this compound in the usa strain eo was . to . %, while its concentration in the eo of spontaneous hemp from hungary was . , . , and . % in the leaves, male, and female flowers, respectively [ ] . the concentration of β-bisabolol (monocyclic sesquiterpenoid) was . to . % in the eo of wild hemp; from n.d. to . % in the eo of the registered cultivars; and . to . % in the eos of the breeding lines (supplementary tables s -s ; table ). overall, β-bisabolol was higher in the eos of cvs. bacalmas, sequieni, and in the wild hemp, and the lowest in the eo of cv. spic ( table ). the concentration of this compound in the usa strain eo was . %. the concentration of α-bisabolol (monocyclic sesquiterpenoid) was . to . % in the eo of wild hemp, n.d to . % in the registered cultivars, and . to . % in the eo of the breeding lines (supplementary tables s -s ). α-bisabolol was statistically the highest in the eo of cv. cs and lower in wild hemp and cvs. bacalmas, sequieni, and spic (table ) . α-bisabolol in the usa strain eo was . to . %. epi-α-bisabolol was reported in the eo of spontaneous hemp from hungary [ ] . the major eo constituents of the usa hemp strain that was grown in close vicinity had a different chemical profile, with major constituents of myrcene ( . to %), β-caryophyllene ( . to . %), limonene ( . to . %), β-(e)-ocimene ( . to . %), and α-bisabolol ( . to . %). some previous reports identified a different number of eo constituents in hemp eo (e.g., eo constituents, with myrcene, α-pinene, and β-pinene as the main monoterpenes, and β-caryophyllene as the main sesquiterpene) [ ] ; eo constituents were identified in wild (spontaneous) hemp by nagy et al. [ ] . the latter authors named these hemp plants spontaneous forms, with the main eo constituents of the leaves, male and female flowers being e-caryophyllene ( . %), α-humulene ( . to . %), β-selinene ( . to . %), and α-selinene ( . to . %) [ ] . apparently, the spontaneous hemp plants from hungary had a different eo chemical profile compared with the eo of the wild hemp of this study that was collected in the northeastern part of serbia. the hydro-distilled leaf eo of wild hemp in austria contained mainly β-caryophyllene ( . %), α-humulene ( . %), β-selinene ( . %), caryophyllenen oxide ( . %), and α-selinene ( . %) [ ] . in the same study, stem eo contained α-pinene ( . %), β-caryophyllene ( . %), β-pinene ( . %), and myrcene ( . %) [ ] . apparently, the eo of wild hemp in austria had a similar composition to some, but not to other wild hemp eos, in this study. previous research has suggested that metabolic fingerprinting can be used for chemotaxonomic purposes in c. sativa [ ] . however, this and earlier reports on wild hemp [ , ] have demonstrated that the eo profile of wild hemp can vary significantly. therefore, genetic analyses may be needed to ascertain if the spontaneous or wild hemps in europe originated from some of the old industrial hemp cultivars that have been grown in europe over the last few centuries or from medical cannabis, or are products of spontaneous crossings between the two groups. in this study, the concentration of cbd in the eo of wild hemp varied from . to . % of the total oil, the cbd in the eo of the registered cultivars was from . to . %, while the cbd concentration in the oil of the breeding lines was from . to . % (supplementary tables s -s ) . however, when we grouped them together, because of the high variation, there were no significant differences between the wild accessions, registered cultivars, and the breeding lines with overall means of . % (table ). the concentration of cbd in the eo of the usa strain (which is commercially grown for cbd production) varied between . and . % of the total eo, which is an interesting result. the cbd concentrations in the eo of wild hemp slavka, kovacica, susara, perez and titelski breg were . , . , . , . , and . %, respectively, while the cbd concentrations in the eo of buro and saykaj were . and . %, respectively. therefore, this and previous studies [ , ] support the notion that wild hemps can be used as a source for the commercial production of cbd-enriched eo. this is the first report on such a high concentration of cbd in hydro-distilled hemp eo. overall, the concentration of thc in the eo was significantly different between the three groups of hemp ( table ). the thc concentration was significantly higher in the eo of wild hemp accessions, with an average of . % of the total oil, and a range between n.d. (in daleka zemlia) and . % (in kovacica) (supplementary tables s -s ). interestingly, it was much higher than the thc ( . %) in the eo of the usa hemp strain, which was actually developed from marijuana type hemp. the thc concentration in the eo of most of the registered cultivars varied from n.d. (e.g., in cvs. spic and bacalmas) to over . % (in the eo of cv. chameleon) ( table ). the thc concentration in the eo of the breeding lines was generally low, n.d., or below . % with the exception of line sk , where it reached . % of the total oil. previous research has shown that hydro-distilled eo from industrial hemp varieties contained cannabidiol (cbd) [ ] . overall, the content of monoterpenes fluctuated from n.d. to . % in the eo of wild hemp, . to % in the eo of the registered cultivars, and . to % of the eo of the breeding lines (supplementary tables s -s ). monoterpene concentration in the eo of the usa hemp strain was to % of the oil. sesquiterpenes were the largest group of chemical constituents. the content of sesquiterpenes was to % of the eo of wild hemp, to % of the eo of the registered cultivars, and to % of the eo of the breeding lines (supplementary tables s -s ). sesquiterpenes constituted to % of the eo in the usa hemp strain. cannabinoids also comprised the second largest group of chemical constituents in the wild hemp. the concentration of cannabinoids was to % of the eo of wild hemp, . to % of the eo of the registered cultivars, and . to % of the eo of breeding lines (supplementary tables s -s ) . cannabinoid concentration in the eo or usa hemp strain were . to . %, surprisingly low. most of the strains for thee commercial production of cbd were selected from marijuana (drug-type hemp with to % of ∆ -thc %), however, they were selected to have < . % total thc in dried biomass in order to be compliant with the current rules and regulations that are evolving [ ] . in a study of spontaneous hemp, nagy et al. [ ] reported that the eo was majorly composed of sesquiterpene hydrocarbons ( . to . %), followed by cannabinoids ( . to . %) and oxygenated sesquiterpenes ( . to . %). overall, the results from this study suggest that wild/spontaneous hemp in europe is chemotaxonomically related to the industrial hemp varieties (cultivars) grown in europe and deviate from the chemical profile of the usa hemp strain that was developed from marijuana-type cannabis for the commercial production of cbd. the usa hemp strain used in this study was started with feminized seed, which guarantees the production of female only plants, which may be one of the reasons for its much higher eo content. the pharmacological power of hemp is based on the content of ∆ -tetrahydrocannabinolic acid (thca) and cannabidiolic acid (cbda) [ ] . other major cannabinoids include cannabinolic acid (cbna), cannabigerolic acid (cbga), cannabichromenic acid (cbca), and cannabinodiolic acid (cbnda) [ , ] . current hemp eo and cannabionoid production systems in the usa have been scaled up from marijuana production. like with marijuana production, hemp 'christmas tree' production systems rely on feminized seed, because female plants with non-fertilized flowers (flower bracts) accumulate significantly higher concentrations of secondary metabolites such as cannabinoids and terpenes compared with fertilized flowers [ ] . currently, hemp chemical production is based on non-registered hemp 'strains' that were originally developed by marijuana breeders, which must meet government regulations for hemp with less than . % ∆ -tetrahydrocannabinolic acid (thca) in the dried biomass. due to the rapidly growing market for non-psychoactive cannabinoids, mainly cannabidiol (cbd), most of production has been focused on cannabidiolic acid (cbda). newer hemp strains bred for these characteristics are more likely to be compliant, and breeding companies are working on the registration of a number of hemp strains with high cbd content as commercial cultivars. the results from this study demonstrate that wild hemp may be a good source for further selection and breeding of cultivars with high concentration of cbd and low concentrations of thc. we used antibiotics as a positive control: cefoxitin for gram-negative (g -) bacteria and gentamicin for gram-positive (g + ) bacteria, and fluconazole for yeast. from gwe used se, salmonella enterica subsp. enterica; pa, pseudomonas aeruginosa; and ye, yersinia enterocolitica. from g + , we used sa, staphylococcus subsp. aureus; ef, enterococcus faecalis; and sp, streptococcus pneumoniae. from yeast, ca, candida albicans; ck, candida krusei; and ct, candida tropicalis were used. the method has been described previously [ ] . the eos of different hemp cultivars, accessions, and strains had differential antimicrobial activity that can be explained with differences in the eo profile. some of the eos had similar (although lower) activity to the antibiotics gentamycin, cetofoxin, and fluconazole. the antibiotic gentamycin was used as a positive control in the data presented in figure . the eo of wild hemp buro was the most potent against staphylococcus subsp. aureus (sa), followed by the eo of wild hemp saykaj ( figure a) . similarly, the eos of wild hemps buro and saykaj showed the highest antimicrobial activity against enterococcus faecalis (ef). the eos of wild hemp buro and cv. dioica were the most potent against streptococcus pneumoniae (sp) ( figure b) . the eos of wild hemp buro and the registered cv. dioica had higher antimicrobial activity against streptococcus pneumoniae compared with that of other eos ( figure c) . furthermore, the eos of wild hemp buro and the registered cv. dioica had higher antimicrobial activity against pseudomonas aeruginosa compared with that of the eos of the other hemps ( figure a ). cetofoxin (antibiotic) was used as a positive control for the data in figure . the eo of wild hemp susara had the highest activity against the g − yersinia enterocolitica ( figure b ). the eo of wild hemp buro had the highest activity against the g − salmonella enterica subsp. enterica, lower than the eos of wild hemps daleka zemlia and saykaj, and the lowest in the eo of other hemps ( figure c ). the antibiotic fluconazole was the positive control for the data presented in figure . the eo of wild hemp saykaj had the highest bioactivity against candida albicans (ca), the bioactivity of eo of wild hemp perez was not significantly different, while the bioactivity of the eos of the other hemp eos was lower than that of saykaj ( figure a ). the eo of wild hemp susara and the eo of registered cv. cs had the highest bioactivity against candida krusei (ck), while the bioactivity of the eo of sequieni was not different from the above ( figure b ). the eo of wild hemp paluka had the highest bioactivity against candida tropicalis (ct) ( figure c) . previously, hemp eo has shown biological activity against several targets of pharmaceutical interest: s. aureus, h. pylori, candida and malassezia spp., enzymes, and cancer cell lines [ ] . in another study, the eos (collected from inflorescences after blooming) of cvs. carmagnola, fibranova, and futura showed significant antimicrobial activity against gram (+) and gram (−) bacteria and yeast, and the effect depended on the cultivar and seeding date [ ] . the antibiotic fluconazole was the positive control for the data presented in figure . the eo of wild hemp saykaj had the highest bioactivity against candida albicans (ca), the bioactivity of eo of wild hemp perez was not significantly different, while the bioactivity of the eos of the other hemp eos was lower than that of saykaj ( figure a) . the eo of wild hemp susara and the eo of registered cv. cs had the highest bioactivity against candida krusei (ck), while the bioactivity of the eo of previously, hemp eo has shown biological activity against several targets of pharmaceutical interest: s. aureus, h. pylori, candida and malassezia spp., enzymes, and cancer cell lines [ ] . in another study, the eos (collected from inflorescences after blooming) of cvs. carmagnola, fibranova, and futura showed significant antimicrobial activity against gram (+) and gram (−) bacteria and yeast, and the effect depended on the cultivar and seeding date [ ] . overall, the findings in this study are consistent with the ones in a recent report [ ] . this study provides new information on the antimicrobial activity of the eos of the registered and wild hemps. overall, the findings in this study are consistent with the ones in a recent report [ ] . this study provides new information on the antimicrobial activity of the eos of the registered and wild hemps. good antimicrobial activity against enterococcus, listeria, and staphylococcus growth were found, which were compared to the conventional antibiotics that were used [ , ] . novak et al. [ ] found that the eo of five different cultivars of hemp had modest antibacterial activity. the gram-positive bacterial strains tested demonstrated high sensitivity toward cannabidiol, with slightly lower effects by cannabidiolic acid [ ] . due to significant antimicrobial potency of cbd against mrsa, the synergy with conventional antibiotics was tested. however, cbd was not able to revert the resistance pattern or demonstrate synergy with any of the conventional antibiotics tested [ , ] . certified industrial hemp (cannabis sativa l.) seeds were obtained from the institute for field and vegetable crops in novi sad, serbia. field experiments were set up at the alternative crops and organic production department in backi petrovac, serbia (n e ) using different cultivars of industrial hemp ( table ) , some of them included in the european list of approved hemp varieties (cultivars) [ ] . the cultivars used in this study included cs (carmagnola selezionata), spic, dioica, helena, carmagnola, squieni, bacalmas, simba, silesia, chameleon, fibrol, futura, and lovrin ( table ). the results from the first eight varieties were used in the statistical analyses and in the tables. in addition, new hemp breeding lines (named sk to sk ) were seeded adjacent to the above experiment and subjected to the same growth conditions. although the botanical classification of hemp is controversial [ ] , wild populations belong to uncultivated narrow leaf cannabis sativa ssp. spontanea vavilov [ ] , which is considered native to central and eastern europe and parts of asia. wild hemp samples in this study were collected from the edges of agricultural fields in the same region that have been used to grow other crops, but not hemp. these were agricultural fields where no hemp has been grown for the last years. however, around - years ago, at some of the collection sites, there were hemp processing factories or hemp seed storage facilities. generally, wild hemp is phenotypically different from plants of either old or new commercial hemp cultivars; wild hemp also has a shorter stature, with fewer branches than plants from registered cultivars. the locations of the wild hemp samples were named after the names of the nearby villages: buro, daleka zemlia, paluka, perez, saykaj, susara, slavka, kovacica, and titelski breg ( table ). the wild hemp samples were dried under the same conditions, and the eo was extracted via the same method and during the same time as the hydro-distillations of other hemp samples. hemp was grown as a rainfed crop without irrigation, as is traditional for the region, and the production technology, which is typical production system for commercial production of hemp in middle and southern europe, was applied. hemp was seeded with a corn planter on march, with cm spacing between rows and at a seeding rate of kg/ha. the soil type was alluvial chernozem with ph . ., previous crop was millet. the soil preparation prior to seeding included deep plowing on november, and fine seedbed preparation on march, . the experimental design was completely randomized with three replicates, with the size of the experimental plots being m × m. fertilizer (npk : : ) was applied as a broadcast treatment at kg/ha before deep plowing. an additional kg/ha of nitrogen was applied in the spring before sowing. weed control was conducted using burnout with glyphosate prior to seeding. mechanized weed control was performed twice during the first four weeks of vegetation stage though the use of sweep-type cultivators. hemp closes the canopy at - weeks after emergence and hence, suppresses weeds very well after that. the trial of the breeding lines was at the same field, approximately m from the main trial, and was subjected to the same agricultural conditions. plants in both trials were cultivated the same way including seeding time, seeding depth, fertilization, and weed control. fresh biomass samples (around kg fresh, in three reps) were obtained on june, at the beginning of flowering of the male plants. hemp tissue samples were generated by cutting the top . feet ( cm) from the top of female plants (male plants were not included in the samples). fresh weight was measured, then the samples were hung in a shady area (tobacco dryer) until constant air-dried weight and then extracted. the eo extraction was conducted via hydro-distillation of hemp air-dried material using -l hydro-distillation clevenger-type units as described previously for another plant material [ ] . the sample size was up to g dry weight (dw) biomass in . l water. all distillations were performed in two replicates, which was sufficient for statistical analyses. beginning of the distillation was noted when the first drop of eo was deposited in the collection part of the clevenger apparatus. all samples were distilled non-stop for min. at the end of the distillation, the heat source was removed, the eos (along with some water) were collected in glass vials, and placed in a freezer. after all distillations were complete, the eo was separated from water, measured on an analytical scale, and kept in a freezer until the gas chromatography (gc) analyses could be performed. hemp volatile compounds were analyzed by gc-fid and gc-ms as described previously [ ] , with the following modifications: the column temperature was initially set at • c, then increased to • c at a rate of • c/min, which was held for min. the flow rate of the carrier gas (he) was maintained at . ml/min. the injection volume was . µl at a split ratio of : . the temperatures of the ionization source, the transfer line, and the injector were • c, • c, and • c, respectively. the msd was operated in full scan mode. all mass spectra were acquired in electron impact (ei) mode with ev in the m/z range of - . the injector and detector temperature (fid) was set at • c and • c, respectively. all constituents present in the eo samples were identified by comparing their linear retention indices (lri) and ms fragmentation patterns with those from the national institute of standards and technology (nist ) and adams mass spectra library. the estimated lri were determined using a mixture of a homologous series of aliphatic hydrocarbons from c to c under the same conditions described above. for the agar disc diffusion method, a µl of cfu/ml bacterial suspension after incubation was spread on the mueller hinton agar (mha, oxoid, basingstoke, uk). filter paper discs ( mm in diameter) were infused with µl of the eo, tested, and placed on the inoculated mha. mha was kept at • c for h and then at • c for h. for yeasts, µl of the yeast suspension was spread on sabouraud agar (sa, oxoid, basingstoke, uk) and agars were cultivated at • c for h. after the incubation period, the diameter of inhibition zones was measured (mm). growth inhibition was compared with the standard drugs. the standard drugs cefoxitin for g − bacteria, gentamycin for g + bacteria and fluconazole for yeasts were used as positive controls. tests were performed in three separate experiments, and the means were calculated. one-way analysis of variance (anova) with two replications was completed to determine the significance of differences among the mean oil content and constituents obtained from nine cultivars. the constituents were: α-pinene, β-pinene, isocaryophyllene (γ-caryophyllene), β-caryophyllene, α-(e)-bergamotene, (z)-β-farnesene, caryophyllene oxide, humulene epoxide , selina- -en- -ol, caryophylla- ( ), ( )-dien- α-ol, caryophylla- ( ), ( )-dien- β-ol, -hydroxy-(z)-caryophyllene, β-bisabolol, α-bisabolol, cbd, and δ -tetrahydrocannabinol (dronabinol). this completely randomized design has two replications. the differences among the mean antimicrobial activities (sa, ef, sp, pa, ye, se, ca, ck, and ct) obtained from the seven registered cultivars (bacalmas, carmagnola, cs, dioica, helena, sequieni, and simba) and seven wild accessions (buro, daleka zemlia, paluka, perez, saykaj, susara, and titelski breg) were also compared using one-way anova with three replications. the analyses were completed using the mixed procedure of sas [ ] . since the effect of cultivar was significant (p-value < . ) or marginally significant ( . ≤ p-value < . ) on most of the constituents, further multiple means comparison was completed using the lsmeans statement of proc mixed (equivalent to fisher's least significant difference [lsd]) at % level of significance and letter groupings were generated. the overall mean was calculated for the constituents where cultivar effect was not significant. for the antimicrobial activities, cultivar effect was highly significant (p-value < . ) on all nine activities, and considering the large number ( ) of cultivars and accessions compared, multiple means comparison was done using tukey's multiple range test, which controls the type ii experiment-wise error rate. for each response variable, the validity of model assumptions was verified by examining the residuals as described in montgomery [ ] . the results from this study demonstrated that the essential oil (eo) of wild hemp from serbia is different in its chemical profile and bioactivity from the eo of registered industrial hemp cultivars, the breeding lines, and the hemp strain grown for cbd production in north america. wild hemp eos were also somewhat different from the eo profile of wild and spontaneous hemps collected in austria and hungary, as reported in the literature. however, although having been named differently, the wild hemp in this study, the wild hemp collected in austria, and the spontaneous hemp from hungary might have common origins; the collection sites were approximately in the same region (with a dimeter of approximately km) although collected in three different countries. these wild/spontaneous hemps have been exposed to significant environmental and agricultural (pesticide) pressure over the last few decades. these populations may all belong to cannabis sativa var. spontanea vavilov, (a synonym of cannabis sativa l.), considered native to central and eastern europe and parts of asia. however, the taxonomy of hemp is still debatable and there is no consensus among taxonomists as to whether it is a single species with several subspecies or multiple species. the concentration of the four major constituents in the industrial hemp lines and wild hemp varied as follows: β-caryophyllene to % and . to . %; α-humulene (α-caryophyllene) . to . % and . to . %; caryophyllene oxide, . to . % and . to . %; humulene epoxide , . to . % and . to . %, respectively. the major eo constituents in the usa hemp strain that was grown in the vicinity of the field trials had different chemical profiles, with the major constituents myrcene ( . to %), β-caryophyllene ( . to . %), limonene ( . to . %), β-(e)-ocimene ( . to . %), and α-bisabolol ( . to . %). overall, the eo of wild hemp has shown greater antimicrobial activity against staphylococcus susp. aureus, enterococcus faecalis, and g−yersinia enterocolitica, salmonella enterica subsp. enterica, and yeasts candida albicans and candida tropicalis compared with the eo of registered cultivars. this is the first report to show that a significant amount of cbd can be accumulated in the eo of wild and registered cultivars of hemp following hydro-distillation. one of the wild hemp eos showed a very high concentration of cbd. the eo of the wild hemp had a significantly higher concentration of thc relative to the one from the registered eu cultivars of industrial hemp breeding lines. in addition, it was interesting to see that wild hemp had a higher concentration of cbd in the eo relative to the eo from a strain grown for commercial production of cbd in the usa therefore, wild hemp collected in serbia could be used for the development of varieties (registered cultivars) with specific desirable chemical composition, and may also provide excellent material for the selection and breeding of hemp cultivars with high cbd for the commercial production of cbd and other high-value chemicals. supplementary materials: the following are available online, table s . essential oil constituents of wild hemp accessions, in % of total oil. table s . essential oil constituents of new hemp breeding lines in % of total oil (area under the curve). table s . essential oil constituents of registered hemp cultivars. cannabis sativa: the plant of the thousand and one molecules organic trade association the field of dreams. in an economic survey of the united states hemp cultivation industry repellent activity of essential oils from seven aromatic plants grown in colombia against sitophilus zeamais motschulsky (coleoptera) taming thc: potential cannabis synergy and phytocannabinoid-terpenoid entourage effects chemistry and analysis of phytocannabinoids and other cannabis constituents method for the analysis of cannabinoids and terpenes in cannabis evolution and classification of cannabis sativa (marijuana, hemp) in relation to human utilization metabolic fingerprinting of cannabis sativa l., cannabinoids and terpenoids for chemotaxonomic and drug standardization purposes fiber hemp inflorescences; from crop-residues to essential oil production terpenoid chemoprofiles distinguish drug-type cannabis sativa l. cultivars in nevada terpene synthases from cannabis sativa chromatographic analyses, in vitro biological activities, and cytotoxicity of cannabis sativa l. essential oil: a multidisciplinary study characterization and antimicrobial activity of essential oils of industrial hemp varieties (cannabis sativa l.) essential oil composition obtained from cannabis sativa growing wild in the urban area of vienna, austria genetics and selection of hemp the crop-residue of fiber hemp cv. futura : from a waste product to a source of botanical insecticides chemical characterization of leaves, male and female flowers from spontaneous cannabis (cannabis sativa l.) growing in hungary new methods for the comprehensive analysis of bioactive compounds in cannabis sativa l. (hemp). molcules cannabis sativa and humulus lupulus essential oils as novel control tools against the invasive mosquito aedes albopictus and fresh water snail physella acuta beta-caryophyllene is a dietary cannabinoid the essential oil from industrial hemp (cannabis sativa l.) by-products as an effective tool for insect pest management in organic crops variation in the compositions of cannabinoid and terpenoids in cannabis sativa derived from inflorescence position along the stem and extraction methods valorizing industrial hemp (cannabis sativa l.) by-products: cannabidiol enrichment in the inflorescence essential oil optimizing sample pre-treatment prior to distillation cannabidiol-enriched hemp essential oil obtained by an optimized microwave-assisted extraction using a central composite design production of ∆ -tetrahydrocannabinolic acid from cannabigerolic acid by whole cells of pichia (komagataella) pastoris expressing ∆ -tetrahydrocannabinolic acid synthase from cannabis sativa l constituents of cannabis sativa. in handbook of cannabis cannabis guide grinding and fractionation during distillation alter hemp essential oil profile and its antimicrobial activity chemical characterization and evaluation of the antibacterial activity of essential oils from fibre-type cannabis sativa l. (hemp) essential oils of different cultivars ofcannabis sativa l. and their antimicrobial activity. flavour fragr isolation, purification, and antimicrobial characterization of cannabidiolic acid and cannabidiol from cannabis sativa l synergistic effects of antimicrobial peptides and antibiotics against clostridium difficile azithromycin synergizes with cationic antimicrobial peptides to exert bactericidal and therapeutic activity against highly multidrug-resistant gram-negative bacterial pathogens agricultural species-varieties. hemp-cannabis sativa controversial taxonomy of hemp cannabis: evolution and ethnobotany chemical composition of the essential oil of the endemic species micromeria frivaldszkyana (degen) velen user's guide sample availability: samples of the essential oils are not available from the authors. © by the authors the authors are grateful to the institute for field and vegetable crops in novi sad, serbia; oregon state university, usa; the global hemp innovation center (ghic) in corvallis, or, usa.; university of novi sad, department of field and vegetable crops; the plant genetic research group, agrobioinstitute in sofia, bulgaria; slovak university of agriculture in nitra, slovakia; the university of rzeszow, poland; and the agricultural university, plovdiv, bulgaria. these institutions provided significant in-kind support such as access to laboratories and fields, and access to other infrastructure and research instrumentation. authors also thank michelle jeliazkova for critically reading and editing the final version of the manuscript. the authors declare no conflict of interest. key: cord- - l m yh authors: ishihara, masayuki; hata, yuuki; hiruma, sumiyo; takayama, tomohiro; nakamura, shingo; sato, yoko; ando, naoko; fukuda, koichi; murakami, kaoru; yokoe, hidetaka title: safety of concentrated bioshell calcium oxide water application for surface and skin disinfections against pathogenic microbes date: - - journal: molecules doi: . /molecules sha: doc_id: cord_uid: l m yh immediately post-production, commercially available bioshell calcium oxide (biscao) water is colorless, transparent, and strongly alkaline (ph . ), and is known to possess deodorizing properties and broad microbicidal activity. however, biscao water may represent a serious safety risk to the living body, given the strong alkalinity. this study aimed to investigate the safety of biscao water for use as an antiseptic/disinfectant despite concerns regarding its high alkalinity. the change over time in ph of biscao water was measured during air contact (stirring biscao water in ambient air). when sprayed on metal, plastic, wood piece, paper, and skin surfaces, the ph of biscao water decreased rapidly, providing a white powder coating upon drying. scanning electron microscopy images, energy dispersive x-ray elemental mapping, and x-ray diffractograms showed that the dried powder residues of biscao water were composed primarily of calcium carbonate. these results suggested that biscao water is a potent reagent that may overcome the obstacles of being strongly alkaline, making this material appropriate for use in disinfection against pathogenic microbes. the recent epidemic of coronavirus disease , caused by a newly discovered coronavirus sars-cov- , is a worldwide crisis [ ] [ ] [ ] . some antiseptics/disinfectants, such as ethanol and naclo, show significant activity with broad microbicidal and antiviral activities, notably, activity against sars-cov- results from disruption of the viral envelope. however, such antiseptics/disinfectants are cytotoxic to cellular and organic components [ ] [ ] [ ] and require high concentrations for disinfection activity [ ] [ ] [ ] . furthermore, chlorine-derived disinfectants are ineffective in the presence of organic compounds and may generate compounds harmful to the environment and living tissue [ ] [ ] [ ] . therefore, novel disinfectants that can decrease the bacterial and viral bioburden without harmful side effects and environmental disruption are still desired for environmental hygiene and public health. biscao water was freeze-dried for sem observation and energy dispersive x-ray (edx) elemental mapping of particles contained. the images and edx spectra are shown in figure . the edx elemental mapping showed that the particles contained in biscao water were composed of oxygen, carbon, and calcium. the dried white powder was insoluble in water, and the ph of the supernatant of a suspension of this powder was below . these results suggested that the observed particles were generated by an interaction between ca + ions in biscao water and co in the air. furthermore, x-ray diffraction analysis revealed that the dried powder obtained from biscao water consisted primarily of calcium carbonate (caco ) (figure ). molecules , , x of biscao water was freeze-dried for sem observation and energy dispersive x-ray (edx) elemental mapping of particles contained. the images and edx spectra are shown in figure . the edx elemental mapping showed that the particles contained in biscao water were composed of oxygen, carbon, and calcium. the dried white powder was insoluble in water, and the ph of the supernatant of a suspension of this powder was below . these results suggested that the observed particles were generated by an interaction between ca + ions in biscao water and co in the air. furthermore, x-ray diffraction analysis revealed that the dried powder obtained from biscao water consisted primarily of calcium carbonate (caco ) (figure ). scanning electron microscopy (sem) and energy dispersive x-ray (edx) elemental mapping images and edx spectra of freeze-dried bioshell calcium oxide (biscao) water. two different regions of the sample were analyzed (left and right). yellow, red, and green pictures show the presence of carbon, oxygen, and calcium, respectively, by edx elemental mapping. when biscao water was exposed to air with stirring for h, the liquid became cloudy. furthermore, the ph decreased to . and . after and h, respectively, as shown in figure . following drying, the resulting white powder was insoluble; the supernatant of a suspension of this white powder had a ph of . . on the other hand, the ph of a biscao suspension ( . wt.% biscao) [ , , ] , dispersion ( . wt.% biscao + . wt.% na hpo ) [ ] , and colloidal dispersion ( . wt.% biscao + . wt.% pp) [ ] did not change during h of stirring in open air. these results suggest that the insoluble powder was caco generated by an interaction between ca + ions in biscao water and co in the air, and that the biscao suspension, dispersion, and colloidal dispersion contained insoluble cao and/or ca(oh) in the form of micro-/nano-particles or precipitates that provide hydroxyl ions (oh -) to maintain the alkaline ph. when biscao water was exposed to air with stirring for h, the liquid became cloudy. furthermore, the ph decreased to . and . after and h, respectively, as shown in figure . following drying, the resulting white powder was insoluble; the supernatant of a suspension of this white powder had a ph of . . on the other hand, the ph of a biscao suspension ( . wt.% biscao) [ , , ] , dispersion ( . wt.% biscao + . wt.% na hpo ) [ ] , and colloidal dispersion ( . wt.% biscao + . wt.% pp) [ ] did not change during h of stirring in open air. these results suggest that the insoluble powder was caco generated by an interaction between ca + ions in biscao water and co in the air, and that the biscao suspension, dispersion, and colloidal dispersion contained insoluble cao and/or ca(oh) in the form of micro-/nano-particles or precipitates that provide hydroxyl ions (oh -) to maintain the alkaline ph. when biscao water was exposed to air with stirring for h, the liquid became cloudy. furthermore, the ph decreased to . and . after and h, respectively, as shown in figure . following drying, the resulting white powder was insoluble; the supernatant of a suspension of this white powder had a ph of . . on the other hand, the ph of a biscao suspension ( . wt.% biscao) [ , , ] , dispersion ( . wt.% biscao + . wt.% na hpo ) [ ] , and colloidal dispersion ( . wt.% biscao + . wt.% pp) [ ] did not change during h of stirring in open air. these results suggest that the insoluble powder was caco generated by an interaction between ca + ions in biscao water and co in the air, and that the biscao suspension, dispersion, and colloidal dispersion contained insoluble cao and/or ca(oh) in the form of micro-/nano-particles or precipitates that provide hydroxyl ions (oh -) to maintain the alkaline ph. biscao water was sprayed onto the surfaces of a plastic plate, a steel plate, a piece of wood, and tissue paper (kimwipe), and the ph of each wet surface was measured over the course of the next min (figure ). for all tested surfaces, the ph immediately after spraying was . ± . , a value that was lower than the original ph of biscao water (i.e., ph . ). the ph of the surfaces fell below after min, and continued falling to less than ph and after and min, respectively. molecules , , x of biscao water was sprayed onto the surfaces of a plastic plate, a steel plate, a piece of wood, and tissue paper (kimwipe), and the ph of each wet surface was measured over the course of the next min (figure ). for all tested surfaces, the ph immediately after spraying was . ± . , a value that was lower than the original ph of biscao water (i.e., ph . ). the ph of the surfaces fell below after min, and continued falling to less than ph and after and min, respectively. in a parallel experiment, biscao water (~ ml) was sprayed onto the back skin of hairless rat, which then were rubbed lightly, and the ph of each wet skin was measured over the course of the following min ( figure ). the ph of the wet skin immediately after spraying was . ± . , a value that was lower than the original ph of biscao water (i.e., ph . ). the phs on the backs subsequently fell to ph . after min, ph . after min, and ph . after min. we confirmed that none of the skin exhibited chapping or inflammation from use of biscao water. in a parallel experiment, biscao water (~ ml) was sprayed onto the back skin of hairless rat, which then were rubbed lightly, and the ph of each wet skin was measured over the course of the following min ( figure ). the ph of the wet skin immediately after spraying was . ± . , a value that was lower than the original ph of biscao water (i.e., ph . ). the phs on the backs subsequently fell to ph . after min, ph . after min, and ph . after min. we confirmed that none of the skin exhibited chapping or inflammation from use of biscao water. molecules , , x of biscao water was sprayed onto the surfaces of a plastic plate, a steel plate, a piece of wood, and tissue paper (kimwipe), and the ph of each wet surface was measured over the course of the next min (figure ). for all tested surfaces, the ph immediately after spraying was . ± . , a value that was lower than the original ph of biscao water (i.e., ph . ). the ph of the surfaces fell below after min, and continued falling to less than ph and after and min, respectively. in a parallel experiment, biscao water (~ ml) was sprayed onto the back skin of hairless rat, which then were rubbed lightly, and the ph of each wet skin was measured over the course of the following min ( figure ). the ph of the wet skin immediately after spraying was . ± . , a value that was lower than the original ph of biscao water (i.e., ph . ). the phs on the backs subsequently fell to ph . after min, ph . after min, and ph . after min. we confirmed that none of the skin exhibited chapping or inflammation from use of biscao water. during incubation of bath tub water with % dulbecco's modified eagle's medium (dmem) and . wt.% bovine serum albumin (bsa) at • c for h the tc and cf values increased from ± colony-forming unit (cfu)/ml and ± cfu/ml (respectively) to . × ± . × cfu/ml and . × ± . × cfu/ml, respectively. the cfu/ml for tc and cf following treatment for min with undiluted (final two-fold diluted) and two-fold diluted (final four-fold diluted) biscao water and . and . wt.% (final . and . wt.%, respectively) of biscao suspension, dispersion, and colloidal dispersion were below the detection limit (< cfu/ml), whereas - and - , cfu/ml of both tc and cf remained viable following treatment with . wt.% (final . wt.%) and . wt.% (final . wt.%) of biscao suspension, dispersion, and colloidal dispersion, respectively ( figure ). in contrast, around , cfu/ml of tc and cf remained viable, even following treatment with undiluted (final -fold diluted) biscao water, when the reagent had been subjected to h of stirring in ambient air. molecules , , x of during incubation of bath tub water with % dulbecco's modified eagle's medium (dmem) and . wt.% bovine serum albumin (bsa) at °c for h the tc and cf values increased from ± colony-forming unit (cfu)/ml and ± cfu/ml (respectively) to . × ± . × cfu/ml and . × ± . × cfu/ml, respectively. the cfu/ml for tc and cf following treatment for min with undiluted (final two-fold diluted) and two-fold diluted (final four-fold diluted) biscao water and . and . wt.% (final . and . wt.%, respectively) of biscao suspension, dispersion, and colloidal dispersion were below the detection limit (< cfu/ml), whereas - and - , cfu/ml of both tc and cf remained viable following treatment with . wt.% (final . wt.%) and . wt.% (final . wt.%) of biscao suspension, dispersion, and colloidal dispersion, respectively ( figure ). in contrast, around , cfu/ml of tc and cf remained viable, even following treatment with undiluted (final -fold diluted) biscao water, when the reagent had been subjected to h of stirring in ambient air. the cfu/ml for tc and cf released from the contaminated wood pieces was . × ± . × cfu/ml and . × ± . × cfu/ml, respectively. although the cfu/ml for tc and cf following treatment with undiluted (final two-fold diluted) biscao water and . (final . wt.%) biscao suspension, dispersion, and colloidal dispersion exhibited high disinfection activities (> log decreases in cfu/ml), a small part of tc (> cfu/ml) and cf (> cfu/ml) remained viable (figure ) . the cfu/ml for tc and cf following treatment with two-fold diluted (final four-fold diluted) biscao water . wt.% (final . wt.%,) biscao suspension, dispersion, and colloidal dispersion exhibited > log decreases, while - cfu/ml of tc and - cfu/ml of cf remained viable. in contrast, biscao water that had been subjected to -h stirring in ambient air exhibited weaker sterilization activity against both tc and cf, with tc (about , cfu/ml) and cf (about cfu/ml) remaining viable even following treatment even with undiluted (final two-fold diluted) biscao water with h stirring (figure ) . on the other hand, in biscao suspension, dispersion, and colloidal dispersion the ph did not decrease at all with stirring for h and their microbicidal activities against tc and cf also did not decrease (data not shown). the cfu/ml for tc and cf released from the contaminated wood pieces was . × ± . × cfu/ml and . × ± . × cfu/ml, respectively. although the cfu/ml for tc and cf following treatment with undiluted (final two-fold diluted) biscao water and . (final . wt.%) biscao suspension, dispersion, and colloidal dispersion exhibited high disinfection activities (> log decreases in cfu/ml), a small part of tc (> cfu/ml) and cf (> cfu/ml) remained viable (figure ) . the cfu/ml for tc and cf following treatment with two-fold diluted (final four-fold diluted) biscao water . wt.% (final . wt.%,) biscao suspension, dispersion, and colloidal dispersion exhibited > log decreases, while - cfu/ml of tc and - cfu/ml of cf remained viable. in contrast, biscao water that had been subjected to -h stirring in ambient air exhibited weaker sterilization activity against both tc and cf, with tc (about , cfu/ml) and cf (about cfu/ml) remaining viable even following treatment even with undiluted (final twofold diluted) biscao water with h stirring (figure ) . on the other hand, in biscao suspension, dispersion, and colloidal dispersion the ph did not decrease at all with stirring for h and their microbicidal activities against tc and cf also did not decrease (data not shown). proper action plans for cleaning and disinfection play a critical role for minimizing the spread of infectious diseases such as covid- during an outbreak. an effective action plan should outline what should be cleaned, the frequency of cleaning, the proper materials and techniques for disinfection, and the training of healthcare workers. the action plans using proper materials and techniques will support a facility's pandemic response [ ] [ ] [ ] , ] . biscao water, which is a colorless and transparent disinfectant with a ph of . , can be sprayed and dried on smooth metal or plastic surfaces, providing a white powder coating. previously, sem observation for the dried powder obtained from biscao water showed the presence of agglomerated microparticles ( - µm) [ ] . in addition, cryo-sem observation has indicated that aggregates ( - nm in size) of nanoparticles ( - nm) were contained in biscao water [ ] . in the present work, elemental mapping of particles from biscao water revealed that the particles in the cloudy, air-exposed reagent, and the dried powder were composed of oxygen, carbon, and calcium ( figure ). the dried white powder obtained from biscao water was insoluble in water, and the ph of the resulting supernatant was less than . furthermore, x-ray diffraction analysis confirmed that the dried powder recovered from biscao water consisted primarily of caco (figure ). when biscao water was exposed to air with stirring, the liquid became cloudy after h. furthermore, the ph decreased to approximately . and . after and h of stirring, respectively ( figure ). in contrast, the ph of a biscao suspension, dispersion, and colloidal dispersion did not decrease when treated similarly. these results suggest that the insoluble powder from biscao water is caco , which presumably is generated by the interaction between ca + ions in biscao water and co in the air, and that a biscao suspension, dispersion, and colloidal dispersion contained insoluble cao and/or ca(oh) in the form of micro-/nano-particles or precipitates that provide hydroxyl ions (oh -) to maintain the elevated ph. biscao water after h stirring, which had decreased ph and contained caco , exhibited much less microbicidal activities than did fresh biscao water (figures and ) . we have previously reported that biscao water was a potent reagent with excellent deodorization and disinfection activities against pathogenic bacteria and viruses including both enveloped and nonenveloped viruses due to the strong alkaline [ ] . however, there has been serious concern about the safety of biscao water for use on living tissues, given that this reagent has a ph exceeding . . this study showed that biscao water, when biscao water was sprayed onto a variety of surfaces (plastic plate, steel plate, wood, and paper), gave an initial ph of . ± . , a value lower than the original value (ph . ) for biscao water, with the ph on the surfaces subsequently and rapidly decreasing to below , , and after , , and min, respectively ( figure ) . thus, the high ph of this fresh reagent rapidly decreases on surfaces and skin due to the generation of caco that lack obstacles following the interaction between ca + ions in biscao water and co in the air. in fact, caco powder is used as a gastric antacid [ ] , an anti-erosive agent [ ] , and a plant fertilizer [ ] . we infer that the ingestion of a small amount of biscao water would not cause problem if swallowed or inhaled, given that caco powder will be converted to a safe and soluble compound (calcium bicarbonate (ca(hco ) )) upon further interaction between caco and co . these results in this study suggest that spraying biscao water onto the surfaces of various materials is safe cleaning procedure in spite of its strong initial alkalinity in addition to the strong disinfectant activity. furthermore, we have already reported that cleansing p. aeruginosa-infected wounds with transparent supernatant liquids of . wt.% biscao suspension for days enhanced removal of the bioburden and wound repair without any complications such as acute inflammation, abscess formation, or seroma accumulation [ ] . similarly, the ph of biscao water sprayed onto hairless rat skin fell to ph . after min and ph . after min of application ( figure ) without apparent harmful effects such as rough skin. thus, biscao water appears to be friendly to skin. nevertheless, it is necessary to perform the safety study for biscao water to apply onto human such as hand sterilization and mouth cleaning with official ethics committee approval on use of human subjects. the world health organization (who) recommends "to ensure that environmental cleaning and disinfection procedures are followed consistently and correctly. thoroughly cleaning environmental surfaces with water and detergent and applying commonly used hospital-level disinfectants (such as sodium hypochlorite) are effective and sufficient procedures" [ ] . although some antiseptics/disinfectants, including ethanol and naclo, exhibit significant activity against sars-cov- by damaging the virus envelope, they are cytotoxic to cellular and organic components and require high concentrations for sufficient activity [ ] [ ] [ ] . furthermore, the presence of organic materials significantly diminishes the activity of chlorine-derived compounds [ , ] . therefore, novel antiseptics/disinfectants without harmful side effects or environmental disruption are still desired for environmental hygiene and public health. we anticipate that biscao water with attractive characteristics, including its apparent safety and virucidal activity against enveloped viruses, may be a trump card against respiratory viruses such as sars-cov- , even though further studies are required to validate the activity of biscao water against critical pathogens, including the coronavirus. scallop shell powder that had been heated at • c for h (average biscao particle diameter of µm) was purchased from plus lab corp., kanagawa, japan. according to the manufacturer, the cao concentration in all biscao preparations exceeded %. na hpo ( - ) and na-polypo (pp) ( - ) were purchased from fuji film wako pure chemical corp., osaka, japan. biscao was added to pure water followed by rotary mixing to generate biscao suspensions, to which a solution of % na hpo or % pp compared with biscao was added to prepare biscao dispersions or biscao colloidal dispersions, respectively [ , ] . biscao water was purchased from plus lab corp. according the manufacturer, biscao water was prepared by adding g of biscao to ml chilled (< • c) clean water; the combination then was gently mixed and allowed to stand for min. the resulting supernatant ( ml) was decanted to a l water tank. another chilled ml of clean water was gently added to the remaining biscao precipitate; again the combination was gently mixed and allowed to stand for min, and the supernatant was decanted into the collection tank. this process was repeated for a total of a hundred times. the resulting biscao water (total l) was obtained commercially from plus lab corp. sem observation and edx elemental mapping of freeze-dried powder of biscao water were performed by outsourcing to jeol, ltd., tokyo, japan. the observations were conducted using a jeol jsm- f microscope (jeol ltd., tokyo, japan) equipped with a jed- edx analysis system at an accelerating voltage of kv at room temperature. the content of dried powder derived from biscao water was determined using an x-ray diffractometer system (phillips x'pert-pro; phillips japan, ltd., japan). this analysis was performed at the kanagawa institute of industrial science and technology. each aliquot ( ml) of biscao water, biscao suspension ( . wt.% biscao) [ , ] , dispersion ( . wt.% biscao + . wt.% na hpo ) [ ] , or colloidal dispersion ( . wt.% biscao + . wt.% pp)) [ ] was dispensed into a -ml beaker and actively stirring using digital stirrer/hotplate (corning japan inc., tokyo, japan) with stirring bar ( mm in diameter × mm) (as one corp. osaka, japan) for h at room temperature in ambient air. the ph of each biscao formulation was measured (using a bench ph meter; f- , horiba, ltd., kyoto, japan) at , , , , , , and h during the process. when an aliquot (approximately ml) of biscao water was sprayed onto a defined surface (about cm × cm) of a culture plate and a steel plate, generations of large and small droplets (diameter: - mm, thickness: . - mm) were observed. the ph of large droplet was measured every min during min using diagonally placed micro tough electrode ( s- d; horiba, ltd.) attached to a bench ph meter (laqua ph meter, f- , horiba, ltd.). similarly, approximately ml of biscao water were sprayed onto the back skin of hairless rats (male, - g) from japan slc inc., shizuoka, japan and maintained under appropriate condition (i.e., • c and % humidity). in the experiment, the rats were placed under general anesthesia by intraperitoneal injection of pentobarbital sodium (dainippon sumitomo parma co., ltd., osaka, japan) [ , , ] . the ph of biscao water on the back skin of the rats was measured every min during min using diagonally placed micro tough electrode attached to the ph bench meter. all animal experiments were approved by the national defense medical college, tokorozawa, saitama, japan, and carried out following the relevant guidelines for animal experimentation (approval number, , / / ). on the other hand, the spraying approximately ml of biscao water onto surface (about cm × cm) of wooden pieces and paper sheets (kimwipe, nippon paper crecia, co., ltd., tokyo, japan) were permeated into them and did not generate droplets. however, the ph of their wet surface could be measured using diagonally placed micro tough electrode attached to the ph bench meter for min. we investigated the microbicidal efficacy of variously dilutions of biscao water and biscao water with -h stirring, as well as various concentrations of biscao suspension, dispersion, and colloidal dispersion. these reagents were tested against a contaminated suspension comprising normal bacterial flora (cf and tc), which was prepared by incubating the leftover bath water with % dmem and . wt% bsa at • c for h [ , , ] . equal volumes of each test sample and the contaminated suspension were mixed well and incubated at room temperature for min, and then the density of cfu/ml per sample was determined. for the disinfection assay of contaminated wood pieces, a wood piece was added to ml of each disinfectant, rinsed gently for min, and then tc and cf were released from the contaminated wood piece in ml of clean sterile water by vigorous vortexing for min. to evaluate cfu, ml of each mixture was gently added to individual petri dishes with pre-aliquoted portions of simple and easy dry medium for tc or cf (nissui pharmaceutical co., ltd., tokyo, japan) [ , , , ] , followed by incubation for h in a • c incubator (a ; ikuta sangyo co., ltd., ueda, nagano, japan). plating and counting experiments were conducted as a set of technical replicates (n = ). biscao water is colorless and transparent and has a ph of . . this reagent has higher deodorization and disinfection activities than do other microbicidal reagents such as ethanol, naclo, and povidone iodine [ , ] . however, concerns have been raised concerning the safety of biscao water when applied to the living body, given the strong alkalinity of this reagent. the present study showed that the high initial ph of biscao water following application to various surfaces and skin of hairless rat, rapidly decreases for areas that lack obstacles with the generation of caco as a result of the interaction between ca + in biscao water and co in the air. thus, the characteristics of biscao water, including both its safety and microbicidal activity, may be valuable for limiting the spread of various pathogenic microbes and viruses. persistence of coronaviruses on inanimate surfaces and their inactivation with biocidal agents covid- coronavirus: recommended personal protective equipment for the orthopaedic and trauma surgeon human coronavirus: insights into environmental resistance and its influence on the development of new antiseptic strategies a toxicity index of skin and wound cleaners used on in vitro fibroblasts and keratinocytes in vitro toxicity of topical antimicrobial agents to human fibroblasts cytotoxicity of silver nanoparticle and chitin-nanofiber sheet composites caused by oxidative stress stability of weakly acidic hypochlorous acid solution with microbicidal activity effects of a low concentration hypochlorous acid nasal irrigation solution on bacteria, fungi, and virus disinfection of pseudomonas aeruginosa-infected wounds in diabetic db/db mice by weakly acidic hypochlorous acid antimicrobial characteristics of heated scallop shell powder and its application antibacterial characteristics of heated scallop-shell nano-particles inactivation of avian influenza virus, newcastle disease virus and goose parvovirus using solution of nano-sized scallop shell powder sporicidal kinetics of bacillus subtilis spores by heated scallop shell powder comparison of antifungal activities of scallop shell, oyster shell and their pyrolyzed products disinfection treatment of heated scallop-shell powder on biofilm of escherichia coli atcc surrogated for e. coli o :h ability of heated scallop-shell powder to disinfect staphylococcus aureus biofilm heated scallop-shell powder treatment for deactivation and removal of listeria sp. biofilm formed at a low temperature comparison of various disinfectants on bactericidal activity under organic matter contaminated environments skin cleansing technique with disinfectant using improved high-velocity steam-air micromist jet spray development of antimicrobial biomaterials produced from chitin-nanofiber sheet/silver nanoparticle composites adsorption of silver nanoparticles onto different surface structures of chitin/chitosan and correlations with antimicrobial activities synthesis and application of silver nanoparticles (ag nps) for the prevention of infection in healthcare workers healing of pseudomonas aeruginosa-infected wounds in diabetic db/db mice by weakly acidic hypochlorous acid cleansing and silver nanoparticle/chitin-nanofiber sheet covering bioshell calcium oxide (biscao) for cleansing and healing of pseudomonas aeruginosa-infected wounds in hairless rats bioshell calcium oxide (biscao) ointment for the disinfection and healing of pseudomonas aeruginosa-infected wounds in hairless rats cleaning technique using high-velocity steam-air micromist jet spray preparation and application of bioshell calcium oxide (biscao) nanoparticles-dispersions with bactericidal activity application of colloidal dispersions of bioshell calcium oxide (biscao) for disinfection concentrated bioshell calcium oxide (biscao) water kills pathogenic microbes: characterization and activity evaluation of calcium hydrogen carbonate mesoscopic crystals as a disinfectant for influenza a viruses inactivation of scrapie prions by the electrically charged disinfectant cac- absorption characteristics of novel compound calcium carbonate granules: effect of gastric acid deficiency and exogenous weak acids anti-erosive effect of calcium carbonate suspensions influence of precipitated calcium carbonate (caco ) on shiitake (lentinula edodes) yield and mushroom size this article is an open access article distributed under the terms and conditions of the creative commons attribution (cc by) license the authors thank forte, inc. (www.forte-science.co.jp), for their english language editing services. the authors declare no conflict of interest. key: cord- - q tk fd authors: zhu, qinchang; liu, ge; kai, masaaki title: dna aptamers in the diagnosis and treatment of human diseases date: - - journal: molecules doi: . /molecules sha: doc_id: cord_uid: q tk fd aptamers have a promising role in the field of life science and have been extensively researched for application as analytical tools, therapeutic agents and as vehicles for targeted drug delivery. compared with rna aptamers, dna aptamers have inherent advantages in stability and facility of generation and synthesis. to better understand the specific potential of dna aptamers, an overview of the progress in the generation and application of dna aptamers in human disease diagnosis and therapy are presented in this review. special attention is given to researches that are relatively close to practical application. dna aptamers are expected to have great potential in the diagnosis and treatment of human diseases. aptamers can be broadly defined as short biomolecules like oligonucleotides and peptides that bind to specific targets with extremely high affinity based on their structural conformations. since the early s, systematic evolution of ligands by exponential enrichment (selex) and similar methods have been reported to efficiently select rna and dna aptamers [ ] [ ] [ ] [ ] . thereafter, nucleic acid aptamers have been extensively researched and applied. nucleic acid aptamers are rna and single-stranded (ss) dna oligonucleotides with lengths typically ranging from to mers, which have the same level of target-binding affinity as monoclonal antibodies (the dissociation constant (k d ) usually ranges from . to nm) [ , ] . compared with antibodies, nucleic acid aptamers have many advantages in their suitability for clinical application and industrialization, including almost no immunogenicity, efficient penetration, less batch variation, easy modification, cost-effectiveness and short production times [ , ] . in the past years, much progress has been made in the use of nucleic acid aptamers, particularly rna aptamers, as therapeutic agents, diagnostic and analytical tools, vehicles for targeted drug delivery, biosensors and even genetic control devices [ , [ ] [ ] [ ] [ ] . until now, although only one aptamer (macugen, pfizer/eyetech) has been approved for the therapeutic use in the clinic [ ] , ten other aptamers are being evaluated at different stages of clinical trials [ , ] . currently, the application in diagnostics, research and development is expected to account for the largest share of the aptamer market. the global aptamer market was estimated to be valued at $ . million in and to reach $ . million by [ ] . dna aptamers are expected to account for the largest share of the global market in [ ] , which indicates an increase in the application of dna aptamers. dna and rna aptamers are functionally similar but have some differences in their stability and accessibility. compared with dna aptamers, rna aptamers are chemically unstable because of the presence of a reactive hydroxyl group (-oh) at the position of the ribose sugar in rna nucleotides. this -oh group easily gets deprotonated in solution, especially in alkaline solutions. the resulting anionic -o´may nucleophilically attack the phosphorus atom of the phosphodiester linkage, leading to the hydrolysis of rna molecules [ ] . the nuclease resistance of rna aptamers was found to increase when the -hydroxyl group was removed from the sugars of rna [ , ] . dna aptamers are less reactive and relatively stable because of the c-h bonds at the position of the deoxyribose sugar of dna nucleotides. this chemical difference gives dna aptamers an inherent advantage in stability over rna aptamers. our previous study also confirmed that dna aptamers are much more stable than natural rna aptamers in % fetal bovine serum (fbs) and human serum [ ] . that is the reason why extra chemical modifications are usually added to rna aptamers to improve their chemical stability [ ] . however, it should be noted that the reactivity of rna nucleotides and non-watson-crick base pairing makes rna oligonucleotides prone to forming more diverse and complex three-dimensional ( d) structures [ , ] , which is helpful for selecting aptamers with high affinity and specificity from rna libraries [ ] . therefore, dna aptamers are usually selected from the libraries containing longer randomized regions for the purpose of obtaining more complicated structures. another potential advantage of dna aptamers over rna aptamers is their relatively simple selection process. the selection of rna aptamers requires reverse transcription and in vitro transcription in every round of selection, as well as an initial transcription for generating the rna library from a dna library in most cases [ , ] . but the selection process of dna aptamers does not require these extra steps [ , ] . moreover, once selected, the cost of producing dna aptamers is lower than that for rna aptamers. since the first ssdna aptamer was selected for human thrombin in [ ] , dna aptamers have been researched and applied in various fields, especially in diagnosis and treatment of human diseases. dna aptamers are usually explained to work in a similar way to rna aptamers. because of the potential advantages described above, dna aptamers have gained increasing attention in recent years. however, they have seldom been reviewed systematically and independently. to better understand the unique potential of dna aptamers, this article will review the recent progress of dna aptamers in regard to their preparation and application in the diagnosis and treatment of human diseases. the advantages and remaining challenges to develop and use dna-aptamer-based diagnostic tools and therapeutics will also be discussed. selex, an interactive in vitro selection procedure, is the basic method used to engineer aptamers, which was first reported for screening of rna aptamers independently by ellington's and tuerk's groups years ago [ , ] . selex was then adapted to generate dna aptamers [ , ] . in principle, aptamers are selected from an initial random ssdna pool based on the binding of the oligonucleotides to the target molecules under optimal conditions. the ssdna pool contains - random sequences of synthetic dna (the typical length is - nucleotides, flanked by two constant regions with primer sites for polymerase chain reaction [pcr] amplification). the unbound sequences are separated from the bound molecules, and the target-bound sequences are amplified by pcr. the amplified products (double stranded dna) are converted to ssdna through various ways [ ] and then used as a new aptamer pool for the next selection round. enriched aptamer sequences are finally cloned and identified by sequencing. usually after - rounds of selection, the specific aptamers with the strongest affinity for the target molecules are obtained. selection of aptamers using conventional in vitro selex requires purified and soluble target proteins. the processes used to obtain purified protein targets are time-consuming. sometimes it is difficult to purify target proteins. moreover, sometimes the aptamers selected using a non-native protein do not interact with the protein in a native conformation. to solve these problems, a strategy using whole live cells as targets for aptamer selection has been developed, which is known as cell-selex. this selex is able to generate dna aptamers that recognize the cell-surface or intracellular target protein in their native conformation, which shows great potential in cell-specific therapeutics and diagnostic applications [ ] [ ] [ ] . this method first selects the specific aptamers that bind to the target cells by a positive selection step, and then eliminates the non-target cell-specific aptamers by a negative selection step using non-target cells, followed by the selex process as summarized in figure a . recently, modified cell-selex methods have been developed to select aptamers targeting specific cells like disease state cells and metastatic cells. they are stimulus-response cell-selex [ ] and metastatic-cell-based selex [ ] . to shorten the time of conventional cell-selex, a two-step stimulus-response cell-selex method has been developed, which utilizes asymmetric pcr or streptavidin-biotin magnetic separation for the generation of single-stranded dna to reduce the selection cycle to two steps [ ] . competitive cell-selex is another approach to improve selection efficiency and affinity, which utilizes a nitrocellulose membrane that contains the target cells and negative control cells to select target aptamers [ ] . this method can reduce the selection time, since the negative selection step is not required. the conventional selex procedure is time-consuming and requires several steps to obtain a specific dna aptamer. to effectively generate highly specific aptamers, several novel methods using different selection approaches have been developed. combining selex with other method is one of the most effective ways to further improve the selection efficiency and binding affinity of dna aptamers. for example, cell-selex coupled with in silico maturation was developed to improve aptamer specificity [ ] . the method consists of cell-selex, a post-selex in silico process and in vitro screening. after the normal cell-selex, an extra in silico process was performed to further evolve improved aptamers from the selex-selected sequences through successive rounds of sequence shuffling and random mutation based on a genetic algorithm. this is followed by in vitro functional screening and selection of enhanced aptamers from the shuffling and mutated sequences. this method permits the evolution of functionally enhanced aptamer sequences recognizing targets of interest. in addition to selex-like methods, other methods have also been developed; magnetic-assisted rapid aptamer selection (maras) is a method that uses magnetic beads and an externally applied rotating magnetic field to provide the competitive mechanism for the rapid selection of aptamers with different affinity to the molecular target as summarized in figure b [ , ] . this method uses biofunctionalized magnetic nanoparticles to separate target-bound dna oligonucleotides from a library, selecting those interactions that survive a disruptive force generated by the movement of the particles in an externally applied rotating magnetic field. it abandons the multi-cycle evolutionary process used in conventional selex and thus can achieve rapid selection (completed in less than one hour). a one-step selection approach is another promising way to increase the rate of aptamer generation. recently, this rapid one-step selection method was developed to select specific dna aptamers using only one pcr step as summarized in figure c [ ] . in this method, a target immobilized on a glass coverslip was subjected to carboxyfluorescein (fam)-labeled nucleic acid pool binding, extensive washing and microscopy examination, followed by pcr enrichment of the selected aptamers. a control experiment used a labeled target and a labeled dna library was used to make sure the specificity of the selection. in the overlay image of the immobilized target labeled with alexa fluor (red color) and target bound fam-labeled dna aptamer (green fluorescence), an orange color is observed for target-specific selection. although the binding affinity of aptamers selected with the current version of the one-step method was in the low micromolar range, it was believed that it can be further improved by using larger targets, increasing the stringency of selection, and by combining it with a capillary electrophoresis separation. this method was described as a user-friendly, low-cost and easy way to select dna aptamers. in particular, this method allows the use of a chemically modified nucleic acid library directly as it requires only one pcr step. moreover, a dna microarray has been utilized to achieve one-step aptamer identification, in which the sequences of interest can be produced on the arrays for selection and pcr, cloning and sequencing are not required [ , ] . molecules , , page-page selected with the current version of the one-step method was in the low micromolar range, it was believed that it can be further improved by using larger targets, increasing the stringency of selection, and by combining it with a capillary electrophoresis separation. this method was described as a user-friendly, low-cost and easy way to select dna aptamers. in particular, this method allows the use of a chemically modified nucleic acid library directly as it requires only one pcr step. moreover, a dna microarray has been utilized to achieve one-step aptamer identification, in which the sequences of interest can be produced on the arrays for selection and pcr, cloning and sequencing are not required [ , ] . the target-coated magnetic beads are incubated with the ssdna pool. the beads with bound sequences are then separated from the unbound sequences with a u-shaped magnet or magnetic stand. after re-dispersion, the beads are put in an externally applied rotating or alternating magnetic field. during this process, the weak and non-specific binding sequences are released and separated. the strong-binding sequences are finally released from the beads by heating and then incubated with the beads without a target for the negative selection. the selected sequences are amplified, cloned and sequenced as usual; (c) one-step selection: a fam-labeled oligonucleotide library is incubated with a target immobilized on a glass coverslip that was coated with n-hydroxysuccinimide (nhs) functionalized polyethylene glycol (peg). unbound sequences are discarded by extensive washing followed by monitoring with fluorescence microscopy. the coverslip is later crushed and the bound sequences are eluted by heating in water. the selected aptamers are amplified, cloned and sequenced as usual. dna aptamers are well known to be more stable than rna aptamers, which allows them to be readily used in the primary stage of developing diagnostic tools. however, the degradation of dna aptamers by nuclease is still a serious problem that limits their clinical application, when they are subjected to complicated biological samples. to improve the nuclease resistance of dna aptamers, one those that do not bind to negative targets are retained and amplified by pcr. the pcr products are separated into ssdna for further rounds. after - cycles, the selected ssdnas are cloned and sequenced for aptamer identification; (b) magnetic-assisted rapid aptamer selection (maras): the target-coated magnetic beads are incubated with the ssdna pool. the beads with bound sequences are then separated from the unbound sequences with a u-shaped magnet or magnetic stand. after re-dispersion, the beads are put in an externally applied rotating or alternating magnetic field. during this process, the weak and non-specific binding sequences are released and separated. the strong-binding sequences are finally released from the beads by heating and then incubated with the beads without a target for the negative selection. the selected sequences are amplified, cloned and sequenced as usual; (c) one-step selection: a fam-labeled oligonucleotide library is incubated with a target immobilized on a glass coverslip that was coated with n-hydroxysuccinimide (nhs) functionalized polyethylene glycol (peg). unbound sequences are discarded by extensive washing followed by monitoring with fluorescence microscopy. the coverslip is later crushed and the bound sequences are eluted by heating in water. the selected aptamers are amplified, cloned and sequenced as usual. dna aptamers are well known to be more stable than rna aptamers, which allows them to be readily used in the primary stage of developing diagnostic tools. however, the degradation of dna aptamers by nuclease is still a serious problem that limits their clinical application, when they are subjected to complicated biological samples. to improve the nuclease resistance of dna aptamers, one of the effective solutions is to use a library with chemically modified dna sequences in the screening process. the modified dna library can be prepared by pcr amplification using specific polymerase and catalysis reactions using modified dna enzymes [ , ] . modification of the aptamers can also be performed after the in vitro selection from natural nucleic acid library. modifications of the sugar phosphate backbone or the pyrimidine are the strategies used to increase the stability and nuclease resistance of aptamers [ ] [ ] [ ] . capping the end of aptamers is also utilized to enhance aptamer stability against nucleases [ ] . incorporation of unnatural nucleotides is another approach to overcome aptamer instability. locked nucleic acid (lna) is one of the most prominent and successful nucleic acid analogues because of their pronounced stability [ ] [ ] [ ] . generation of "mirror aptamers", which are also known as spiegelmers, is another approach to improve aptamer stability against nucleases [ ] . generation of highly specific aptamers with a long lifetime is very important for their therapeutic application. because most dna aptamers have a small molecular weight (ranging from - kda), dna aptamers are readily removed via renal filtration and metabolic processes, limiting their therapeutic application. one of the effective ways to control the lifetime of dna aptamer in vivo is to conjugate the aptamers with bioavailable materials. conjugation of dna aptamers with polyethylene glycol (peg) is commonly used to prolong their circulation in the bloodstream [ , ] . coating dna aptamers with other nanomaterials such as nanoparticles [ , ] , liposomes [ ] and copolymers [ ] has been successfully used to improve the lifetime of dna aptamers. once selected to bind to disease-related biomarkers or pathogens, dna aptamers can be developed as biosensors through chemical modification with luminophores or linkage to nanoparticles in various formats [ ] . for example, in an earlier study, dna aptamers selected for bacillus anthracic spores were developed to detect anthrax spores in an aptamer-magnetic bead-electrochemiluminescence (am-ecl) sandwich mode [ ] . the dynal m- magnetic beads were covered with the aptamer and used as the capturer to capture the spores, while another biotinylated aptamer was used as the reporter and the signal was finally transduced through the streptavidin-ru(bpy) + ecl. since the first dna aptamer was selected, dozens of dna aptamers have been selected for the use of disease-related detection. although there are no aptamer-based diagnostic tools that are in clinical use at the moment, many preclinical studies indicate that dna aptamers have great potential to be used in this way. to profile the features of recent studies that use dna aptamers as a diagnostic tool for human diseases, we listed some dna aptamers selected for this purpose in table , with a special attention to parameters like the sensitivity and specificity, which have a great impact on their potential for clinical use. in theory, aptamers can be selected for developing diagnostic tools for various diseases, as long as definite targets, such as disease-specific biomarkers or pathogens, are available. to date, dna aptamers have been explored mainly for the diagnosis of infectious diseases, cancer and cardiovascular diseases. because of the convenience of cell-selex, which does not require preparation and purification of target molecules, many reported dna aptamers are selected with cancer cells, aiming to diagnose and image cancer tissue. these cancer cells include pancreatic [ ] , colon [ ] , liver [ , ] , cholangio [ ] , gastric [ , ] , prostate [ ] , breast [ ] and glioblastoma [ ] cancer cells. usually, the selection consists of positive selection with cancer cells and negative selection with normal cells from the same organs, as described in section . . . it is worth noting that, because the diagnosis and monitoring of metastasis of cancer cells is important for the treatment of cancer, several dna aptamers have been selected with metastatic cancer cells and negatively selected with non-metastatic cancer cells [ , , ] . target-induced dissociation (aunp) . µm % (n = amino acids) [ ] ns.: non-specified. for example, li et al. selected dna aptamers for metastatic colon cancer cells using sw cells derived from a metastatic site lymph node in the positive selection and sw cells from a primary colon adenocarcinoma of the same patient in the negative selection [ ] . the resulting aptamer (xl- ) was found to possess specific affinity to the metastatic colon cancer cells (k d = . nm). its truncated form (xl- - ) was used to image the cancer tissue after labeling with fluorescein amidite (fam), displaying an . % detection rate against colon cancer tissue with metastasis in regional lymph nodes and . % specificity against nonmetastatic colon cancer tissue. the biomarker molecules that specifically express or overexpress on the cancer cells were also used for the selection of diagnostic dna aptamers. in addition to the previously known prostate-specific membrane antigen (psma) [ ] and mucin (muc ) [ ] , other biomarkers like epidermal growth factor receptor variant iii (egfrviii) [ ] , epithelial cell adhesion molecule (epcam) [ ] and vascular endothelial growth factor (vegf ) [ ] have also been used to select dna aptamers for cancer detection. by directly using cancer biomarkers to select cancer-cell-recognized aptamers, you obtain highly specific and affinitive aptamers, but the potential risk is that the aptamers might not recognize the natural biomarkers on the surface of cancer cells because of the difference in the d structure of the purified biomarkers and the natural biomarkers. for the diagnosis of infectious agents, lots of dna aptamers have been selected predominantly with cell-selex and magnetic beads-based methods by targeting various viruses and bacteria or their antigen protein [ ] , including targets such as norovirus, influenza virus, severe acute respiratory syndrome coronavirus (sars-cov), hepatitis c virus (hcv), hepatitis b virus (hbv), human immunodeficiency virus (hiv), human papillomavirus (hpv), salmonella typhimurium, and pathogenic e. coli. because of the low cost and short production time of dna aptamers, the dna aptamer based diagnostic tools hold particular advantages for the diagnosis of infections that need point-of-care testing, such as infections caused by highly pathogenic pandemic influenza virus, hiv, sars-cov and malaria. avian influenza virus h n is a highly pathogenic subtype of the influenza virus, which can also infect humans and has a high mortality rate. wang et al. selected a dna aptamer targeting h n using both the virus antigen and the whole virus particle using the selex method [ ] . for the first four selection cycles, the purified virus antigen hemagglutinin (ha) was used as the target protein, while for the remaining eight cycles the entire h n virus particles were used as the targets. the selected aptamer showed high affinity (k d = . nm) and specificity to the h n virus (table ). using such a mixed selection mode might be a good strategy to obtain ideal aptamers, which can overcome the drawbacks of selection using only free biomarkers or cells. in the case of cardiovascular diseases, markers in the blood, such as myoglobin, c-reactive protein, l-homocysteine and thrombin have been used to select dna aptamer for the diagnosis of related diseases. myoglobin increases after acute myocardial infarction, which is an important early marker in urgent diagnosis of cardiovascular diseases. wang et al. selected dna aptamers against myoglobin using a fluidic chip method [ ] . the dna aptamer with the lowest k d value ( . nm) was subjected to the development of different biosensors for the detection of myoglobin, including the myoglobin-induced structural switching supersandwich biosensor [ ] and the antibody-myoglobin-aptamer sandwich biosensor [ ] . human c-reactive protein (crp) is a homopentameric oligoprotein, which has been validated as a powerful predictor and risk factor of inflammation and cardiovascular disease [ ] . yang et al. selected a dna aptamer (k d = . nm) targeting crp and used it to develop a sensor based on surface plasmon resonance technology [ ] . l-homocysteine is an amino acid intermediate, whose elevated level in the blood is associated with coronary heart disease [ ] . mckeague et al. selected dna aptamers targeting l-homocysteine and developed an aptamer-aunp sensor, in which the dna aptamers first coil around the surface of the gold particles and release the particles after binding to the homocysteine, which leads to salt-induced aggregation and colorization of the particles [ ] . thrombin is a serine protease that plays a critical role in the formation of obstructive blood clots, or thrombosis, which is involved in various diseases including chronic cardiovascular diseases [ ] . the first reported dna aptamer is the thrombin aptamer. since then, thrombin aptamers have been used as model aptamers to explore the mode of aptamer-based biosensors. it is the most frequently used dna aptamer for the demonstration of the proof-of-principle of various detection methods [ ] . the general modalities and assay formats for using aptamers in sensors have been well summarized in many comprehensive reviews [ ] [ ] [ ] [ ] [ ] . they can be roughly classified into four types based on the way the signal is generated: ( ) direct binding-based mode; ( ) target-induced structural switching mode; ( ) sandwich-like mode; and ( ) target-induced assembly or dissociation mode. these protocols are illustrated in figure . in the direct binding-based mode (figure a ), aptamers labeled with signal molecules directly bind to the target. the signal molecules on the aptamer-bound target can be detected directly. the aptamers for cancer cells imaging usually use this mode (table ) . it is a simple and direct mode, which is especially suitable for the situations like in vivo imaging that do not allow complex detection. but it does not contain signal amplification, which makes it difficult to increase the detection sensitivity. in target-induced structural switching mode, the binding of the target will cause a specific conformational change to the aptamer, which will "switch on or switch off" the signal generation [ , ] . for example, in a bio-chromophoric target-induced structural switching approach, a fluorophore-labeled dna aptamer forms a partial duplex with a small oligonucleotide modified with a quenching moiety (denoted qdna) in the absence of the target, bringing the fluorophore (f) and the quencher (q) into close proximity for maximum fluorescence quenching. when the target is introduced, the aptamer prefers to form the aptamer-target complex. the binding of the target to the aptamer will change the structure of the aptamers and release the partially complementary quencher, which will trigger the increase of the signal ( figure b ). this mode is suited for the development of aptamer-based reporters for real-time sensing applications. but the limitation of this mode is the decreased aptamer binding affinity because of competition from the qdna [ ] . in the sandwich-like mode, the target is "the meat" inserted within "two pieces of bread" consisting of an aptamer/aptamer or antibody/aptamer, which is very similar to the enzyme-linked immunosorbent assay (elisa) ( figure c ). aptamers in this mode function as reporter and/or capturer. this method permits double recognition of the target and signal amplification, which can greatly improve the specificity and sensitivity of aptamer-based detection. moreover, because of the small size of the aptamer, the aptamer sandwich mode can conquer the potential limitation of antibody elisa in detecting small molecules [ ] . but this mode requires extensive washing during the detection. in the target-induced assembly or dissociation mode, binding of the target to the aptamer will trigger the assembly of split aptamers or dissociation of bound aptamers, along with the change of detectable signals [ , ] (figure d ). in addition, it should be noted that various detection methods have been applied by dna aptamer based biosensors, including electrochemical, chemiluminescence, fluorescence, colorimetric, quantum dots-based and mass-sensitive detections [ ] . for imaging of cancer cells, most of the dna aptamer based methods use fluorophore-labeled aptamers to recognize the cancer cells directly (table ) . for the small targets like microorganisms and the marker protein, methods based on different modes have been explored extensively. the sandwich-like mode is frequently used ( figure c ) [ , ] . in the example of leishmania detection [ ] , the aptamer (lmwc- r) targeting the promastigote of leishmania was immobilized on the surface of the m magnetic beads. after the target leishmania promastigote were captured by lmwc- r, another -biotinylated reporter aptamer (lmhsp- b/ r) that targets the hydrophilic surface protein (hsp) of leishmania promastigote was added. then the beads with aptamer-captured leishmania promastigote and reporters were collected on the magnetic rack. streptavidin-horseradish peroxidase (sav-hrp) and the substrate ample red were added to transduce and display the signal. another frequently used mode is the target-induced dissociation mode, in which the system was in a non-signal status until the single-stranded aptamer dissociated from the initial state as a result of binding to the target [ , , ] . the detection of l-homocysteine mentioned above is a good example of the target-induced dissociation mode [ ] (figure d ). dna aptamers coil around the surface of the gold nanoparticles (aunps) and prevent the salt-induced aggregation of aunps. when the target molecules appear, the dna aptamers fold into a rigid structure and bind to the targets, and therefore release from the aunps. the aunps without the coverage of aptamers aggregate in the salt solution and change color from red to purple. the signal-molecule-labeled aptamers directly bind to the immobilized or free target. the signal molecules on the target are detected directly; (b) target-induced structural switching mode: a fluorophore-labeled dna aptamer forms a partial duplex with a small oligonucleotide modified with a quenching moiety (qdna) in the absence of the target, bringing the fluorophore (f) and the quencher (q) into close proximity for maximum fluorescence quenching. when the target is introduced, the aptamer prefers to form the aptamer-target complex. the binding of the target to the aptamer will change the structure of the aptamers and release the partially complementary quencher, which will trigger the increase of the signal; (c) sandwich-like mode: the aptamer or antibody is immobilized on the solid phase as the capturer. the captured target is reported by the biotinylated aptamer, which displays the signal through further binding to the streptavidin-conjugated hrp or aunp; (d) target-induced dissociation mode: dna aptamers coil around the surface of the gold nanoparticles (aunps) and stop the salt-induced aggregation of aunps. when the target appears, the dna aptamers bind to the target and release from the aunps. the aunps without the cover of aptamers aggregate in the salt solution and change color from red to purple. one of the biggest challenges for aptamer-based diagnostic tools and their clinical use is the possible invalidation in a real biological environment as a result of degradation from nucleases or interference from other matrix factors. using chemical modified dna aptamers is an effective way to prolong the lifetime of dna aptamer in the biological samples. for ultimate success, sufficient studies with clinical samples or in vivo studies are indispensable. currently, although most of the studies about dna aptamer based diagnostic tools for human diseases are still in the primary stage of laboratory studies, some of them have gone further by testing them with clinical samples and animal studies in vivo [ , , , ] . the dna aptamer xq- d ( table ) that targets the pancreatic ductal adenocarcinomas (pdac) cells has been tested with clinical pdac tissue sections and in vivo imaging of pdac tumor-bearing mice [ ] . out of pdac tissues and two out of eight normal pancreatic tissues were detected by the cy -labeled xq- d. in the in vivo imaging test, cy -labeled xq- d was injected into the balb/c-nude mice grafted with pdac through the tail vein. it was found to illuminate the tumor site up to h post-injection, while the cy -labeled library control did not give any signal. for this study, both phosphorothioate and ′-o-methyl modifications were tried for the dna aptamer. the four bases at the ′ and ′ termini of xq- d were replaced by ether phosphorothioate oligonucleotides or ′-o-methyl oligonucleotides. however, the phosphorothioate modification was the signal-molecule-labeled aptamers directly bind to the immobilized or free target. the signal molecules on the target are detected directly; (b) target-induced structural switching mode: a fluorophore-labeled dna aptamer forms a partial duplex with a small oligonucleotide modified with a quenching moiety (qdna) in the absence of the target, bringing the fluorophore (f) and the quencher (q) into close proximity for maximum fluorescence quenching. when the target is introduced, the aptamer prefers to form the aptamer-target complex. the binding of the target to the aptamer will change the structure of the aptamers and release the partially complementary quencher, which will trigger the increase of the signal; (c) sandwich-like mode: the aptamer or antibody is immobilized on the solid phase as the capturer. the captured target is reported by the biotinylated aptamer, which displays the signal through further binding to the streptavidin-conjugated hrp or aunp; (d) target-induced dissociation mode: dna aptamers coil around the surface of the gold nanoparticles (aunps) and stop the salt-induced aggregation of aunps. when the target appears, the dna aptamers bind to the target and release from the aunps. the aunps without the cover of aptamers aggregate in the salt solution and change color from red to purple. one of the biggest challenges for aptamer-based diagnostic tools and their clinical use is the possible invalidation in a real biological environment as a result of degradation from nucleases or interference from other matrix factors. using chemical modified dna aptamers is an effective way to prolong the lifetime of dna aptamer in the biological samples. for ultimate success, sufficient studies with clinical samples or in vivo studies are indispensable. currently, although most of the studies about dna aptamer based diagnostic tools for human diseases are still in the primary stage of laboratory studies, some of them have gone further by testing them with clinical samples and animal studies in vivo [ , , , ] . the dna aptamer xq- d ( table ) that targets the pancreatic ductal adenocarcinomas (pdac) cells has been tested with clinical pdac tissue sections and in vivo imaging of pdac tumor-bearing mice [ ] . out of pdac tissues and two out of eight normal pancreatic tissues were detected by the cy -labeled xq- d. in the in vivo imaging test, cy -labeled xq- d was injected into the balb/c-nude mice grafted with pdac through the tail vein. it was found to illuminate the tumor site up to h post-injection, while the cy -labeled library control did not give any signal. for this study, both phosphorothioate and -o-methyl modifications were tried for the dna aptamer. the four bases at the and termini of xq- d were replaced by ether phosphorothioate oligonucleotides or -o-methyl oligonucleotides. however, the phosphorothioate modification was found to weaken the binding ability of the aptamer, whereas the -o-methyl modification did not significantly affect the binding ability. moreover, the -o-methyl modified xq- d could last for h in % fbs medium, but the unmodified xq- d was completely degraded after -h incubation. an important application of aptamers is their use as small-molecule therapeutic agents. dna aptamers exhibit significant advantages in therapeutic application and have been developed as attractive therapeutic agents in competition with antibodies. generally, dna aptamers used for therapeutic applications function in two ways. first, dna aptamers can inhibit protein-protein interactions by specifically binding to the target protein and thereby functioning as antagonists. second, dna aptamers can function as agonists, which promotes the function of the target protein upon binding to the target protein. although dna aptamers act similarly to antibodies, the non-immunogenicity of dna aptamers make it more notable in therapeutics. compared with antibodies, dna aptamers are easier to uptake because of their small size. importantly, aptamers can specifically recognize a wide range of targets including small molecules, proteins and cells. moreover, given that dna aptamers can be designed and selected in vitro, they have lower production cost than antibodies. the properties and advantages of dna aptamers mentioned above facilitate the promising application of dna aptamers in the field of therapeutics. although there are currently no dna aptamer therapeutics in clinical use, four dna aptamers are being evaluated in clinical trials for their effect on hematological disease, macular degeneration disease and cancer ( table ) . as (antisoma) is a guanine-rich aptamer with a guanine quadruplex structure, targeting nucleolin. nucleolin is a eukaryotic nucleolar phosphoprotein that is involved in the synthesis and maturation of ribosomes and has been reported as a target for anti-cancer therapies. the guanine quadruplex structure can help to enhance the nuclease degradation resistance and cell uptake of as . as possesses anti-cancer activity against breast cancer cells [ ] , metastatic renal cell carcinoma [ ] and acute myeloid leukemia [ ] . the phase clinical trial for using as to treat renal cell carcinoma was completed in (nct ), while another phase clinical trial for treating acute myeloid leukemia was completed in (nct and nct ). arc (achemix), a pegylated dna aptamer, recognizes platelet ligand receptor von willebrand factor that mediates platelet recruitment. arc blocks the binding between von willebrand factor and the platelet, thereby inducing an antithrombotic effect. the efficacy of arc in platelet inhibition has been demonstrated and a phase clinical trial for treating von willebrand factor related platelet function disorders (nct ) has been completed. recent studies show that arc can effectively prevent thromboembolism [ ] . a phase clinical study shows that arc exhibits favorable pharmacokinetic, pharmacodynamic and safety properties in patients with congenital thrombotic thrombocytopenic purpura [ ] . nu (arca biopharma) is an unmodified dna aptamer targeting thrombin, which can prolong blood clotting. the phase clinical trial using nu as anticoagulation agent in patients undergoing off-pump coronary artery bypass graft (cabg) surgery (snap-cabg-off) has been completed (nct ). e (ophthotech), a pegylated dna aptamer, functions as an antagonist of platelet-derived growth factor. e in combination with anti-vegf agent can effectively prevent angiogenesis [ ] . currently, a phase clinical study of e in combination with ranibizumab (lucentis ) for wet age-related macular degeneration treatment is ongoing (nct ). besides the dna aptamers in clinical trials, many promising dna aptamers are in preclinical studies for treating various diseases, such as virus infections, tumors and central nervous system diseases. a dna aptamer recognizing the receptor-binding region of influenza a hemagglutinin was found to inhibit viral infection in an animal model against different influenza strains, as manifested by a %- % reduction of the virus burden in the lungs of treated mice [ ] . dna aptamer nas- was found to bind to vimentin and then cause apoptosis of mouse ascites adenocarcinoma cells in vitro and in vivo [ ] . recently, dna aptamers targeting human epidermal growth factor receptor (erbb- /her ) was demonstrated to retard the tumorigenic growth of gastric cancer in mice with more effective activity than anti-erbb- /her monoclonal antibody [ ] . moreover, remyelination was induced by a dna aptamer in a mouse model of multiple sclerosis (inflammatory disease of the central nervous system), which highlights the potential therapeutic application of dna aptamers in the treatment of multiple sclerosis [ ] . cell-specific drug delivery can help to increase the efficiency of a drug and reduce side effects. in addition to functioning as therapeutic agents, dna aptamers have also been explored as delivery vehicles in targeted delivery of drugs or small oligonucleotides such as small interference rna (sirna) and microrna (mirna). the ability of aptamers to specifically recognize the target and to be readily modified makes it a potential targeted delivery tool. dna aptamers are used in two ways for targeted delivery of drugs: ( ) directly linked to the drug molecules; ( ) in combination with nanoparticles to form the delivery platform. conjugating drug molecules directly to specific dna aptamers is a potential way to deliver the drugs specifically, and thus reduce the risk of off-target drugs. by linking dna aptamers to drugs or packing the drug into an aptamer-folded structure, dna aptamers-drug conjugates can efficiently deliver drugs to target cells with increased specificity. many dna aptamers have been selected to efficiently deliver chemotherapy drugs in vitro or in vivo, such as doxorubicin (dox) [ ] , fluorouracil [ ] and epirubicin [ ] . dimeric or dendrimer dna aptamers in conjugation with drugs have been developed to further enhance the efficiency of target delivery [ , ] . with regard to small oligonucleotides delivery, dna aptamers were directly linked to the small oligonucleotides to form a dna aptamer-oligonucleotide chimera, which could help to prevent non-specific internalization as well as decrease the cellular toxicity towards non-target cells. we have reported that a dna aptamer-sirna chimera could specifically enter into cd (+) t cells and efficiently decrease the expression of exogenous the hiv protease gene [ ] . an anti-mucin dna aptamer covalently linked to mirna- b was found to deliver mirna- b into ovarian cancer cells specifically and induce significant apoptosis of the cancer cells [ ] . dna aptamers in combination with nanoparticles as a delivery vehicle is another promising targeted delivery approach. a number of nanomaterials have been explored to conjugate with dna aptamers to form the delivery platform [ , ] . dna aptamer conjugated liposome likely has the highest potential as a delivery system, and liposome-based drug delivery systems have been evaluated in clinical trials. for example, as aptamer conjugated liposome was found be able to enhance the delivery specificity and uptake of dox in tumor cells, as well as to increase the accumulation of dox in the tumor tissues with reduced cardiotoxicity in vivo [ ] . micelles, aggregation of lipid molecules, are also used in dna aptamer-nanoparticles delivery systems. recently, as aptamer conjugated peg-poly(lactic-co-glycolic acid) (plga) nanoparticles have been developed to facilitate antiglioma delivery of paclitaxel in vivo [ ] . this aptamer-peg-plga delivery system can prolong circulation time and enhance target accumulation of paclitaxel, which facilitates tumor inhibition. gold nanoparticles are another attractive material used in drug delivery system, because of their high stability, low or no toxicity and facile conjugation. a dna aptamer (sgc c) conjugated gold nanoparticle system has been found to increase the uptake of dox into cancer cells [ ] . other nanomaterials such as silica, carbon nanotubes and quantum dots have also been used in dna aptamer-nanoparticles delivery systems to enhance the specificity and prolong the circulation of drug molecules [ ] . by summarizing the progress of the generation of dna aptamers and their application in human disease diagnosis and therapy, we have shown that dna aptamers have great potential to be used as an alternative to antibodies. since the first monoclonal antibody was produced in s, antibodies have been successfully and extensively used in the diagnosis and therapy of human diseases. aptamers are expected to achieve a similar success to antibodies. because of their stability, low cost and facile manipulation, dna aptamers will continue to be extensively studied and applied. although modified rna aptamers have enhanced stability, the high cost of chemically modified rna might limit their study and application. however, rna aptamers hold an advantage in providing more complex and diverse d structures, which is helpful for selecting aptamers with high affinity for complex targets needed for disease therapy. therefore, dna aptamers might have more promising application in diagnosis and in vivo imaging, while modified rna aptamers might have more promising application in therapy. in the era of personalized medicine, dna aptamer-based therapeutics and diagnostics are believed to have great potential for extensive application because of their flexibility to specifically bind to any molecule targets. before they can be widely applied, there are still many problems that remain to be addressed. problems like nuclease degradation, quick renal excretion and potential cross-reactivity of aptamers should be analyzed in future studies. systematic evolution of ligands by exponential enrichment: rna ligands to bacteriophage t dna polymerase in vitro selection of rna molecules that bind specific ligands selection in vitro of single-stranded dna molecules that fold into specific ligand-binding structures selection of single-stranded dna molecules that bind and inhibit human thrombin aptamers as therapeutics single-stranded dna aptamers against pathogens and toxins: identification and biosensing applications a highlight of recent advances in aptamer technology and its application aptamers and their biological applications applications of aptamers for chemistry analysis, medicine and food security outlook for aptamers after twenty five years rna aptamers as genetic control devices: the potential of riboswitches as synthetic elements for regulating gene expression pegaptanib (macugen): treating neovascular age-related macular degeneration and current role in clinical practice aptamer nanomedicine for cancer therapeutics: barriers and potential for translation aptamers market-global forecast to post-selex chemical optimization of a trypanosome-specific rna aptamer building oligonucleotide therapeutics using non-natural chemistries inhibition of hiv- protease expression in t cells owing to dna aptamer-mediated specific delivery of sirna improving the stability of aptamers by chemical modification influence of the -hydroxyl group conformation on the stability of a-form helices in rna oligonucleotide aptamers: new tools for targeted cancer therapy in vitro selection of functional nucleic acids aptamers overview: selection, features and applications single-stranded dna (ssdna) production in dna aptamer generation development of a novel dna aptamer ligand targeting to primary cultured tumor endothelial cells by a cell-based selex method dna aptamer evolved by cell-selex for recognition of prostate cancer development of an efficient targeted cell-selex procedure for dna aptamer reagents a two-step stimulus-response cell-selex method to generate a dna aptamer to recognize inflamed human aortic endothelial cells as a potential in vivo molecular probe for atherosclerosis plaque detection evolution of dna aptamers through in vitro metastatic-cell-based systematic evolution of ligands by exponential enrichment for metastatic cancer recognition and imaging in silico maturation of binding-specificity of dna aptamers against proteus mirabilis a novel protocol for generating high-affinity ssdna aptamers by using alternating magnetic fields magnetic-assisted rapid aptamer selection (maras) for generating high-affinity dna aptamer using rotating magnetic fields rapid one-step selection method for generating nucleic acid aptamers: development of a dna aptamer against alpha-bungarotoxin array-based evolution of dna aptamers allows modelling of an explicit sequence-fitness landscape single-round patterned dna library microarray aptamer lead identification the application of a modified nucleotide in aptamer selection: novel thrombin aptamers containing -( -pentynyl)- -deoxyuridine molecular evolution of functional nucleic acids with chemical modifications novel combinatorial selection of phosphorothioate oligonucleotide aptamers chemically modified nucleic acid aptamers for in vitro selections: evolving evolution effect of -end capping of aptamer with various , -bridged nucleotides: enzymatic post-modification toward a practical use of polyclonal aptamers application of locked nucleic acids to improve aptamer in vivo stability and targeting function locked nucleic acids: a promising molecular family for gene-function analysis and antisense drug development selection of lna-containing dna aptamers against recombinant human cd synthesis and properties of mirror-image dna simple peg modification of dna aptamer based on copper ion coordination for tumor targeting a multivalent dna aptamer specific for the b-cell receptor on human lymphoma and leukemia affinity analysis of dna aptamer-peptide interactions using gold nanoparticles aptamer-functionalized peg-plga nanoparticles for enhanced anti-glioma drug delivery selective delivery of an anticancer drug with aptamer-functionalized liposomes to breast cancer cells in vitro and in vivo dna aptamer-micelle as an efficient detection/delivery vehicle toward cancer cells applications of aptasensors in clinical diagnostics in vitro selection of dna aptamers to anthrax spores with electrochemiluminescence detection dna aptamer selected against pancreatic ductal adenocarcinoma for in vivo imaging and clinical tissue recognition cell-selex based selection and characterization of dna aptamer recognizing human hepatocarcinoma molecular recognition of human liver cancer cells using dna aptamers generated via cell-selex cell-selex based selection and optimization of dna aptamers for specific recognition of human cholangiocarcinoma qbc- cells a cell-based single-stranded dna aptamer specifically targets gastric cancer a dna aptamer with high affinity and specificity for molecular recognition and targeting therapy of gastric cancer in vitro selection of dna aptamers for metastatic breast cancer cell recognition and tissue imaging selection of dna aptamers against glioblastoma cells with high affinity and specificity dna aptamers that target human glioblastoma multiforme cells overexpressing epidermal growth factor receptor variant iii in vitro selection of dna aptamers against epithelial cell adhesion molecule for cancer cell imaging and circulating tumor cell capture probing high affinity sequences of dna aptamer against vegf selection of dna aptamers against vegf( ) using a protein competitor and the aptamer blotting method in vitro selection of dna aptamers to glioblastoma multiforme selection and characterization of dna aptamers for use in detection of avian influenza virus h n development of a fluorescent enzyme-linked dna aptamer-magnetic bead sandwich assay and portable fluorometer for sensitive and rapid leishmania detection in sandflies label-free detection of prion protein with its dna aptamer through the formation of t-hg +-t configuration application of a novel in vitro selection technique to isolate and characterise high affinity dna aptamers binding mammalian prion proteins structural basis for discriminatory recognition of plasmodium lactate dehydrogenase by a dna aptamer screening of dna aptamers against myoglobin using a positive and negative selection units integrated microfluidic chip and its biosensing application development of a dna aptamer for direct and selective homocysteine detection in human serum dna aptamer-based detection of prostate cancer dna aptamers against the muc tumour marker: design of aptamer-antibody sandwich elisa for the early diagnosis of epithelial tumours sensitive point-of-care monitoring of cardiac biomarker myoglobin using aptamer and ubiquitous personal glucose meter c-reactive protein, inflammation and coronary heart disease dna aptamer-based surface plasmon resonance sensing of human c-reactive protein homocysteine level and coronary heart disease incidence: a systematic review and meta-analysis thrombin, inflammation, and cardiovascular disease: an epidemiologic perspective aptamer binding assays for proteins: the thrombin example-a review design strategies for aptamer-based biosensors aptamer-based molecular recognition for biosensor development aptamer in bioanalytical applications aptamer-based biosensors for biomedical diagnostics structure-switching signaling aptamers structure-switching signaling aptamers: transducing molecular recognition into fluorescence signaling enzyme-linked small-molecule detection using split aptamer ligation target-induced conjunction of split aptamer fragments and assembly with a water-soluble conjugated polymer for improved protein detection an aptamer-based biosensor for sensitive thrombin detection the nucleolin targeting aptamer as destabilizes bcl- messenger rna in human breast cancer cells a phase ii trial of as (a novel nucleolin-targeted dna aptamer) in metastatic renal cell carcinoma randomized phase ii trial of the nucleolin targeting aptamer as combined with high-dose cytarabine in relapsed/refractory acute myeloid leukemia (aml) the von willebrand inhibitor arc reduces cerebral embolization after carotid endarterectomy: a randomized trial a dose ranging phase i/ii trial of the von willebrand factor inhibiting aptamer arc in patients with congenital thrombotic thrombocytopenic purpura emerging pharmacologic therapies for wet age-related macular degeneration as- , a guanosine-rich oligonucleotide aptamer targeting nucleolin for the potential treatment of cancer, including acute myeloid leukemia arc- , a pegylated aptamer antagonist of von willebrand factor for potential use as an anticoagulant or antithrombotic agent a dna aptamer prevents influenza infection by blocking the receptor binding region of the viral hemagglutinin dna-aptamer targeting vimentin for tumor therapy in vivo aptamer to erbb- /her enhances degradation of the target and inhibits tumorigenic growth remyelination induced by a dna aptamer in a mouse model of multiple sclerosis molecular assembly of an aptamer-drug conjugate for targeted drug delivery to tumor cells automated modular synthesis of aptamer-drug conjugates for targeted drug delivery epirubicin loaded super paramagnetic iron oxide nanoparticle-aptamer bioconjugate for combined colon cancer therapy and imaging in vivo dimeric dna aptamer complexes for high-capacity-targeted drug delivery using ph-sensitive covalent linkages a controllable aptamer-based self-assembled dna dendrimer for high affinity targeting, bioimaging and drug delivery anticancer role of muc aptamer-mir- b chimera in epithelial ovarian carcinoma cells through regulation of pten methylation aptamer-conjugated nanomaterials and their applications aptamer-mediated targeted delivery of chemotherapeutic drugs and sirna for cancer therapy an as aptamer-conjugated liposomal system containing a bubble-generating agent for tumor-specific chemotherapy that overcomes multidrug resistance dna and aptamer stabilized gold nanoparticles for targeted delivery of anticancer therapeutics cell-type-specific, aptamer-functionalized agents for targeted disease therapy the authors would like to thank the nagasaki university for covering the cost to publish this paper in open access. the funder had no role in decision to publish and preparation of the manuscript.author contributions: mk, qz and gl conceived the review. qz and gl wrote the manuscript, and then mk revised the manuscript. the authors declare no conflict of interests. key: cord- - pp lv authors: qiu, yingshan; lam, jenny k. w.; leung, susan w. s.; liang, wanling title: delivery of rnai therapeutics to the airways—from bench to bedside date: - - journal: molecules doi: . /molecules sha: doc_id: cord_uid: pp lv rna interference (rnai) is a potent and specific post-transcriptional gene silencing process. since its discovery, tremendous efforts have been made to translate rnai technology into therapeutic applications for the treatment of different human diseases including respiratory diseases, by manipulating the expression of disease-associated gene(s). similar to other nucleic acid-based therapeutics, the major hurdle of rnai therapy is delivery. pulmonary delivery is a promising approach of delivering rnai therapeutics directly to the airways for treating local conditions and minimizing systemic side effects. it is a non-invasive route of administration that is generally well accepted by patients. however, pulmonary drug delivery is a challenge as the lungs pose a series of anatomical, physiological and immunological barriers to drug delivery. understanding these barriers is essential for the development an effective rna delivery system. in this review, the different barriers to pulmonary drug delivery are introduced. the potential of rnai molecules as new class of therapeutics, and the latest preclinical and clinical studies of using rnai therapeutics in different respiratory conditions are discussed in details. we hope this review can provide some useful insights for moving inhaled rnai therapeutics from bench to bedside. lung diseases are among the leading causes of death worldwide. current treatment of lung diseases such as lung cancer [ ] , respiratory infections [ , ] , inflammatory diseases [ , ] and pulmonary fibrosis [ ] have limited efficacy. almost two decades ago, double-stranded rnas (dsrnas) were discovered to play an important role in regulating gene functions by a sequence-specific post-transcriptional gene silencing mechanism called rna interference (rnai) [ ] . the therapeutic potential of rnai was soon realized. it presents a new and powerful approach to treat or prevent many diseases including respiratory disorders by modulating gene expression [ ] [ ] [ ] [ ] [ ] [ ] . rnai can be mediated by various types of rna molecules, including long dsrna, short interfering rna (sirna), short hairpin rna (shrna) and microrna (mirna). the mechanism of rnai is illustrated in figure , and the general properties of different types of rnai molecules are summarized in table . long dsrnas (around - nucleotides in length) have been employed to study gene functions. after the exogenous dsrna is introduced into the cytoplasm of the cells, it is cleaved by rnase iii enzyme dicer into short dsrna called sirna. the sirna which is - nucleotides in length is loaded into a protein complex called the rna-induced silencing complex (risc). the sirna is then unwound, and the sense strand (also known as the passenger strand) of the sirna is degraded, whereas the remaining antisense strand (also known as the guide strand) guides the activated rics to the target messenger rna (mrna) through full complementary binding. the mrna is then cleaved by argonaute (ago ) in the risc, leading to the silencing of the target gene [ , ] . since long dsrna is known to trigger immunostimulatory response through the activation of dicer-related antiviral pathways and induction of type interferon (ifn) [ ] , it is less suitable for therapeutic use. in contrast, synthetic sirna is a more promising gene silencing mediator because of the lower risk of immune response. it is also the most widely investigated rnai molecule for therapeutic applications, with over clinical trial studies being initiated since [ ] . shrna is a sequence of rna transcribed in the nucleus of the cells from a dna vector by either rna polymerase ii or iii. the primary transcript is called primary shrna (pri-shrna), which contains a hairpin like stem-loop structure. the pri-shrna is processed into a - nucleotides long loop-stem precursor shrna (pre-shrna) by a protein complex containing the rnase iii nuclease drosha and the dsrna binding domain protein dgcr . it is then transported to the cytoplasm through a specialized nuclear membrane protein, exportin- (exp ). the loop sequence of the pre-shrna is cleaved by the dicer to form a double-stranded sirna. this endogenously produced sirna is loaded into risc and induce rnai through the similar process as the synthetic sirna [ , ] . since shrna expression unit can be incorporated into viral vectors and continuously synthesized by the host cell, it can induce long-lasting gene silencing effect. the risc-loading process of shrna is about ten times more efficient than sirna, implicating that lower dose of shrna is required to maintain the therapeutic efficacy with less off-target effect [ ] . however, the shrna approach is a dna-based strategy depending on the expression of shrna encoding gene which often requires viral vectors. from the delivery perspective, the introduction of synthetic sirna to the cytoplasm is a more straightforward way to induce rnai. the use of viral vectors for delivery poses safety concerns in its therapeutic applications [ ] . mirna is a naturally occurring non-coding rna that plays a key role in regulating gene expression. primary mirna (pri-mirna) is transcribed by rna polymerase ii from endogenous mirna gene in the nucleus. the hairpin-containing pri-mirna is structurally similar to the pri-shrna, therefore the maturation pathway of mirna is also similar to shrna [ ] . the pri-mrna is converted by drosha/dcgr complex into precursor mirna (pre-mirna), which contains - nucleotides with interspersed mismatches and adopts a loop structure. the pre-mirna is subsequently transported to the cytoplasm by exp , and is processed by the dicer into mature mirna of - nucleotides in length [ ] . as opposed to sirna, the antisense strand of mirna is only partially complementary to the target mrna, leading to gene silencing via translational repression and/or mrna deadenylation. position to of the end of the mirna is the "seed sequence", which is essential for target recognition, and the mirna binding sites of mrna are located in the untranslated region (utr) [ ] . there are two major approaches of mirna-based therapeutics: (i) mirna inhibition [ , ] , which suppresses the action of the endogenous mirna by antisense oligonucleotide (antimir); and (ii) mirna replacement [ , ] , which introduces synthetic mirna (mirna mimic) to restore the functions of the endogenous mirna. only the latter is discussed in this review. compared to sirna-based therapeutics, mirna has the potential to target multiple genes due to the imperfect binding to the target mrna [ ] . rnai therapeutics offer several advantages over the traditional small molecules and protein-based drugs. they could virtually target any genes with high selectivity, including those "undruggable" targets. the design and synthesis of rnai molecules are relatively simple because they do not need a cellular expression system, complex protein purification, or refolding schemes [ ] . compared to the antisense oligonucleotides, rnai is more potent and specific [ ] . despite its huge therapeutic potential, delivery remains to be a major barrier to the clinical application of rnai therapeutics [ , ] . for the treatment of respiratory disorders, pulmonary route of administration can deliver rnai molecules directly to the site of action, thereby lowering the dose required while minimizing systemic adverse effects. the rnai molecules can also avoid the rapid clearance by serum nuclease in the bloodstream. moreover, inhalation is a non-invasive route of administration that is easily accepted by patients compared to parenteral administration, leading to better patient compliance [ , ] . however, pulmonary drug delivery is a challenge with several key biological barriers, which are highlighted in figure . rnai therapeutics offer several advantages over the traditional small molecules and protein-based drugs. they could virtually target any genes with high selectivity, including those "undruggable" targets. the design and synthesis of rnai molecules are relatively simple because they do not need a cellular expression system, complex protein purification, or refolding schemes [ ] . compared to the antisense oligonucleotides, rnai is more potent and specific [ ] . despite its huge therapeutic potential, delivery remains to be a major barrier to the clinical application of rnai therapeutics [ , ] . for the treatment of respiratory disorders, pulmonary route of administration can deliver rnai molecules directly to the site of action, thereby lowering the dose required while minimizing systemic adverse effects. the rnai molecules can also avoid the rapid clearance by serum nuclease in the bloodstream. moreover, inhalation is a non-invasive route of administration that is easily accepted by patients compared to parenteral administration, leading to better patient compliance [ , ] . however, pulmonary drug delivery is a challenge with several key biological barriers, which are highlighted in figure . human respiratory tract is divided into the conducting region (nasal cavity, pharynx, trachea, bronchi and bronchioles) and the respiratory region (respiratory bronchioles and alveoli). the former is the pathway for gases conduction whereas the latter is the site of gaseous exchange [ ] . the extensive branched structure of the airways with varying length and diameter presents the primary hurdle in pulmonary drug delivery. the mechanisms of inhaled particle deposition in the lungs include inertial impaction, gravitational sedimentation, diffusion, interception and electrostatic precipitation [ ] . as illustrated in figure , the first three mechanisms are the major mechanisms which are greatly influenced by the aerodynamic size of the particles [ ] . the optimal particle size for lung deposition is between and µm [ , ] . larger particles are likely to be impacted and trapped on the upper airway wall at bifurcations while the small particles from . to µm are easily exhaled during normal breathing. smaller particles under nm may be successfully deposited in the alveolar space because of the increasing diffusional mobility [ ] . however, aerosols under nm are difficult to produce, which make their application less favorable. human respiratory tract is divided into the conducting region (nasal cavity, pharynx, trachea, bronchi and bronchioles) and the respiratory region (respiratory bronchioles and alveoli). the former is the pathway for gases conduction whereas the latter is the site of gaseous exchange [ ] . the extensive branched structure of the airways with varying length and diameter presents the primary hurdle in pulmonary drug delivery. the mechanisms of inhaled particle deposition in the lungs include inertial impaction, gravitational sedimentation, diffusion, interception and electrostatic precipitation [ ] . as illustrated in figure , the first three mechanisms are the major mechanisms which are greatly influenced by the aerodynamic size of the particles [ ] . the optimal particle size for lung deposition is between and µm [ , ] . larger particles are likely to be impacted and trapped on the upper airway wall at bifurcations while the small particles from . to µm are easily exhaled during normal breathing. smaller particles under nm may be successfully deposited in the alveolar space because of the increasing diffusional mobility [ ] . however, aerosols under nm are difficult to produce, which make their application less favorable. the presence of fluid layers in the airways, including the mucus and the pulmonary surfactant, also creates a barrier to the delivery of drug molecules to the lungs. mucus is a gel of three-dimensional network structure. it acts as a sieve that filters out large molecules (> nm) [ ] . mucins, which are the major components of mucus, contain the hydrophilic glycosylated residues rich in serine and threonine and the hydrophobic non-glycosylated cysteine-rich domains. this characteristic allows electrostatic, hydrophilic and hydrophobic interactions between mucus and drug particles [ ] . since mucus is continuously produced, shed and replaced, and together with the mucociliary clearance action, the fast turnover rate of mucus leads to the rapid clearance of the entrapped drug molecules, preventing them from reaching the epithelium [ , ] . moreover, cough clearance that occurs when the mucus reaches the throat is another removal mechanism of rnai molecules from the airways. on the other hand, the role of pulmonary surfactant in nucleic acid delivery is controversial. pulmonary surfactant is composed of approximately % lipids and % proteins [ ] . it has been suggested that the lipids and proteins in the lung surfactant interact with the non-viral cationic lipid-based delivery system, leading to the premature release of nucleic acids [ , ] . conversely, polymer-based delivery systems were found to be more compatible with pulmonary surfactant. commercial pulmonary surfactant was employed in the preparation of some polymeric nanoparticles to facilitate the cellular uptake of sirna to improve gene silencing effect [ , ] . apart from the physical barriers, the rnai molecules may undergo enzymatic degradation following pulmonary delivery. rna is extremely susceptible to nuclease activity. naked sirna has a short half-life of less than min in serum [ ] . due to the lack of serum in the lungs, the half-life of unmodified sirna in the airways can reach up to three hours in mouse [ ] . rnai molecules may also be taken up and removed by alveolar macrophages upon lung deposition. the alveolar macrophages are responsible for engulfing and digesting foreign particles in the lower airways via phagocytosis. unless the target of rnai molecules is located inside the macrophages, such as mycobacterium tuberculosis which is the causative agent of tuberculosis that typically reside in the alveolar macrophages [ ] , otherwise the rnai molecules are subjected to degradation inside the macrophages before reaching their target sites. the presence of fluid layers in the airways, including the mucus and the pulmonary surfactant, also creates a barrier to the delivery of drug molecules to the lungs. mucus is a gel of three-dimensional network structure. it acts as a sieve that filters out large molecules (> nm) [ ] . mucins, which are the major components of mucus, contain the hydrophilic glycosylated residues rich in serine and threonine and the hydrophobic non-glycosylated cysteine-rich domains. this characteristic allows electrostatic, hydrophilic and hydrophobic interactions between mucus and drug particles [ ] . since mucus is continuously produced, shed and replaced, and together with the mucociliary clearance action, the fast turnover rate of mucus leads to the rapid clearance of the entrapped drug molecules, preventing them from reaching the epithelium [ , ] . moreover, cough clearance that occurs when the mucus reaches the throat is another removal mechanism of rnai molecules from the airways. on the other hand, the role of pulmonary surfactant in nucleic acid delivery is controversial. pulmonary surfactant is composed of approximately % lipids and % proteins [ ] . it has been suggested that the lipids and proteins in the lung surfactant interact with the non-viral cationic lipid-based delivery system, leading to the premature release of nucleic acids [ , ] . conversely, polymer-based delivery systems were found to be more compatible with pulmonary surfactant. commercial pulmonary surfactant was employed in the preparation of some polymeric nanoparticles to facilitate the cellular uptake of sirna to improve gene silencing effect [ , ] . apart from the physical barriers, the rnai molecules may undergo enzymatic degradation following pulmonary delivery. rna is extremely susceptible to nuclease activity. naked sirna has a short half-life of less than min in serum [ ] . due to the lack of serum in the lungs, the half-life of unmodified sirna in the airways can reach up to three hours in mouse [ ] . rnai molecules may also be taken up and removed by alveolar macrophages upon lung deposition. the alveolar macrophages are responsible for engulfing and digesting foreign particles in the lower airways via phagocytosis. unless the target of rnai molecules is located inside the macrophages, such as mycobacterium tuberculosis which is the causative agent of tuberculosis that typically reside in the alveolar macrophages [ ] , otherwise the rnai molecules are subjected to degradation inside the macrophages before reaching their target sites. once the rnai molecules overcome the aforementioned extracellular barriers and reach the target cells, they need to overcome a set of intracellular barriers. the ultimate site of actions depends on the types of rnai molecules. for synthetic sirna and mirna mimic, they need to reach the cytoplasm where the risc locates, whereas dna encoding shrna or mirna need to enter the nucleus in order for transcription to take place. surface adhesion is the first step of cellular entry. there are various endocytic mechanisms involved in the cellular uptake of macromolecules, which subsequently influence their intracellular trafficking and delivery efficiency [ ] [ ] [ ] [ ] [ ] [ ] . the clathrin-dependent endocytosis is the most common route of cellular entry for macromolecules [ ] . following cell entry, the rnai molecules are entrapped in the early endosomes where progressive acidification occurs. the late endosomes then fuse with the lysosomes, which contain hydrolases that degrade the rnai molecules [ , ] . the rnai molecules must escape from endosome at early stage to exert their biological effect. strategies of endosomal escape have been reviewed in other literatures [ ] [ ] [ ] [ ] . for shrna or mirna vectors, nuclear entry is an additional barrier which is particularly challenging due to the presence of nuclear membranes that are impermeable to most substances except small non-polar molecules [ ] . non-viral vectors are inefficient in delivering dna into the nucleus. some viruses are evolved to transport their genome into the nucleus; hence, they become attractive dna carriers to improve transduction efficiency. however, poor safety profile, high production cost and the risk of immunogenicity [ ] limited the applications of viral vector mediated rnai to laboratory tool. a delivery vector or carrier is generally necessary to overcome the aforementioned barriers by promoting cellular uptake and offering protection to the rnai molecules. rnai delivery system can be categorized into viral and non-viral vectors according to their nature, and these delivery systems have been extensively reviewed [ ] [ ] [ ] [ ] [ ] . the viral vectors refer to the of use of viruses to deliver genetic materials to the cells. they are extremely efficient in transducing cells and providing either transient or long-term gene expression, depending on the type of viruses employed. most of the in vivo studies used the adenoviruses, adeno-associated virus (aav) and retroviruses to express the plasmid dna encoding shrna or mirna to induce rnai [ ] . the most attractive property of viral vectors is that they have the ability to access the nucleus of the cells. thus, they have high efficacy to express the rna and subsequently regulate the gene expression [ ] [ ] [ ] [ ] . however, the clinical application of viral vectors is limited by the toxicity, insertional mutagenesis (associated with retroviruses) and immunogenicity (associated with adenoviruses) [ ] . in addition, the smaller size of avv limits the amount of therapeutic gene that can be inserted. compared to viral vectors, non-viral vectors generally have better safety profile and lower production cost. they have been investigated for the pulmonary delivery of the rnai molecules. lipid-based, polymer-based and peptide-based are three major non-viral delivery systems [ ] . the lipid-based vectors are the most commonly used vectors for rnai delivery. they include cationic liposomes, solid lipid nanoparticles, solid nanostructured lipid carriers, lipidoids and ph-responsive lipids [ ] . many commercially available transfection agents (i.e., lipofectamine, oligofectamine, transit-tko and dharmafect) are lipid-based systems [ ] [ ] [ ] [ ] . despite the promising transfection efficacy, the major challenges of using the lipids are the immune response and cytotoxicity [ ] . in addition to the lipids, biocompatible and biodegradable natural polymers such as chitosan and dextran, as well as synthetic polymers such as poly lactic-co-glycolic acid (plga), polyethylenimine (pei) and pamam dendrimer are used for the preparation of polymeric nanoparticles for rna delivery [ ] [ ] [ ] [ ] [ ] [ ] . the polymers have relatively low toxicity compared to the lipid-based vectors and are more versatile for chemical modification. for peptide-based delivery systems, peptides such poly(l-lysine) (pll), cell penetrating peptides (cpps) and ph-responsive peptides have been investigated for the delivery of the rnai molecules [ ] [ ] [ ] [ ] [ ] . different types of peptides mediate cellular transfection via different mechanisms such as facilitating cellular uptake and promoting endosomal escape. peptides can be used to carry the rnai molecules alone or act as a functional component in other carriers. currently, efficient peptides with low toxicity and high transfection efficacy are still under development. it is noted that for pulmonary delivery, the reduced nuclease activity in the lungs makes successful delivery of unmodified naked rna become possible [ , ] . however, the mechanism of how naked sirna could overcome the extracellular and intracellular barrier following pulmonary administration still remains to be understood. the therapeutic potential of rnai-based therapy targeting respiratory disorders has been widely explored in the past decade. in the following sections, the pre-clinical and clinical studies that involved the delivery of rnai molecules to the airways for the treatment of lung diseases are discussed. lung cancer is a leading cause of cancer death in the world. the mainstay of treatment is chemotherapy, which is indicated for around % of newly diagnosed patients with local and advanced metastatic disease [ ] . other treatment options include surgery and radiotherapy [ ] . angiogenesis inhibitors [ ] and epidermal growth factor receptor (egfr) inhibitors [ , ] are new medications licensed for the treatment of lung cancer. despite various treatment options, the prognosis of lung cancer is poor. the major problems of chemotherapy are toxicity, lack of specificity and drug resistance [ ] . in recent years, pulmonary delivery of rnai molecules is being explored as a new treatment for lung cancer [ ] . genes that are associated with tumor cell growth and drug resistance are potential targets. table summarizes some recent in vivo studies of pulmonary rnai delivery for lung cancer treatment. genes that are essential for tumor growth and progression, such as akt (rac-alpha serine/threonine-protein kinase b) and c-myc, are evaluated as rnai targets. akt is an important mediator of cell growth, proliferation and survival of non-small cell lung cancer (nsclc). activation of akt pathway is observed in most nsclc patients. it is found to reduce chemo-and radiation-induced apoptosis to extend the survival of tumor cells [ , ] . sorbitol diacrylatepolyethylenimine (sda-pei) was employed to deliver shrna targeting akt (shakt ) and cdna encoding programmed cell death protein (pdcd ) in a dual expression vector via pulmonary route to murine lung cancer model. simultaneous akt inhibition and pdcd overexpression could induce potent anticancer effect. the total tumor number and the tumor size larger than mm in the lung were significantly reduced after eight doses [ ] . glycerol triacrylate-spermine (gt-spe) polyspermine [ ] and lentivirus [ ] were also employed as carriers of shrna targeting akt signaling pathway for pulmonary delivery. although repeated aerosol delivery of lentiviral-based shrna can achieve potent knockdown of the target, inhalation is a procedure that generates large number of viral particles. there are safety concerns regarding the use of viral vectors for inhalation, including the potential mutagenicity, immunogenicity and the risk of aerosol-transmitted infections [ ] . therefore, aerosol delivery of rnai molecules using viral vectors must go through vigorous biosafety evaluation for clinical use. notes: aimp -dx , aminoacyl-trna synthetases (ars)-interacting multifunctional protein lacking exon ; akt , rac-alpha serine/threonine-protein kinase b; bcl- , b-cell lymphoma ; dox: doxorubicin; egfp, enhanced green fluorescence protein; gpt-spe: glycerol propoxylate triacrylate and spermine; gt-spe, glycerol triacrylate-spermine; mrp , multidrug resistance protein ; npt b: sodium-dependent phosphate co-transporter b; pdcd : programmed cell death protein ; pei: polyethylenimine; rgd, arginine-glycine-aspartic acid peptide; rpn , ribophorin ii; nps, nanoparticles; sda-pei, sorbitol diacrylatepolyethylenimine; tax, paclitaxel. c-myc oncogene is a downstream conduit for most oncogenic signals. it is necessary for the growth control, differentiation and apoptosis. abnormal expression of c-myc is associated with many tumors. de-activation of myc is efficacious for lung tumor therapy [ ] . conde et al. delivered sirna targeting c-myc to the lung of mouse using rgd-surface-modified gold nanoparticles (aunps) by intratracheal instillation. rgd peptide (arg-gly-asp) is a cell adhesion peptide responsible to mediate cell adhesion and proliferation by binding to the integrin avb receptor family, a specific marker for angiogenesis and up-regulated in the endothelium. this strategy successfully suppressed c-myc expression level and tumor cell proliferation, resulting in approximately % increase in survival of orthograft mouse tumor models [ ] . genes associated with mdr were also evaluated as target of rnai, such as ribophorin ii (rpn ) [ ] , b-cell lymphoma (bcl- ) [ ] and multidrug resistance protein (mrp ) [ ] . rpn is known to be associated with the apoptosis in docetaxel-resistant human breast cancer cells [ ] , and its involvement in nsclc was investigated recently [ ] . a novel rnai molecule (pnkrna) targeting rpn was developed and delivered to mice bearing lung cancer by intrapulmonary delivery. this new class of rnai agent has a unique helical structure containing a central stem and two loops, and is claimed to be stable against nuclease degradation. inhibition of lung tumor growth was observed without any signs of toxicity after treatment. the antitumor effect was triggered by pnkrna targeting rpn without the use of other anticancer drugs. it could be attributed to the multiple activities of rpn , which is anti-apoptotic and involved in the regulation of tumor survival [ ] . however, the mechanism of how the naked the pnkrna molecules overcame the extracellular and intracellular barriers to initiate rnai was not elucidated. besides the inhibition of mdr associated proteins alone, the co-delivery of anticancer drugs with rnai therapeutics is as an alternative approach to tackle mdr in various types of cancers [ , [ ] [ ] [ ] . combination therapy offers the potential advantages of synergistic activity, overcoming the drug resistance and reducing the side effects of chemotherapy. bcl- is a main player of nonpump cellular resistance [ ] and mrp is responsible for drug efflux in cancer cells [ ] . they were investigated in the combination approach. taratula et al. prepared a lipid-based nanoparticles containing sirna targeting bcl- and mrp , as well as anticancer drugs (doxorubicin or paclitaxel). the nanoparticles were delivered to the lung of tumor mouse model using a nose only exposure chamber for inhalation. almost complete disappearance of lung tumor was observed in animals treated with paclitaxel plus the sirnas. in contrast, treatment with paclitaxel alone only slowed down the growth of the tumor [ ] . xu et al. developed a polyethylenimine (pei)-based delivery system that simultaneously delivered sirna targeting bcl- and doxorubicin to the lungs of mice with metastatic lung cancer. synergistic antitumor efficacy was achieved compared with the delivery of doxorubicin or sirna alone [ ] . these studies indicated that suppression of resistance gene by rnai molecules via inhalation could restore the sensitivity of chemotherapeutic drugs, leading to the death of tumor cells. respiratory infection can be caused by a wide range of microorganisms in the airways including bacteria and viruses [ ] . it is one of the most common reasons for hospitalization among adults [ ] . due to the rise of antimicrobial resistance, many infectious diseases including respiratory infections have become difficult to treat. rnai therapeutics appears to be an attractive approach to fight against infections [ ] . the pre-clinical studies of rnai therapeutics to combat against respiratory infections are summarized in table . since rnai was first reported to inhibit respiratory syncytial virus (rsv) in [ ] , an increasing number of studies have been initiated to explore the potential of rnai technology to treat other respiratory viral infections including influenza [ , , , ] and severe acute respiratory syndrome (sars) [ ] . rnai molecules offer several advantages as antiviral therapeutics. they can specifically target the conserved region of mrna sequence in the viral genome, making it effective against mutated viral strains. in addition, rnai molecules can be designed rapidly to target viral genes, shortening the lead time of developing new antiviral agents, which is particularly useful in response to an infection outbreak. to achieve effective antiviral effect, the viral targets must be essential for the pathogenesis of viral infection and/or replication cycle. it is also desirable that the target genes share similar conserved sequence among different strains to achieve broad viral inhibition. furthermore, the viral gene should be substantially different with human gene to minimize any undesirable side effects. rsv infection is one of the most common infections in children [ , ] . in adult, rsv infection usually leads to self-limited upper respiratory illness [ ] . however, severe rsv infection has been observed in elderly patients with lung diseases or in immunocompromised patients [ ] . current management of rsv includes bronchodilators, corticosteroids, antibiotics and supportive care [ ] , but their efficacy is not satisfactory. to date, there is no licensed vaccine available to prevent this disease [ ] . there is an unmet need to develop effective rsv therapeutics and vaccines in both the pediatric and adult population. in , bitko et al. reported the inhibition of rsv and parainfluenza virus (piv) replication by sirna targeting viral phosphoprotein (p protein), a crucial subunit of the viral rna-dependent rna polymerase. since rsv replicate primarily in the superficial layer of the respiratory epithelium, local delivery of rnai molecules to the lungs is a rational approach to inhibit rsv replication [ ] . in the study, transit-tko was employed as transfection agent and the sirna was administered to the lungs of balb/c mice by intranasal instillation h before viral challenge. the rsv and piv titer was reduced by over % five days after infection. the use of naked sirna also showed substantial inhibition of infection, with approximately %- % efficiency achieved as compared to complexed sirna. interestingly, the administration of sirna before and concomitant with rsv infection was more effective in reducing viral load than treatment after infection [ ] . the prophylactic regimen may not be a clinically attractive approach. to achieve optimal therapeutic benefit, fast detection and early intervention are crucial for the prognosis of rsv infection. it is desirable to initiate the antiviral rnai therapy as quickly as possible once rsv infection is confirmed in clinical practice. p protein may not be an ideal target as it is limited by its specificity to a particular viral strain [ ] . another sirna, aln-rsv , was designed to target the highly conserved region of the mrna encoding viral nucleocapsid protein (n protein). n protein is a core protein in the rna polymerase and plays a key role in the replication cycle of rsv [ ] . after intranasal administration of aln-rsv to the lungs of mouse at h prior to viral infection, . -to . -log-unit reductions in viral load was observed in comparison with the mismatch sirna control. for the treatment regimen, comparable antiviral efficacy was achieved in multiple daily doses of aln-rsv at day , and post-infection [ ] . the phase i clinical study of aln-rsv was reported in [ ] . intranasal administration of aln-rsv was well tolerated and the side effect profile was similar to placebo. later in , the antiviral effect of aln-rsv was evaluated in a phase ii trial. aln-rsv was administered by nasal spray daily to healthy adults for two days before and for three days after rsv inoculation. the group treated with aln-rsv had a significantly lower number of rsv infection compared to the placebo group [ ] . another phase ii b clinical trial of aln-rsv in rsv-infected lung transplant patients was also completed [ ] . the primary endpoint of the study was to evaluate the effect of aln-rsv on the incidence of new or progressive bronchiolitis obliterans syndrome (bos) in rsv-infected lung transplant patients. even though the study marginally missed the primary endpoint statistically (p = . ), inhaled aln-rsv treatment induced a clinically meaningful reduction in the incidence of bos, reinforcing the potential of inhaled rnai as new antiviral therapeutics. seasonal influenza viruses, especially influenza type a virus, cause annual epidemics that led to millions of death worldwide [ ] . it is one of the major public health problems. two categories of antiviral drugs are currently approved to treat influenza, including m ion-channel protein inhibitors (adamantanes) and neuraminidase inhibitors (zanamivir and oseltamivir). however, some influenza a virus strains have shown to be resistant to these drugs [ , ] , making the development of new antiviral drug urgently needed. a number of studies were carried out to investigate the use of rnai to inhibit viral gene expression and protect cells from influenza infection. the most commonly investigated targets are the nucleocapsid protein (np), polymerase acidic protein (pa) and polymerase basic protein (pb). these proteins are indispensable to influenza viral replication. more importantly, they are highly conserved across different subtypes of influenza virus strains [ ] . the first animal study applying rnai against influenza was reported by ge et al. [ ] . sirna targeting np or pa were complexed with pei. the complexes were injected intravenously to the mice h before or h after influenza virus (pr h n ) infection. the antiviral effect of sirna targeting np was dose-dependent and combination of sirnas targeting np and pa enhanced the effect. in the same study, shrna targeting np or pb was also investigated. the dna was mixed with infasurf, a commercial available pulmonary surfactant. after intranasal instillation, a significant reduction of virus titer was detected h after infection although the effect was less prominent compared with intravenous injection of pei complexes. infasurf may play a role as nucleic acid carrier. little is known about the interaction between dna and pulmonary surfactant in the airspace. it could be possible that the dna interacted with the charged lipid components in the pulmonary surfactant leading to the efficient spread and absorption in the airways [ ] . another study reported the administration of sirna targeting np and/or pa in lipid carrier to influenza-infected mice by hydrodynamic injection and intranasal administration. the combined sirna delivery method effectively protected animals against lethal challenge of highly pathogenic avian influenza a viruses, including the h n , h n and h n subtypes [ ] . since then, the potential of sirna for the treatment of influenza virus infection was continuously evaluated in vitro [ , [ ] [ ] [ ] and in vivo [ ] [ ] [ ] . however, the exploration of rnai for the treatment of influenza appears to reach the bottleneck. it could be partly attributed to the criticism of the antiviral effect induced by sirna, which was due to innate immune response rather than viral inhibition through rnai. development of chemically modified rnai molecules with minimal immunostimulatory effect could be helpful to delineate the mechanism of the antiviral effects [ ] . tuberculosis (tb) is a bacterial lung infection caused by mycobacterium tuberculosis (mtb). because of the emergence of multidrug-resistant (mdr) and extensively drug-resistant (xdr) tb, the slow development of new anti-tb agents and the inefficiency of vaccination, tb is still a major global health problem. who reported that . million people suffered from tb in and . million die annually because of the disease [ ] . a new and effective tb therapeutics is highly sought after. the approach of rnai-based therapeutics against tb aims to modulate the gene expression of the host instead of the bacillus because bacteria do not contain the requisite machinery for rnai. currently, rnai has been investigated for tb treatment in two directions: (i) targeting the host factors that are essential for the survival of mycobacteria; and (ii) modulating the host immune response to facilitate the elimination of mycobacterium [ ] . autophagy is a host defense mechanism against mtb. boosting autophagy could be an effective strategy to promote the eradication of the mycobacteria. hence, a number of host factors that are involved in the regulation of autophagy were explored as the target of rnai, including bfl- /a [ ] , rap a [ ] , ras homologue enriched in brain (rheb) [ ] and uv radiation resistance-associated gene (uvrag) [ ] . down-regulation of these factors promoted autophagosome formation and reduced the bacterial growth. however, the existing studies of using rnai to suppress host factors are preliminary and in vivo data is lacking. moreover, it was recently reported that autophagy pathway may not be essential for restricting mtb growth [ ] . further investigations are required to understand the relationship between mtb infection and autophagy pathway, and to better position the approach of rnai for the control of tuberculosis infection. another strategy is to modulate host immune response to improve the antimicrobial ability of the host [ ] . one of the key characteristics of tb infection is the formation of granuloma, which is a compact, organized aggregate of immune cells. granuloma is previously considered as a host-protective structure to sequester the infecting mycobacteria, but recent work revealed that the pathogens could take advantage of granuloma formation to facilitate their proliferation and dissemination in the host [ ] , as it provides a hospitable environment for mycobacteria to survive and replicate over a long period of time. there is a complicated chemokines and cytokines network involved in the granulomatous response. rnai can be used to target immunosuppressive cytokines in order to inhibit the growth of mtb. among them, tumor necrosis factor (tnf)-α and ifn-γ are critical in activating macrophages and triggering the formation of granuloma [ ] . xcl (lymphotactin) is a chemokine produced by activated cd + t cells during chronic tb infection. it was suggested that xcl regulates ifn-γ production by cd + t cells and contribute to the stability of the granuloma [ ] . when a single dose of sirna targeting xcl was delivered to the lungs of mice that were chronically infected with tb, the expression of xcl was effectively inhibited. the suppression of xcl expression in the lungs was associated with the decreasing number of t lymphocytes, reduction in the ifn-γ response, disorganized granulomatous lesions and higher fibrosis [ ] . although the inhibition effect elicited by sirna was transient and did not provide significant therapeutic benefit, this proof-of-concept study demonstrated that the local-environment and immunopathology of the lungs can be modulated to favor host response via pulmonary delivery of sirna. other targets of rnai-based immunotherapy include the transforming growth factor β (tgf-β) and interleukin (il- ) [ ] . both of them are found to be elevated in tb patients, and their high level of expression is connected with tb reactivation [ ] . during chronic infection, these cytokines restrain inflammation through affecting helper t cell (t h ) response and macrophage activation, leading to substantial reduction of their antimicrobial activity [ ] . in that study, sirna targeting tgf-β was administered through the intrapulmonary route to il- knockout mice at days post-infection. tgf-β gene silencing resulted in the increased expression of the antimicrobial mediators nitric oxide (no) and inducible nitric oxide synthase (inos), and significantly reduced the bacterial load for at least four weeks after treatment. a synergistic effect at improving the antimicrobial activity in the lungs was observed when both il- and tgf-β were targeted simultaneously. although the inhibition of bacterial load in this study was modest, it showed that sirna immunotherapy is a feasible approach to enhance the host antimicrobial capacity. moreover, mdr and xdr tb pose huge challenges for the current chemotherapeutic agents. combined immunotherapy targeting immunosuppressive cytokines and chemotherapy to target drug resistance bacteria population is a direction that is worth future investigation. respiratory inflammatory diseases impose substantial healthcare burden worldwide as many of the patients are inadequately controlled by current therapies [ , , ] . these diseases are characterized by inflammation in the respiratory tract, which is manifested with increased expression of inflammatory cytokines and chemokines [ , ] . the common lung inflammatory diseases include asthma, chronic obstructive pulmonary disease (copd) and acute lung injury (ali). table lists the major in vivo studies that examine the effects of rnai molecules in respiratory inflammatory disease. notes: ahr, airway hyperresponsiveness; ali, acute lung injury; balf, bronchoalveolar lavage fluid; cd , cluster of differentiation ; c-kit, a stem cell factor receptor; dcs, dendritic cells; hmgb a, high mobility group box- a peptide; ifu, infectious unit; lps, lipopolysaccharide; mpl, myeloproliferative leukemia virus oncogene; ova, ovalbumin; r v , an arginine-rich peptide; rip , receptor-interacting protein ; rsv, respiratory syncytial virus; s plyase, sphingosine- -phosphate lyase, socs, suppressors of cytokine signaling protein ; stat , signal transducer and activator of transcription factor ; syk, spleen tyrosine kinase; tf-pei, transferrin polyethylenimine; t h , t helper cells; tnf-α, tumor necrosis factor-α; vegfr, vascular endothelial growth factor. asthma is characterized by the variable and reversible airflow obstruction, airway hyperresponsiveness (ahr), mucus hypersecretion and chronic inflammation [ ] . the most common form of asthma is allergic asthma, which is triggered by environmental stimuli such as house dust and seasonal pollen [ ] . according to who, it is estimated that around million peoples have asthma worldwide [ ] . the mainstay treatments of asthma are corticosteroids, β -adrenergic receptor agonists and leukotriene receptor antagonists, either alone or in combination. although these medications can manage the conditions in most of the patients, there are still challenges of poor patient compliance, intolerability and long term adverse effects [ ] . for persistent allergic asthmatic patients, omalizumab which is an anti-immunoglobulin e (ige) monoclonal antibody can be used, but the high cost and parenteral administration make it less favorable [ ] . alternative anti-inflammatory drugs are needed. many inflammatory mediators are identified to be involved in the pathogenesis of asthma and they are investigated as potential targets of rnai therapy. these include cytokines/chemokines [ ] , transcription factors [ ] , tyrosine kinases/tyrosine kinase receptors [ ] and co-stimulatory molecules [ ] . in allergic asthma, there is predominant activation of cd + t helper (t h ) cell-driven inflammatory response [ ] . t cell activation and t h cytokine expression are increased in the airways of atopic asthma patients after exposure to allergen [ ] . t h -type cytokines, interleukin- (il- ), il- and il- , are the main players in the pathology of allergic airway inflammation, contributing to allergic sensitization, ige production, eosinophil infiltration and ahr respectively [ , ] . clinical studies that examine the therapeutic potential of using monoclonal antibodies (mabs) to neutralize the bioactivity of il- [ ] , il- [ , ] and il- [ ] or target their receptors are in progress. similarly, rnai technology can be employed to target these cytokines [ , ] . targeting suppressor of cytokine signaling (socs) is an alternative approach [ ] . socs proteins are negative regulators of cytokine signaling pathway, which are involved in t h cells mediated allergic response via controlling the balance between t h and t h cells [ ] . the expression of socs is a pathological marker of allergic disease. after sirna targeting, socs was intranasally administered to the lungs of chronic asthmatic mouse model [ ] , the silencing of socs down-regulated the expression of t h cell associated cytokines, il- , il- and il- , leading to substantial reduction of airway inflammation, ahr as well as ige production. reduction of mucus secretion and collagen deposition was also detected in the airways. besides t h cell, other t cells subsets, such as t h and t h cells, also contribute the inflammatory response in asthma by releasing il- and il- a. these molecules may also be the potential targets for rnai therapy [ ] . furthermore, the combination of inflammatory and antiviral sirnas is a prospective therapy for patients with virus-induced exacerbation of severe uncontrolled asthma, in view of the report that respiratory viral infection is related to % of asthma exacerbation in children and adults [ ] . the therapeutic potential of targeting il- and p protein of rsv simultaneously by sirnas was examined in a mouse model of rsv-induced asthma exacerbation [ ] . the intranasal delivery of the two sirnas was able to: (i) significantly suppress il- mrna expression and rsv replication in the lungs; (ii) reduce eosinophil and neutrophil infiltration in bronchoalveolar lavage fluid (balf); (iii) inhibit ahr and inflammation; and (iv) increase interferon (ifn)-γ expression. many transcription factors including nuclear factor-κb (nf-κb) [ ] , signal transducer and activator of transcription factor (stat ) [ ] and gata-binding protein (gata ) [ ] are involved in the production t h cytokine in the airways of patients with asthma. they were investigated as targets for rnai in the management of asthmatic inflammation. nf-κb plays a key role in the expression of many pro-inflammatory proteins [ ] . inhibition of nf-κb pathway in the lungs was reported to be beneficial in asthma model [ , ] . one approach to inhibit the activation of nf-κb signaling pathway is to target receptor-interacting protein (rip ), which is a transcriptional product and an activator of nf-κb. suppression of rip expression was achieved in mice by intratracheal delivery of sirna, resulting in the inhibition of ovalbumin (ova)-induced cytokine release, inflammatory cell infiltration, mucus hypersecretion and serum ige level. this result suggested that rip could be a novel therapeutic target for the treatment of asthma [ ] . consistent with this finding, suppression of stat [ ] and gata [ , ] in murine model of allergen-induced asthma by rnai significantly inhibited the expression of downstream t h cytokines. these studies support the hypothesis of using rnai molecules targeting transcription factors to manage airway inflammation and ahr of asthma. tyrosine kinases, which are broadly classified into receptor and non-receptor types, play an important role in activating the transcription factors for inflammatory gene expression upon activation by the inflammatory signals [ , ] . the key receptor tyrosine kinases involved in the inflammatory response in asthma include epidermal growth factor receptor (egfr), c-kit (a stem cell factor receptor) and vascular endothelial growth factor receptor (vegfr), while the non-receptor forms are spleen tyrosine kinase (syk), src and janus kinase (jak) [ ] . syk is a pivotal intracellular signaling molecule involved in the activation of several pro-inflammatory transcription factors [ ] . inhibition of syk expression by antisense oligonucleotides has proven effective in suppression of tracheal contraction and pulmonary inflammation in animal models of ova-induced asthma [ ] . when naked sirna targeting syk was administered to the lungs of allergen-induced asthma mice model via nasal instillation, the recruitment of inflammatory cells in the balf was reduced [ ] . similarly, intranasal administration of sirna targeting c-kit also induced a promising result in reducing mucus secretion and airway inflammation in experimental asthma mouse model [ ] . moreover, clinical studies have been performed on a sirna targeting syk (excellair™ developed by zabecor pharmaceuticals, usa) for the treatment of asthma. in the phase i study, % of asthma patients receiving excellair™ by inhalation for consecutive days showed improvement in breathing or reduced the usage of rescue inhaler compared to placebo. the drug was well tolerated by patients without eliciting serious side effects. a phase ii clinical trial excellair™ has been initiated in ; the results are not yet available during the preparation of this review [ , ] . targeting dendritic cells (dcs) in the airways by rnai is another strategy for the treatment of asthma. dcs are antigen presenting cells that could be found in the airways [ , ] . they are actively involved in the differentiation of t h cells and hence contribute to the progress of airway inflammation [ ] . dcs express co-stimulatory molecules, cluster of differentiation (cd) and , on the surface. the binding of cd /cd with cd and cytotoxic t lymphocyte-associated antigen- (ctla- ) molecules on t cells is necessary for priming naive t cells into t h cells [ ] . blocking cd and/or cd activity with pharmacological inhibitors can inhibit t cell activation and alleviate ahr and pulmonary inflammation in mice exposed to aerosolized allergen challenge [ ] . likewise, intratracheal administration of sirna targeting cd significantly reduced its expression in the airway mucosa of mouse model of allergic asthma. cd sirna treatment also contributed to the amelioration of ova-induced airway eosinophilia, hyperresponsiveness, the elevations of ige in the sera and the production of inflammatory cytokines in balf [ ] . copd is a persistent lung disease characterized by irreversible chronic airflow limitation. the disease is usually progressive and associated with an increased chronic inflammatory response in the lungs [ , ] . it is the result of long term exposure to air pollutants and usually associated with cigarette smoking [ ] . it affects around % of the population in the world. it is the fourth leading cause of death in most of the industrialized countries and expected to become the third by [ ] . three classes of medications used to manage copd are bronchodilators (including β -adrenergic receptor and anticholinergic drugs), corticosteroids and phosphodiesterase- inhibitor. they are effective in relieving the symptoms of copd, but none of them can reverse the progressive deterioration in lung function or cure the disease [ , ] . it is desirable to develop a novel therapy that can simultaneously control the symptoms and improve the long-term prognosis of the disease. despite similar clinical features, the mechanisms of underlying airway inflammation in asthma and copd are different with distinct patterns of inflammatory cells and mediators being involved [ ] . the mechanisms and the mediators that drive the progression of chronic inflammation and emphysema in copd are far from fully understood [ ] . the major signaling factors involved in the pathogenesis of copd include tnf-α, ccl , cxcl , tgf-β and vegf [ ] . many of the cytokines and chemokines secreted in the airways of copd patients are modulated by the nf-κb pathway [ ] . therefore, rnai targeting nf-κb pathway can be a strategy for the management of copd [ , ] . current research work on rnai therapy for copd remains mainly in the in vitro stage, likely due to the insufficient understanding of the highly complex mechanisms underlying the pathology of the disease [ ] . moreover, the degree of airway inflammatory response is determined both by genetic and epigenetic factors. since multiple inflammatory mediators are involved in the disease process, blocking a single type of molecule is unlikely to exert significant clinical impact [ ] . mirna has the advantage of targeting multiple genes simultaneously. it is involved in the regulation of the inflammatory process of copd by modulating the transcription and translation of genes. differentiated expression of several mirnas, like let- c, mir- b, mir- b [ ] and mir- a- p [ ] were identified in copd patients compared to subjects without copd. modulation the expression of multiple genes using mirna could be a direction of rnai therapy for the treatment of copd. ali is a severe form of diffuse lung disease that is commonly caused by acute systemic inflammatory disorders, such as sepsis, pneumonia, trauma and pancreatitis [ , ] . it is a progressive disease, which can lead to acute respiratory distress syndrome (ards) or more critically, multiple organ failure and even death [ ] . its pathological changes include the release of pro-inflammatory cytokines and chemokines, and the destruction of the epithelium-capillary interface; the latter can facilitate the extravasation of protein-rich fluid and the infiltration of neutrophils, thereby exacerbating the condition [ , ] . since the molecular mechanism underlying the pathology of ali is not well understood, there is no pathophysiologic-driven therapeutic approach available [ ] . rnai targeting the essential inflammatory mediators could offer protection against the progression of the disease. in an in vivo study, systemic delivery of sirna targeting tnf-α significantly reduced the expression of tnf-α in the lungs and lung injury in a shock-induced ali mouse model [ ] . the findings suggested that pulmonary vascular cells, but not the epithelial cells, contribute to the release of tnf-α leading to lung injury following a systemic inflammatory insult. although these findings are contradictory to the others who suggest that pulmonary epithelial cells are the major players for causing lung injury induced by a systemic disease [ , ] , nevertheless, they provide evidence that support the use of sirna targeting tnf-α in the management of ali. increase microvascular permeability is a major cause of amplification of the inflammatory responses in ali, leading to the progression to severe pathological condition [ ] . the integrity of pulmonary and systemic endothelial barrier is maintained by the bioactive lipid, sphingosine- phosphate (s p), which can be irreversibly degraded by an enzyme called sphingosine- -phosphate lyase (s plyase) [ ] . therefore, suppressing endothelial s plyase expression by sirna is a potential therapeutic approach to maintain the integrity of the endothelial barrier [ ] . nanoparticles containing sirna targeting s plyase and recombinant high mobility group box- a peptide (hmgb a) were delivered to the lungs of lps-induced ali mouse model by intratracheal instillation. hmgb a peptide is an antagonist of the pro-inflammatory cytokine hmgb and provides additional anti-inflammatory effect in ali. an arginine-rich r v peptide was used as a carrier. s plyase level was significantly inhibited in the balf, leading to a significantly reduction of inflammatory cytokine (tnf-α and il- ) and inflammatory response in the lungs of the treated animal [ ] . although several in vivo studies demonstrated promising results of using rnai therapeutics for the management of respiratory inflammatory diseases, there are several concerns that need to be taken into consideration with this approach. the distribution of naked sirna following intranasal and intratracheal administration was investigated. it was shown that the sirna molecules were predominantly accumulated in the peribronchial epithelial cells within h post-administration [ ] . since the inflammatory mechanism of diseases like asthma appears to be driven by the activated t h lymphocytes [ ] , it is highly desirable for rnai molecules to target the activated t cells in the airways to achieve therapeutic benefit. however, specific targeting to t cells is extremely difficult [ ] . effective t cells transfection in vitro was achieved by electroporation [ ] , an approach that is not clinically feasible. recently, a ligand-polymer conjugate sirna delivery system, transferrin-polyethylenimine (tf-pei), has been developed to target t cells in the lungs [ ] . the rational of such a design was based on the increased expression of transferrin receptors on t cells after activation. biodistribution study showed that tf-pei polyplexes can selectively deliver the fluorescently labeled sirna to the activated t cells in a murine asthma model. however, the therapeutic effect of the system was not evaluated. therefore, targeting delivery of rnai therapeutics to specific cell types associated with the disease is a field that certainly required further exploration. most of the key signaling molecules involved in the pathogenesis of respiratory inflammatory disorders have multiple functions and participate in normal physiological activities to maintain homeostasis. short-term regimen (up to four days) was employed in the majority of the animal studies. the duration of treatment may not be sufficient to observe any long-term effect. since many inflammatory diseases are chronic disorders, long-term therapy is often required to control the disease. therefore, it is crucial to evaluate the long-term effect of any rnai therapeutics. inhaled corticosteroid is commonly used to control the symptoms of lung inflammatory disorders. steroid treatment reduces airway inflammation, partially through down-regulation of the key effectors molecules, such as c-kit [ ] and gata [ ] . for this reason, it is necessary to determine the effect of concurrent medications on the expression of targeting molecules before initiating the rnai therapy. given that lung inflammatory diseases are heterogeneous diseases that are categorized into different subtypes according to the inflammatory profiles and responses to treatment, it is desirable to identify the patients' profile in order to maximize the benefit of rnai therapy. pulmonary fibrosis is a respiratory disorder characterized by the progressive and irreversible destruction of lung architecture, which ultimately leads to organ malfunction, disruption of gas exchange and even death [ ] . it is initiated by lung tissue damage resulting from autoimmune disorders or exposure to external irritants. the damage in turn triggers inflammatory reactions for tissue repair in the attempt to restore the normal tissue architecture and functions [ ] . under pathological conditions, extracellular matrix (ecm) components, which are released by the activated myofibroblasts during the wound healing process, are excessively produced and accumulate at the site of tissue injury, resulting in fibrosis [ ] . there are many forms of pulmonary fibrosis. all of them are lethal diseases with high mortality rate but with very limited therapeutic options [ ] . thus pulmonary fibrosis is a lung disease with huge unmet medical need. rnai has been exploited to suppress the production of key molecules involved in the fibrotic process, such as tgf-β [ , ] , amphiregulin (ar) and connective transforming growth factor (ctgf) [ , ] (table ) . clinical trials examining the effect of neutralized antibodies to block the activity of tgf-β are currently ongoing [ ] . since lung epithelial cells are the key cell types in the pathogenesis of pulmonary fibrosis, local administration of rnai targeting tgf-β to the lungs can ensure high local concentration of rnai molecules to produce therapeutic effects. intratracheal delivery of sirna targeting tgf-β could effectively inhibit pulmonary fibrosis, improved lung function, and prolonged survival in human tgf-β transgenic mice [ ] . in another in vivo study, intranasal delivery of mirna- mimic, which was designed to suppresses the expression of tgf-β and other profibrotic genes, significantly reduced tgf-β expression level and further alleviated the fibrotic feature in the bleomycin-induced pulmonary fibrosis [ ] . the results suggest the potential of targeting tgf-β or its signaling pathway using rnai technology for the management of lung fibrosis. notes: ar, amphiregulin; ccl , chemokine (c-c motif) ligand /monocyte chemoattractant protein- ; ctgf, connective tissue growth factor; pdgf, platelet-derived growth factor; pdmaema, poly(dimethylamino)ethylmethacrylate; pmapeg: poly(methylether-methacrylate-ethyleneglycol); samirna, self-assembled micelle interfering rna; tgf-β, transforming growth factor β. one complication of using tgf-β as the molecular target of rnai is related to its physiological role in maintaining immune and cellular homeostasis. it has a role in tumor suppression as suggested by the finding that specific inhibition of tgf-β in mouse stromal fibroblasts results in spontaneous carcinoma formation in the adjacent epithelium [ ] . as a result, targeting the downstream effectors of tgf-β may offer the therapeutic benefit of inhibiting the tgf-β-mediated fibrotic response while preserving the major physiological function of the profibrotic cytokine [ ] . ctgf is a key molecule that regulates fibroblast mitosis and promotes collagen deposition in pulmonary fibrosis induced by tgf-β. a non-viral delivery system consists of poly(dimethylamino)ethylmethacrylate (pdmaema) and its copolymer with poly(methylether-methacrylate-ethyleneglycol) [pmapeg] was used to incorporate and form complexes with sirna targeting ctgf. after intratracheal administration, significant reduction of ctgf expression, collagen deposition and inflammatory cytokines (il- , tnf-α and tgf-β) production were observed in bleomycin-induced lung fibrosis animal model. more importantly, the survival rate of the animals treated with sirna targeting ctgf was significantly increased in comparison with the group treated with scrambled sirna [ ] . another downstream mediator of tgf-β is amphiregulin (ar), which is an epidermal growth factor receptor (egfr) ligand primarily induced by tgf-β [ ] . sirnas targeting ar and ctgf were delivered to the lung of pulmonary fibrosis mouse model via either intratracheal or intravenous administration. to improve the efficiency and cell-specific delivery, a modified self-assembled micelle interfering rna nanoparticles (samirna) was developed. the nanoparticles contain hydrophilic polymer and hydrophobic lipid on each ends of sirna and can spontaneously form micelle in solution. after administration of ar or ctgf samirnas, the expression of ar and ctgf, and collagen accumulation in the lung of pulmonary fibrosis model were significantly reduced compared to control samirna [ ] . rnai technology has been under rapid development for the last decade. the therapeutic potential of rnai molecules in airway diseases has been demonstrated in several clinical studies (table ) , and they could be the solution to some unmet medical needs. no rnai therapeutics has gained approval by regulatory authority at the time this manuscript was prepared. while delivery is generally considered as the major obstacle to rnai therapeutics development, it is interesting to notice that sirnas or mirna mimics do not need a carrier to generate therapeutic effects following pulmonary delivery. the cellular uptake mechanism of naked rna in the airways is not clear, but this is certainly an attractive feature that allows simple formulation without the need of delivery vectors. chemically modified sirna or mirna is necessary to improve the stability of the rna molecules. on the other hand, the delivery of shrna or mirna encoding plasmids requires viral vectors to ensure efficient transduction and the subsequent expression of rnai molecules. however, the use of viral vectors could raise safety concerns for clinical applications, especially when they are used for pulmonary delivery. the employment of liposomal or polymer based nanoparticle delivery technology could be a promising approach. whether the nanoparticles or naked rna are used, it is important to evaluate the pharmacokinetic profile of each delivery system to ensure the rnai therapeutics are localized at the site of action but not quickly distributed to other organs in the body. in terms of formulation of rnai therapeutics, dry powder aerosol is preferred over the liquid aerosol due to higher stability and the dry powder inhalers are easier to operate with better lung deposition [ ] . this would be the direction for rnai formulation development. among the different types of rnai molecules for therapeutic development, sirna has made significant progress, with a couple of clinical studies investigate the pulmonary delivery of sirna to treat respiratory diseases. there are also a growing number of clinical pipelines involving the use of sirna and other rnai molecules. several key questions are raised that need to be answered. for infectious diseases, as demonstrated in several in vivo studies, it appears that rnai is more effective in prophylactic regimen than in therapeutic regimen. how can we improve the therapeutic efficacy of rnai in combating infections to make it clinically relevant? for inflammatory diseases, long-term therapy is expected. how can we minimize the undesirable effects when cytokines/chemokines are being targeted? when the respiratory functions are compromised in patients with lung disease, is pulmonary delivery a suitable route for administration? as complicated signaling pathway is involved in the pathogenesis of different lung disorders, a thorough investigation of the underlying pathological mechanisms of the disease is a prerequisite for identifying the target of rnai. despite the challenges and obstacles, we believe that the pulmonary delivery of rnai molecules targeting airway diseases is promising. a systematic review of the clinical effectiveness of first-line chemotherapy for adult patients with locally advanced or metastatic non-small cell lung cancer respiratory syncytial virus-a comprehensive review novel drugs against tuberculosis: a clinician's perspective the copd pipeline asthma: pathogenesis and novel drugs for treatment a review of current and novel therapies for idiopathic pulmonary fibrosis potent and specific genetic interference by double-stranded rna in caenorhabditis elegans self-assembled micelle interfering rna for effective and safe targeting of dysregulated genes in pulmonary fibrosis histological quantification of gene silencing by intratracheal administration of dry powdered small-interfering rna/chitosan complexes in the murine lung development of pre-clinical models for evaluating the therapeutic potential of candidate sirna targeting stat combined delivery of hmgb- box a peptide and s plyase sirna in animal models of acute lung injury gene silencing of socs by sirna intranasal delivery inhibits asthma phenotype in mice local pulmonary immunotherapy with sirna targeting tgfβ enhances antimicrobial capacity in mycobacterium tuberculosis infected mice pulmonary delivery of therapeutic sirna rnai therapeutics: principles, prospects and challenges immune responses to dsrna: implications for gene silencing technologies preclinical and clinical development of sirna-based therapeutics in vivo delivery aspects of mirna, shrna and sirna subcellular fate and off-target effects of sirna, shrna, and mirna shrna: similarities and differences sirna versus mirna as therapeutics for gene silencing regulation of microrna biogenesis systemic delivery of anti-mirna for suppression of triple negative breast cancer utilizing rna nanotechnology lipid nanoparticle delivery of a microrna- inhibitor improves experimental pulmonary hypertension microrna- mimics delivery inhibits lung tumor progression developing an effective therapeutic by delivery of synthetic microrna- e in lung cancer treatment development of small rna delivery systems for lung cancer therapy a protocol for designing sirnas with high functionality and specificity comparison of antisense oligonucleotides and sirnas in cell culture and in vivo rna interference (rnai)-based therapeutics: delivering on the promise? rna-based drugs and vaccines polyethylenimines for rnai-mediated gene targeting in vivo and sirna delivery to the lung nanoparticle-mediated pulmonary drug delivery: a review influence of particle size on regional lung deposition-what evidence is there? in vivo, in vitro and ex vivo models to assess pulmonary absorption and disposition of inhaled therapeutics for systemic delivery inhaling medicines: delivering drugs to the body through the lungs kissel, t. sirna delivery to the lung: what's new? airway mucus function and dysfunction structure and function of the polymeric mucins in airways mucus regulation of mammalian ciliary beating mucus-penetrating nanoparticles for drug and gene delivery to mucosal tissues composition, structure and mechanical properties define performance of pulmonary surfactant membranes and films pulmonary surfactant inhibits cationic liposome-mediated gene delivery to respiratory epithelial cells in vitro the influence of natural pulmonary surfactant on the efficacy of sirna-loaded dextran nanogels cellular uptake mechanism and knockdown activity of sirna-loaded biodegradable deapa-pva-g-plga nanoparticles bio-inspired pulmonary surfactant-modified nanogels: a promising sirna delivery system inhibition of rnase a family enzymes prevents degradation and loss of silencing activity of sirnas in serum biokinetic studies of non-complexed sirna versus nano-sized pei f -lmw/sirna polyplexes following intratracheal instillation into mice potential and development of inhaled rnai therapeutics for the treatment of pulmonary tuberculosis clathrin-independent pathways of endocytosis targeting of nanoparticles to the clathrin-mediated endocytic pathway dna internalized via caveolae requires microtubule-dependent, rab -independent transport to the late endocytic pathway for delivery to the nucleus high density of octaarginine stimulates macropinocytosis leading to efficient intracellular trafficking for gene expression flotillin-dependent endocytosis and a phagocytosis-like mechanism for cellular internalization of disulfide-based poly(amido amine)/dna polyplexes on the cellular processing of non-viral nanomedicines for nucleic acid delivery: mechanisms and methods gateways for the intracellular access of nanocarriers: a review of receptor-mediated endocytosis mechanisms and of strategies in receptor targeting image-based analysis of lipid nanoparticle-mediated sirna delivery, intracellular trafficking and endosomal escape breaking down the barriers: sirna delivery and endosome escape enhancing endosomal escape for nanoparticle mediated sirna delivery endosomal escape pathways for delivery of biologicals endosomal escape pathways for non-viral nucleic acid delivery systems. in molecular regulation of endocytosis nuclear entry of nonviral vectors pulmonary administration of small interfering rna: the route to go? aerosol delivery of sirna to the lungs. part : nanocarrier-based delivery systems in vivo gene delivery by nonviral vectors: overcoming hurdles & quest delivery materials for sirna therapeutics non-viral vectors for gene-based therapy viral vector-mediated rna interference sustained mirna-mediated knockdown of mutant aat with simultaneous augmentation of wild-type aat has minimal effect on global liver mirna profiles construction of sh-rps lentivirus vectors and its effect on proliferation in lung adenocarcinoma a cell lines. sichuan da xue xue bao yi xue ban influence of suppression of epstein-barr virus-encoded latent membrane protein by raav vector mediated rna interference on metastatic ability of nasopharyngeal cancer cells in vivo pseudotyped adeno-associated virus / -delivered ccl shrna alleviates lung inflammation in an allergen-sensitized mouse model. hum aerosol delivery of sirna to the lungs. part : rationale for gene delivery systems lipid-based vectors for sirna delivery attenuation of fibrosis in vitro and in vivo with sparc sirna advanced transfection with lipofectamine reagent: primary neurons, sirna, and high-throughput applications small interfering rna (sirna) targeting vegf effectively inhibits ocular neovascularization in a mouse model antibody-directed cell-type-specific delivery of sirna toxicity of cationic lipids and cationic polymers in gene delivery recent developments in nucleic acid delivery with polyethylenimines polyethylenimines for sirna and mirna delivery in vivo chitosan-coated plga nanoparticles for dna/rna delivery: effect of the formulation parameters on complexation and transfection of antisense oligonucleotides tat-bmps-pamam conjugates enhance therapeutic effect of small interference rna on u glioma cells in vitro and in vivo biodegradable dextran nanogels for rna interference: focusing on endosomal escape and intracellular sirna delivery rna interference in vitro and in vivo using a chitosan/sirna nanoparticle system formulation of ph responsive peptides as inhalable dry powders for pulmonary delivery of nucleic acids peptides used in the delivery of small noncoding rna intracellular delivery of molecular cargo using cell-penetrating peptides and the combination strategies polyester analogue of poly-l-lysine as a soluble poly-l-lysine functionalized large pore cubic mesostructured silica nanoparticles as biocompatible carriers for gene delivery intratracheal administration of small interfering rna targeting fas reduces lung ischemia-reperfusion injury microrna- regulates profibrotic functions of transforming growth factor-β in pulmonary fibrosis rnai therapeutic platforms for lung diseases metastatic non-small-cell lung cancer (nsclc): esmo clinical practice guidelines for diagnosis, treatment and follow-up fda drug approval summary: bevacizumab (avastin ® ) plus carboplatin and paclitaxel as first-line treatment of advanced/metastatic recurrent nonsquamous non-small cell lung cancer approval summary: erlotinib maintenance therapy of advanced/metastatic non-small cell lung cancer (nsclc) fda drug approval summary: gefitinib (zd )(iressa ® ) tablets nanomedicine as an emerging platform for metastatic lung cancer therapy aerosol gene delivery using viral vectors and cationic carriers for in vivo lung cancer therapy akt/protein kinase b is constitutively active in non-small cell lung cancer cells and promotes cellular survival and resistance to chemotherapy and radiation dual expression of shakt and pdcd suppresses lung tumorigenesis in k-ras la mice suppression of lung cancer progression by biocompatible glycerol triacrylate-spermine-mediated delivery of shakt lentivirus-aimp -dx shrna suppresses cell proliferation by regulating akt signaling pathway in the lungs of aimp +/-mice factors involved in the aerosol transmission of infection and control of ventilation in healthcare premises pulmonary codelivery of doxorubicin and sirna by ph-sensitive nanoparticles for therapy of metastatic lung cancer a novel platform to enable inhaled naked rnai medicine for lung cancer in vivo tumor targeting via nanoparticle-mediated therapeutic sirna coupled to inflammatory response in lung cancer mouse models knockdown of the sodium-dependent phosphate co-transporter b (npt b) suppresses lung tumorigenesis gene silencing in a mouse lung metastasis model by an inhalable dry small interfering rna powder prepared using the supercritical carbon dioxide technique nanostructured lipid carriers as multifunctional nanomedicine platform for pulmonary co-delivery of anticancer drugs and sirna modelling myc inhibition as a cancer therapy co-delivery of doxorubicin and bcl- sirna by mesoporous silica nanoparticles enhances the efficacy of chemotherapy in multidrug-resistant cancer cells rpn gene confers docetaxel resistance in breast cancer prognostic and therapeutic impact of rpn -mediated tumor malignancy in non-small-cell lung cancer co-delivery of chemosensitizing sirna and an anticancer agent via multiple monocomplexation-induced hydrophobic association polypeptide cationic micelles mediated co-delivery of docetaxel and sirna for synergistic tumor therapy co-delivery of sirnas and anti-cancer drugs using layered double hydroxide nanoparticles bcl- inhibitors: targeting mitochondrial apoptotic pathways in cancer therapy multidrug resistance proteins mrp , mrp , and mrp in lung cancer correlation of protein levels with drug response and messenger rna levels sirna-based therapies for pulmonary diseases bacterial complications of respiratory tract viral illness: a comprehensive evaluation rna interference-based therapeutics: new strategies to fight infectious disease inhibition of respiratory viruses by nasally administered sirna rna interference-mediated silencing of the respiratory syncytial virus nucleocapsid defines a potent antiviral strategy inhibition of influenza virus production in virus-infected mice by rna interference protection against lethal influenza virus challenge by rna interference in vivo full deacylation of polyethylenimine dramatically boosts its gene delivery efficiency and specificity to mouse lung effective small interfering rnas targeting matrix and nucleocapsid protein gene inhibit influenza a virus replication in cells and mice rna interference of avian influenza virus h n by inhibiting viral mrna with sirna expression plasmids intrapulmonary delivery of xcl -targeting small interfering rna in mice chronically infected with mycobacterium tuberculosis phenotypic silencing of cytoplasmic genes using sequence-specific double-stranded short interfering rna and its application in the reverse genetics of wild type negative-strand rna viruses use of sirnas to prevent and treat influenza virus infection expression of a single sirna against a conserved region of np gene strongly inhibits in vitro replication of different influenza a virus strains of avian and swine origin using sirna in prophylactic and therapeutic regimens against sars coronavirus in rhesus macaque clinical practice guideline: the diagnosis, management, and prevention of bronchiolitis respiratory syncytial virus infection in adults respiratory syncytial virus infection in elderly and high-risk adults strategic priorities for respiratory syncytial virus (rsv) vaccine development. vaccine evaluation of the safety, tolerability and pharmacokinetics of aln-rsv , a novel rnai antiviral therapeutic directed against respiratory syncytial virus (rsv) a randomized, double-blind, placebo-controlled study of an rnai-based therapy directed against respiratory syncytial virus complete results of our aln-rsv phase iib study influenza a viruses: new research developments adamantane-resistant influenza a viruses in the world ( - ): frequency and distribution of m gene mutations influenza virus resistance to neuraminidase inhibitors rna interference of influenza virus production by directly targeting mrna for degradation and indirectly inhibiting all viral rna transcription barrier or carrier? pulmonary surfactant and drug delivery targeted small interfering rna-immunoliposomes as a promising therapeutic agent against highly pathogenic avian influenza a (h n ) virus infection small interfering rna targeting m gene induces effective and long term inhibition of influenza a virus replication inhalable dry powder formulations of sirna and ph-responsive peptides with antiviral activity against h n influenza virus misinterpreting the therapeutic effects of small interfering rna caused by immune stimulation. hum who world health organization: tuberculosis-global tb programme core functions bfl- /a acts as a negative regulator of autophagy in mycobacteria infected macrophages higher order rab programming in phagolysosome biogenesis microrna- promotes autophagy to eliminate intracellular mycobacteria by targeting rheb microrna- a inhibits autophagy activation and antimicrobial responses during mycobacterial infection tuberculosis: autophagy is not the answer revisiting the role of the granuloma in tuberculosis the tuberculous granuloma: an unsuccessful host defence mechanism providing a safety shelter for the bacteria? xcl (lymphotactin) chemokine produced by activated cd t cells during the chronic stage of infection with mycobacterium tuberculosis negatively affects production of ifn-γ by cd t cells and participates in granuloma stability enhanced production of tgf-beta by blood monocytes from patients with active tuberculosis and presence of tgf-beta in tuberculous granulomatous lung lesions down-modulation of lung immune responses by interleukin- and transforming growth factor β (tgf-β) and analysis of tgf-β receptors i and ii in active tuberculosis pathogenesis of indirect (secondary) acute lung injury immunology of asthma and chronic obstructive pulmonary disease acute lung injury small interfering rnas targeted to interleukin- and respiratory syncytial virus reduce airway inflammation in a mouse model of virus-induced asthma exacerbation. hum small interfering rna against transcription factor stat inhibits allergic airway inflammation and hyperreactivity in mice vegf controls lung t h inflammation via the mir- -mpl (myeloproliferative leukemia virus oncogene)-p-selectin axis receptor-interacting protein gene silencing attenuates allergic airway inflammation let- microrna-mediated regulation of il- and allergic airway inflammation lentiviral-mediated gata- rnai decreases allergic airway inflammation and hyperresponsiveness small interfering rna against cd during allergen challenge blocks experimental allergic asthma effect of locally administered syk sirna on allergen-induced arthritis and asthma intranasal sirna targeting c-kit reduces airway inflammation in experimental allergic asthma targeted delivery of sirna to activated t cells via transferrin-polyethylenimine (tf-pei) as a potential therapy of asthma the role and source of tumor necrosis factor-α in hemorrhage-induced priming for septic lung injury targeting the nf-κb pathway in asthma and chronic obstructive pulmonary disease asthma: defining of the persistent adult phenotypes international ers/ats guidelines on definition, evaluation and treatment of severe asthma investigating the value of omalizumab in the treatment of severe persistent allergic asthma: a systematic review of cost-effectiveness studies sirnas targeted to il- and rsv reduce airway inflammation in a mouse model of virus-induced asthma exacerbation. hum pulmonary delivery of sirna via polymeric vectors as therapies of asthma functions of t cells in asthma: more than just th cells the role of t lymphocytes in the pathogenesis of asthma dupilumab in persistent asthma with elevated eosinophil levels a phase , randomized, placebo-controlled, dose-escalation study of an anti-il- monoclonal antibody in healthy subjects and mild asthmatics efficacy and safety of tralokinumab in patients with severe uncontrolled asthma: a randomised, double-blind, placebo-controlled, phase b trial benralizumab, an anti-interleukin receptor α monoclonal antibody, versus placebo for uncontrolled eosinophilic asthma: a phase b randomised dose-ranging study suppressor of cytokine signaling (socs ) in th cells evokes th cytokines, ige, and eosinophilia. curr role of viral respiratory infections in asthma and asthma exacerbations nf-κb: a key role in inflammatory diseases persistent activation of nuclear factor-κb signaling pathway in severe uncontrolled asthma treatment of allergic airway inflammation and hyperresponsiveness by antisense-induced local blockade of gata- expression inhibitors of the tyrosine kinase signaling cascade for asthma tyrosine kinase inhibitors: a new approach for asthma the potential use of tyrosine kinase inhibitors in severe asthma syk activation in dendritic cells is essential for airway hyperresponsiveness and inflammation inhibition of allergic inflammation in the airways using aerosolized antisense to syk kinase clinical status of duplex rna current progress of sirna/shrna therapeutics in clinical trials dendritic cells and epithelial cells: linking innate and adaptive immunity in asthma essential role of dendritic cell cd /cd costimulation in the induction, but not reactivation, of t h effector responses in a mouse model of asthma tracking and treating activated t cells essential role for both cd and cd costimulation, but not cd interactions, in allergen-induced th cytokine production from asthmatic bronchial tissue: role for αβ, but not γδ, t cells global strategy for the diagnosis, management, and prevention of chronic obstructive pulmonary disease: gold executive summary cellular and molecular mechanisms of chronic obstructive pulmonary disease immunologic aspects of chronic obstructive pulmonary disease recent advances in pre-clinical mouse models of copd regulation of airway muc ac expression by il- β and il- a; the nf-κb paradigm gene expression networks in copd: microrna and mrna regulation microrna- a- p is associated with hypoxia-inducible factor- α expression in lungs from patients with copd acute lung injury review the acute respiratory distress syndrome the acute respiratory distress syndrome silencing of fas, but not caspase- , in lung epithelial cells ameliorates pulmonary apoptosis, inflammation, and neutrophil influx after hemorrhagic shock and sepsis tnf-induced death signaling triggers alveolar epithelial dysfunction in acute lung injury protection of lps-induced murine acute lung injury by sphingosine- -phosphate lyase suppression innate and adaptive immune responses in asthma high-efficiency transfection of primary human and mouse t lymphocytes using rna electroporation stem cell factor and its receptor c-kit as targets for inflammatory diseases suppression of gata- nuclear import and phosphorylation: a novel mechanism of corticosteroid action in allergic disease integrating mechanisms of pulmonary fibrosis pulmonary fibrosis: pathogenesis, etiology and regulation development and preclinical efficacy of novel transforming growth factor-β short interfering rnas for pulmonary fibrosis noncovalenly pegylated ctgf sirna/pdmaema complex for pulmonary treatment of bleomycin-induced lung fibrosis tgf-ß signaling in fibroblasts modulates the oncogenic potential of adjacent epithelia modifiers of tgf-β effector function as novel therapeutic targets of pulmonary fibrosis amphiregulin, an epidermal growth factor receptor ligand, plays an essential role in the pathogenesis of transforming growth factor-β-induced pulmonary fibrosis dry powder formulation of plasmid dna and sirna for inhalation the promise, pitfalls and progress of rna-interference-based antiviral therapy for respiratory viruses rna interference therapy in lung transplant patients infected with respiratory syncytial virus the authors declare no conflict of interest. key: cord- -blycbi u authors: saadeh, haythem a.; sweidan, kamal a.; mubarak, mohammad s. title: recent advances in the synthesis and biological activity of -hydroxyquinolines date: - - journal: molecules doi: . /molecules sha: doc_id: cord_uid: blycbi u compounds containing the -hydroxyquinoline ( -hq) nucleus exhibit a wide range of biological activities, including antimicrobial, anticancer, and antifungal effects. the chemistry and biology of this group have attracted the attention of chemists, medicinal chemists, and professionals in health sciences. a number of prescribed drugs incorporate this group, and numerous -hq- based molecules can be used to develop potent lead compounds with good efficacy and low toxicity. this review focusses on the recent advances in the synthesis of -hq derivatives with different pharmacological properties, including anticancer, antiviral, and antibacterial activities. for this purpose, recent relevant references were searched in different known databases and search engines, such as medline (pubmed), google scholar, science direct, scopus, cochrane, scientific information database (sid), scifinder, and institute for scientific information (isi) web of knowledge. this review article provides a literature overview of the various synthetic strategies and biological activities of -hq derivatives and covers the recent related literature. taken together, compounds containing the -hq moiety have huge therapeutic value and can act as potential building blocks for various pharmacologically active scaffolds. in addition, several described compounds in this review could act leads for the development of drugs against numerous diseases including cancer. -hydroxyquinoline derivatives are an important group of compounds with rich and diverse biological activities. these compounds incorporate the -hydroxyquinoline ( -hq) moiety, which is a bicyclic compound that consists of a pyridine ring fused to phenol, in which the hydroxyl group is attached to position [ ] . in this respect, the pyridine ring maintains its properties as an electron-deficient entity with a basic nitrogen. in addition, and due to the presence of the phenolic group, -hq displays typical phenolic properties that make it susceptible to numerous chemical reactions and structural modifications, such as electrophilic aromatic substitution, diazonium coupling, and molecular rearrangements. the close proximity of the hydroxyl group to the heterocyclic nitrogen makes -hydroxyquinolines good monoprotic bidentate chelating agents, which form four-and six-covalent complexes with a wide range of metal ions, including cu + , zn + , bi + , mn + , mg + , cd + , ni + , fe + , and al + [ ] . figure is a schematic representation of the different sites of reactions of -hq. all of the aforementioned properties make -hq a privileged structure with a variety of structural modifications, possessing a rich diversity of physical, chemical and biological properties. -hydroxyquinoline has attracted the attention of chemists, medicinal chemists, and people in health sciences due to its unique physical and chemical properties. the interest in this compound and its derivatives has increased considerably in the last two decades [ ] . -hydroxyquinoline and many of its derivatives have a wide range of pharmacological applications, e.g., as iron-chelators for neuroprotection, as anticancer agents, as inhibitors of og-dependent enzymes, as chelators of metalloproteins, as anti-hiv agents, as antifungal agents, as antileishmanial agents, as antischistosomal agents, as mycobacterium tuberculosis inhibitors, and as botulinum neurotoxin inhibitors [ ] . furthermore, these compounds are used as electron carriers in organic light-emitting diodes (oleds) and as fluorescent chemosensors for metal ions [ , ] . on the basis of the preceding discussion, and owing to the importance of -hq from a chemistry point of view and to the wide range of biological activities and pharmacological applications of -hq and derivatives, this review focuses on current knowledge of the synthetic methods of novel derivatives, along with the derivatives' biological activities and medicinal applications. in addition, this review summarizes the most recent literature pertaining to the synthesis and bioactivity of -hq and its derivatives, covering the period ranging from to the present. in addition, structure-activity relationships (sars) of these derivatives are discussed to provide directions for further development of novel -hq-based bioactive agents. for this purpose, recent relevant references have been obtained from different databases, such as medical literature retrieval analysis and retrieval system online (medline) (pubmed), google scholar, science direct, scopus, cochrane, sid, and scifinder. our intention is that the content and organization of this review will be valuable to the field and will greatly help researchers. below are details about the recent documented synthesis methods of -hq and its derivatives, as well as their biological activities against different diseases and disorders. due to the coronavirus (covid- ) pandemic and its devastating economic, social, and health effects, we decided to start this study with the antiviral activities of some recent -hq derivatives. a few publications have dealt with the antiviral activities of -hq and its derivatives. de la guardia et al. described the synthesis of novel -hydroxyquinoline derivatives (scheme ) and investigated their activity against dengue virus [ ] . these researchers prepared a number of compounds from -hydroxyquinoline n-oxide , which upon treatment with copper-catalyzed grignard reagents (rmgx; r = i-pr and i-bu) gave -alkyl- -hydroxyquinoline . subsequent chlorination using n-chlorosuccinimide (ncs) under acidic conditions afforded the corresponding -alkyl- , -dichloro- -hydroxyquinoline in a good yield. scheme . synthesis of -isopropy, -isobutyl- , -dichloro- -hydroxyquinoline . -hydroxyquinoline has attracted the attention of chemists, medicinal chemists, and people in health sciences due to its unique physical and chemical properties. the interest in this compound and its derivatives has increased considerably in the last two decades [ ] . -hydroxyquinoline and many of its derivatives have a wide range of pharmacological applications, e.g., as iron-chelators for neuroprotection, as anticancer agents, as inhibitors of og-dependent enzymes, as chelators of metalloproteins, as anti-hiv agents, as antifungal agents, as antileishmanial agents, as antischistosomal agents, as mycobacterium tuberculosis inhibitors, and as botulinum neurotoxin inhibitors [ ] . furthermore, these compounds are used as electron carriers in organic light-emitting diodes (oleds) and as fluorescent chemosensors for metal ions [ , ] . on the basis of the preceding discussion, and owing to the importance of -hq from a chemistry point of view and to the wide range of biological activities and pharmacological applications of -hq and derivatives, this review focuses on current knowledge of the synthetic methods of novel derivatives, along with the derivatives' biological activities and medicinal applications. in addition, this review summarizes the most recent literature pertaining to the synthesis and bioactivity of -hq and its derivatives, covering the period ranging from to the present. in addition, structure-activity relationships (sars) of these derivatives are discussed to provide directions for further development of novel -hq-based bioactive agents. for this purpose, recent relevant references have been obtained from different databases, such as medical literature retrieval analysis and retrieval system online (medline) (pubmed), google scholar, science direct, scopus, cochrane, sid, and scifinder. our intention is that the content and organization of this review will be valuable to the field and will greatly help researchers. below are details about the recent documented synthesis methods of -hq and its derivatives, as well as their biological activities against different diseases and disorders. in a similar fashion, kos and coworkers reported the microwave-assisted synthesis of thirty-two mono-, di-, and tri-substituted -hydroxyquinoline- -carboxanilides, as shown in scheme [ ] . condensation of activated -hydroxyquinoline- -carboxylic acid ( ) with substituted aniline in the presence of by phosphorus trichloride yielded the desired target compound in good amounts ( - %) . molecules , , x for peer review of the antiviral activities of the two novel quinoline derivatives (r = i-pr and i-bu) were evaluated in vitro against the dengue virus serotype (denv ). both exhibited significant inhibitory activities against this virus. the results indicated that the iso-pr-substituted derivative exhibits a half-maximal inhibitory concentration (ic ) of . µm and a half-maximal cytotoxic concentration (cc ) of . µm, for an estimated selectivity index (si) of . . on the other hand, the iso-bu derivative was also active, showing a higher si value of . , with an ic of . µm and cc of . µm. the mechanism of action was also investigated and the results showed that these two derivatives are not virucidal, but appear to act at an early stage of the virus lifecycle, reducing the intracellular production of the envelope glycoprotein and the yield of infectious virions in treated and infected cells. in a similar fashion, kos and coworkers reported the microwave-assisted synthesis of thirty-two mono-, di-, and tri-substituted -hydroxyquinoline- -carboxanilides, as shown in scheme [ ] . condensation of activated -hydroxyquinoline- -carboxylic acid ( ) with substituted aniline in the presence of by phosphorus trichloride yielded the desired target compound in good amounts ( - %). these prepared compounds were subjected to bioactivity screening against the highly pathogenic h n avian influenza viruses, with the results expressed as percentages of growth inhibition, and to cytotoxicity evaluation against the a cell line. the lipophilicity and electronic properties were the molecular parameters used to determine the structure-activity relationship. the lipophilicity of the studied compounds was determined as a log k value using reversed-phase highperformance liquid chromatography (rp-hplc). based on the presented results, most monosubstituted derivatives did not exert any antiviral activity or demonstrated only moderate activity. for example, -hydroxy-n-( -nitrophenyl)quinoline- -carboxamide showed optimum virus growth inhibition activity and cytotoxicity values of . % and %, respectively. furthermore, the results show that the antiviral activity is influenced by increasing the electron-withdrawing properties of substituents on the anilide ring and is positively influenced by increasing the lipophilicity; in this manner, the derivative showing values of r = -no and log k = ca. . showed maximal activity with insignificant cytotoxicity. di-and tri-substituted derivatives (r = , -cl, , , -cl, -cl- -f, and , -no ) showed higher inhibition of h n growth and simultaneous low cytotoxicity. for example, -cl- -f and , , -cl exhibited virus growth inhibition activity values of . and . % and cytotoxicity values of . and . %, respectively; the log k values for these derivatives were . and . , respectively. the study indicated that the antiviral activity linearly increases with increasing lipophilicity and is positively influenced by increasing the electron-withdrawing properties of substituents on the anilide ring. with the frantic search for drugs to treat covid- patients and ultimately for a covid- vaccine, the -hq nucleus could play a role in this due to its promising antiviral activity; however, more research is required in this area. the spread of the antibiotic-resistant bacterial strains constitutes a serious threat to public health. some of these strains have even become resistant to many antibiotics and chemotherapeutic agents; these prepared compounds were subjected to bioactivity screening against the highly pathogenic h n avian influenza viruses, with the results expressed as percentages of growth inhibition, and to cytotoxicity evaluation against the a cell line. the lipophilicity and electronic properties were the molecular parameters used to determine the structure-activity relationship. the lipophilicity of the studied compounds was determined as a log k value using reversed-phase high-performance liquid chromatography (rp-hplc). based on the presented results, most mono-substituted derivatives did not exert any antiviral activity or demonstrated only moderate activity. for example, -hydroxy-n-( -nitrophenyl)quinoline- -carboxamide showed optimum virus growth inhibition activity and cytotoxicity values of . % and %, respectively. furthermore, the results show that the antiviral activity is influenced by increasing the electron-withdrawing properties of substituents on the anilide ring and is positively influenced by increasing the lipophilicity; in this manner, the derivative showing values of r = -no and log k = ca. . showed maximal activity with insignificant cytotoxicity. di-and tri-substituted derivatives (r = , -cl, , , -cl, -cl- -f, and , -no ) showed higher inhibition of h n growth and simultaneous low cytotoxicity. for example, -cl- -f and , , -cl exhibited virus growth inhibition activity values of . and . % and cytotoxicity values of . and . %, respectively; the log k values for these derivatives were . and . , respectively. the study indicated that the antiviral activity linearly increases with increasing lipophilicity and is positively influenced by increasing the electron-withdrawing properties of substituents on the anilide ring. with the frantic search for drugs to treat covid- patients and ultimately for a covid- vaccine, the -hq nucleus could play a role in this due to its promising antiviral activity; however, more research is required in this area. the spread of the antibiotic-resistant bacterial strains constitutes a serious threat to public health. some of these strains have even become resistant to many antibiotics and chemotherapeutic agents; hence, the term "multidrug resistance" has been introduced in the literature. thus, the development of existing drugs and the discovery of new leads are major strategies used to combat the threat of these multidrug-resistant strains. in this context, hu and coworkers prepared new derivatives of the known potent antibacterial agent , -difluoro- -hydroxybenzamide (dfmba) [ ] . the substituted -hq was alkylated with , -dibromopropane in the presence of an aqueous sodium hydroxide solution and tetrabutylammonium iodide (tbai) in dichloromethane to afford a mono-halogenated intermediate . target compound was obtained via alkylation of dfmba with the corresponding -hq mono bromide (scheme ). molecules , , x for peer review of hence, the term "multidrug resistance" has been introduced in the literature. thus, the development of existing drugs and the discovery of new leads are major strategies used to combat the threat of these multidrug-resistant strains. in this context, hu and coworkers prepared new derivatives of the known potent antibacterial agent , -difluoro- -hydroxybenzamide (dfmba) [ ] . the substituted -hq was alkylated with , -dibromopropane in the presence of an aqueous sodium hydroxide solution and tetrabutylammonium iodide (tbai) in dichloromethane to afford a mono-halogenated intermediate . target compound was obtained via alkylation of dfmba with the corresponding -hq mono bromide (scheme ). scheme . synthesis of , -difluoro- -hydroxybenzamide- -hydroxyquinoline derivatives. the antibacterial activity of these derivatives was evaluated against several gram-positive and gram-negative bacteria using the zone-of-inhibition test. inhibition zones were compared to the standard antibiotic pc ( ) , which is an effective bactericidal cell division inhibitor that targets cell division protein (ftsz) in several gram-positive bacteria. the results showed that the antibacterial activities were lower than that of pc . in addition, the highest inhibition zone ratios (inhibition zone of the test compound/inhibition zone of the standard) of . and . against s. aureus were observed for derivatives where r = -cl and , -dicl, respectively. moreover, when comparing the activity of -hq derivatives with -hq naphthalene analogues, the former were more active; this is probably due to the ring nitrogen, which may increase the polarity and water solubility of the compounds-factors that are required for antibacterial activity. krishna reported the synthesis of a series of new -amino- -bromoquinolin- -yl sulfonates in good yield using a multistep synthesis method [ ] . bromination of -hydroxquinoline with nbromosuccinimide (nbs) in chloroform afforded -bromoquinolin- -ol, which upon treatment with nano /hcl followed by reduction with na s o in : tetrahydrofuran (thf) and water gave amino- -bromoquinolin- -ol . the target sulfonate derivatives were obtained from -amino- bromoquinolin- -ol by reaction with various sulfonyl chlorides in dry thf in the presence of triethylamine (tea) (scheme ). the antibacterial activity of these derivatives was evaluated against several gram-positive and gram-negative bacteria using the zone-of-inhibition test. inhibition zones were compared to the standard antibiotic pc ( ) , which is an effective bactericidal cell division inhibitor that targets cell division protein (ftsz) in several gram-positive bacteria. the results showed that the antibacterial activities were lower than that of pc . in addition, the highest inhibition zone ratios (inhibition zone of the test compound/inhibition zone of the standard) of . and . against s. aureus were observed for derivatives where r = -cl and , -dicl, respectively. moreover, when comparing the activity of -hq derivatives with -hq naphthalene analogues, the former were more active; this is probably due to the ring nitrogen, which may increase the polarity and water solubility of the compounds-factors that are required for antibacterial activity. krishna reported the synthesis of a series of new -amino- -bromoquinolin- -yl sulfonates in good yield using a multistep synthesis method [ ] . bromination of -hydroxquinoline with n-bromosuccinimide (nbs) in chloroform afforded -bromoquinolin- -ol, which upon treatment with nano /hcl followed by reduction with na s o in : tetrahydrofuran (thf) and water gave -amino- -bromoquinolin- -ol . the target sulfonate derivatives were obtained from -amino- -bromoquinolin- -ol by reaction with various sulfonyl chlorides in dry thf in the presence of triethylamine (tea) (scheme ). the newly synthesized sulfonate derivatives were tested against several bacterial strains, such as staphylococcus aureus and bacillus megaterium (gram-positive bacteria), klebsiella pneumoniae and pseudomonas aeruginosa (gram-negative bacteria), and against two gram-negative, antibioticresistant e. coli bacterial strains, namely mutant e. coli (streptomycin-resistant) and donor e. coli (rifampin-resistant), using the agar well diffusion method; amoxiclav (ac) was used as the standard drug. among the synthesized compounds, derivatives with an aryl group showed potent activity. for example, the compound with ar = biphenyl exhibited potent activity against staphylococcus aureus, with an inhibition zone of mm compared to the reference drug ( mm). on the other hand, derivatives with ar = -fph and -oh- -no ph displayed potent activity against pseudomonas aeruginosa, with inhibition zones of mm and mm, respectively, compared to the standard drug ( mm), whereas the derivative with r = -ch ph was potent against klebsiella pneumonia, with an inhibition zone of mm compared to the standard drug ( mm these compounds were tested as antibacterial agents against six pathogenic strains, including e. cloacae, e. coli, k. pneumoniae, p. aeruginosa, s. aureus, and a. baumanii. minimal inhibitory concentrations of these compounds were calculated and compared with three standard antibiotics, namely penicillin g, norfloxacin, and erythromycin, using the disk diffusion technique. some of these prepared compounds exhibited remarkable antibacterial activity against gram-positive and gramnegative strains compared to the standard antibiotics used. one derivative (r = no , r′ = h) was more potent than the standard drugs and showed activity against e. cloacae, k. pneumoniae, s. aureus, the newly synthesized sulfonate derivatives were tested against several bacterial strains, such as staphylococcus aureus and bacillus megaterium (gram-positive bacteria), klebsiella pneumoniae and pseudomonas aeruginosa (gram-negative bacteria), and against two gram-negative, antibiotic-resistant e. coli bacterial strains, namely mutant e. coli (streptomycin-resistant) and donor e. coli (rifampin-resistant), using the agar well diffusion method; amoxiclav (ac) was used as the standard drug. among the synthesized compounds, derivatives with an aryl group showed potent activity. for example, the compound with ar = biphenyl exhibited potent activity against staphylococcus aureus, with an inhibition zone of mm compared to the reference drug ( mm). on the other hand, derivatives with ar = -fph and -oh- -no ph displayed potent activity against pseudomonas aeruginosa, with inhibition zones of mm and mm, respectively, compared to the standard drug ( mm), whereas the derivative with r = -ch ph was potent against klebsiella pneumonia, with an inhibition zone of mm compared to the standard drug ( mm) . a study published by rbaa the newly synthesized sulfonate derivatives were tested against several bacterial strains, such as staphylococcus aureus and bacillus megaterium (gram-positive bacteria), klebsiella pneumoniae and pseudomonas aeruginosa (gram-negative bacteria), and against two gram-negative, antibioticresistant e. coli bacterial strains, namely mutant e. coli (streptomycin-resistant) and donor e. coli (rifampin-resistant), using the agar well diffusion method; amoxiclav (ac) was used as the standard drug. among the synthesized compounds, derivatives with an aryl group showed potent activity. for example, the compound with ar = biphenyl exhibited potent activity against staphylococcus aureus, with an inhibition zone of mm compared to the reference drug ( mm). on the other hand, derivatives with ar = -fph and -oh- -no ph displayed potent activity against pseudomonas aeruginosa, with inhibition zones of mm and mm, respectively, compared to the standard drug ( mm), whereas the derivative with r = -ch ph was potent against klebsiella pneumonia, with an inhibition zone of mm compared to the standard drug ( mm) . a study published by rbaa these compounds were tested as antibacterial agents against six pathogenic strains, including e. cloacae, e. coli, k. pneumoniae, p. aeruginosa, s. aureus, and a. baumanii. minimal inhibitory concentrations of these compounds were calculated and compared with three standard antibiotics, namely penicillin g, norfloxacin, and erythromycin, using the disk diffusion technique. some of these prepared compounds exhibited remarkable antibacterial activity against gram-positive and gramnegative strains compared to the standard antibiotics used. one derivative (r = no , r′ = h) was more potent than the standard drugs and showed activity against e. cloacae, k. pneumoniae, s. aureus, these compounds were tested as antibacterial agents against six pathogenic strains, including e. cloacae, e. coli, k. pneumoniae, p. aeruginosa, s. aureus, and a. baumanii. minimal inhibitory concentrations of these compounds were calculated and compared with three standard antibiotics, namely penicillin g, norfloxacin, and erythromycin, using the disk diffusion technique. some of these prepared compounds exhibited remarkable antibacterial activity against gram-positive and gram-negative strains compared to the standard antibiotics used. one derivative (r = no , r = h) was more potent than the standard drugs and showed activity against e. cloacae, k. pneumoniae, s. aureus, a. baumanii, e. coli, e. cloacae, and e. cloaca, with minimum inhibitory concentration (mic) values (mg/ml) of × − , × − , × − , × − , and × − , respectively, while the mic for the standard drug was × − . derivatives with r = r = h and r = h, r = cl displayed similar activities against s. aureus, with mic values of × − . bioinformatic petra, osiris, and molinspiration (pom) analyses indicated that all compounds exhibit good bioavailability and less toxic profiles when compared with penicillin g. interestingly, two research groups have reported on the hybridization of -hydroxyquinoline with ciprofloxacin in a single step and evaluated the antibacterial activity of the hybrid molecule. fu et al. reported the synthesis of -chloro- -hydroxyquinoline-ciprofloxacin via the mannich reaction (scheme ). thus, -chloro- -hydroxyquinoline reacted with ciprofloxacin in the presence of paraformaldehyde in ethanol to afford the hybrid product at % yield [ ] . interestingly, two research groups have reported on the hybridization of -hydroxyquinoline with ciprofloxacin in a single step and evaluated the antibacterial activity of the hybrid molecule. fu et al. reported the synthesis of -chloro- -hydroxyquinoline-ciprofloxacin via the mannich reaction (scheme ). thus, -chloro- -hydroxyquinoline reacted with ciprofloxacin in the presence of paraformaldehyde in ethanol to afford the hybrid product at % yield [ ] . the hybrid was screened against both gram-positive and gram-negative (staphylococcus epidermidis, s. aureus, enterococci faecalis, and e. faecium) bacterial strains, and the minimum inhibitory concentrations (mics) were determined via the agar dilution method. the results indicated that the hybrid shows exciting promise against both gram-positive and gram-negative bacteria. it displayed significant effects against both susceptible and drug-resistant strains, with mic values of - µg/ml. these values were lower than those of standard drug (ciprofloxacin), which has mic values of . - . µg/ml. however, the authors speculated that the introduction of a quinolone skeleton might be an effective way to modify these kinds of compounds into a novel class of broad-spectrum antibacterial agents with a specific mode of action. on the other hand, vu and coworkers prepared a hybrid compound at % yield through coupling of -hydroxyquinoline- -carboxylic acid and ciprofloxacin by using -( h-benzotriazole- -yl)- , , , -tetramethylaminium tetrafluoroborate (tbtu) and n,n-diisopropylethylamine (diea) (scheme ) [ ] . the hybrid was screened against both gram-positive and gram-negative (staphylococcus epidermidis, s. aureus, enterococci faecalis, and e. faecium) bacterial strains, and the minimum inhibitory concentrations (mics) were determined via the agar dilution method. the results indicated that the hybrid shows exciting promise against both gram-positive and gram-negative bacteria. it displayed significant effects against both susceptible and drug-resistant strains, with mic values of - µg/ml. these values were lower than those of standard drug (ciprofloxacin), which has mic values of . - . µg/ml. however, the authors speculated that the introduction of a quinolone skeleton might be an effective way to modify these kinds of compounds into a novel class of broad-spectrum antibacterial agents with a specific mode of action. on the other hand, vu and coworkers prepared a hybrid compound at % yield through coupling of -hydroxyquinoline- -carboxylic acid and ciprofloxacin by using -( h-benzotriazole- -yl)- , , , -tetramethylaminium tetrafluoroborate (tbtu) and n,n-diisopropylethylamine (diea) (scheme ) [ ] . interestingly, two research groups have reported on the hybridization of -hydroxyquinoline with ciprofloxacin in a single step and evaluated the antibacterial activity of the hybrid molecule. fu et al. reported the synthesis of -chloro- -hydroxyquinoline-ciprofloxacin via the mannich reaction (scheme ). thus, -chloro- -hydroxyquinoline reacted with ciprofloxacin in the presence of paraformaldehyde in ethanol to afford the hybrid product at % yield [ ] . the hybrid was screened against both gram-positive and gram-negative (staphylococcus epidermidis, s. aureus, enterococci faecalis, and e. faecium) bacterial strains, and the minimum inhibitory concentrations (mics) were determined via the agar dilution method. the results indicated that the hybrid shows exciting promise against both gram-positive and gram-negative bacteria. it displayed significant effects against both susceptible and drug-resistant strains, with mic values of - µg/ml. these values were lower than those of standard drug (ciprofloxacin), which has mic values of . - . µg/ml. however, the authors speculated that the introduction of a quinolone skeleton might be an effective way to modify these kinds of compounds into a novel class of broad-spectrum antibacterial agents with a specific mode of action. on the other hand, vu and coworkers prepared a hybrid compound at % yield through coupling of -hydroxyquinoline- -carboxylic acid and ciprofloxacin by using -( h-benzotriazole- -yl)- , , , -tetramethylaminium tetrafluoroborate (tbtu) and n,n-diisopropylethylamine (diea) (scheme ) [ ] . the hybrid was screened for cell toxicity against hela cells, a tumor cell line, primary cell cultures of mouse fibroblasts, and against non-cancerous mammalian cell line. it showed no cell toxicity at the concentrations tested ( - µm). then, the antibacterial activity was tested against gram-negative and gram-positive bacteria, including pathogenic bacteria present in the hospital environment that are difficult to treat (enterococcus faecium, staphylococcus aureus, klebsiella pneumoniae, acinetobacter baumannii, pseudomonas aeruginosa, and enterobacter species). the results indicated that that hybrid exhibits more potent activity than the standard drug against staphylococcus aureus, with an mic (mg/ml) value of . (mic of the standard drug = . ). however, the hybrid was less active against the rest of the pathogens as compared to the standard drug. in general, the two aforementioned hybrids were systematically less active than the parent antibiotic, ciprofloxacin. from these published data, it is obvious that -hq could be an important motif for future antibacterial drugs, especially against antibiotic-resistant strains. however, more research is required, including experiments with animals using newly synthesized antibacterial agents. fungal resistance to antifungal agents has become a problem and a challenge to scientists in recent years. this resistance has serious implications for morbidity, mortality, and health care costs worldwide. hence, attention has been given to understanding the mechanism of antifungal resistance in order to develop new treatments and strategies to manage infections caused by resistant organisms. along this line, le pan and colleagues reported the synthesis of conjugates of -hydroxycoumarin (umbelliferone) with several heterocylic rings, including -and -hydroxyquinolines [ ] . -hydroxycoumarin was mono-alkylated in moderate yields via an excess of br-(ch ) n -br (n = , ) in refluxing acetone in the presence of k co -naoh ( : ). bromoalkylated coumarins were then reacted with -and -hydroxyquinoline in the presence of equivalent of koh and catalytic amounts of ki and tetra-n-butylammonium bromide (tbab) as a phase transfer catalyst in refluxing acetonitrile to give the corresponding conjugates at - % yields (scheme ). molecules , , x for peer review of indicated that that hybrid exhibits more potent activity than the standard drug against staphylococcus aureus, with an mic (mg/ml) value of . (mic of the standard drug = . ). however, the hybrid was less active against the rest of the pathogens as compared to the standard drug. in general, the two aforementioned hybrids were systematically less active than the parent antibiotic, ciprofloxacin. from these published data, it is obvious that -hq could be an important motif for future antibacterial drugs, especially against antibiotic-resistant strains. however, more research is required, including experiments with animals using newly synthesized antibacterial agents. fungal resistance to antifungal agents has become a problem and a challenge to scientists in recent years. this resistance has serious implications for morbidity, mortality, and health care costs worldwide. hence, attention has been given to understanding the mechanism of antifungal resistance in order to develop new treatments and strategies to manage infections caused by resistant organisms. along this line, le pan and colleagues reported the synthesis of conjugates of hydroxycoumarin (umbelliferone) with several heterocylic rings, including -and hydroxyquinolines [ ] . -hydroxycoumarin was mono-alkylated in moderate yields via an excess of br-(ch )n-br (n = , ) in refluxing acetone in the presence of k co -naoh ( : ). bromoalkylated coumarins were then reacted with -and -hydroxyquinoline in the presence of equivalent of koh and catalytic amounts of ki and tetra-n-butylammonium bromide (tbab) as a phase transfer catalyst in refluxing acetonitrile to give the corresponding conjugates at - % yields (scheme ). the antifungal activity of these new conjugates against four phytopathogenic fungi (a. alternate, a. solani, b. cinerea, and f. oxysporum) was evaluated by measuring the mycelial inhibition of radial growth on potato dextrose agar (pda) media compared to the commercial fungicide carbendazim. the inhibition percentages of radial growth against all of these fungi for umbelliferone- hydroxyquinoline (n = ), umbelliferone- -hydroxyquinoline (n = ), and umbelliferone- hydroxyquinoline (n = ) were - , - , and - %, respectively, compared to carbendazim ( - %) at µg/ml concentration. when these derivatives were tested at a series of lower concentrations to measure the half-maximal effective concentration (µg/ml), umbelliferone- hydroxyquinoline (n = ) was the most active compared to the reference drug at . and . µg/ml, respectively. the rest of the -hydroxyquinoline derivatives exhibited weak activity. the results also showed that umbelliferone derivatives with a spacer of carbon atoms exhibit better antifungal the antifungal activity of these new conjugates against four phytopathogenic fungi (a. alternate, a. solani, b. cinerea, and f. oxysporum) was evaluated by measuring the mycelial inhibition of radial growth on potato dextrose agar (pda) media compared to the commercial fungicide carbendazim. the inhibition percentages of radial growth against all of these fungi for umbelliferone- -hydroxyquinoline (n = ), umbelliferone- -hydroxyquinoline (n = ), and umbelliferone- -hydroxyquinoline (n = ) were - , - , and - %, respectively, compared to carbendazim ( - %) at µg/ml concentration. when these derivatives were tested at a series of lower concentrations to measure the half-maximal effective concentration (µg/ml), umbelliferone- -hydroxyquinoline (n = ) was the most active compared to the reference drug at . and . µg/ml, respectively. the rest of the -hydroxyquinoline derivatives exhibited weak activity. the results also showed that umbelliferone derivatives with a spacer of carbon atoms exhibit better antifungal activity than those with a spacer of carbon atoms. the chain length may contribute to the activity by influencing the flexibility of the molecules. in addition, the position of the oh group affects the antifungal activity; derivatives with oh at position were more active than those with oh at position . the structure-activity relationship suggests that modification of the umbelliferone- -hysroxyquinoline analogues could help to develop highly selective and low phytotoxic fungicides. the phytotoxicity of effective compounds was evaluated at mg/l with l. sativa, with the results showing that umbelliferone- -hysroxyquinoline (n = ) had no phytotoxic effects on the seedling growth of lettuce. yurras and coworkers have reported on the multistep synthesis of novel , , -trisubstituted triazole derivatives bearing -hydroxyquinoline , evaluating their antimicrobial activity [ ] . synthesis was achieved by first reacting -hydroxyquinoline with ethyl -chloroacetate in refluxing acetone in the presence of a base. treatment of the formed ester with hydrazine hydrate in ethanol followed by phenyl isocyanate afforded the corresponding thiourea . ring closure using koh in refluxing ethanol led to the formation of -mercapto- , , -triazole derivative . reaction of -mercapto- , , -triazole with -chloro-n-(substituted (benzo)/thiazole)acetamide afforded the target compounds, as depicted in scheme . molecules , , x for peer review of activity than those with a spacer of carbon atoms. the chain length may contribute to the activity by influencing the flexibility of the molecules. in addition, the position of the oh group affects the antifungal activity; derivatives with oh at position were more active than those with oh at position . the structure-activity relationship suggests that modification of the umbelliferone- hysroxyquinoline analogues could help to develop highly selective and low phytotoxic fungicides. the phytotoxicity of effective compounds was evaluated at mg/l with l. sativa, with the results showing that umbelliferone- -hysroxyquinoline (n = ) had no phytotoxic effects on the seedling growth of lettuce. yurras and coworkers have reported on the multistep synthesis of novel , , -trisubstituted triazole derivatives bearing -hydroxyquinoline , evaluating their antimicrobial activity [ ] . synthesis was achieved by first reacting -hydroxyquinoline with ethyl -chloroacetate in refluxing acetone in the presence of a base. treatment of the formed ester with hydrazine hydrate in ethanol followed by phenyl isocyanate afforded the corresponding thiourea . ring closure using koh in refluxing ethanol led to the formation of -mercapto- , , -triazole derivative . reaction of mercapto- , , -triazole with -chloro-n-(substituted (benzo)/thiazole)acetamide afforded the target compounds, as depicted in scheme . some sulfonates reported by krishna (scheme ) were also tested in vitro against aspergillus niger and penicillium spinulosum fungal strains by the poison plate technique; fluconazole was used as a standard drug for antifungal activity. two compounds with ar = biphenyl and ar = -nitro- -hydroxybenzene exhibited the most potent antifungal activity against aspergillus niger, with inhibition zones of and mm, respectively, compared to fluconazole ( mm); and against penicillium spinulosum, with inhibition zones of and mm, respectively, compared to fluconazole ( mm). cancer is a dreadful disease that has become a global burden, causing thousands of deaths per year, despite the technological and pharmaceutical improvements over the past several years. it has emerged as one of the leading causes of mortality worldwide [ ] . cancer treatments include surgery, radiotherapy, and anticancer drugs (chemotherapy), in addition to other specialized techniques. much scientific effort is being made every day in the fight against cancer, but successful treatment of some cancer types is still a challenge that needs more work [ ] . on the synthetic front in the fight against cancer, two new series of derivatives have been prepared where the bioactive quinolone motif is incorporated, as shown in scheme [ ] . -bromo- -methoxyquinoline ( ) was prepared according to a published procedure [ ] . afterwards, compound was subjected to a suzuki cross-coupling reaction with p-formphenylboronic ( ) acid to afford synthon in excellent yield. all target products ( ) were synthesized via a simple and effective method using a one-pot mannich-type reaction that involves a reaction of amines, carbonyl compounds, and dialkylphosphonate. molecules , , x for peer review of some sulfonates reported by krishna (scheme ) were also tested in vitro against aspergillus niger and penicillium spinulosum fungal strains by the poison plate technique; fluconazole was used as a standard drug for antifungal activity. two compounds with ar = biphenyl and ar = -nitro- hydroxybenzene exhibited the most potent antifungal activity against aspergillus niger, with inhibition zones of and mm, respectively, compared to fluconazole ( mm); and against penicillium spinulosum, with inhibition zones of and mm, respectively, compared to fluconazole ( mm). cancer is a dreadful disease that has become a global burden, causing thousands of deaths per year, despite the technological and pharmaceutical improvements over the past several years. it has emerged as one of the leading causes of mortality worldwide [ ] . cancer treatments include surgery, radiotherapy, and anticancer drugs (chemotherapy), in addition to other specialized techniques. much scientific effort is being made every day in the fight against cancer, but successful treatment of some cancer types is still a challenge that needs more work [ ] . on the synthetic front in the fight against cancer, two new series of derivatives have been prepared where the bioactive quinolone motif is incorporated, as shown in scheme [ ] . -bromo- -methoxyquinoline ( ) was prepared according to a published procedure [ ] . afterwards, compound was subjected to a suzuki crosscoupling reaction with p-formphenylboronic ( ) acid to afford synthon in excellent yield. all target products ( ) were synthesized via a simple and effective method using a one-pot mannichtype reaction that involves a reaction of amines, carbonyl compounds, and dialkylphosphonate. the cytotoxicity of these prepared compounds against esophageal (eca ) and hepatocellular (huh ) cancer cell lines was evaluated using sunitinib as a positive control. the results showed that most of these compounds exhibit moderate to high activity, with ic values ranging from . to . µmol/l for the two most promising derivatives containing -methylphenyl and -methylphenyl groups for r and iso-propyl for r ; some of these compounds exhibited inhibition activities comparable to those of sunitinib, which showed ic values of . and . µmol/l towards eca and huh , respectively. furthermore, the results indicated that ethyl and isopropyl substituents of phosphonate have no major effects on the cytotoxicity activity, while substituents on the phenyl ring showed significant influence on the bioactivity. the cytotoxicity of these prepared compounds against esophageal (eca ) and hepatocellular (huh ) cancer cell lines was evaluated using sunitinib as a positive control. the results showed that most of these compounds exhibit moderate to high activity, with ic values ranging from . to . µmol/l for the two most promising derivatives containing -methylphenyl and -methylphenyl groups for r and iso-propyl for r ; some of these compounds exhibited inhibition activities comparable to those of sunitinib, which showed ic values of . and . µmol/l towards eca and huh , respectively. furthermore, the results indicated that ethyl and isopropyl substituents of phosphonate have no major effects on the cytotoxicity activity, while substituents on the phenyl ring showed significant influence on the bioactivity. faydy and coworkers described the synthesis of new derivatives of -hydroxyquinoline ( - ) ( figure ) in a multistep approach [ ] according to published procedures [ ] [ ] [ ] . the antioxidant activity of prepared products, along with l-ascorbic acid, was evaluated by means of the free radical scavenging method using the , -diphenyl- -picryhydrazyl (dpph) assay. the results revealed that all products showed low antioxidant activity, with ic values of . - . mg/ml, whereas the ic value of -ascorbic acid was . mg/ml. furthermore, the results showed that as the number of hydroxyl groups increases, the inhibition activity increases too. molecules , , x for peer review of faydy and coworkers described the synthesis of new derivatives of -hydroxyquinoline ( - ) ( figure ) in a multistep approach [ ] according to published procedures [ ] [ ] [ ] . the antioxidant activity of prepared products, along with l-ascorbic acid, was evaluated by means of the free radical scavenging method using the , -diphenyl- -picryhydrazyl (dpph) assay. the results revealed that all products showed low antioxidant activity, with ic values of . - . mg/ml, whereas the ic value of -ascorbic acid was . mg/ml. furthermore, the results showed that as the number of hydroxyl groups increases, the inhibition activity increases too. [ ] following a published procedure [ ] . the structures of prepared compounds were confirmed with the aid of a panel of spectroscopic methods, including h nmr, the prepared compounds were then subjected to cell viability evaluation against hela, mcf- , a- , and mda-mb- (triple-negative breast cancer cell line) cell lines using the mtt assay [ ] [ ] [ ] at a concentration of mm. the results revealed that only compounds designated with r = phenyl, , -dimethylphenyl, -fluorophenyl, and -trifluoromethylphenyl exhibited significantly reduced cell viability percentages towards the tested cancer cell lines. the ic values for these compounds were between . and . mm against selected cancer cell lines, whereas the ic of docetaxel (positive control) was in the range of . - . mm. a new series of glycoconjugates composed of various sugar units ( , ) (d-glucose or dgalactose) and -hydroxyquinolines ( , ) was prepared in an effective and simple method [ ] . the connection between these units was accomplished by o-glycosidic bond or via o-methylene , , -triazole linker, as shown in scheme . sugar derivatives of and were prepared according to published procedures [ ] [ ] [ ] ; sugar was used to enhance the bioavailability and solubility of potential drugs. (figure ) in moderate to excellent yields [ ] following a published procedure [ ] . the structures of prepared compounds were confirmed with the aid of a panel of spectroscopic methods, including h nmr, c nmr, ir, and hrms. molecules , , x for peer review of faydy and coworkers described the synthesis of new derivatives of -hydroxyquinoline ( - ) ( figure ) in a multistep approach [ ] according to published procedures [ ] [ ] [ ] . the antioxidant activity of prepared products, along with l-ascorbic acid, was evaluated by means of the free radical scavenging method using the , -diphenyl- -picryhydrazyl (dpph) assay. the results revealed that all products showed low antioxidant activity, with ic values of . - . mg/ml, whereas the ic value of -ascorbic acid was . mg/ml. furthermore, the results showed that as the number of hydroxyl groups increases, the inhibition activity increases too. (figure ) in moderate to excellent yields [ ] following a published procedure [ ] . the structures of prepared compounds were confirmed with the aid of a panel of spectroscopic methods, including h nmr, ( , ) was prepared in an effective and simple method [ ] . the connection between these units was accomplished by o-glycosidic bond or via o-methylene , , -triazole linker, as shown in scheme . sugar derivatives of and were prepared according to published procedures [ ] [ ] [ ] ; sugar was used to enhance the bioavailability and solubility of potential drugs. ( , ) was prepared in an effective and simple method [ ] . the connection between these units was accomplished by o-glycosidic bond or via o-methylene , , -triazole linker, as shown in scheme . sugar derivatives of and were prepared according to published procedures [ ] [ ] [ ] ; sugar was used to enhance the bioavailability and solubility of potential drugs. all prepared derivatives were tested against different cancer cell lines, including hela, hct , and mcf- , in addition to a normal human dermal neonatal fibroblast (nhdf-neo). compound , designated with r = r = h and r = oac, was the most active, with ic values of . , . , and . mm against hela, hct , and mcf- , respectively. other derivatives exhibited low bioactivity. this could be attributed to the use of the quinolone hydroxyl group to form a glycosidic linkage, which impedes the chelation of metal ions due to steric hindrance. in this respect, it should be stated that the presence of the , , -triazole moiety improves the activity of glycoconjugates, whereas the type of sugar fragment did not affect the activity significantly. finally, conjugates with a sugar moiety and free hydroxyl group exerted better inhibitory potential than acetylated analogs. all prepared derivatives were tested against different cancer cell lines, including hela, hct , and mcf- , in addition to a normal human dermal neonatal fibroblast (nhdf-neo). compound , designated with r = r = h and r = oac, was the most active, with ic values of . , . , and . mm against hela, hct , and mcf- , respectively. other derivatives exhibited low bioactivity. this could be attributed to the use of the quinolone hydroxyl group to form a glycosidic linkage, which impedes the chelation of metal ions due to steric hindrance. in this respect, it should be stated that the presence of the , , -triazole moiety improves the activity of glycoconjugates, whereas the type of sugar fragment did not affect the activity significantly. finally, conjugates with a sugar moiety and free hydroxyl group exerted better inhibitory potential than acetylated analogs. fouda published a paper dealing with the synthesis, characterization, and cytotoxicity of new derivatives of halogenated -amino- -aryl- -pyrano[ , -h]quinolone- -carbonitrile ( ) derivatives (scheme ) [ ] . these derivatives were prepared through interactions of various hydroxyquinolines ( ) with α-cyanocinnamonitriles ( ) . these prepared compounds were screened for potential anticancer activity against mcf- , hct , hepg- , and a using the mmt assay. structure-activity relationship (sar) results revealed that -chloroanalogues were the most active, whereas the -methylanaloguess were the least potent. in addition, the lipophilicity of the products increased in the presence of halogen atom substituents at positions , , and . the ic values (mg/ml) of these compounds were in the ranges of . - . all prepared derivatives were tested against different cancer cell lines, including hela, hct , and mcf- , in addition to a normal human dermal neonatal fibroblast (nhdf-neo). compound , designated with r = r = h and r = oac, was the most active, with ic values of . , . , and . mm against hela, hct , and mcf- , respectively. other derivatives exhibited low bioactivity. this could be attributed to the use of the quinolone hydroxyl group to form a glycosidic linkage, which impedes the chelation of metal ions due to steric hindrance. in this respect, it should be stated that the presence of the , , -triazole moiety improves the activity of glycoconjugates, whereas the type of sugar fragment did not affect the activity significantly. finally, conjugates with a sugar moiety and free hydroxyl group exerted better inhibitory potential than acetylated analogs. fouda published a paper dealing with the synthesis, characterization, and cytotoxicity of new derivatives of halogenated -amino- -aryl- -pyrano[ , -h]quinolone- -carbonitrile ( ) derivatives (scheme ) [ ] . these derivatives were prepared through interactions of various hydroxyquinolines ( ) with α-cyanocinnamonitriles ( ) . these prepared compounds were screened for potential anticancer activity against mcf- , hct , hepg- , and a using the mmt assay. structure-activity relationship (sar) results revealed that -chloroanalogues were the most active, whereas the -methylanaloguess were the least potent. in addition, the lipophilicity of the products increased in the presence of halogen atom substituents at positions , , and . the ic values (mg/ml) of these compounds were in the ranges of . - . , . - . , . - . , and . - . against mcf- , hct , hepg , and a , respectively; while the colchicine reference showed ic values of . , . , . , and . mg/ml, respectively. in a similar fashion, chhabra et al. ( ) described an amberlite ira (oh)-mediated synthesis of novel benzothiazole-quinoline conjugates with excellent yields [ ] . a synthetic procedure (scheme ) involved a condensation reaction between the amino group (nh ) in and the carbonyl group in salicylic aldehyde ( ), followed by intramolecular cyclization under a these prepared compounds were screened for potential anticancer activity against mcf- , hct , hepg- , and a using the mmt assay. structure-activity relationship (sar) results revealed that -chloroanalogues were the most active, whereas the -methylanaloguess were the least potent. in addition, the lipophilicity of the products increased in the presence of halogen atom substituents at positions , , and . the ic values (mg/ml) of these compounds were in the ranges of . - . , . - . , . - . , and . - . against mcf- , hct , hepg , and a , respectively; while the colchicine reference showed ic values of . , . , . , and . mg/ml, respectively. in a similar fashion, chhabra et al. ( ) described an amberlite ira (oh)-mediated synthesis of novel benzothiazole-quinoline conjugates with excellent yields [ ] . a synthetic procedure (scheme ) involved a condensation reaction between the amino group (nh ) in and the carbonyl group in salicylic aldehyde ( ), followed by intramolecular cyclization under a microwave approach to form the benzothiazole ( ) . the reaction between and , -dialkyl- -hydroxyquinoline as a phenolate ion in the presence of a catalytic amount of amberlite ira and an excess amount of dibromoalkane under microwave conditions afforded the target product ; dibromoalkanes were employed as linkers between the two fragments, -hydroxyquinoline and -benzothiazol- -ylphenol. the prepared compounds were then subjected to a cytotoxicity study along with cisplatin (a positive control) against a panel of cancer cell lines, including hela, mcf- , a , and human ovarian carcinoma (a ), using the mmt assay. most of the prepared compounds were more potent than cisplatin, with ic values in the ranges of - , - , - , and - mm against mcf- , hela cells, a , and a , respectively. in addition, the target products were - -fold more selective in cancer cell lines than normal fibroblasts. molecules , , x for peer review of microwave approach to form the benzothiazole ( ) . the reaction between and , -dialkyl- hydroxyquinoline as a phenolate ion in the presence of a catalytic amount of amberlite ira and an excess amount of dibromoalkane under microwave conditions afforded the target product ; dibromoalkanes were employed as linkers between the two fragments, -hydroxyquinoline and benzothiazol- -ylphenol. the prepared compounds were then subjected to a cytotoxicity study along with cisplatin (a positive control) against a panel of cancer cell lines, including hela, mcf- , a , and human ovarian carcinoma (a ), using the mmt assay. most of the prepared compounds were more potent than cisplatin, with ic values in the ranges of - , - , - , and - mm against mcf- , hela cells, a , and a , respectively. in addition, the target products were - -fold more selective in cancer cell lines than normal fibroblasts. gayathri and coworkers prepared a novel compound (figure ) bearing three quinolinone moieties in a simple procedure that involved a reaction of , -bis(bromomethyl)- -chloroquinoline and -hydroxyquinoline at a ratio of : in acetone (aprotic polar solvent) under reflux conditions. the target product was fully characterized by using various spectroscopic techniques and singlecrystal x-ray diffraction method. this compound was screened against mcf- and hela cancer cell in addition, shamsi and coworkers prepared quinoline-based , , -oxadiazole-triazole derivatives (scheme ) based on the hybrid strategy of nitrogen-containing heterocyclic scaffolds [ ] . these compounds were considered to be high-impact motifs with a wide range of biological activities [ , ] . the synthetic strategy was based on the treatment of with carbonate to produce the phenolate anion, which reacts with ethyl chloroacetate under sn conditions to afford the corresponding intermediate. reaction of this intermediate with hydrazine produced , which undergoes intramolecular cyclization in the presence of carbon disulfide and the base (koh) to give compound ( , , -oxadiazole) after acidification. the thiol group (ar-sh) in is acidic and gives the ar-sanion upon treatment with a base. then, the corresponding anion reacts with propargyl bromide to afford compound . finally, reaction of various azide derivatives of yielded the target derivative through a ( + ) cycloaddition mechanism. scheme . synthesis of novel benzothiazole-quinoline derivatives. gayathri and coworkers prepared a novel compound (figure ) bearing three quinolinone moieties in a simple procedure that involved a reaction of , -bis(bromomethyl)- -chloroquinoline and -hydroxyquinoline at a ratio of : in acetone (aprotic polar solvent) under reflux conditions. the target product was fully characterized by using various spectroscopic techniques and single-crystal x-ray diffraction method. this compound was screened against mcf- and hela cancer cell lines using mmt assay, giving ic values of . and . mm, respectively [ ] . molecules , , x for peer review of microwave approach to form the benzothiazole ( ) . the reaction between and , -dialkyl- hydroxyquinoline as a phenolate ion in the presence of a catalytic amount of amberlite ira and an excess amount of dibromoalkane under microwave conditions afforded the target product ; dibromoalkanes were employed as linkers between the two fragments, -hydroxyquinoline and benzothiazol- -ylphenol. the prepared compounds were then subjected to a cytotoxicity study along with cisplatin (a positive control) against a panel of cancer cell lines, including hela, mcf- , a , and human ovarian carcinoma (a ), using the mmt assay. most of the prepared compounds were more potent than cisplatin, with ic values in the ranges of - , - , - , and - mm against mcf- , hela cells, a , and a , respectively. in addition, the target products were - -fold more selective in cancer cell lines than normal fibroblasts. gayathri and coworkers prepared a novel compound (figure ) bearing three quinolinone moieties in a simple procedure that involved a reaction of , -bis(bromomethyl)- -chloroquinoline and -hydroxyquinoline at a ratio of : in acetone (aprotic polar solvent) under reflux conditions. the target product was fully characterized by using various spectroscopic techniques and singlecrystal x-ray diffraction method. this compound was screened against mcf- and hela cancer cell lines using mmt assay, giving ic values of . and . mm, respectively in addition, shamsi and coworkers prepared quinoline-based , , -oxadiazole-triazole derivatives (scheme ) based on the hybrid strategy of nitrogen-containing heterocyclic scaffolds [ ] . these compounds were considered to be high-impact motifs with a wide range of biological activities [ , ] . the synthetic strategy was based on the treatment of with carbonate to produce the phenolate anion, which reacts with ethyl chloroacetate under sn conditions to afford the corresponding intermediate. reaction of this intermediate with hydrazine produced , which undergoes intramolecular cyclization in the presence of carbon disulfide and the base (koh) to give compound ( , , -oxadiazole) after acidification. the thiol group (ar-sh) in is acidic and gives the ar-sanion upon treatment with a base. then, the corresponding anion reacts with propargyl bromide to afford compound . finally, reaction of various azide derivatives of yielded the target derivative through a ( + ) cycloaddition mechanism. in addition, shamsi and coworkers prepared quinoline-based , , -oxadiazole-triazole derivatives (scheme ) based on the hybrid strategy of nitrogen-containing heterocyclic scaffolds [ ] . these compounds were considered to be high-impact motifs with a wide range of biological activities [ , ] . the synthetic strategy was based on the treatment of with carbonate to produce the phenolate anion, which reacts with ethyl chloroacetate under s n conditions to afford the corresponding intermediate. reaction of this intermediate with hydrazine produced , which undergoes intramolecular cyclization in the presence of carbon disulfide and the base (koh) to give compound ( , , -oxadiazole) after acidification. the thiol group (ar-sh) in is acidic and gives the ar-s − anion upon treatment with a base. then, the corresponding anion reacts with propargyl bromide to afford compound . finally, reaction of various azide derivatives of yielded the target derivative through a ( + ) cycloaddition mechanism. the anticancer activities of all of these derivatives were examined against four different human cancel cell lines, namely human lung carcinoma (a- ), hepatocellular carcinoma (hepg ), human cervical carcinoma-hpv (hela), and human cervical carcinoma-hpv (siha), using the mmt colorimetric assay; normal cells were used as controls, whereas doxorubicin was used as the reference drug. the results indicated that the product with an o-chloro substitution on the phenyl ring was the most potent, with an ic value of . mm against the a- cell line, which is higher than that of doxorubicin (ic = . mm). interestingly, this compound was not toxic towards normal cells (up to mm concentration). the anticancer activities of all of these derivatives were examined against four different human cancel cell lines, namely human lung carcinoma (a- ), hepatocellular carcinoma (hepg ), human cervical carcinoma-hpv (hela), and human cervical carcinoma-hpv (siha), using the mmt colorimetric assay; normal cells were used as controls, whereas doxorubicin was used as the reference drug. the results indicated that the product with an o-chloro substitution on the phenyl ring was the most potent, with an ic value of . mm against the a- cell line, which is higher than that of doxorubicin (ic = . mm). interestingly, this compound was not toxic towards normal cells (up to mm concentration). a series of styrylquinolines with various substituents was prepared as shown in scheme [ ] . in the first step of the synthesis, the hydroxyl group in was protected by conversion into the acetyl analogue . then, a condensation reaction between a methyl group at position of protected hydroxyquinoline and appropriate aromatic aldehydes as carried out using microwave heating or conventional procedures [ ] . finally, deprotection of the acyl group was achieved with carbonate anion-methanol or pyridine-water mixture afforded the target products . a series of styrylquinolines with various substituents was prepared as shown in scheme [ ] . in the first step of the synthesis, the hydroxyl group in was protected by conversion into the acetyl analogue . then, a condensation reaction between a methyl group at position of protected -hydroxyquinoline and appropriate aromatic aldehydes as carried out using microwave heating or conventional procedures [ ] . finally, deprotection of the acyl group was achieved with carbonate anion-methanol or pyridine-water mixture afforded the target products . the anticancer activities of all of these derivatives were examined against four different human cancel cell lines, namely human lung carcinoma (a- ), hepatocellular carcinoma (hepg ), human cervical carcinoma-hpv (hela), and human cervical carcinoma-hpv (siha), using the mmt colorimetric assay; normal cells were used as controls, whereas doxorubicin was used as the reference drug. the results indicated that the product with an o-chloro substitution on the phenyl ring was the most potent, with an ic value of . mm against the a- cell line, which is higher than that of doxorubicin (ic = . mm). interestingly, this compound was not toxic towards normal cells (up to mm concentration). a series of styrylquinolines with various substituents was prepared as shown in scheme [ ] . in the first step of the synthesis, the hydroxyl group in was protected by conversion into the acetyl analogue . then, a condensation reaction between a methyl group at position of protected hydroxyquinoline and appropriate aromatic aldehydes as carried out using microwave heating or conventional procedures [ ] . finally, deprotection of the acyl group was achieved with carbonate anion-methanol or pyridine-water mixture afforded the target products . all prepared compounds were screened for anticancer activity against the wild-type hct p +/+ and hct p −/− cells and for cytotoxicity against normal cell fibroblasts. the results revealed that derivatives that have hydroxyl or acyloxy groups at position of the quinolone moiety exhibit moderate activities ( . - . mm) and ( . - . mm) towards p +/+ and p −/− , respectively. analogues that were based on dichloroquinone and oxyacyl groups were the most active in this series towards p +/+ and p −/− ( . - . [ ] . their synthetic procedure involved a knoevenagel condensation between malonitrile ( ) and the respective aryl aldehyde to afford the corresponding arylidene-malonitrile intermediates , as shown in scheme . then, the phenolate anion attacks the β-carbon via c- to produce an acyclic michael adduct, which undergoes cyclization reaction ( -exo-dig) and tautomerization to afford the target product . all prepared derivatives in the study (along with compound ly used as the standard) have been tested against various cancer cell lines, namely pancreatic carcinoma a, colon carcinoma cells ht- , dld- , hct , cervix carcinoma kb-v vbl , and mcf- tobo breast carcinoma cell lines, in addition to non-malignant fibroblasts. the results revealed that that these derivatives exhibit remarkable activities, with ic values in nanomolar concentrations, meaning these results are even better than the activity of ly . two compounds designated with r = ch , r = r = h, r = r = f and r = ch , r = r = r = h, r = no were the most active among all examined molecules. the first one had ic values of . and nm against mcf- and kb-v vbl , respectively. on the other hand, the second compound had an ic value of nm against kb-v vbl , which is even better than the reference. in this regard, it is worth mentioning that the mechanism of action of these compounds may be associated with tubulin polymerization interference and ros formation, in which the molecule-induced ros generation could be responsible for their cytotoxicity, since ros overproduction may induce endoplasmic reticulum stress. all prepared compounds were screened for anticancer activity against the wild-type hct p +/+ and hct p −/− cells and for cytotoxicity against normal cell fibroblasts. the results revealed that derivatives that have hydroxyl or acyloxy groups at position of the quinolone moiety exhibit moderate activities ( . - . mm) and ( . - . mm) towards p +/+ and p −/− , respectively. analogues that were based on dichloroquinone and oxyacyl groups were the most active in this series towards p +/+ and p −/− ( . - . [ ] . their synthetic procedure involved a knoevenagel condensation between malonitrile ( ) and the respective aryl aldehyde to afford the corresponding arylidene-malonitrile intermediates , as shown in scheme . then, the phenolate anion attacks the β-carbon via c- to produce an acyclic michael adduct, which undergoes cyclization reaction ( -exo-dig) and tautomerization to afford the target product . all prepared derivatives in the study (along with compound ly used as the standard) have been tested against various cancer cell lines, namely pancreatic carcinoma a, colon carcinoma cells ht- , dld- , hct , cervix carcinoma kb-v vbl , and mcf- tobo breast carcinoma cell lines, in addition to non-malignant fibroblasts. the results revealed that that these derivatives exhibit remarkable activities, with ic values in nanomolar concentrations, meaning these results are even better than the activity of ly . two compounds designated with r = ch , r = r = h, r = r = f and r = ch , r = r = r = h, r = no were the most active among all examined molecules. the first one had ic values of . and nm against mcf- and kb-v vbl , respectively. on the other hand, the second compound had an ic value of nm against kb-v vbl , which is even better than the reference. in this regard, it is worth mentioning that the mechanism of action of these compounds may be associated with tubulin polymerization interference and ros formation, in which the molecule-induced ros generation could be responsible for their cytotoxicity, since ros overproduction may induce endoplasmic reticulum stress. matrix metalloproteinases (mmps) play significant roles in cancer diseases, with mmp- and mmp- being important types among the various mmps. for instance, they could induce the release of cell membrane precursors of growth factors (e.g., epidermal growth factor receptor) ligands, which promote tumor proliferation [ , ] . along this line, chen and colleagues described the synthesis of two series of -hydroxyquinolines, as shown in schemes and [ ] . in scheme , the first step involved protecting the amino group in with tert-butyloxycarbonyl (boc), then the free carboxylic acid group in reacted with the amino groups in various substrates leading, to the formation of carboxamide . other steps involved deprotection to liberate the free primary amine [ ] , which matrix metalloproteinases (mmps) play significant roles in cancer diseases, with mmp- and mmp- being important types among the various mmps. for instance, they could induce the release of cell membrane precursors of growth factors (e.g., epidermal growth factor receptor) ligands, which promote tumor proliferation [ , ] . along this line, chen and colleagues described the synthesis of two series of -hydroxyquinolines, as shown in schemes and [ ] . in scheme , the first step involved protecting the amino group in with tert-butyloxycarbonyl (boc), then the free carboxylic acid group in reacted with the amino groups in various substrates leading, to the formation of carboxamide . other steps involved deprotection to liberate the free primary amine [ ] , which reacts with -chloro- -hydroxyquinoline- -carboaldehyde or -hydroxycarbaldehyde through reduction amination to yield target product . furthermore, in scheme , the phenolic hydroxyl group in was protected and reaction of the primary amino group in with substituted carboxylic acids afforded derivative . finally, deprotection of the hydroxyl group yielded the target derivative . molecules , , x for peer review of reacts with -chloro- -hydroxyquinoline- -carboaldehyde or -hydroxycarbaldehyde through reduction amination to yield target product . furthermore, in scheme , the phenolic hydroxyl group in was protected and reaction of the primary amino group in with substituted carboxylic acids afforded derivative . finally, deprotection of the hydroxyl group yielded the target derivative . ho all of these derivatives were screened as potential mmp- / inhibitors. the results revealed that compounds (of series ) that have substituents at c- on the quinolone moiety showed ic values (mm) in the range of . - , while for those with substituents at c- , the ic values ranged from . to . on the other hand, derivatives belonging to series showed ic values in the range of . - against mmp- . as for mmp- , using the same sequence, the ic values (mm) were in the ranges of . - , . - , and ˃ . some selected compounds (those with substituents at c- ) were further screened against a panel of cancer cell lines (hl , k , kg , a , pc- , and mcf- ), along with human umbilical vein endothelial cells. the results indicated that most of the tested compounds exhibited good bioactivity, with ic values in the range of . - mm. finally, the positive control, all of these derivatives were screened as potential mmp- / inhibitors. the results revealed that compounds (of series ) that have substituents at c- on the quinolone moiety showed ic values (mm) in the range of . - , while for those with substituents at c- , the ic values ranged from . to . on the other hand, derivatives belonging to series showed ic values in the range of . - against mmp- . as for mmp- , using the same sequence, the ic values (mm) were in the ranges of . - , . - , and ˃ . some selected compounds (those with substituents at c- ) were further screened against a panel of cancer cell lines (hl , k , kg , a , pc- , and mcf- ), along with human umbilical vein endothelial cells. the results indicated that most of the tested compounds exhibited good bioactivity, with ic values in the range of . - mm. finally, the positive control, all of these derivatives were screened as potential mmp- / inhibitors. the results revealed that compounds (of series ) that have substituents at c- on the quinolone moiety showed ic values (mm) in the range of . - , while for those with substituents at c- , the ic values ranged from . to . on the other hand, derivatives belonging to series showed ic values in the range of . - against mmp- . as for mmp- , using the same sequence, the ic values (mm) were in the ranges of . - , . - , and > . some selected compounds (those with substituents at c- ) were further screened against a panel of cancer cell lines (hl , k , kg , a , pc- , and mcf- ), along with human umbilical vein endothelial cells. the results indicated that most of the tested compounds exhibited good bioactivity, with ic values in the range of . - mm. finally, the positive control, the hydroxamate-based mmp inhibitor nngh [ ] , showed ic values of - mm for antiproliferation activities against various cancer cell lines, and against mmp- and mmp- showed values of . and . mm, respectively. Ökten and coworkers described the synthesis of , -dibromo- -hydroxyquinoline [ ] in excellent yield via reaction of -hydroxyquinoline with two equivalents of bromine in chloroform. the target molecule exhibited ic values (mg/ml) of . , . , . , . , . , and > against a , fl, hela, ht , mcf , and hep b, respectively. from all of these studies, one can see the importance of the -hq moiety in potential anticancer drugs. in addition, some of the prepared compounds could be leads towards the development of potent and safe drugs. however, more work is required in this category, which could involve the use of animals and possibly human subjects to evaluate the efficacy and safety profiles of the prepared compounds. alzheimer's disease (ad), characterized by a loss of cognitive ability and severe behavioral irregularities, is a chronic neurodegenerative disorder. it is most common among the elderly, and can be described as an irreversible brain disorder that breaks down memory and reduces the ability of a patient to carry out simple mental and cognitive functions, such as comprehension, solving simple problems, and trivial calculations. this disease is becoming a universal health problem, and it can eventually lead to death [ ] . statistics indicate the presence of approximately . to . million alzheimer's disease patients in the united states, and and million worldwide [ ] . published research findings indicate that cholinergic dysfunction could be associated with selective and irreversible deficiency of the neurotransmitter acetylcholine, which is controlled by hydrolysis of acetylcholine via acetylcholinestrase (ache) and butyrylcholinestrase (bche). additionally, it was suggested that ache predominates in a healthy brain, whereas bche is considered to play a minor role in regulating the brain's ach levels [ ] . the approved prescribed commercial drugs for the treatment of ad, which provide slight improvements in memory, include donepezil, rivastigmine, and others; their action is based on the inhibition of acetylcholinesterase. it is also worth mentioning that other factors contribute to ad, such as β-amyloid (a β) deposits and oxidative stress. in this respect, several studies have shown that levels of redox-active metal ions, including cu + and zn + , are observed in the brains of ad patients [ ] . these metal ions can interact with β-amyloid peptides to form insoluble plaques [ , ] . in the search for potent buche and ache inhibitors, hirbod et al. ( ) designed and prepared eight novel compounds incorporating coumarin and -hydroxyquinoline moieties ( ), as shown in the [ ] . these researchers used various dibromoalkanes (n = - ) as cross-linkers between -hydroxyquinoline and coumarin rings in the presence of an aprotic solvent (n,n-dimethylformamide). in addition, the activity levels of these prepared compounds were evaluated against buche and ache using ellman's method. the results demonstrated that some of the prepared compounds exhibited potent ache and buche inhibition activities, with half-maximal inhibitory concentration (ic ( and ) , as shown in figure [ ] . the target compounds were prepared by chloromethylation of -hq to give -chloromethyl- -hydroxyquinoline. then, the former compound was reacted with tert-butylpiperazine- -carboxylate followed by trifluoroacetic acid to remove the protected boc group. finally, the resulting compound was reacted with the appropriate cinnamic or hydroxycinnamic acids, using -ethyl- -( -dimethylaminopropyl)carbodiimide (edc). in this context, β-amyloid (aβ) significantly contributed to the progression of alzheimer's disease (ad), where elevated levels of aβ have been detected in the brains of ad patients. moreover, aβ a aggregation can be clearly observed in the presence of cu + , zn + , and fe + ions, since these ions can readily bind to aβ through some of its specific residues. in addition, aβ can aggregate by itself. th prepared target compounds were tested for their inhibition of aβ aggregation using the thioflavon t-binding assay [ , ] . furthermore, chelating studies of cu + , zn + , fe + , and fe + were conducted using the former prepared derivatives by means of a uv-vis spectrophotometer. the results revealed that the compound designated with r = h, r , r = och showed the maximum percentage inhibition of aβ → aggregation of . % with an ic of . mm compared to resveratrol, which showed corresponding values of . % and . mm. in addition, the previous compound was selected as a representative to examine its metal chelation behavior; it exhibited significant activity in this context, producing even better results than clioquinol. molecules , [ ] . synthesis of these compounds involved protection of the phenolic group in using boc group to allow the reaction of the primary aromatic amine in with substituted benzoyl chlorides in the presence of triethylamine (tea) as the base and catalyst to form the carboxamide group . in the final step, deprotection of the boc group was accomplished with hydrogen chloride to afford the desired products as salt . regarding scheme , the phenoxide anion in was formed in situ by reaction of the phenolic group with potassium carbonate (base), which then reacts with benzyl bromide. the methyl group in was then subjected to pinnick oxidation using seo to afford the corresponding acid . reaction of the carboxylic acid group in with thionyl chloride and then with -(cyclopentyloxy)- -methoxyaniline or , -dimethoxyaniline afforded upon removal of bn with hydrogen gas in the presence of the palladium catalyst yielded the desired product . on the other hand, synthesis of compound was achieved by converting into tert-butyldimethylsilyl (tbs)-protected -aminoquinolin- -ol ( ). this process was carried out by oxidation of the starting material using m-chloroperbenzoic acid (mcpba) to form n-oxide, which was subsequently followed by refluxing with dimethylsulfate, treatment with nh oh, and finally protection with tert-butyldimethylsilyl chloride (tbscl). the obtained derivative was treated with the corresponding benzoyl chlorides, then went through the deprotection process using tetrabutylammoniumfluoride (tbaf) to afford the phenolic group in the target derivative (scheme ). compounds , , and are considered hybrids of clioquinol-rolipram and roflumilast as multitarget-directed ligands for the treatment of ad in terms of inhibition of phosphodiesterase d (pde d), the oxygen radical absorbance capacity (orac) value, and the experimental potential of the blood-brain barrier (bbb) permeability (pe) of the selected compounds using parallel artificial membrane permeation assay (pampa). in this respect, pde d is involved in the process of longterm potentiation and memory consolidation. one of the derivatives of , for which x = h, r = cf h, [ ] . synthesis of these compounds involved protection of the phenolic group in using boc group to allow the reaction of the primary aromatic amine in with substituted benzoyl chlorides in the presence of triethylamine (tea) as the base and catalyst to form the carboxamide group . in the final step, deprotection of the boc group was accomplished with hydrogen chloride to afford the desired products as salt . regarding scheme , the phenoxide anion in was formed in situ by reaction of the phenolic group with potassium carbonate (base), which then reacts with benzyl bromide. the methyl group in was then subjected to pinnick oxidation using seo to afford the corresponding acid . reaction of the carboxylic acid group in with thionyl chloride and then with -(cyclopentyloxy)- -methoxyaniline or , -dimethoxyaniline afforded upon removal of bn with hydrogen gas in the presence of the palladium catalyst yielded the desired product . on the other hand, synthesis of compound was achieved by converting into tert-butyldimethylsilyl (tbs)-protected -aminoquinolin- -ol ( ). this process was carried out by oxidation of the starting material using m-chloroperbenzoic acid (mcpba) to form n-oxide, which was subsequently followed by refluxing with dimethylsulfate, treatment with nh oh, and finally protection with tert-butyldimethylsilyl chloride (tbscl). the obtained derivative was treated with the corresponding benzoyl chlorides, then went through the deprotection process using tetrabutylammoniumfluoride (tbaf) to afford the phenolic group in the target derivative (scheme ). during ad [ ] . the pe values of selected compounds were evaluated using pampa. the results indicated that a derivative of with r = ch and r = cyclopentylmethyl exhibited a maximum value of pe . (± . ) × cm s - , whereas other tested compounds showed values higher than . × cm s - , indicating that these compounds may cross the bbb. the pe values for rolipram, roflumilast, and clioquinol were evaluated as ( . ± . ), ( . ± . ), and ( . ± . ) x cm s - , respectively. during ad [ ] . the pe values of selected compounds were evaluated using pampa. the results indicated that a derivative of with r = ch and r = cyclopentylmethyl exhibited a maximum value of pe . (± . ) × cm s - , whereas other tested compounds showed values higher than . × cm s - , indicating that these compounds may cross the bbb. the pe values for rolipram, roflumilast, and clioquinol were evaluated as ( . ± . ), ( . ± . ), and ( . ± . ) x cm s - , respectively. figure [ ] . compounds and were prepared from -hq by reaction first with formaldehyde in the presence of hydrogen chloride, followed by reaction with triethylphosphite to afford diethyl (( -hydroxyquinolin- -yl)methyl)phosphonate [ ] . in this reaction, the phenolic group was protected via reaction with chloro(methoxy)methane to yield diethyl (( -(methoxymethoxy)quinolin- -yl)methyl)-phosphonate. subsequent reaction of the last compound with various -nitrobenzaldehydes in the presence of sodium hydride yielded derivatives of -(methoxymethoxy)- -( -nitrostyryl)quinolone. reductive cyclization of -nitrostyrenes with carbon monoxide followed by removal of the protecting group (methoxymethane) by hydrochloride solution led to the formation of the final product. for compound , diethyl (( -chloro- - compounds , , and are considered hybrids of clioquinol-rolipram and roflumilast as multitarget-directed ligands for the treatment of ad in terms of inhibition of phosphodiesterase d (pde d), the oxygen radical absorbance capacity (orac) value, and the experimental potential of the blood-brain barrier (bbb) permeability (pe) of the selected compounds using parallel artificial membrane permeation assay (pampa). in this respect, pde d is involved in the process of long-term potentiation and memory consolidation. one of the derivatives of , for which x = h, r = cf h, r = cyclopentylethyl, exhibited an ic of . mm against pde d compared to rolipram as the reference compound (ic . mm), whereas the derivative showing x = h, r = cyclopentylmethyl of family exhibited the highest value among all tested compounds in the orac assay (its value is . expressed as trolox equivalents). in this respect, the orac value of antioxidant activities increases the ability of a given compound as the antioxidant becomes better [ ] . clioquinol, rolipram, and roflumilast showed values of . , . , and . , respectively. oxidative stress has a major effect in the production of excess free radicals, which lead to cell death and cytosceletal damage in ad [ ] . finally, the bbb has a major role in the generation of chronic brain inflammation during ad [ ] . the pe values of selected compounds were evaluated using pampa. the results indicated that a derivative of with r = ch and r = cyclopentylmethyl exhibited a maximum value of pe . (± . ) × cm s − , whereas other tested compounds showed values higher than . × cm s − , indicating that these compounds may cross the bbb. the pe values for rolipram, roflumilast, and clioquinol were evaluated as ( . ± . ), ( . ± . ), and ( . ± . ) × cm s − , respectively. an earlier paper by wang et al. ( ) described the synthesis of new -hydroxyquinoline derivatives, as shown in figure [ ] . compounds and were prepared from -hq by reaction first with formaldehyde in the presence of hydrogen chloride, followed by reaction with triethylphosphite to afford diethyl (( -hydroxyquinolin- -yl)methyl)phosphonate [ ] . in this reaction, the phenolic group was protected via reaction with chloro (methoxy)methane to yield diethyl (( -(methoxymethoxy) quinolin- -yl)methyl)-phosphonate. subsequent reaction of the last compound with various -nitrob enzaldehydes in the presence of sodium hydride yielded derivatives of -(methoxymethoxy)- -( -nitro styryl)quinolone. reductive cyclization of -nitrostyrenes with carbon monoxide followed by removal of the protecting group (methoxymethane) by hydrochloride solution led to the formation of the final product. for compound , diethyl (( -chloro- -hydroxyquinolin- -yl)methyl)-phosphonate was prepared by reacting diethyl (( -hydroxyquinolin- -yl)methyl)phosphonate with sodium hypochlorite, followed by the same previous steps. on the other hand, compound was prepared through a series of steps that involved reacting -methyl- -hydroxyqunolines with acetic anhydride, then with -nitrobenzaldehydes, followed by reductive cyclization of -nitrostyerenes in the presence of the palladium (ii) acetate catalyst under carbon monoxide gas, and finally with a base (carbonate or methoxide ions, depending on the nature of x). compounds , , and were tested against the orac assay, bbb permeability assay, and inhibition activity towards amyloid beta (aβ) self-induced aggregation. the results revealed that compounds (r = oh), (x = h, r = oh, r = h), and (x = h, r = oh, r = ch ) exhibited the highest activities among all prepared compounds ( . , . , . , and . , respectively). the presence of the phenolic hydroxyl group at c- of the indole moiety enhances the activity. on the other hand, the presence of a chlorine atom in compound lowers the activity compared to a hydrogen. the reference standards, including clioquinol, melatonin, and a mixture of clioquinol and melatonin, exhibit the values of . , . , and . , respectively. this test was performed based on a fluorescein (orac-fl) method with a trolox as the internal standard. similarly, permeation of the bbb is considered an important parameter for potential central nervous system (cns) candidates; this assay was accomplished using the parma method. the results showed that most of the target products could effectively permeate the bbb through passive diffusion. two compounds of series , designated as x = h, r = oh, r = h and x = h, r = oh, r = och , showed higher bbb permeability activity (pe values . and . , respectively) compared to other derivatives that have a hydrogen atom instead of the phenolic hydroxyl group. in contrast, compounds and , bearing the indole moiety at position of the quinolone scaffold, gave pe values of . and . , respectively, even in the presence of a phenolic hydroxyl group. aβ represents the major component of amyloid plaques found in the brains of alzheimer's patients [ ] . inhibition of aβ self-induced aggregation for the prepared -hydroxyquinoline-indole derivatives was examined using thioflavin fluorescence. one of the derivatives of , for which x = h, r = oh, r = h, caused . % percent inhibition, which was the highest percentage among the prepared compounds. on the other hand, compounds and caused . % and . % inhibition, respectively; whereas clioquinol, curcumin, and resveratrol showed . %, . %, and . % inhibition, respectively. in a recent publication, prati and coworkers [ ] described the synthesis of a new series of -hydroxyquinoline derivatives ( ) for which the products possess structural features of two commercial drugs, namely donepezil and clioquinol. depicted in scheme are the steps involved in the synthesis of these compounds. in a recent publication, prati and coworkers [ ] this synthesis was a multicomponent mannich reaction that involved a mixture containing piperazine ( ), paraformaldehyde, and -hq or its -chloroanalogue under a microwave-assisted procedure to afford -(piperazin- -ylmethyl)- -hydroxyquinolines ( ). this synthon was then reacted with various benzyl chlorides in dmf. compound and its derivatives were assayed for potential inhibitory activity towards human anti-hache and anti-hbche. the results indicated that at a concentration of mm, all derivatives exhibited inhibition, with values ranging from . to . % and . to . % for -chloro- -hydroxyquinoline and -hydroxyquinoline derivatives, respectively, against hbche. however, these compounds were inactive or showed very weak inhibition activity against hache, suggesting selectivity of the target products towards hbche. in addition, these results highlighted the effect of the chlorine atom at position of the hydroxyquinoline moiety on the activity compared to the hydrogen atom. the chemical references this synthesis was a multicomponent mannich reaction that involved a mixture containing piperazine ( ), paraformaldehyde, and -hq or its -chloroanalogue under a microwave-assisted procedure to afford -(piperazin- -ylmethyl)- -hydroxyquinolines ( ). this synthon was then reacted with various benzyl chlorides in dmf. compound and its derivatives were assayed for potential inhibitory activity towards human anti-hache and anti-hbche. the results indicated that at a concentration of mm, all derivatives exhibited inhibition, with values ranging from . to . % and . to . % for -chloro- -hydroxyquinoline and -hydroxyquinoline derivatives, respectively, against hbche. however, these compounds were inactive or showed very weak inhibition activity against hache, suggesting selectivity of the target products towards hbche. in addition, these results highlighted the effect of the chlorine atom at position of the -hydroxyquinoline moiety on the activity compared to the hydrogen atom. the chemical references donepezil, tacrine, and galantamine exhibited inhibition towards hbche, with values of . , > , and . %, respectively. on the other hand, the inhibition activity of compound and its derivatives towards the aβ antiaggregating property was evaluated. the obtained results demonstrated that the inhibition potencies of all derivatives ranged from . to . % at the concentration of mm; both series (x = h, cl) had close activity rates. in addition, the metal chelating ability of the selected compounds was examined using cu + and zn + in phosphate buffer solution (ph . ). spectroscopic data showed that there is a bathochromic shift of about nm from the original band ( nm) upon complex formation; as the metal ion concentration increases ( . to mm), the intensity of the absorption band also increases. finally, all derivatives showed antioxidant activity, whereby some prepared compounds exhibited higher activity than trolox. raj and padhi reported that the condensation of -hydroxyquinoline- -carbaldehyde ( ) with aromatic diamines ( ) afforded quinoline-based benzimidazole ( ) followed by intracyclization reaction to produce derivatives of . with aliphatic diamines ( ), compound produced bis-imines ( ) without undergoing intracyclization reaction, as shown in scheme [ ] . products of these reactions have been well-characterized using various techniques, including ft-ir, nmr, ms, and single-crystal x-ray diffraction method. in the first step of both reactions, one equivalent of -hydroxyquinoline- -carbaldehyde underwent a condensation reaction with one equivalent of primary amine to form the mono-imine product (schiff bases). however, in the second step and with the presence of aromatic amine, an intramolecular ring cyclization occurred, where the resulting precursor reacts further with the second equivalent of -hydroxyquinoline- -carbaldehyde, followed by migration of hydride to afford compound . in the case of an aliphatic amine, the second step represents a second condensation reaction with another molecule of -hydroxyquinoline- -carbaldehyde to give the final product . in a recent publication, prati and coworkers [ ] described the synthesis of a new series of hydroxyquinoline derivatives ( ) for which the products possess structural features of two commercial drugs, namely donepezil and clioquinol. depicted in scheme are the steps involved in the synthesis of these compounds. this synthesis was a multicomponent mannich reaction that involved a mixture containing piperazine ( ), paraformaldehyde, and -hq or its -chloroanalogue under a microwave-assisted procedure to afford -(piperazin- -ylmethyl)- -hydroxyquinolines ( ). this synthon was then reacted with various benzyl chlorides in dmf. compound and its derivatives were assayed for potential inhibitory activity towards human anti-hache and anti-hbche. the results indicated that at a concentration of mm, all derivatives exhibited inhibition, with values ranging from . to . % and . to . % for -chloro- -hydroxyquinoline and -hydroxyquinoline derivatives, respectively, against hbche. however, these compounds were inactive or showed very weak inhibition activity against hache, suggesting selectivity of the target products towards hbche. in addition, these results highlighted the effect of the chlorine atom at position of the hydroxyquinoline moiety on the activity compared to the hydrogen atom. the chemical references scheme . synthesis of new derivatives of -hydroxyquinolie-based benzimidazole. on the other hand, kong et al. ( ) prepared novel barbituroquinoline derivatives and in a one-pot procedure by combining three components in water, namely , -thiobarbituric acid ( ), and aldehyde (isatin) [ ] , as shown in scheme . this reaction was conducted under mild experimental conditions and without a catalyst. nitriles or acetonitrile in aqueous potassium hydroxide solution yielded , , -triazole-based quinolone ( on the other hand, kong et al. ( ) prepared novel barbituroquinoline derivatives and in a one-pot procedure by combining three components in water, namely , -thiobarbituric acid ( ), and aldehyde (isatin) [ ] , as shown in scheme . this reaction was conducted under mild experimental conditions and without a catalyst. the -hydroxyquinoline moiety can act as a building block for various pharmacologically active scaffolds. in the present work, we have reviewed the recent literature pertaining to the synthesis and bioactivity of numerous -hq derivatives as anticancer, antiviral, antimicrobial, antibacterial, antifungal, and anticancer agents. the results obtained from this review highlight the importance of numerous derivatives of -hq as possible chemotherapeutic agents and as possible leads towards the development of new drugs to treat various diseases, including cancer. we hope that data presented in this review could help researchers in the fields of medicinal chemistry and pharmacology in designing new active compounds and in the modification of existing compounds in the search for new drug leads. the development of drugs, either natural or synthetic, is gaining popularity in the fight against diseases, such as cardiovascular disorders, cancer insurgence, and immune dysfunction. there are certain nuclei such as -hq that are important building blocks in the medicinal arena. therefore, new synthetic methods for bioactive -hq derivatives should be pursued. in this respect, more biological testing, including in vivo studies, should accompany these syntheses. studies should also involve different pharmacokinetic parameters related to the safety profiles of potent derivatives. in this review, we have shown different synthetic strategies for pharmaceutically important chemicals that incorporate the -hq moiety. these compounds exhibited a wide range of biological activities and could be used as therapeutic agents against different diseases, including cancer. some of these compounds could be envisioned as leads in the development of drugs. compound then reacted with alkenes or alkynes containing electron-withdrawing substituents under the mechanism of , -dipolarcycloaddition and in the presence of a base (k co ) to afford . on the other hand, the reaction of with aromatic nitriles or acetonitrile in aqueous potassium hydroxide solution yielded , , -triazole-based quinolone ( ) -hydroxyquinoline and its derivatives: synthesis and applications immiscible polymers in double spin-coated electroluminescent devices containing phenyl-substituted tris ( -hydroxyquinoline) aluminum derivatives soluble in a host polymer -hydroxyquinolines in medicinal chemistry: a structural perspective -hydroxyquinoline: a privileged structure with a broad-ranging pharmacological potential synthesis, characterization, and anti-corrosion properties of an -hydroxyquinoline derivative synthesis and characterization of -hydroxyquinoline complexes of tin (iv) and their application in organic light emitting diode antiviral activity of novel quinoline derivatives against dengue virus serotype -hydroxyquinoline- -carboxanilides as antiviral agents against avian influenza virus synthesis and antibacterial activity of -benzylamide derivatives as ftsz inhibitors chemoselective synthesis of -amino- -bromoquinolin- -yl sulfonate derivatives and their antimicrobial evaluation synthesis, antibacterial properties and bioinformatics computational analyses of novel -hydroxyquinoline derivatives synthesis and biological evaluation of quinoline derivatives as a novel class of broad-spectrum antibacterial agents in vitro activities of a new fluoroquinolone derivative highly active against chlamydia trachomatis antifungal activity of umbelliferone derivatives: synthesis and structure-activity relationships synthesis of some novel , , -trisubstituted triazole derivatives bearing quinoline ring and evaluation of their antimicrobial activity a comprehensive review on the chemotherapeutic potential of piceatannol for cancer treatment, with mechanistic insights a novel potent nicotinamide phosphoribosyltransferase inhibitor synthesized via click chemistry synthesis and structure-activity relationships study of a-aminophosphonate derivatives containing a quinoline moiety an extremely stable and orthogonal dna base pair with a simplified three-carbon backbone synthesis and investigation of antibacterial and antioxidants properties of some new -subsituted- -hydroxyquinoline derivatives synthesis, spectroscopic characterization, x-ray analysis, and dft-hf calculations of -ethoxymethyl- -hydroxyquinoline synthesis and antimicrobial activities of sulfonohydrazide-substituted -hydroxyquinoline derivative and its oxinates bioconjugation via azide-staudinger ligation: an overview synthesis and biological evaluation of -hydroxyquinoline-hydrazones for anti-hiv- and anticancer potential design, synthesis and biological evaluation of -substituted quinolines as potential antileishmanial agents design, synthesis and biological evaluation of , , -trisubstituted b-carboline derivatives for cytotoxic and anti-leishmanial potential co-delivery of docetaxel and gemcitabine by anacardic acid modified self-assembled albumin nanoparticles for effective breast cancer management novel gemcitabine conjugated albumin nanoparticles: a potential strategy to enhance drug efficacy in pancreatic cancer treatment synthesis of -hydroxyquinoline glycoconjugates and preliminary assay of their b , -galt inhibitory and anti-cancer properties synthesis and characterization of a new cationic galactolipid with carbamate for gene delivery click'assembly of glycoclusters and discovery of a trehalose analogue that retards aβ aggregation and inhibits aβ -induced neurotoxicity chemoselective ligation of maleimidosugars to peptides/protein for the preparation of neoglycopeptides/neoglycoprotein halogenated -amino- h-pyrano [ , -h] quinoline- -carbonitriles as antitumor agents and structure-activity relationships of the -, -, and -positions amberlite ira (oh) mediated green synthesis of novel benzothiazole-quinoline conjugates as cancer theranostics comparative theoretical and experimental study on novel tri-quinoline system and its anticancer studies synthesis, anticancer evaluation and dna-binding spectroscopic insights of quinoline-based , , -oxadiazole- , , -triazole conjugates synthesis and anti-breast cancer activities of substituted quinolines design, synthesis and biological evaluation of new -( -substituted-anilino) quinoline derivatives as anticancer agents the synthesis and anticancer activity of -styrylquinoline derivatives. a p independent mechanism of action an efficient microwave-assisted synthesis of structurally diverse styrylquinolines new pyranoquinoline derivatives as vascular-disrupting anticancer agents matrix metalloproteinase- degrades all insulin-like growth factor binding proteins and facilitates insulin-like growth factor bioavailability review on epidermal growth factor receptor (egfr) structure, signaling pathways, interactions, and recent updates of egfr inhibitors design, synthesis and preliminary bioactivity evaluations of -hydroxyquinoline derivatives as matrix metalloproteinase (mmp) inhibitors synthesis of tetracyclic pyrrolidine/isoxazolidine fused pyrano [ , -h] quinolines via intramolecular , -dipolar cycloaddition in ionic liquid discovery of cgs a, a non-peptidic, potent, and orally active stromelysin inhibitor that blocks cartilage degradation in rabbits quinoline-based promising anticancer and antibacterial agents, and some metabolic enzyme inhibitors acetylcholinesterase inhibition by flavonoids from agrimonia pilosa coumarin derivatives as acetyl-and butyrylcholinestrase inhibitors: an in vitro, molecular docking, and molecular dynamics simulations study cholinesterase inhibitors and beyond pet imaging of copper trafficking in a mouse model of alzheimer disease nanoprobing of the effect of cu + cations on misfolding, interaction and aggregation of amyloid b peptide coumarin derivatives bearing benzoheterocycle moiety: synthesis, cholinesterase inhibitory, and docking simulation study novel -hydroxyquinoline derivatives targeting b-amyloid aggregation, metal chelation and oxidative stress against alzheimer's disease design, synthesis, and evaluation of multitarget-directed resveratrol derivatives for the treatment of alzheimer's disease design, synthesis, and evaluation of orally available clioquinol-moracin m hybrids as multitarget-directed ligands for cognitive improvement in a rat model of neurodegeneration in alzheimer's disease synthesis and evaluation of clioquinol-rolipram/roflumilast hybrids as multitarget-directed ligands for the treatment of alzheimer's disease profiling donepezil template into multipotent hybrids with antioxidant properties antioxidant capacity in the lipophilic fraction of alzheimer's brain tissues inflammatory events at blood-brain barrier in neuroinflammatory and neurodegenerative disorders: implications for clinical disease design, synthesis, and evaluation of orally bioavailable quinoline-indole derivatives as innovative multitarget-directed ligands: promotion of cell proliferation in the adult murine hippocampus for the treatment of alzheimer's disease the amyloid beta peptide: a chemist's perspective. role in alzheimer's and fibrillization novel -hydroxyquinoline derivatives as multitarget compounds for the treatment of alzheimer's disease synthesis, characterization, and structure of quinoline-based benzimidazole derivatives synthesis of pyrazolo-and [ , , ] triazolo-[ , -a] quinolin- -ols by cycloaddition to -hydroxyquinoline n-imide o-mesitylenesulfonylhydroxylamine and related compounds-powerful aminating reagents convenient one-pot synthesis of thiobarbituro-quinoline derivatives via catalyst-free multicomponent reactions in water this article is an open access article distributed under the terms and conditions of the creative commons attribution (cc by) license funding: this research received no external funding. the authors declare no conflict of interest. key: cord- -zz fpvz authors: tian, yong-qi; lin, xiu-ping; wang, zhen; zhou, xue-feng; qin, xiao-chu; kaliyaperumal, kumaravel; zhang, tian-yu; tu, zheng-chao; liu, yonghong title: asteltoxins with antiviral activities from the marine sponge-derived fungus aspergillus sp. scsio xws f date: - - journal: molecules doi: . /molecules sha: doc_id: cord_uid: zz fpvz two new asteltoxins named asteltoxin e ( ) and f ( ), and a new chromone ( ), together with four known compounds were isolated from a marine sponge–derived fungus, aspergillus sp. scsio xws f . the structures of the compounds ( – ) were determined by the extensive d- and d-nmr spectra, and hresims spectrometry. all the compounds were tested for their antiviral (h n and h n ) activity. compounds and showed significant activity against h n with the prominent ic( ) values of . ± . and . ± . μm, respectively. in addition, compound also exhibited inhibitory activity against h n with an ic( ) value of . ± . μm. in , viral pathogenesis became an intriguing hot topic among human society. this is exemplified by the recent ebola virus outbreak in western africa and middle east respiratory syndrome (mers) in south korea [ ] . meanwhile, influenza viruses are still a great threat to human health [ , ] . so far, only two classes of antiviral drugs, which include amantadine and the neuraminidase inhibitors, are currently used as anti-influenza therapeutic drugs, but both of them have some adverse side effects in humans as well as the resistance of the virus towards this drug [ , ] . hence, a concerted effort is aimed at discovering new antiviral agents to treat and eradicate these infectious agents [ , ] . marine sponge-derived fungus tends to produce structurally unique and biologically active natural products which have been documented in recent years; however, only a handful of reports have described new metabolites which have antiviral activities [ , ] . to further the scope of this particular research theme, a fungus, aspergillus sp. scsio xws f , attracted our attention because its crude etoac extract exhibited potential antiviral activity. further isolation yielded three new compounds: asteltoxin e ( ), asteltoxin f ( ) and -hydroxy- -( -hydroxypropyl)- -pentylchro mone ( ), together with four known compounds: asteltoxin ( ) [ ] , sterigmatocystin ( ) [ , ] , dihydrosterigmatocystin ( ), together with four known compounds: asteltoxin ( ) [ ] , sterigmatocystin ( ) [ , ] , dihydrosterigmatocystin ( ) [ , ] , diorcinol ( ) [ , ] (figure ). the antiviral (h n and h n ) activities of these compounds were individually evaluated. herein, we described the isolation, structural elucidation, and bioactivity screening of these metabolites - from aspergillus sp. scsio xws f . compound was obtained as a yellow amorphous solid. the molecular formula of was established as c h o by the hresims data (m/z . [m + h] + ), requiring degrees of unsaturations. the uv spectrum showed maxima at , , , nm, indicating the presence of an extended conjugated moiety. the ir spectrum exhibited absorptions for a hydroxy group at cm − and an α,β-unsaturated carbonyl group at cm − [ ] . close inspection of the h-and c-nmr spectra (table , figures s and s ) of using dept and h- c correlation spectroscopy (hsqc) revealed the presence of six methyls (one oxygenated), two methylenes including one oxygen-bearing, methines including three oxygenated methines, one double oxygenated methine (δh . , δc . ) and eight olefinic methines ,as well as two sp quaternary carbons (one oxygenated), four sp quaternary carbons, including two oxygenated carbons (δc . , . ) and two ester carbonyls (δc . , . ). comparing uv-vis and nmr data of compound with those of asteltoxin ( ) [ , , ] revealed a high degree of similarity and a difference in the substituents of c- . hmbc correlations observed from h - /c- , c- , c- , h - /c- and h - /c- , c- , c- indicated the existence of a methyl crotonate moiety. in addition to this, the hmbc correlation of h - /c- and the chemical shift values of c- (δc . ) and c- (δc . ) suggested that c- was linked with c- through an oxygen bridge. based on these analyses, the planar structure of was determined to be as shown in figure . the relative stereochemistry of ( ) was deduced from noesy correlations as shown in figure . the double-bond geometry of the c- /c- of substituent was deduced from the noesy correlations of h - /h - and h- /h - , which suggested e-configurations. the noesy correlations of h- /h - revealed the cis fusion of the , -dioxabi cyclo [ . . ] octane. the noesy correlations from h- to h- ; h- to h- , h - suggested that h- , h- , h- and h - are positioned on the same side. the noesy correlation of h- /h - , as well as the lack of noesy correlation between h - /h - , indicated that -oh was an α-configuration. the noesy correlation of h - /h- indicated that the ethyl group at c- was in the α-configuration. so, the relative configuration of would be identical with that of an asteltoxin and named asteltoxin e [ ] . compound was obtained as a yellow amorphous solid. the molecular formula of was established as c h o by the hresims data (m/z . [m + h] + ), requiring degrees of unsaturations. the uv spectrum showed maxima at , , , nm, indicating the presence of an extended conjugated moiety. the ir spectrum exhibited absorptions for a hydroxy group at cm´ and an α,β-unsaturated carbonyl group at cm´ [ ] . close inspection of the h-and c-nmr spectra (table , figures s and s ) of using dept and h- c correlation spectroscopy (hsqc) revealed the presence of six methyls (one oxygenated), two methylenes including one oxygen-bearing, methines including three oxygenated methines, one double oxygenated methine (δ h . , δ c . ) and eight olefinic methines ,as well as two sp quaternary carbons (one oxygenated), four sp quaternary carbons, including two oxygenated carbons (δ c . , . ) and two ester carbonyls (δ c . , . ). comparing uv-vis and nmr data of compound with those of asteltoxin ( ) [ , , ] revealed a high degree of similarity and a difference in the substituents of c- . hmbc correlations observed from h - /c- , c- , c- , h - /c- and h - /c- , c- , c- indicated the existence of a methyl crotonate moiety. in addition to this, the hmbc correlation of h - /c- and the chemical shift values of c- (δ c . ) and c- (δ c . ) suggested that c- was linked with c- through an oxygen bridge. based on these analyses, the planar structure of was determined to be as shown in figure . the relative stereochemistry of ( ) was deduced from noesy correlations as shown in figure . the noesy correlation of h- /h - , as well as the lack of noesy correlation between h - /h - , indicated that -oh was an α-configuration. the noesy correlation of h - /h- indicated that the ethyl group at c- was in the α-configuration. so, the relative configuration of would be identical with that of an asteltoxin and named asteltoxin e [ ] . the molecular formula of compound was determined as c h o by its hresims ion peak (m/z . [m + h] + ), which corresponded to units of unsaturations. a comparison of uv-vis and nmr data with those of revealed that compound also was an analogue of asteltoxin. the only difference was the absence of methylene between c- and c- . this was proved by the hmbc spectrum showing correlation of h - with c- , c- and the splitting pattern of h - (δ h . , d, j = . hz) and h- (δ h . , q, j = . hz). based on the noesy experiments, the relative configuration of was also suggested to be analogous to that of an asteltoxin and named asteltoxin f [ ] . compound was obtained as needle crystal. the molecular formula of was established as c [ , ] . the h-nmr spectrum showed proton signals, two sp aliphatic methy protons (δ h . and . ), five sp methylene protons (δ h . , . , . , . and . ), one oxymethine proton (δ h . ), and three sp methine protons (δ h . , . and . ). the c-nmr spectra showed signals, comprising two methyls, five methylenes, four methines and six quaternary carbon atoms. accounting for five of the seven degrees of unsaturation suggested the presence of two rings. the h-and c-nmr spectroscopic data of compound indicate that it is a chromone analogue. the only difference from -carbomethoxy methyl- heptyl- -hydroxy chromone was the substituent of c- [ ] . in addition, the hmbc correlation of h - /c- , c- and c- suggested that the pentyl was located at c- ( figure and table ). the molecular formula of compound was determined as c h o by its hresims ion peak (m/z . [m + h] + ), which corresponded to units of unsaturations. a comparison of uv-vis and nmr data with those of revealed that compound also was an analogue of asteltoxin. the only difference was the absence of methylene between c- and c- . this was proved by the hmbc spectrum showing correlation of h - with c- , c- and the splitting pattern of h - (δh . , d, j = . hz) and h- (δh . , q, j = . hz). based on the noesy experiments, the relative configuration of was also suggested to be analogous to that of an asteltoxin and named asteltoxin f [ ] . compound was obtained as needle crystal. the molecular formula of was established as c h o by the hresims ion peak at m /z . [m + h] + (calculated for c h o , . ) requiring seven degrees of unsaturation. the strong absorptions in the ir spectrum at and cm - showed the existence of a hydroxy group and a carbonyl group [ , ] . the h-nmr spectrum showed proton signals, two sp aliphatic methy protons (δh . and . ), five sp methylene protons (δh . , . , . , . and . ), one oxymethine proton (δh . ), and three sp methine protons (δh . , . and . ). the c-nmr spectra showed signals, comprising two methyls, five methylenes, four methines and six quaternary carbon atoms. accounting for five of the seven degrees of unsaturation suggested the presence of two rings. the h-and c-nmr spectroscopic data of compound indicate that it is a chromone analogue. the only difference from -carbomethoxy methyl- heptyl- -hydroxy chromone was the substituent of c- [ ] . the h- h cosy correlation of h - /h - , h - /h - and hmbc correlations of h - /c- and c- , h - /c- and c- indicated the presence of a pentyl moiety. in addition, the hmbc correlation of h - /c- , c- and c- suggested that the pentyl was located at c- ( figure and table ). the absolute configuration of c- was established by comparison of the optical rotation of compound rαs d = + . (c . , meoh) with that of -hydroxy- -( -hydroxypropyl)- -methyl chromone: rαs d = + (c . , meoh) [ , ] . thus, this establishes the absolute configuration of c- to be s. thus, the structure of compound was determined and named as -hydroxy- -( -hydroxypropyl)- -pentylchromone. the antiviral (h n and h n ) activities of these compounds were individually evaluated through cytopathic effect (cpe) inhibition assay. compounds and showed significant inhibitory activities against the h n strain with the prominent ic values of . ˘ . and . ˘ . µm, respectively. in addition, compound also exhibited significant activity against the h n strain with an ic value of . ˘ . µm. the fungal strain scsio xws f , which was isolated from a sponge callyspongia sp. collected from the sea area near xuwen county, guangdong province, china, exhibited potential antiviral activity in our previous screening tests. after seven days of growth on mb (malt extract agar base) medium at ˝c, colonies were mm to mm in diameter, showed good sporulation, and were light green, whereas the color of the reverse was the same as that of the surface ( figure b ). light microscopy revealed that the stipes were erecting hyphae. spherical expansion (vesicles) were formed at the end of the stipes, on which there were green conidial chains. a teleomorphic state was not observed ( figure c,d) . the its - . s-its sequence region ( base pairs (bp), accession number kt ) of strain scsio xws f was amplified by pcr and sequenced. a phylogenetic tree was procured by the neighbor-joining method as per a similarity based off a -bp consensus length of the its - . s-its sequence ( figure ). strain scsio xws f was found to belong to a clade related to a. austroafricanus nrrl (accession number jq ), with a sequence identity of . %. on the basis of its properties of culture and morphology, and its phylogenetic analysis, strain scsio xws f was identified as a member of the aspergillus genus, and was named as aspergillus sp. scsio xws f . [ , ] . thus, this establishes the absolute configuration of c- to be s. thus, the structure of compound was determined and named as -hydroxy- -( hydroxypropyl)- -pentylchromone. the antiviral (h n and h n ) activities of these compounds were individually evaluated through cytopathic effect (cpe) inhibition assay. compounds and showed significant inhibitory activities against the h n strain with the prominent ic values of . ± . and . ± . μm, respectively. in addition, compound also exhibited significant activity against the h n strain with an ic value of . ± . μm. the fungal strain scsio xws f , which was isolated from a sponge callyspongia sp. collected from the sea area near xuwen county, guangdong province, china, exhibited potential antiviral activity in our previous screening tests. after seven days of growth on mb (malt extract agar base) medium at °c, colonies were mm to mm in diameter, showed good sporulation, and were light green, whereas the color of the reverse was the same as that of the surface ( figure b ). light microscopy revealed that the stipes were erecting hyphae. spherical expansion (vesicles) were formed at the end of the stipes, on which there were green conidial chains. a teleomorphic state was not observed (figure c,d) . the its - . s-its sequence region ( base pairs (bp), accession number kt ) of strain scsio xws f was amplified by pcr and sequenced. a phylogenetic tree was procured by the neighbor-joining method as per a similarity based off a -bp consensus length of the its - . s-its sequence ( figure ). strain scsio xws f was found to belong to a clade related to a. austroafricanus nrrl (accession number jq ), with a sequence identity of . %. on the basis of its properties of culture and morphology, and its phylogenetic analysis, strain scsio xws f was identified as a member of the aspergillus genus, and was named as aspergillus sp. scsio xws f . the nmr spectra were measured on a bruker ac mhz nmr (bruker, fällanden, switzerland) spectrometer with tms as an internal standard. high resolution mass spectra (hresims) were recorded on a bruker micro tof-qii mass spectrometer (bruker, fällanden, switzerland). cd spectra were measured with a chirascan circular dichroism spectrometer (applied photophysics, surrey, uk). size exclusion chromatography was done on sephadex lh- gel (ge healthcare, uppsala, sweden). column chromatography (cc) was carried out on silica gel ( - mesh); (qingdao marine chemical factory, qingdao, china). spots were detected on tlc under uv light or by heating after spraying with % h so and enough vanillin in h o. the fungal strain scsio xws f was isolated from the sponge callyspongia sp., which was collected from the sea area near xuwen county, guangdong province, china, during august . the isolate was stored on mb agar (malt extract g, sea salt g, agar g) slants at °c and then deposited at cas key laboratory of tropical marine bio-resources and ecology. the cultural properties and the morphological features of the spores and mycelia of strain scsio xws f were examined on mb agar medium after culturing at °c for day. the samples were observed with a b series biological microscope (chongqing optec instrument co., ltd., chongqing, china) light microscope using a previously described cover technique. the mycelia of strain scsio xws f cultured in sabouraud's dextrose broth (consisting of g dextrose, g peptone, . g nacl, and ml distilled water, ph . ) were sampled and powdered in a mixer mill after liquid nitrogen was added. dna was isolated through the hpure the nmr spectra were measured on a bruker ac mhz nmr (bruker, fällanden, switzerland) spectrometer with tms as an internal standard. high resolution mass spectra (hresims) were recorded on a bruker micro tof-qii mass spectrometer (bruker, fällanden, switzerland). cd spectra were measured with a chirascan circular dichroism spectrometer (applied photophysics, surrey, uk). size exclusion chromatography was done on sephadex lh- gel (ge healthcare, uppsala, sweden). column chromatography (cc) was carried out on silica gel ( - mesh); (qingdao marine chemical factory, qingdao, china). spots were detected on tlc under uv light or by heating after spraying with % h so and enough vanillin in h o. the fungal strain scsio xws f was isolated from the sponge callyspongia sp., which was collected from the sea area near xuwen county, guangdong province, china, during august . the isolate was stored on mb agar (malt extract g, sea salt g, agar g) slants at ˝c and then deposited at cas key laboratory of tropical marine bio-resources and ecology. the cultural properties and the morphological features of the spores and mycelia of strain scsio xws f were examined on mb agar medium after culturing at ˝c for day. the samples were observed with a b series biological microscope (chongqing optec instrument co., ltd., chongqing, china) light microscope using a previously described cover technique. the mycelia of strain scsio xws f cultured in sabouraud's dextrose broth (consisting of g dextrose, g peptone, . g nacl, and ml distilled water, ph . ) were sampled and powdered in a mixer mill after liquid nitrogen was added. dna was isolated through the hpure fungal dna kit (guangzhou genebase bioscience co., guangzhou, china) according to the manufacturer's protocol. the its region of strain scsio xws f was amplified by polymerase chain reaction with the primer pair its -its . the amplified product was purified with a tiangel mini purification kit (tiangen biotech, beijing, china). pure pcr product was submitted for sequencing together with the primer its to a commercial service (shanghai majorbio bio-pharm technology co., ltd., shanghai, china). the derived its region sequence was compared against the genbank database (ncbi) through blast-algorithmus. similarity analysis was performed using clustal w program. the phylogenetic tree of strain scsio xws f was constructed using neighbor-joining method. a. ochraceoroseus nrrl t was used as an out group. the nucleotide sequence of the its region reported in this article was assigned the genbank accession number kt . aspergillus sp. was cultured on mb-agar plates at ˝c for seven days. the seed medium consisted of malt extract: g, sea salt: g, distilled water: ml, ph . - . , and was inoculated with strain scsio xws f and incubated at ˝c for h on a rotating shaker ( rpm). mass scale fermentation of fungal isolate scsio xws f was carried out using solid rice medium in ml flasks (rice: g, sea salt: . g, distilled water: ml), and inoculated with ml of seed solution. flasks were incubated at ˝c under normal day-night cycle. after days, cultures from flasks were harvested and subjected for organic extraction using ethyl acetate (etoac). the etoac extracts of rice solid media of aspergillus sp. scsio xws f were partitioned between petroleum ether, and % aqueous meoh, the resulting meoh phase was fractionated using silica column, sephadex lh- , and then semi-preparative reversed-phase hplc to yield compounds - (figure ). the culture of solid rice medium was soaked in acetone and cut into small pieces and kept for day. the content was filtered and evaporated under vacuum using a buchner funnel and extracted with etoac until exhaustion and this process was repeated thrice. the organic phase was collected and evaporated to obtain a dark brown oil crude extract ( . g). the crude etoac extract was subjected to silica gel column chromatography (cc) eluted with petroleum ether/ etoac in a gradient eluent (v/v, : , : , : , : , : , : , : ) to obtain fractions (fractions - ) on the basis of tlc. fr. ( . g) was purified by sephadex lh- (chcl /meoh, : ) to afford compound ( . g). fr. ( . mg) was purified by semi-preparative reversed-phase (sp-rp) hplc using a c column (ymc-pack, ods-a s- µmˆ nm ˆ mm i.d., ml/min) eluting with meoh/h o ( : ) to afford compound ( . mg). fr. ( . g) was purified by sephadex lh- (chcl /meoh, : ) yielding three sub-fractions (fr. the antiviral activities against h n and h n were evaluated by the cpe inhibition assay in duplicate assay [ ] . confluent mdck cell monolayers were firstly incubated with influenza virus at ˝c for h. after removing the virus dilution, cells were maintained in infecting media (rpmi , µg/ml of trypsin) containing different concentrations of test compounds. after h incubation at ˝c, the cells were fixed with µl of % formaldehyde for min at room temperature. after removal of the formaldehyde, the cells were stained with . % crystal violet for min. the plates were washed and dried, and the intensity of crystal violet staining for each well was measured in a microplate reader (bio-rad, hercules, ca, usa) at nm. the ic was calculated as the compounds concentration required to inhibit influenza virus yield at h post-infection by %. oseltamivir was used as the positive control with ic values of . and . nm, respectively. in conclusion, three new compounds, asteltoxin e ( ), asteltoxin f ( ) and -hydroxy- -( -hydroxypropyl)- -pentyl chromone ( ), together with four known compounds were isolated from a marine sponge-derived fungus, aspergillus sp. compounds and are analogues of asteltoxin. compound is a new chromone derivative and its pentylbenzene moiety is a rare incidence in nature. compound is a liver carcinogen and forms dna adducts after metabolic activation to an epoxide at the furofuran ring [ ] . it is noteworthy that compound was obtained in a scale of . g and a new dimensional pharmacokinetic research of this compound is a part of our ongoing mission. in the screening to search for seeds of antiviral agents, compounds and showed significant activity against h n with the prominent ic value of . ˘ . and . ˘ . µm; respectively. in addition; compound also exhibited inhibitory activity against h n with ic value of . ˘ . µm. compounds and were published online on november as new compounds after the date we submitted on november [ ] . supplementary materials: supplementary materials can be accessed at: http://www.mdpi.com/ - / / / /s . viruses and disease: emerging concepts for prevention, diagnosis and treatment the influenza viruses continual reintroduction of human pandemic h n influenza a viruses into swine in the united states the emergence of adamantane resistance in influenza a(h ) viruses in australia and regionally in glycyrrhizic acid derivatives as influenza a/h n virus inhibitors enzyme-assisted extraction of bioactive material from chondrus crispus and codium fragile and its effect on herpes simplex virus (hsv- ) marine compounds and their antiviral activities marine natural products total synthesis of (+)-asteltoxin seven new and two known lipopeptides as well as five known polyketides: the activated production of silent metabolites in a marine-derived fungus by chemical mutagenesis strategy using diethyl sulphate three xanthones from a marine-derived mangrove endophytic fungus purification and characterization of o-methyltransferase i involved in conversion of demethylsterigmatocystin to sterigmatocystin and of dihydrodemethy lsterigmatocystin to dihydrosterigmatocystin during aflatoxin biosynthesis sydoxanthone c and acremolin b produced by deep-sea-derived fungus aspergillus sp. scsio ind f synergistic and drug-resistant reversing effects of diorcinol d combined with fluconazole against candida albicans dysideanones a-c, unusual sesquiterpene quinones from the south china sea sponge dysidea avara stereoselective synthesis of the bis-tetrahydrofuran fragment (c- -c- ) of asteltoxin total synthesis of (+/´)-asteltoxin asteltoxins from the entomopathogenic fungus pochonia bulbillosa -h- xanthone derivatives from aspergillus sydowii, an endophytic fungus from the liverwort scapania ciliata s. lac and their immunosuppressive activities cytotoxic and antibacterial angucyclineand prodigiosin-analogues from the deep-sea derived streptomyces sp. scsio chromones from the endophytic fungus pestalotiopsis sp. isolated from the chinese mangrove plant rhizophora mucronata a new polyunsaturated acid from the marine-derived streptomyces violans (no. htta-f ) cytotoxic and antiviral nitrobenzoyl sesquiterpenoids from the marine-derived fungus aspergillus ochraceus jcma f catechol formation: a novel pathway in the metabolism of sterigmatocystin and -methoxysterigmatocystin avertoxins a-d, prenylasteltoxin derivatives from aspergillus versicolor y , an endophytic fungus of huperziaserrata author contributions: yong-qi tian contributed to extraction, isolation, identification and manuscript preparation; xue-feng zhou contributed to structure elucidation and nmr analysis; xiu-ping lin contributed to the isolation of the fungus; zheng-chao tu, tian-yu zhang, zhen wang, xiao-chu qin contributed to the bioactivities test; kumaravel kaliyaperumal contributed to revising the paper; yong-hong liu was the project leader organizing and guiding the experiments and writing the manuscript. the authors declare no conflict of interest.molecules , , key: cord- -co tju u authors: yu, sheng; yan, hui; zhang, li; shan, mingqiu; chen, peidong; ding, anwei; li, sam fong yau title: a review on the phytochemistry, pharmacology, and pharmacokinetics of amentoflavone, a naturally-occurring biflavonoid date: - - journal: molecules doi: . /molecules sha: doc_id: cord_uid: co tju u amentoflavone (c( )h( )o( )) is a well-known biflavonoid occurring in many natural plants. this polyphenolic compound has been discovered to have some important bioactivities, including anti-inflammation, anti-oxidation, anti-diabetes, and anti-senescence effects on many important reactions in the cardiovascular and central nervous system, etc. over plants have been found to contain this bioactive component, such as selaginellaceae, cupressaceae, euphorbiaceae, podocarpaceae, and calophyllaceae plant families. this review paper aims to profile amentoflavone on its plant sources, natural derivatives, pharmacology, and pharmacokinetics, and to highlight some existing issues and perspectives in the future. amentoflavone (c h o ) is a common biflavonoid chemically named as -[ -( , -dihydroxy- -oxo- h-chromen- -yl)- -hydroxyphenyl]- , -dihydroxy- -( -hydroxyphenyl)- h-chromen- -one, which naturally occurs in many plants. it is also considered as an apigenin dimer linked by a c -c covalent bond (figure ). this compound was firstly isolated by okigawa and his colleagues in from three plants of the selaginella species (selaginella tamariscina (beauv.) spring, selaginella nipponica, and selaginella pachystachys) [ ] . from then on, phytochemical researchers have isolated and identified this biflavonoid from more than plants, some of which have been used as traditional folk medicines in many regions of the world for even thousands of years. with the development of modern pharmacology, more and more evidence has proved many of the bioactivities of amentoflavone, including anti-oxidant [ ] , anti-inflammatory [ ] , anti-senescence [ ] , anti-tumor [ ] , anti-virus [ ] , and anti-fungal [ ] effects, as well as therapeutic effects on the central nervous system [ ] and cardiovascular system [ ] , etc. with its good pharmacological performance and high content, amentoflavone is even listed as the chemical marker of selaginellae herba ("juanbai" in chinese, which represents the whole plants of selagenella tamariscina or selaginella pulvinata) for quality evaluation in the chinese pharmacopoeia [ ] . due to its large range of bioactivities and originating from nature, amentoflavone has attracted increasing focus from a number of research fields. here, in this paper, we aim to provide a review this naturally-occurring biflavonoid, describing its sources, natural derivatives, pharmacological effects, and pharmacokinetics, and to help researchers understand and utilize it in a better way. as a polyphenolic compound, amentoflavone exists in a large number of plants (table ) . to our knowledge, the major sources are the plants of calophyllaceae, clusiaceae, cupressaceae, euphorbiaceae, and selaginellaceae families, and calophyllum, garcinia, and selaginella species, etc. some of these plants have been used as folk phytomedicines for a very long time, such as gingko biloba, lobelia chinensis, polygala sibirica, ranunculus ternatus, selaginella pulvinata, selagenella tamariscina for traditional chinese medicines (tcms), calophyllum inophyllum, selaginella bryopteris for traditional indian medicines, byrsonima intermedia for traditional american medicine, and cnestis ferruginea and drypetes gerrardii for traditional african medicines. as a polyphenolic compound, amentoflavone exists in a large number of plants (table ) . to our knowledge, the major sources are the plants of calophyllaceae, clusiaceae, cupressaceae, euphorbiaceae, and selaginellaceae families, and calophyllum, garcinia, and selaginella species, etc. some of these plants have been used as folk phytomedicines for a very long time, such as gingko biloba, lobelia chinensis, polygala sibirica, ranunculus ternatus, selaginella pulvinata, selagenella tamariscina for traditional chinese medicines (tcms), calophyllum inophyllum, selaginella bryopteris for traditional indian medicines, byrsonima intermedia for traditional american medicine, and cnestis ferruginea and drypetes gerrardii for traditional african medicines. to obtain amentoflavone from plants as much as possible, and to fully utilize these plant sources, some studies have been carried out to optimize the extraction technology. a central composite design (ccd) method was used to optimize the extraction technology of amentoflvone from taxus chinensis by supercritical-co fluid extraction (sfe-co ) with methanol as a co-solvent. the highest yield reached . mg/g when the plant was extracted with . % ethanol at • c under a pressure of mpa for . h [ ] . with % water in chcl/ , -butanediol ( : ) as the extraction solvent, . mg/g of amentoflavone could be extracted from chamaecyparis obtusa leaves at • c for min with a solid/liquid ratio of . g/ml, which was optimized by a response surface methodology [ ] . like other phytochemicals, separation and isolation of amentoflavone were mainly performed with conventional thin layer chromatography [ , ] and column chromatography, in which silica gel [ , , ] , polyamide [ ] , macroporous adsorption resin [ , ] , octadecyl silane [ , ] , middle chromatogram isolation (mci) gel [ ] , and gel (sephadex lh- ) [ , , ] were used as stationery phases. in most cases, some of the above methods were combined for use [ , , , , , ] . additionally, as a novel isolation method, high-speed counter-current chromatography (hsccc) has been widely used to isolate this bioflavonoid. a preparative isolation method with hsccc was adopted to isolate amentoflavone from selaginella doederleinii. the mixed solvent consisting of n-hexane:ethyl acetate:methanol:water ( : : . : . , v/v/v/v) was employed for hsgcc of ethyl acetate extract of this plant. as a result, with an approximate yield of . mg from g of crude plant, amentoflavone of . % purity was obtained [ ] . in another experiment, with hsccc and n-hexane:ethyl acetate:methanol:water ( . : . : : , v/v/v), . mg amentoflavone ( % purity) was isolated from approximately . g of selaginella tamariscina [ ] . there are also a large number of derivatives with different substitution positions and types in the natural plants ( figure ). in most cases, they exist in the same plant with amentoflavone. amentoflavone is considered as a dimer of two apigenins with six hydroxyl groups on the positions of c , c , c , c , c , and c in its structure ( figure ). among these groups the c -, c -, c -, or c -hydroxyl group is easily substituted by a methoxyl group table . in the structure of amentoflavone, carbon-carbon double bonds of c -c and c -c are easily hydrogenated, too. in a large number of plants, the hydrogenation products present and their glycosides (table ) . table . hydrogenation derivatives of amentoflavone. as a ubiquitous biflavonoid, amentoflavone has been found with a large number of pharmacological functions, such as anti-inflammation, anti-oxidation, anti-tumor, anti-senescence, anti-virus, anti-diabetes, neuroprotective activities, and effects on cardiovascular system and central nervous system. oxidative stress response is one part of inflammatory response. amentoflavone, isolated from garcinia brasiliensis, exhibited inhibitory effects on the productions of superoxide anion and total reactive oxygen species (ros) inphorbol -myristate -acetate-stimulated human neutrophils. in human erythrocytes induced by , -azobis( -amidinopropane) hydrochloride, it also inhibited the oxidant hemolysis and lipid peroxidation [ ] . in rat astrocytoma cell line, lipopolysaccharide (lps) could increase no, ros, malondialdehyde (mda), and decrease reduced-glutathione (gsh), while tumor necrosis factor-α (tnf-α) was increased by lps in a human monocytic leukemia cell line. all of the changes above were attenuated by amentoflavone significantly. however, there were no notable effects on the cells [ ] . in raw . cells stimulated with lps, amentoflavone was observed to suppress the production of no, prostaglandin e- (pge- ), and the nuclear translocation of c-fos, a subunit of activator protein (ap)- . additionally, extracellular signal-regulated kinase (erk), which mediated c-fos translocation, was inhibited by the active biflavonoid [ ] . in the supernatant media of human peripheral blood mononuclear cells (pbmcs), amentoflavne could inhibit the increases of interleukin- β (il- β), il- , tnf-α, and pge induced by phytohaemagglutinin (pha) [ ] . the ic amentoflavone exerted good cytotoxic effect on cervical adenocarcinoma (hela) cells with ic values of . µm [ ] . after breast cancer mcf- cells were treated with amentoflavone, there were some cellular changes, including dna and nuclear fragmentation, and down-regulation of calcium and intracellular reactive oxygen species. additionally, some marks of mitochondrial-mediated apoptosis were observed, such as the activation of caspase , the reduction of mitochondrial inner-membrane potential, and the release of cytochrome c from mitochondria [ ] . amentoflavone also could significantly inhibit solid tumor development that was induced by b f- melanoma in c bl/ mice. the mechanism might be related to inhibiting cell progression from g /g to s phase and to regulating genes which were involved in cell cycle and apoptosis, such as p , p , bax, caspase- , etc. [ ] . recently, fatty acid synthase (fasn) has been considered as a potential target to treat cancer. some studies indicated that amentoflavone could inhibit fasn expression in human epidermal growth factor receptor (her )-positive human breast carcinoma skbr cells. the inhibition decreased the translocation of sterol regulatory element-binding protein (srebp- ) in skbr cells. the biflavonoid was also found to down-regulate her protein and mrna, to up-regulate polyoma enhancer activator (pea ), a transcriptional repressor of her and to inhibit phosphorylation of protein kinase b (pkb), mechanistic target of rapamycin (mtor) and c-jun n-terminal kinases (c-jnk) [ ] . in another experiment, amentoflavone was observed to increase the cleavage-activity of caspase- , to suppress skbr cell activity, and to have no effect on fasn-nonexpressed nih- t normal cell growth [ ] . ultraviolet b (uvb) irradiation was found to increase the levels of lamin a and phospho-h ax protein in normal human fibroblasts. these cases were present in premature aging diseases or normally old individuals. an investigation indicated amentoflavone was able to ameliorate these damages and to protect nuclear aberration significantly, which showed the anti-senescence activity for some skin aging processes related with uvb [ ] . another investigation in uvb-induced normal human fibroblasts found that amentoflavone could inhibit the activation of erk without affecting erk protein level, p , and jnk activation. in addition, the biflavonoid could decrease phospho-c-jun and c-fos protein expressions, which were ap- transcription factor components. the findings suggested the potential of amentoflavone to prevent or treat skin photoaging [ ] . amentoflavone was observed to ameliorate glucose disorder, regulate insulin secretion, and restore the pancreas in streptozotocin-induced diabetic mice and the optimum dose was mg/kg [ ] . in another anti-diabetes study, this active biflavonoid showed its activities against α-glucosidase (ic . ± . µm) and α-amylase (ic . ± . µm) [ ] . inhibition of protein tyrosine phosphatase b (ptp b) has been considered as a strategy to treat type diabetes. amentoflavone was screened to inhibit ptp b with ic value of . ± . µm and proved to be a non-competitive inhibitor of ptp b by kinetic study. there was a dose-dependent increase in tyrosine phosphorylation of insulin receptor (ir) after d cells with overexpression of ir were treated with amentoflavone [ ] . amentoflavone exhibited its anti-dengue potential in a screening experiment, which may be mediated by inhibiting dengue virus ns rna-dependent rna polymerase [ ] . among the isolated twelve components from torreya nucifera with a bioactivity guide, amentoflavone was proved as the most active one to inhibit severe acute respiratory syndrome coronavirus (sars-coa) with ic value of . µm. the effect was concluded relative to the inhibition of chymotrypsin-like protease ( cl pro ) [ ] . amentoflavone was also found to decrease coxsackievirus b (cvb ) replication by inhibiting fatty acid synthase (fas) expression [ ] . moreover, in cases of human immunodeficiency virus (hiv) and respiratory syncytial virus (rsv), amentoflavone showed good performance with ic values of µm [ ] and . µg/ml [ ] , respectively. after amentoflavone was isolated from cnestis ferruginea, ishola et al. carried out some investigations about its effects on central nervous system. in one pharmacological investigation, oral administration of amentoflavone was proved to attenuate depression induced by metergoline ( -ht receptor antagonist), prazosin (α -adrenoceptor antagonist), or yohimbine (α -adrenoceptor antagonist), and to ameliorate anxiety stimulated by flumazenil (ionotropic gaba receptor antagonist). these findings suggested that the active biflavonoid showed the antidepressant and anxiolytic effects through interactions with the receptors above [ ] . in another study, it was found that the naturally-occurring biflavonoid could prevent scopolamine-induced memory impairment, inhibit ache and enhance antioxidant enzyme activity in mice, which exhibited its protection against memory deficits [ ] . in glutamate injured ht hippocampal cells, amentoflavone showed neuroprotective activity. the active compound was able to restore the reduced superoxide dismutase (sod) activity, glutathione reductase (gr) activity and glutathione content induced by glutamate. additionally, it was found to prevent the phosphorylation of erk / [ ] . amentoflavone also exerted neuroprotective activity in pilocarpine-induced epileptic mice. after preventive administration of the biflavonoid for three consecutive days, the model mice showed some signs of improvement, including reduction of epileptic seizures, shortened attack time, reduction in hippocampal neuron loss and apoptosis, and suppressed nuclear factor-kappa b (nf-κb) activation and expression [ ] . amentoflavone was tested to have a vasorelaxant effect on thoracic aortic blood vessels of rats in vitro, which was concluded as being endothelium-dependent and involved with no [ ] . amentoflavone also had a protective effect on vascular endothelial cells. the viability of human umbilical vein endothelial cells (huvecs) was promoted and the ratio of cells at s phase was increased by treatment with this biflavonoid [ ] . some results of cell studes indicated that amentoflavone could increase the no content, decrease the levels of vcam- , e-selectin, il- , il- , and et- , enhance sod activity, reduce mda content, downregulate the protein expressions of vcam- , e-selectin, and nf-κb p , up-regulate iκbα, and attenuate the nf-κb p transfer to the cell nucleus, which proved its protection on vascular endothelial cells [ ] . cyclic adenosine monophosphate (camp) phosphodiesterase (pde) inhibitor has been found to inhibit the activity of camp-pde- in myocardial cells and vascular smooth muscle cells, which could enhance myocardial contraction, expand peripheral vessels, and improve hemodynamics of heart failure patients. amentoflavone showed a potent inhibitory function on camp-pde [ ] . the effect study of amentoflavone on isolated rat heart exhibited that the phytochemical significantly increased the beat rate at dosage of - µg/ml [ ] . amentoflavone was investigated to have antifungal activity against several pathogenic fungal strains, including candida albicans, saccharomyces cerevisiae, and trichosporon beigelii. in candida albicans, it could stimulate the intracellular trehalose accumulation and disrupt the dimorphic transition, which meant a stress response to the component [ ] . further research on its antifungal mechanism of candida albicans suggested that this active phytochemical arrested cell cycles during the s-phase and inhibited cell proliferation and division [ ] . the anti-candida activity was proved to be related to apoptotic cell death, which may be associated with the mitochondrial dysfunction. additionally, hydroxyl radicals induced by amentoflavone may play a significant role in apoptosis [ ] . in addition to the pharmacological functions above, significant evidence showed its other bioactivities (table ) , such as anti-hyperlipidemia [ ] , anti-hypertrophic scar [ ] , anti-psoriasis [ ] , anti-ulcerative colitis [ ] , hepatoprotection [ ] , osteogenesis effect [ ] and radioprotection [ ] . in recent years, pharmacokinetic studies of extracts and bioactive compounds from traditional chinese medicine and natural medicine have become research highlights. as a representative biflavonoid with several pharmacological functions, amentoflavone was not an exception. in a pharmacokinetic investigation, amentoflavone was administrated to rats with different types including oral gavage (po, mg/kg), intravenous (iv, mg/kg) and intraperitoneal (ip, mg/kg) injection. as a result, . % of the total amentoflavone was discovered to circulate as conjugated metabolites after po administration. in the plasma of rats with iv and ip injection, . % ± . % and . % ± . % of the total amentoflavone was present as conjugated metabolites. in addition, the bioavailability of this compound with po administration was . % ± . %, much lower than that with ip injection ( . % ± . %) [ ] . pharmacokinetic characteristic of amentoflavone individually or together with other components in normal rats and hyperlipidemic model rats have been studied and compared [ ] . in the case of oral administration of only this biflavonoid, t / and t max of amentoflavone were determined as . h ± . h, . h ± . h in normal rats and . h ± . h, . h ± . h in model rats, respectively. shixiao san is a famous tcm formula containing amentoflavone [ ] . after oral administration of a shixiao san decotion, t / and t max of amentoflavone were determined as . h ± . h, . h ± . h in normal rats, and . h ± . h, . h ± . h in model rats. from the contents above, we could conclude that amentoflavone is a bioactive biflavonoid with a variety of pharmacological effects, which has been derived from many natural plants. emerging pharmacological evidence has proved the effects of amentoflavone on various aspects, including anti-inflammation, anti-oxidation, anti-diabetes, anti-senescence, anti-virus, anti-tumor activities, and effects on the central nervous system and cardiovascular system. however, the majority of these bioactivity data came from studies involved with cells in vitro, while the number of studies with model animals in vivo was very low. as we know, bioactivity in vitro is unable to represent and explain biological effect in vivo, while pharmacological investigations in model animals are indispensable prior to clinical use. thus, some bioactivities in vitro should be confirmed and proved by integral animal experiments in the future. in terms of present pharmacokinetic study, the findings have suggested that amentoflavone metabolism procedure was very rapid and there was also a very low bioavailability after oral administration of this biflavonoid in rats. this may be one reason why fewer animal model experiments have been performed. we speculate that improving the bioavailability with introduction of structural modification, precursor synthesis, or particular pharmaceutical necessities may be one focus of amentoflavone studies. meanwhile, since there are some differences of pharmacokinetics between normal and model animals, concerning the specific pharmacological effects, the pharmacokinetic investigations on corresponding model animals should also be carried out. amentoflavone has been found, isolated, and identified in over natural plants, which exhibited its rich plant source. the content of any phytochemical varies very much in different species or in different regions. among plants from selaginella species, the biflavonoid was found with the high contents between . % and . % in selaginella sinensis, selaginella davidii, and selaginella mollendorfii from some specific production areas, while the contents were no more than . % in the rest, and even below . % in some [ , ] . it is well-known that extraction yield will be lower than the determined content. in addition, most of the sources are perennial plants and their recovery or reproduction will last not a short time. thus, at present, plant-derived preparation seems to cost too much. this may be another reason of fewer animal model experiments, which would need much higher amounts of the biflavonoid than cell experiments. we must find some solutions to get the sufficient quantity for studies in the future, such as looking for other plants with much higher contents, biological synthesis, and even chemical synthesis. taken together, since amentoflavone is a promising and naturally-occurring biflavonoid with so many bioactivities, its systematic druggability research as a candidate drug is obviously necessary, including its preparation study (extraction and isolation from plants, chemical synthesis, or biological synthesis), structural modification study, absorption-distribution-metabolism-excretion (adme) study in normal animals and animal models, acute and chronic toxicological studies. thus, we can make full use of amentoflavone as a drug and employ it in the prevention and treatment of diseases. in summary, this paper has provided a full-scale profile of amentoflavone on its plant sources, natural derivatives, pharmacology, and pharmacokinetics, and also proposed some issues and perspectives which may be of concern in the future. we believe this literature review will help us more comprehensively understand, and take advantage more fully, the naturally-occurring biflavonoid amentoflavone. biflavones in selaginella species redox-active biflavonoids from garcinia brasiliensis as inhibitors of neutrophil oxidative burst and human erythrocyte membrane damage anti-inflammatory activity of flavonoids from chrozophora tinctoria protective effects of amentoflavone on lamin a-dependent uvb-induced nuclear aberration in normal human fibroblasts cytotoxic flavonoids and other constituents from the stem bark of ochna schweinfurthiana structure-activity relationship study of biflavonoids on the dengue virus polymerase denv-ns rdrp amentoflavone stimulates mitochondrial dysfunction and induces apoptotic cell death in candida albicans amentoflavone protects hippocampal neurons: anti-inflammatory, antioxidative, and antiapoptotic effects protection effect of amentoflavone in selaginella tamariscina against tnf-α-induced vascular injure of endothelial cells chinese pharmacopeia commission. pharmacopoeia of the people's republic of china isolation and structural elucidation of chemical constituents of amanoa almerindae constituents and antiulcer effect of alchornea glandulosa: activation of cell proliferation in gastric mucosa during the healing process phenolic compounds in leaves of alchornea triplinervia: anatomical localization, mutagenicity, and antibacterial activity flavonoids and other constituents from aletris spicata and their chemotaxonomic significance antimalarial and vasorelaxant constituents of the leaves of allanblackia monticola the separation and indentification of biflavonoids from androsace umbellata tirucallane glycoside from the leaves of antidesma bunius and inhibitory no production in bv cells and raw . macrophages squalene and amentoflavone from antidesma laciniatum amentoflavone from biophytum sensitivum and its effect on cox- /cox- catalysed prostaglandin biosynthesis flavonoids from biota semipervirens flavonoids and antiulcerogenic activity from byrsonima crassa leaves extracts mutagenic evaluation and chemical investigation of byrsonima intermedia a. juss. leaf extracts occurrence of biflavones in leaves of caesalpinia pyramidalis specimens -epiisocommunic acid and amentoflavone from callitris rhomboidea two new compouuds from the leaves of calocedrus microlepic var. formosana cechinel, v. chemical composition and analgesic activity of calophyllum brasiliense leaves cytotoxic and antibacterial activities of constituents from calophyllum ferrugineum ridley bioguided fractionation and isolation of natural inhibitors of advanced glycation end-products (ages) from calophyllum flavoramulum four new -substituted coumarins from calophyllum incrassatum and their biological activities neoflavonoid and biflavonoid constituents of calophyllum inophylloide occurrence of xanthonolignoids in guttifereous plants chemical constituents in the roots of calophyllum membranaceum gardn chemical constituents from leaves of calophyllum membranaceum gardn constituents of the cuban endemic species calophyllum pinetorum antileishmanial activity of leaf extract from calophyllum rivulare against leishmania amazonensis α-glucosidase and -lipoxygenase inhibitory activities of phytochemicals from calophyllum symingtonianum biflavonoids of calophyllum venulosum structure and dynamic of three indole alkaloids from the campylospermum genus (ochnaceae) biflavonoid constituents of campylospermum mannii isolation and structure elucidation of phenolic compounds in chinese olive (canarium album l.) fruit. eur study on chemical constituents of the leaves of canarium album (lour.) raeusch chemical constituents from canarium pimela fruits phenolic metabolites from the seeds of canarium schweinfurthii podophyllotoxin-type lignans as major constituents of the stems and leaves of casearia clarkei effects of some compounds isolated from celaenodendron mexicanum standl (euphorbiaceae) on seeds and phytopathogenic fungi studies on biflavonoids of the leaves of cephalotaxus fortunei hook. f. var. alpina native to china osteoblast differentiation stimulating activity of biflavonoids from cephalotaxus koreana biflavones from chamaecyparis obtusa bioactivity guided isolation of analgesic and anti-inflammatory constituents of cnestis ferruginea vahl ex dc (connaraceae) root study on chemical constituents of the branches and leaves of cunninghamia lanceolata antifungal biflavones from cupressocyparis leylandii phytochemical investigation and hepatoprotective activity of cupressus sempervirens l. leaves growing in egypt studies on phytochemicals, part . a new biflavonold from cycas beddomei phytochemical investigation of cycas circinalis and cycas revoluta leaflets: moderately active antibacterial biflavonoids chemical constituents of cycas panzhihuaensis anti-diabetic molecules from cycas pectinata griff. traditionally used by the maiba-maibi biflavones of decussocarpus rospigliosii as phosphodiesterases inhibitors antibacterial constituents of extracts of the aerial parts of discocleidion rufescens antimicrobial activity of the crude extracts and five flavonoids from the twigs of dorstenia barteri (moraceae) antiplasmodial activity of compounds from drypetes gerrardii chemical constituents from drypetes hainanensis stems and leaves kouno, i. isolation, purification and identification of chemical constituents from elateriospermum tapos chemical studies on the galeobdolon chinense biflavonoids, main constituents from garcinia bakeriana leaves new flavonoid c-o-c dimers and other chemical constituents from garcinia brevipedicellata stem heartwood chemical constituents from fruits of garcinia cowa trypanocidal constituents in plants . leaves of garcinia intermedia and heartwood of calophyllum brasiliense antibacterial activity of two biflavonoids from garcinia livingstonei leaves against mycobacterium smegmatis benzophenones and biflavonoids from garcinia livingstonei fruits tetraoxygenated xanthones and biflavanoids from the twigs of garcinia merguensis isolation of six isoprenylated biflavonoids from the leaves of garcinia subelliptica bioactive benzophenones from garcinia xanthochymus fruits isolation of amentoflavone from ginkgo biloba herpes virus inhibitory substances from hypericum connatum lam., a plant used in southern brazil to treat oral lesions isolation of i , ii -biapigenin (amentoflavone) from hypericum perforatum cechinel, v. chemical composition and antinociceptive properties of hyeronima alchorneoides leaves phytochemical study on american plants i. two new phenol glucosides, together with known biflavones and diterpene, from leaves of juniperus occidentalis hook anti-inflammatory phenolics isolated from juniperus rigida leaves and twigs in lipopolysaccharide-stimulated raw . macrophage cells taxonomic status of lanaria lanata and isolation of a novel biflavone chemical constituents of lobelia chinensis chemical constituents in aerial parts of lonicera chrysantha turcz (ii) a biflavonoid from stems and leaves of lonicera macranthoides study on the chemical constituents of lonicera similes new biflavonoid and other constituents from luxemburgia nobilis (eichl) new flavonoids from lysimachia christinae hance chemical constituents of mangifera indica leaves (i) chemical constituents from the stems of cassava (manihot esculenta) in hainan the chemical composition of microbiota decussata isolation of ametoflavone and new glycosides from leaves of nandina domestica thunb , "-biisokaempferide, a cytotoxic biflavonoid and other chemical constituents of nanuza plicata (velloziaceae) proposed active compounds from ouratea parviflora biflavonoids and a glucopyranoside derivative from ouratea semiserrata antimicrobial biflavonoids from the aerial parts of ouratea sulcata chemical constituents from edible part of pistacia chinensis the chemical constituents from podocarpus imbricadus chemical constituents from n-butanol extract of aerial part of polygala sibirica studies on chemical constituents of flavonoids and glycosides in ranunculus ternatus biflavones from the leaves of retrophyllum rospigliosii biflavones from rhus species with affinity for the gaba(a)/benzodiazepine receptor in vitro anti-hiv activity of biflavonoids isolated from rhus succedanea and garcinia multiflora chemical constituents in twigs and leaves of sabina pingii var study on chemical constituents of sabina sinoalpina flavonoids from the leaves of sabina vulgaris antoine structurally unique biflavonoids from selaginella chrysocaulos and selaginella bryopteris cytotoxic biflavonoids from selaginella delicatula biflavonoids of selaginella denticulata growing in spain phenolic constituents of selaginella doederleinii study on the chemical constituents of selaginella involvens spring and antibacterial activity bioactive compounds of inhibiting xanthine oxidase from selaginella labordei selective cytotoxicity of ginkgetin from selaginella moellendorffii evaluation of the diuretic activity in two mexican medicinal species: selaginella nothohybrida and selaginella lepidophylla and its effects with ciclooxigenases inhibitors study on the chemical constituents of selaginella pulvinata maxim biflavonoid constituents from selaginella remotifolia spring new biflavonoid from selaginella rupestris isolation of amentoflavone from selaginella rupestris and its pharmacological activity on central nervous system, smooth muscles and isolated frog heart preparations amentoflavone from selaginella sanquinolenta the biflavonoid pattern of selaginella selaginoides antiviral amentoflavone from selaginella sinensis studies on chemical constituents of selaginella stauntoniana (i) vasorelaxation by amentoflavone isolated from selaginella tamariscina new , "-linked biflavonoids from selaginella uncinata displaying protective effect against anoxia cytotoxic biflavonoids from selaginella willdenowii flavonoids from speranskia tuberculata flavones from struthiola argentea with anthelmintic activity in vitro antifungal activity of biflavones from taxus baccata and ginkgo biloba isoquercitrin is the most effective antioxidant in the plant thuja orientalis and able to counteract oxidative-induced damage to a transformed cell line free radical scavenging and antielastase activities of flavonoids from the fruits of thuja orientalis presence of amentoflavone in tmesipteris tannensis biflavonoids from torreya nucifera displaying sars-cov cl(pro) inhibition chemical constituents from amentotaxus yunnanensis and torreya yunnanensis iridoid glucosides from viburnum chinshanense biflavones and furanone glucosides from zabelia tyaihyonii optimization of process parameters of extraction of amentoflavone, quercetin and ginkgetin from taxus chinensis using supercritical co plus co-solvent evaluation of alcohol-based deep eutectic solvent in extraction and determination of flavonoids with response surface methodology optimization flavonoids from selaginella uncinata preparative isolation of six anti-tumour biflavonoids from selaginella doederleinii hieron by high-speed counter-current chromatography rapid screening and detection of xod inhibitors from s. tamariscina by ultrafiltration lc-pda-esi-ms combined with hpccc in vitro peroxynitrite scavenging activity of -hydroxykynurenic acid and other flavonoids from gingko biloba yellow leaves antiplasmodial and leishmanicidal activity of biflavonoids from indian selaginella bryopteris chemical constituents of selaginella mollendorfii bioactive compounds from celaenodendron mexicanum study on chemical constituents of podocarpus brevifolius seco-terpenoids and other constituents from elateriospermum tapos the chemical constituents in dacrydium pierrei selaginellin a and b, two novel natural pigments isolated from selaginella tamariscina chemical constituents from selaginella doederleinii and their bioactivities structrue identification of biflavones and determination of taxol from taxus madia antimicrobial activity, toxicity and selectivity index of two biflavonoids and a flavone isolated from podocarpus henkelii (podocarpaceae) leaves the chemical constituents from podocarpus nagi (ii) a rare biflavone from taxus baccata biflavonoids from the wollemi pine, wollemia nobilis (araucariaceae) biflavonoids isolated from selaginella tamariscina regulate the expression of matrix metalloproteinase in human skin fibroblasts inhibition of inducible nitric oxide synthase by sumaflavone isolated from selaginella tamariscina the structures of amentoflavone glycosides isolated from psilotum nudum a novel cytotoxic c-methylated biflavone from the stem of cephalotaxus wilsoniana two new dihydroamentoflavone glycosides from cycas revoluta study on biflavonoids from selaginella uncinata (desv.) spring. chin biflavonoids from cycas beddomei chemical constituents from dysoxylum cauliflorum (meliaceae) soluble epoxide hydrolase inhibitory constituents from selaginella tamariscina evaluation of amentoflavone isolated from cnestis ferruginea vahl ex dc (connaraceae) on production of inflammatory mediators in lps stimulated rat astrocytoma cell line (c ) and thp- cells extracellular signal-regulated kinase is a direct target of the anti-inflammatory compound amentoflavone derived from torreya nucifera amentoflavone protects against hydroxyl radical-induced dna damage via antioxidant mechanism amentoflavone induces cell-cycle arrest and apoptosis in mcf- human breast cancer cells via mitochondria-dependent pathway effect of amentoflavone, a phenolic component from biophytum sensitivum, on cell cycling and apoptosis of b f- melanoma cells fatty acid synthase inhibition by amentoflavone suppresses her /neu (erbb ) oncogene in skbr human breast cancer cells fatty acid synthase inhibition by amentoflavone induces apoptosis and antiproliferation in human breastcancer cells amentoflavone inhibits uvb-induced matrix metalloproteinase- expression through the modulation of ap- components in normal human fibroblasts anti-diabetic activity of amentoflavone in selaginella tamariscina in diabetic mice protein tyrosine phosphatase b inhibitory activity of amentoflavone and its cellular effect on tyrosine phosphorylation of insulin receptors inhibition of fatty acid synthase by amentoflavone reduces coxsackievirus b replication antidepressant and anxiolytic effects of amentoflavone isolated from cnestis ferruginea in mice protective effect of cnestis ferruginea and its active constituent on scopolamine-induced memoryimpairment in mice: a behavioral and biochemical study neuroprotective biflavonoids of chamaecyparis obtusa leaves against glutamate-induced oxidative stress in ht hippocampal cells experiment study on vasodilative effects of amentoflavone ethyl acetate extract of selaginella tamariscina effects of total flavonoids and amentoflavone isolated from selaginella tamariscina on human umbilical vein endothelial cells proliferation and vegf expression inhibition of camp-phosphodiesterase by biflavones of ginkgo biloba in rat adipose tissue safety of dietary supplements: chronotropic and inotropic effects on isolated rat atria antifungal effect of amentoflavone derived from selaginella tamariscina s-phase accumulation of candida albicans by anticandidal effect of amentoflavone isolated from selaginella tamariscina lowering blood lipid and hepatoprotective activity of amentoflavone from selaginella tamariscina in vivo amentoflavone inhibits angiogenesis of endothelial cells and stimulates apoptosis in hypertrophic scar fibroblasts amentoflavone protects against psoriasis-like skin lesion through suppression of nf-κb-mediated inflammation and keratinocyte proliferation amentoflavone inhibits inos, cox- expression and modulates cytokine profile, nf-κb signal transduction pathways in rats with ulcerative colitis amentoflavone enhances osteogenesis of human mesenchymal stem cells through jnk and p mapk pathways amentoflavone acts as a radioprotector for irradiated v cells by regulating reactive oxygen species(ros), cell cycle and mitochondrial mass. asian pac liquid chromatography-tandem mass spectrometry determination and pharmacokinetic analysis of amentoflavone and its conjugated metabolites in rats simultaneous determination of five free and total flavonoids in rat plasma by ultra hplc-ms/ms and its application to a comparative pharmacokinetic study in normal and hyperlipidemic rats hplc fingerprint of shixiao san determination f biflavonoids in selaginellae plants by micellar electrokinetic capillary electrophoresis determination of biflavones from selaginella by hplc. chin all the authors declare no conflict of interest. key: cord- -tm yj sn authors: itumoh, emeka j.; data, shailja; leitao, erin m. title: opening up the toolbox: synthesis and mechanisms of phosphoramidates date: - - journal: molecules doi: . /molecules sha: doc_id: cord_uid: tm yj sn this review covers the main synthetic routes to and the corresponding mechanisms of phosphoramidate formation. the synthetic routes can be separated into six categories: salt elimination, oxidative cross-coupling, azide, reduction, hydrophosphinylation, and phosphoramidate-aldehyde-dienophile (pad). examples of some important compounds synthesized through these routes are provided. as an important class of organophosphorus compounds, the applications of phosphoramidate compounds, are also briefly introduced. phosphoramidates (p-n) are a class of organophosphorus compounds known for the presence of a single covalent bond between the tetracoordinate p(v) atom and n(iii) atom. there are generally three types of phosphoroamidates, which are distinguished according to the substitution on the p and n atoms ( figure ) [ ] . another feature of these organophosphorous compounds, defined as (ro) p(o)nr' (r, r' = h, alkyl, aryl, heteroaryl), is a stable phosphoryl bond (p=o). both p and n atoms are key physiological elements present in genetic material, energy transfer, enzymes, and other biomolecules and are required for various life processes. as such, molecules containing p-n linkages are found in a large array of biologically active natural products ( - ) ( figure ) [ , ] . for example, microcin c ( ) (figure ) is an antibiotic produced by escherichia coli [ ] . dinogunellin ( ) (figure ), which is produced in the roe of some fishes, is a natural toxin [ ] . phosphoarginine and phosphocreatine ( and ) ( figure ) are important biological molecules used as sources of stored energies in invertebrates and vertebrates, respectively [ ] . phosphoramidon ( ) (figure ), derived from streptomyces tanashiensis, inhibits the thermolysin enzyme, which is a key factor in the development of various diseases [ , ] . lastly, phosmidosine and agrocin ( and ) (figure ) are nucleotide antibiotics isolated from streptomyces durhameusis and agrobacterium radiobacter and are used to control gray mold disease and crown gall disease in plants, respectively [ , ] . another feature of these organophosphorous compounds, defined as (ro) p(o)nr' (r, r' = h, alkyl, aryl, heteroaryl), is a stable phosphoryl bond (p=o). both p and n atoms are key physiological elements present in genetic material, energy transfer, enzymes, and other biomolecules and are required for various life processes. as such, molecules containing p-n linkages are found in a large array of biologically active natural products ( - ) ( figure ) [ , ] . for example, microcin c ( ) (figure ) is an antibiotic produced by escherichia coli [ ] . dinogunellin ( ) (figure ), which is produced in the roe of some fishes, is a natural toxin [ ] . phosphoarginine and phosphocreatine ( and ) ( figure ) are important biological molecules used as sources of stored energies in invertebrates and vertebrates, respectively [ ] . phosphoramidon ( ) (figure ), derived from streptomyces tanashiensis, inhibits the thermolysin enzyme, which is a key factor in the development of various diseases [ , ] . lastly, phosmidosine and agrocin ( and ) (figure ) are nucleotide antibiotics isolated from streptomyces due to the inherent physiochemical properties of the phosphorus atom including its polarizability, multivalency, varying oxidation states, and low coordination number, a diverse range of compounds containing the p-n motifs have been synthesized [ ] . these p-ns are widely used in medicine for the control and treatment of various diseases, in agriculture as pesticides for crop protection, in industry as novel fire-retardant compounds to delay the flammability of polymeric compounds, and in the fields of analytical and coordination chemistry. therefore, synthetic p-n compounds are not only researched out of academic interest, but they also have commercial applications. uses for p-ns can be separated into main classes: agriculture, industry, analytical chemistry, synthetic chemistry, and medicinal chemistry ( figure ) [ ] [ ] [ ] [ ] [ ] [ ] [ ] . this class of organophosphates (with common names such as cruformate, fenamiphos, and fosthietan) has been widely used in pest management for crop protection (figure ) [ ] [ ] [ ] . p-ns for pest control affect the nervous system of pests/insects by inhibiting acetylcholinesterase (ache), a major neurotransmitter [ ] [ ] [ ] [ ] . in addition, these p-n compounds have been utilized as urease inhibitors [ , ] . urease is a nickel-based enzyme that catalyzes the hydrolysis of urea on soil surfaces into volatile ammonia and carbon dioxide (scheme ) [ , ] . thus, on inhibiting the activity of this metalloenzyme, the availability and performance of urea is enhanced, which is a vital nitrogenous fertilizer for plant growth. p-ns have been used as additives in both oil and water-based lubricants ( figure ). it has been proposed that phosphorus acts as a key element in enhancing the tribological properties of water/oil-based fluids by forming a protective layer containing phosphates and/or polyphosphates. thus, they possess anti-wear and friction-reducing properties [ , ] . furthermore, these p-ns have been found to be a promising alternative to halogenated flame retardants (frs), which are being phased out as they have been recognized as global contaminants due to their ill effects on health and the environment [ ] . p-n fire retardants are less volatile and relatively thermally stable with this class of organophosphates (with common names such as cruformate, fenamiphos, and fosthietan) has been widely used in pest management for crop protection (figure ) [ ] [ ] [ ] . p-ns for pest control affect the nervous system of pests/insects by inhibiting acetylcholinesterase (ache), a major neurotransmitter [ ] [ ] [ ] [ ] . in addition, these p-n compounds have been utilized as urease inhibitors [ , ] . urease is a nickel-based enzyme that catalyzes the hydrolysis of urea on soil surfaces into volatile ammonia and carbon dioxide (scheme ) [ , ] . thus, on inhibiting the activity of this metalloenzyme, the availability and performance of urea is enhanced, which is a vital nitrogenous fertilizer for plant growth. this class of organophosphates (with common names such as cruformate, fenamiphos, and fosthietan) has been widely used in pest management for crop protection (figure ) [ ] [ ] [ ] . p-ns for pest control affect the nervous system of pests/insects by inhibiting acetylcholinesterase (ache), a major neurotransmitter [ ] [ ] [ ] [ ] . in addition, these p-n compounds have been utilized as urease inhibitors [ , ] . urease is a nickel-based enzyme that catalyzes the hydrolysis of urea on soil surfaces into volatile ammonia and carbon dioxide (scheme ) [ , ] . thus, on inhibiting the activity of this metalloenzyme, the availability and performance of urea is enhanced, which is a vital nitrogenous fertilizer for plant growth. p-ns have been used as additives in both oil and water-based lubricants ( figure ). it has been proposed that phosphorus acts as a key element in enhancing the tribological properties of water/oil-based fluids by forming a protective layer containing phosphates and/or polyphosphates. thus, they possess anti-wear and friction-reducing properties [ , ] . furthermore, these p-ns have been found to be a promising alternative to halogenated flame retardants (frs), which are being phased out as they have been recognized as global contaminants due to their ill effects on health and the environment [ ] . p-n fire retardants are less volatile and relatively thermally stable with scheme . hydrolysis of urea on soil in the presence of urease. p-ns have been used as additives in both oil and water-based lubricants ( figure ). it has been proposed that phosphorus acts as a key element in enhancing the tribological properties of water/oil-based fluids by forming a protective layer containing phosphates and/or polyphosphates. thus, they possess anti-wear and friction-reducing properties [ , ] . furthermore, these p-ns have been found to be a promising alternative to halogenated flame retardants (frs), which are being phased out as they have been recognized as global contaminants due to their ill effects on health and the environment [ ] . p-n fire retardants are less volatile and relatively thermally stable with enhanced char formation, and they are more efficient and sustainable than their halogenated molecules , , of counterparts [ , ] . recently, compounds containing both p and n atoms in combination have been reported to have a synergistic effect, as they can act in both vapor phase and condensed phase, thus offering improved flame retardancy on various substrates such as polyurethane foams, cotton, cellulose, epoxy resins, textiles, etc. [ ] [ ] [ ] [ ] [ ] [ ] [ ] . for example, novel p-n-based intumescent flame retardants have been incorporated into polycarbonates (pc) to impart flame retardancy to the composite by forming an expandable protective char [ ] . maldi-tof ms is a prominent tool for analyzing biomolecules (figure ) [ ] . for the detection of low molecular weight such as amino acids and peptides, conventional matrices yield a lot of matrix-related ions in the low m/z range of the spectrum, thus interfering with analyte signals. to tackle this, various approaches such as using additives in the organic matrix, high mass matrix molecules, or matrices based on inorganic compounds have been employed [ ] . furthermore, to improve analysis, synthesizing derivatives of small analyte molecules by n-phosphorylation labeling has been done. the neutral phosphoryl group attached at the n-terminal of amino acids suppresses the matrix signals and favors the transfer of energy from matrix to analyte for ionization by secondary ion-molecule reactions. therefore, p-ns have been used for analysis of biomolecules to improve the ionization efficiency and suppress matrix background signals [ ] . in addition to this, p-ns have found novel applications as task-specific ionic liquids (tsils), which are tailored ionic liquids having a specific functionality to complex metal ions. recently, these tsils have been used for the efficient extraction of uranium metal, which is an important element in nuclear fuel, and the extraction of uranium metal from the spent/used nuclear fuel is vital for the sustainable economy [ ] . the enhanced extraction capabilities using tsils in contrast to liquid-liquid extraction could be attributed to synergistic effects as a result of complexation and h-bonding [ ] . to this end, these tailored ionic liquids have been found to be selective for the micellar extraction of uranium metal from aqueous waste samples as even the ultra-trace levels of uranium are toxic [ ] . p-ns act as , -n,o-chelating ligands (e.g., figure , ligand) and have diverse applications in the field of coordination chemistry for bond activation, catalysis, and metal ligand cooperativity [ ] [ ] [ ] . for example, a p-n-tantalum complex has been developed as a pre-catalyst for hydroaminoalkylation reactions [ ] . in addition, the synthesis of medium and large n-based heterocycles has been achieved with the aid of p-ns, as the phosphoryl group improves intramolecular cyclization, and it can also be easily removed afterwards [ ] . furthermore, p-ns have been used as directing groups for arylation reactions, as they selectively activate c-h bonds, and thus, desired functionality can be achieved [ ] . p-ns have garnered considerable interest in the field of biomedicine due to their potential applications as antimicrobial [ ] [ ] [ ] , antioxidant [ ] , anticancer [ ] , antimalarial [ ] , antiviral [ , ] , and anti-hiv [ ] drugs and prodrugs against various ailments ( figure ). the protide approach, pioneered by the mcguigan group in for drug delivery, is a powerful tool to enhance the efficacy, intracellular delivery, and therapeutic potential of the parent drugs [ , ] . recently, remdesivir ( figure , drug candidate), which has a p-n moiety, has been evaluated as a therapeutic option for the treatment of covid- -affected patients and has attracted a lot of attention in the clinical field [ ] . additionally, these p-n compounds have been used in gene therapy and nucleoside treatment [ ] [ ] [ ] . moreover, many p-ns have been found to be hydrolytically degradable under acidic conditions and are comparatively stable at neutral and higher phs. the acid labile p-n bond is selectively cleaved due to protonation at n, which then undergoes nucleophilic attack by a molecule of water to eliminate the corresponding phosphates and amines [ , ] . thus, these ph responsive p-ns have notable applications in controlled drug release [ , ] . for example, the p-n-based prodrug of l-dopa has been synthesized for its controlled release for the treatment of parkinson's disease [ ] . similarly, p-n derivatives of the antiherpetic drug, acyclovir (acv), hydrolyses at ph into acv monophosphate and can be a promising antiviral drug due to increased cell penetration and therapeutic potential relative to the parent drug [ ] . although p-n biomolecules such as phosphocreatine have been known since the work of fiske and subbarow in , synthetic routes to p-n compounds prior to have been limited [ , ] . these early routes involve the preparation of and use of toxic reagents over multiple synthetic steps and often resulted in the elimination of stoichiometric waste. however, with an increase in the number of uses of p-ns, the need to develop one-pot synthetic routes that are more environmentally benign has driven researchers to discover catalytic strategies. accordingly, all the known synthetic routes to p-ns can be separated into six categories: salt elimination, oxidative cross-coupling, azide, reduction, hydrophosphinylation, and phosphoramidate-aldehyde-dienophile (pad). each of the categories will be discussed in detail in the coming sections, with an emphasis on conditions, scope, and mechanism. this discussion will be preceded by a brief introduction to the early synthetic routes. based on the available literature, one of the earliest syntheses of p-ns involved the works of audrieth and toy [ , ] . audrieth and toy treated phosphoryl trichloride with phenol in the presence of pyridine to obtain a mixture of diphenylchlorophosphate (dpcp) and phenyl dichlorophosphate (pdcp). upon reacting the mixture with gaseous or aqueous nh in or primary amine in , p-n and n-p-n were formed (scheme route a, r = ph). in , p-ns were made by atherton, openshaw, and todd through a salt elimination process using disubstituted h-phosphonates (h-p), amines, and chlorinating agents (scheme route b) [ ] . subsequently, the atherton-todd group synthesized p-ns via the same method using different polyhalogen solvents in [ ] and using alternative phosphorylating agents in [ ] . in , wagner-jauregg et al. synthesized p-n compounds from diisopropylchlorophosphate (dprcp) or tetraethylpyrophosphate (tepyp) and amines (scheme route a, r = i-pr and route c, respectively) [ ] . sodium anilide or sodium diphenylamide was refluxed with triphenylphosphate (tppo) to eliminate phenol and give diphenyl n-phenylphosphoramidate or diphenyl n,n-diphenylphosphoramidate in (scheme route d) [ ] . in , sundberg reacted -ethyl- -nitrobenzene and triethyl phosphite to obtain a phosphorimidate-product (n=p) that was converted to p-n on silica or alumina columns [ ] . cadogan and coworkers reportedly synthesized and investigated the nucleophilicity of various p-ns in [ ] . two years later, a mixture of n-arylphosphoramidates, dialkyl phosphoramidates, and phosphonates were obtained when nitrosoand nitro-compounds were reduced in the presence of (ro) p (r = alkyl) [ ] . in , appel and einig [ ] reported a new synthesis to p-ns by reacting phosphoric acid and an amine in the presence of triphenylphosphine (tpp) and ccl . the same year, phase-transfer catalysis was introduced by zwierzak to synthesize p-ns through the phosphorylation of amines [ ] . the selective alkylation of (eto) p(o)nh via silylation was achieved by zwierzak in using [n-bu n]br catalyst (scheme route e) [ ] . in , zwierzak and osowska-pacewicka reported the synthesis of various p-ns via inorganic salt elimination [ ] and the direct conversion of phosphoric acid [ ] . in , riesel et al. reacted primary amines or nh with (ro) p (r = alkyl) and ccl as an effective route to p-ns [ ] . nielsen and caruthers reported the first iodine-mediated reaction of phosphite and n-butylamine to synthesize p-ns in (scheme route f) [ ] . at about the same time, p-ns were synthesized from a reaction of azide and (ro) p (r = alkyl) in a one-pot process [ ] . a magnesium chloride-catalyzed reaction of , -dimethylphenol and phosphorus oxychloride was used to generate phosphorochloridate, which was used for the phosphorylation of amines to synthesize p-ns [ ] . the aforementioned syntheses demonstrate the diversity of organophosphorus synthons available for p-n chemistry up until and highlight the main pioneers in the field (figure ). the research in these initial studies has focused on reducing waste, limiting toxic reagents, improving selectivity, expanding scope, and searching for effective and efficient catalytic p-n bond-making solutions. the aforementioned syntheses demonstrate the diversity of organophosphorus synthons available for p-n chemistry up until and highlight the main pioneers in the field (figure ). the research in these initial studies has focused on reducing waste, limiting toxic reagents, improving selectivity, expanding scope, and searching for effective and efficient catalytic p-n bond-making solutions. the aforementioned syntheses demonstrate the diversity of organophosphorus synthons available for p-n chemistry up until and highlight the main pioneers in the field (figure ). the research in these initial studies has focused on reducing waste, limiting toxic reagents, improving selectivity, expanding scope, and searching for effective and efficient catalytic p-n bond-making solutions. a synthetic p-n was fortuitously prepared by atherton, openshaw, and todd in by the salt elimination method using h-phosphonates (h-p) ((ro) p(o)h; r = et, i-pr, bn) and ammonia in the presence of a base (scheme route b) [ ] . the scope of this method was investigated using different amines (e.g., morpholine, cyclohexylamine, -phenylethan- -amine and benzylamine, -methylmorpholine) or a mixture of aniline and n,n-dimethylcyclohexanamine, as well as different solvents (e.g., ccl , cl cccl , and hcl cccl ). isolated yields of - % were obtained. the atherton-todd method was prominent for its in situ generation of (ro) p(o)cl (dacp; r = alkyl, benzyl), which eliminates the use of moisture-and air-sensitive dacp. however, the major drawbacks of the method include the use of halogen sources such as ccl , cl cccl , and hcl cccl and extensive work-up/purification of the products. in , chen et al. reported a modified atherton-todd reaction for the synthesis of p-ns using air as a radical initiator (scheme ) [ ] . the modified method explored p-n synthesis using (h-p) ((ro) p(o)h; r = et, i-pr, bu, i-bu) and benzylamine with yields in the range of - %. although this method offered advantages such as using air as a radical initiator at room temperature, the use of relatively expensive , , , , , , , -octahydropyrimido[ , -a]azepine (dbu) as a base in the process, the elimination of large amounts of stoichiometric waste, and comparatively lower yields of products are some of the limitations. [ ] . the scope of this method was investigated using different amines (e.g., morpholine, cyclohexylamine, -phenylethan- -amine and benzylamine, -methylmorpholine) or a mixture of aniline and n,n-dimethylcyclohexanamine, as well as different solvents (e.g., ccl , cl cccl , and hcl cccl ). isolated yields of - % were obtained. the atherton-todd method was prominent for its in situ generation of (ro) p(o)cl (dacp; r = alkyl, benzyl), which eliminates the use of moisture-and air-sensitive dacp. however, the major drawbacks of the method include the use of halogen sources such as ccl , cl cccl , and hcl cccl and extensive work-up/purification of the products. in , chen et al. reported a modified atherton-todd reaction for the synthesis of p-ns using air as a radical initiator (scheme ) [ ] . the modified method explored p-n synthesis using (h-p) ((ro) p(o)h; r = et, i-pr, bu, i-bu) and benzylamine with yields in the range of - %. although this method offered advantages such as using air as a radical initiator at room temperature, the use of relatively expensive , , , , , , , -octahydropyrimido[ , -a]azepine (dbu) as a base in the process, the elimination of large amounts of stoichiometric waste, and comparatively lower yields of products are some of the limitations. with reference to the limitations (such as low yield and the formation of organophosphorus anhydrides side products) in the method reported by appel and einig [ ] , zwierzak and osowska-pacewicka [ ] proposed an alternative method that uses −oh group activator, hexamethyltriaminodibromophosphorane ((me n) pbr ). in the reported method, (me n) pbr was prepared in situ in a one-pot two-step synthesis of p-ns from (eto) p(o)oh and amines (scheme ) [ ] . the synthesis was explored using various amines such as aniline, phenylmethanamine, dibutylamine, butan- -amine, dipropylamine, and hept- -en- -amine. this method was favorable with improved yields of p-n products ( - %) after workup and eliminated the route(s) for anhydride formation. nevertheless, the method used potentially harmful bromine in a multi-step tedious process with low atom economy. with reference to the limitations (such as low yield and the formation of organophosphorus anhydrides side products) in the method reported by appel and einig [ ] , zwierzak and osowska-pacewicka [ ] proposed an alternative method that uses −oh group activator, hexamethyltriaminodibromophosphorane ((me n) pbr ). in the reported method, (me n) pbr was prepared in situ in a one-pot two-step synthesis of p-ns from (eto) p(o)oh and amines (scheme ) [ ] . the synthesis was explored using various amines such as aniline, phenylmethanamine, dibutylamine, butan- -amine, dipropylamine, and hept- -en- -amine. this method was favorable with improved yields of p-n products ( - %) after workup and eliminated the route(s) for anhydride formation. nevertheless, the method used potentially harmful bromine in a multi-step tedious process with low atom economy. [ ] . the scope of this method was investigated using different amines (e.g., morpholine, cyclohexylamine, -phenylethan- -amine and benzylamine, -methylmorpholine) or a mixture of aniline and n,n-dimethylcyclohexanamine, as well as different solvents (e.g., ccl , cl cccl , and hcl cccl ). isolated yields of - % were obtained. the atherton-todd method was prominent for its in situ generation of (ro) p(o)cl (dacp; r = alkyl, benzyl), which eliminates the use of moisture-and air-sensitive dacp. however, the major drawbacks of the method include the use of halogen sources such as ccl , cl cccl , and hcl cccl and extensive work-up/purification of the products. in , chen et al. reported a modified atherton-todd reaction for the synthesis of p-ns using air as a radical initiator (scheme ) [ ] . the modified method explored p-n synthesis using (h-p) ((ro) p(o)h; r = et, i-pr, bu, i-bu) and benzylamine with yields in the range of - %. although this method offered advantages such as using air as a radical initiator at room temperature, the use of relatively expensive , , , , , , , -octahydropyrimido[ , -a]azepine (dbu) as a base in the process, the elimination of large amounts of stoichiometric waste, and comparatively lower yields of products are some of the limitations. with reference to the limitations (such as low yield and the formation of organophosphorus anhydrides side products) in the method reported by appel and einig [ ] , zwierzak and osowska-pacewicka [ ] proposed an alternative method that uses −oh group activator, hexamethyltriaminodibromophosphorane ((me n) pbr ). in the reported method, (me n) pbr was prepared in situ in a one-pot two-step synthesis of p-ns from (eto) p(o)oh and amines (scheme ) [ ] . the synthesis was explored using various amines such as aniline, phenylmethanamine, dibutylamine, butan- -amine, dipropylamine, and hept- -en- -amine. this method was favorable with improved yields of p-n products ( - %) after workup and eliminated the route(s) for anhydride formation. nevertheless, the method used potentially harmful bromine in a multi-step tedious process with low atom economy. in another salt elimination route, this time using inorganic bases and [n-bu n]br as a catalyst, zwierzak and osowska-pacewicka synthesized p-ns from (eto) p(o)h (diethyl h-p) and various amines (scheme a) [ ] . aromatic amines such as aniline and -chloroaniline, aliphatic amines such as cyclohexylamine, n-butylamine, and ethanamine, and alkanolamines such as -aminoethan- -ol, -aminopropan- -ol, and bis( -(-ethoxy)ethyl)amine were explored to demonstrate the broad scope of this synthetic transformation. the p-ns were isolated in - % yield after recrystallization. in a similar synthesis, using [bnet n]cl as the catalyst instead of [n-bu n]br, zwierzak accomplished the phosphorylation of amines using dialkyl h-phosphonate (ro) p(o)h (r = et, bn, t-bu; scheme b) [ ] . a range of amines, such as aniline, benzylamine, cyclohexylamine, ethylamine, and diethylamine were used. the crude p-ns were recrystallized to afford pure products in - % yield. due to the difficulty encountered in phosphorylating aromatic amines, as mentioned by zwierzak [ , ] , lukanov et al. proposed a similar synthetic route using n-phenylformamide or -chloro-n-phenylacetamide as the n-source, which offered less steric hindrance and better n-h acidity (scheme c) [ ] . derivatives of n-phenylformamide or -chloro-n-phenylacetamide (r'r"nc(o)r"') were used containing a wide range of aryl-substituents (e.g., r" = ph, -meoph, -clph, -meoph, , -cl ph, -meph, and , -(c h ) ph and , -(ch ) ph). either column chromatography (on silica) or recrystallization were used to purify the p-ns to obtain isolated yields in the range of - %. in another salt elimination route, this time using inorganic bases and [n-bu n]br as a catalyst, zwierzak and osowska-pacewicka synthesized p-ns from (eto) p(o)h (diethyl h-p) and various amines (scheme a) [ ] . aromatic amines such as aniline and -chloroaniline, aliphatic amines such as cyclohexylamine, n-butylamine, and ethanamine, and alkanolamines such as -aminoethan- -ol, -aminopropan- -ol, and bis( -(-ethoxy)ethyl)amine were explored to demonstrate the broad scope of this synthetic transformation. the p-ns were isolated in - % yield after recrystallization. in a similar synthesis, using [bnet n]cl as the catalyst instead of [n-bu n]br, zwierzak accomplished the phosphorylation of amines using dialkyl h-phosphonate (ro) p(o)h (r = et, bn, t-bu; scheme b) [ ] . a range of amines, such as aniline, benzylamine, cyclohexylamine, ethylamine, and diethylamine were used. the crude p-ns were recrystallized to afford pure products in - % yield. due to the difficulty encountered in phosphorylating aromatic amines, as mentioned by zwierzak [ , ] , lukanov et al. proposed a similar synthetic route using n-phenylformamide or -chloro-n-phenylacetamide as the n-source, which offered less steric hindrance and better n-h acidity (scheme c) [ ] . derivatives of n-phenylformamide or -chloro-n-phenylacetamide (r'r''nc(o)r''') were used containing a wide range of aryl-substituents (e.g., r'' = ph, -meoph, -clph, -meoph, , -cl ph, -meph, and , -(c h ) ph and , -(ch ) ph). either column chromatography (on silica) or recrystallization were used to purify the p-ns to obtain isolated yields in the range of - %. inorganic salt elimination methods stimulated an era of catalytic transformations of dialkyl h-p ((ro) p(o)h; r = alkyl) to (ro) p(o)cl (dacp; r = alkyl) in the presence of readily available inorganic bases, thereby eliminating the use of et n. however, this method has limitations including a pre-functionalization step, the production of large amounts of stoichiometric waste, and the use of hazardous halogenating reagents. a one-pot synthesis of p-ns was reported by gupta et al. (scheme a) [ ] . according to the authors, the chlorination of (ro) p(o)h (r = me, et, pr, i-pr; eq.) to afford (ro) p(o)cl (dacp; r = alkyl) was possible upon reacting with trichloroisocyanuric acid (tcic-a; eq.), which was then treated with an equal mixture of amines, r'r"nh (r' = me, et, pr; r" = h) and et n, to form the p-n in - % yield, which was determined as conversions by p nmr spectroscopy. however, the isolation of (ro) p(o)nr'r" (r = et, r', r" = et, pr) product gave % yield. a similar method, under base-free reaction conditions, was achieved by kaboudin et al. (scheme b) [ ] . the scope of the synthesis was explored with (h-p) ((ro) p(o)h; r = et, i-pr) and amine (e.g., n-methylaniline, naphthalen- -amine, -phenylethan- -amine, -chloro- -methylaniline, -nitroaniline, benzylamine, or aniline). the crude product was purified by column chromatography (on silica) to yield isolated pure products in the range of - %. alkyl) was possible upon reacting with trichloroisocyanuric acid (tcic-a; eq.), which was then treated with an equal mixture of amines, r'r''nh (r' = me, et, pr; r'' = h) and et n, to form the p-n in - % yield, which was determined as conversions by p nmr spectroscopy. however, the isolation of (ro) p(o)nr'r" (r = et, r', r" = et, pr) product gave % yield. a similar method, under base-free reaction conditions, was achieved by kaboudin et al. (scheme b) [ ] . the scope of the synthesis was explored with (h-p) ((ro) p(o)h; r = et, i-pr) and amine (e.g., n-methylaniline, naphthalen- -amine, -phenylethan- -amine, -chloro- -methylaniline, -nitroaniline, benzylamine, or aniline). the crude product was purified by column chromatography (on silica) to yield isolated pure products in the range of - %. jang and coworkers reported a facile synthetic route to form p-ns using diphenyl phosphoric acid (dppa) and an amine in the presence of both a chlorinating agent (cl ccn) and a base (et n), which was added to sequester the acid formed during the reaction (scheme c) [ ] . the applicability of the method was explored using prop- -en- -amine, diethylamine, -methylpropan- -amine, cyclohexylamine, piperidine, morpholine, and aniline. the crude product was purified by column chromatography (on silica) to obtain isolated yields of p-n products in the range of - %. a similar chlorination method reacting an amine (e.g., aniline, hexylamine, diethylamine, and methanamine) with phosphoric acid ((r p(o)oh; r = oet, obu, obn) in the presence of triphenylphosphine and ccl was also used to synthesize p-ns (scheme d) [ ] . the isolated yields of the products in this case were in the range of - %. p-n synthesis via oxidative cross-coupling using chlorinating agents offers a short reaction time, low temperature synthesis, and a diverse choice of phosphorylating reagents. nevertheless, the methods use hazardous chlorinating agents such as ccl and cl ccn, generate large stoichiometric wastes, and involve a pre-functionalization step in the synthetic process. the synthesis of p-ns from amines (aromatic and aliphatic) and symmetrical trialkyl phosphite (p(or) ; r= me, et, i-pr) in the presence i was reported by chen et al. [ ] . crude products were purified by column chromatography (on silica) and resulted in yields in the range of - %. for the low yielding derivatives, the reaction was more selective to three identified by-products, h-phosphonate, trialkyl phosphate, and tetraalkyl diphosphate, as well as some other unidentified jang and coworkers reported a facile synthetic route to form p-ns using diphenyl phosphoric acid (dppa) and an amine in the presence of both a chlorinating agent (cl ccn) and a base (et n), which was added to sequester the acid formed during the reaction (scheme c) [ ] . the applicability of the method was explored using prop- -en- -amine, diethylamine, -methylpropan- -amine, cyclohexylamine, piperidine, morpholine, and aniline. the crude product was purified by column chromatography (on silica) to obtain isolated yields of p-n products in the range of - %. a similar chlorination method reacting an amine (e.g., aniline, hexylamine, diethylamine, and methanamine) with phosphoric acid ((r p(o)oh; r = oet, obu, obn) in the presence of triphenylphosphine and ccl was also used to synthesize p-ns (scheme d) [ ] . the isolated yields of the products in this case were in the range of - %. p-n synthesis via oxidative cross-coupling using chlorinating agents offers a short reaction time, low temperature synthesis, and a diverse choice of phosphorylating reagents. nevertheless, the methods use hazardous chlorinating agents such as ccl and cl ccn, generate large stoichiometric wastes, and involve a pre-functionalization step in the synthetic process. the synthesis of p-ns from amines (aromatic and aliphatic) and symmetrical trialkyl phosphite (p(or) ; r= me, et, i-pr) in the presence i was reported by chen et al. [ ] . crude products were purified by column chromatography (on silica) and resulted in yields in the range of - %. for the low yielding derivatives, the reaction was more selective to three identified by-products, h-phosphonate, trialkyl phosphate, and tetraalkyl diphosphate, as well as some other unidentified by-products. important compounds such as phosphoryl amino acid esters ( ) ( ) ( ) ( ) were synthesized using the iodine-mediated method ( figure ). molecules , , x for peer review of by-products. important compounds such as phosphoryl amino acid esters ( ) ( ) ( ) ( ) were synthesized using the iodine-mediated method ( figure ). structures ( ) synthesized by iodinating agents. iodine (i ) has also been used as a catalyst under solvent-free conditions to couple aryl amines (phnh and derivatives containing oh, oet, cn, ome, no , f, cl, or br substituents on ph) with (ro) p(o)h (dialkyl h-p; r = me, et) affording dialkyl p-ns (scheme b) [ ] . then, the crude products were purified by column chromatography (on silica) to obtain yields of product in - %. the best yielding conditions were shorter reaction times without solvent. reactions performed in polar non-protic solvents (e.g., ch cl ), non-polar solvents (e.g., hexane), or protic solvents (e.g., meoh) all suffered from reduced efficiency and selectivity. furthermore, aniline with substituents at ortho-and meta-positions were observed to have reacted faster and were higher yielding than aniline containing substituents at the para-position. i was used in the presence of h o as a catalyst at °c for the dehydrogenative cross-coupling of diethyl h-p and amine or sulfoximines to form p-ns (scheme c) [ ] . purification of the products by column chromatography (on silica) afforded p-ns with isolated yields of - %, with the lower yielding products reported from sulfoximine. both alkyl and aryl h-p ((ro) p(o)h, r = et, i-pr, ph) were used as the substrate along with cyclic aromatic secondary amines (e.g., indole), derivatives of heterocyclic amines (e.g., furfurylamine), aliphatic cyclic secondary amines (e.g., -benzhydrylpiperazine), tertiary amines (e.g., n-methylhomopiperazine), or sulfoximine. according to the article, using other sources of i such as ki or n-bromosuccinimide (nbs) for the reaction resulted in decreased yield and trace quantities of the desired products, respectively. iodine-mediated oxidative cross-coupling methods offer better routes to p-n compounds by eliminating toxic halogenating agents such as ccl in the synthetic processes. the method also uses inexpensive oxidants without additives and generates water or alkanol as stoichiometric waste. however, the synthetic routes are prone to the formation of many undesired side products, suffer from low yields, and sometimes have limited applicability. iodine (i ) has also been used as a catalyst under solvent-free conditions to couple aryl amines (phnh and derivatives containing oh, oet, cn, ome, no , f, cl, or br substituents on ph) with (ro) p(o)h (dialkyl h-p; r = me, et) affording dialkyl p-ns (scheme b) [ ] . then, the crude products were purified by column chromatography (on silica) to obtain yields of product in - %. the best yielding conditions were shorter reaction times without solvent. reactions performed in polar non-protic solvents (e.g., ch cl ), non-polar solvents (e.g., hexane), or protic solvents (e.g., meoh) all suffered from reduced efficiency and selectivity. furthermore, aniline with substituents at orthoand meta-positions were observed to have reacted faster and were higher yielding than aniline containing substituents at the para-position. iodine (i ) has also been used as a catalyst under solvent-free conditions to couple aryl amines (phnh and derivatives containing oh, oet, cn, ome, no , f, cl, or br substituents on ph) with (ro) p(o)h (dialkyl h-p; r = me, et) affording dialkyl p-ns (scheme b) [ ] . then, the crude products were purified by column chromatography (on silica) to obtain yields of product in - %. the best yielding conditions were shorter reaction times without solvent. reactions performed in polar non-protic solvents (e.g., ch cl ), non-polar solvents (e.g., hexane), or protic solvents (e.g., meoh) all suffered from reduced efficiency and selectivity. furthermore, aniline with substituents at ortho-and meta-positions were observed to have reacted faster and were higher yielding than aniline containing substituents at the para-position. i was used in the presence of h o as a catalyst at °c for the dehydrogenative cross-coupling of diethyl h-p and amine or sulfoximines to form p-ns (scheme c) [ ] . purification of the products by column chromatography (on silica) afforded p-ns with isolated yields of - %, with the lower yielding products reported from sulfoximine. both alkyl and aryl h-p ((ro) p(o)h, r = et, i-pr, ph) were used as the substrate along with cyclic aromatic secondary amines (e.g., indole), derivatives of heterocyclic amines (e.g., furfurylamine), aliphatic cyclic secondary amines (e.g., -benzhydrylpiperazine), tertiary amines (e.g., n-methylhomopiperazine), or sulfoximine. according to the article, using other sources of i such as ki or n-bromosuccinimide (nbs) for the reaction resulted in decreased yield and trace quantities of the desired products, respectively. iodine-mediated oxidative cross-coupling methods offer better routes to p-n compounds by eliminating toxic halogenating agents such as ccl in the synthetic processes. the method also uses inexpensive oxidants without additives and generates water or alkanol as stoichiometric waste. however, the synthetic routes are prone to the formation of many undesired side products, suffer from low yields, and sometimes have limited applicability. i was used in the presence of h o as a catalyst at • c for the dehydrogenative cross-coupling of diethyl h-p and amine or sulfoximines to form p-ns (scheme c) [ ] . purification of the products by column chromatography (on silica) afforded p-ns with isolated yields of - %, with the lower yielding products reported from sulfoximine. both alkyl and aryl h-p ((ro) p(o)h, r = et, i-pr, ph) were used as the substrate along with cyclic aromatic secondary amines (e.g., indole), derivatives of heterocyclic amines (e.g., furfurylamine), aliphatic cyclic secondary amines (e.g., -benzhydrylpiperazine), tertiary amines (e.g., n-methylhomopiperazine), or sulfoximine. according to the article, using other sources of i such as ki or n-bromosuccinimide (nbs) for the reaction resulted in decreased yield and trace quantities of the desired products, respectively. iodine-mediated oxidative cross-coupling methods offer better routes to p-n compounds by eliminating toxic halogenating agents such as ccl in the synthetic processes. the method also uses inexpensive oxidants without additives and generates water or alkanol as stoichiometric waste. however, the synthetic routes are prone to the formation of many undesired side products, suffer from low yields, and sometimes have limited applicability. the aerobic oxidative coupling of amines and disubstituted h-phosphonates (h-p) (ro) p(o)h (r = me, et, i-pr; eq.) in the presence of copper catalysts to synthesize p-ns was reported by hayes and coworkers (table , route a) [ ] . the authors reported that the ligands on the copper catalyst were important in the reaction, but not the initial oxidation state of copper catalyst, and that the primary amines coupled better than the secondary and tertiary amines due to the reduced steric hindrance. the method was slightly modified for the synthesis of some important phosphoryl amino compounds such as ethyl (diethoxyphosphoryl)glycinate, etc. ( - ) ( figure ). the aerobic oxidative coupling of amines and disubstituted h-phosphonates (h-p) (ro) p(o)h (r = me, et, i-pr; eq.) in the presence of copper catalysts to synthesize p-ns was reported by hayes and coworkers (table , route a) [ ] . the authors reported that the ligands on the copper catalyst were important in the reaction, but not the initial oxidation state of copper catalyst, and that the primary amines coupled better than the secondary and tertiary amines due to the reduced steric hindrance. the method was slightly modified for the synthesis of some important phosphoryl amino compounds such as ethyl (diethoxyphosphoryl)glycinate, etc. ( - ) ( figure ). (table , route b) [ ] . although paraor meta-substituted aryl amines were very effective substrates in this reaction, the conversions observed with ortho-substituted aryl amines or aryl amines with strong electron-withdrawing another copper-catalyzed aerobic oxidative cross-coupling reaction reported by wang et al. in involved the coupling of aryl amines ( . eq.) and dialkyl h-phosphonates (ro) p(o)h (r = et, i-pr, bu; eq.) in the presence of cubr ( mol%) to give p-ns (table , route b) [ ] . although paraor meta-substituted aryl amines were very effective substrates in this reaction, the conversions observed with ortho-substituted aryl amines or aryl amines with strong electron-withdrawing substituents (e.g., -nitroaniline) as well as alkylamines were very poor. the effectiveness of the reaction with diaryl h-phosphonates was not reported. in , fe o @mgo nanoparticles were used as a catalyst to synthesize p-n from primary amines and h-p via the atherton-todd coupling reaction with ccl ( table , route c) [ ] . after column chromatography (on silica), p-ns were isolated in - % yield. the fe o @mgo nanoparticles were recycled by washing successively with h o and meoh and drying before reuse. although the scope of the transformation was not challenged by investigating aromatic h-ps, this route offers some advantages, such as the use of a low-cost reusable catalyst with proven activity for up to four consecutive reactions. purohit et al. reported a one-pot synthesis of p-ns from (ro) p(o)h (r = et, pr, i-pr; eq.) and amine in the presence of cucl and cs co ( table , route d) [ ] . the reported yields for the variety of desired products were in the range of - % as determined by p nmr spectroscopy. a cu(ii) catalyst, a base, and air as an oxidant were used to synthesize n-acylphosphoramidates (acp-n) from (ro) p(o)h (r = me, et, i-pr, bu) and an amide via an oxidative cross-coupling reaction according to mizuno et al. (table , route e) [ ] . the scope of the reaction was investigated by changing the r substituent as well as the nitrogen nucleophiles (e.g., urea, oxazolidinone, indole, pyrrolidinone, lactam, and sulfonamide derivatives). column chromatography (on silica) using different solvent mixtures was used to purify the desired p-ns in - % yield. through the reagent screening, mizuno et al. [ ] found that cu(otf) gave similar cross-coupling results as cu(oac) , while other cu catalysts such as cucl and cuso and other transition metal catalysts (e.g., co(oac) . h o, fe(oac) ) were not selective or completely ineffective. another copper-catalyzed synthesis of p-n at • c in the presence of air, reported by zhou et al., involved reacting (ro) p(o)h (r = me, et, i-pr, ph) with primary or secondary amines (butylamine, dibutylamine, hexylamine, cylohexylamine, and methyl alaninate; table , route f) [ ] . the crude p-ns were extracted and then concentrated under vacuum to obtain pure products with excellent isolated yields ( - %). similar to other copper-catalyzed p-n synthesis, the authors reported a low yield ( %) when dibutylamine and diisopropyl h-p were used as substrates, which could be attributed to steric effects. in addition, the method was not effective when diphenylphosphine oxide (dppo) was reacted with butylamine ( % isolated yield) due to oxidative conversion to phosphonic acid (pp-a). using this aerobic copper-catalyzed dehydrocoupling method, zhou et al. synthesized a phosphoryl amino compound ( ) (figure ). (butylamine, dibutylamine, hexylamine, cylohexylamine, and methyl alaninate; table , route f) [ ] . the crude p-ns were extracted and then concentrated under vacuum to obtain pure products with excellent isolated yields ( - %). similar to other copper-catalyzed p-n synthesis, the authors reported a low yield ( %) when dibutylamine and diisopropyl h-p were used as substrates, which could be attributed to steric effects. in addition, the method was not effective when diphenylphosphine oxide (dppo) was reacted with butylamine ( % isolated yield) due to oxidative conversion to phosphonic acid (pp-a). using this aerobic copper-catalyzed dehydrocoupling method, zhou et al. synthesized a phosphoryl amino compound ( ) (figure ). phenylboronic acid or ester-based substrates were converted in situ to an organic azide using a cu-based catalyst and subsequently used for the synthesis of p-ns via a coupling reaction, according to dangroo et al. (table , route g) [ ] . triethyl and trimethyl phosphate were used as the p-sources while phenylboronic acids or esters with substituents such as cn, no , oh, ome and halides were used for azide conversion. the reaction was very tolerant to compounds with unsaturated boron-carbon bonds giving isolated yields of - %. however, this reaction is not effective for compounds containing saturated boron-carbon bonds such as butyl boronic acid or cyclopentyl boronic acid. transition metal-catalyzed oxidative cross-coupling methods for p-n synthesis have become phenylboronic acid or ester-based substrates were converted in situ to an organic azide using a cu-based catalyst and subsequently used for the synthesis of p-ns via a coupling reaction, according to dangroo et al. (table , route g) [ ] . triethyl and trimethyl phosphate were used as the p-sources while phenylboronic acids or esters with substituents such as cn, no , oh, ome and halides were used for azide conversion. the reaction was very tolerant to compounds with unsaturated boron-carbon bonds giving isolated yields of - %. however, this reaction is not effective for compounds containing saturated boron-carbon bonds such as butyl boronic acid or cyclopentyl boronic acid. transition metal-catalyzed oxidative cross-coupling methods for p-n synthesis have become attractive in recent years because of the advantages of using readily available inexpensive catalysts, for example cux n (x = cl, br, (oac), etc., n = , ), and air as an oxidant. in addition, this route uses environmentally friendly h-p (ro) p(o)h (r = alkyl, ph) and amines as starting materials in a one-pot single-step process without any need for purification of the starting material, thereby making the process cost-and time-effective. apart from eliminating hazardous starting materials, the method uses fewer chemical additives and generates mainly water as a stoichiometric waste. furthermore, the methods offer a possibility of catalyst recycling, which is an important attribute for any green chemical process in terms of mass production. however, a notable limitation of the synthetic route is the formation of various undesired by-products from the reactivities of h-p and amine with the catalysts under oxidative conditions [ ] . guo et al. used visible light and organic dyes as catalysts for the dehydrogenative cross-coupling reaction of (eto) p(o)h (h-p) and amines (aniline, aniline derivatives with cn, x, me, ome and -naphthylamine) to form the corresponding p-ns (scheme ) [ ] . after an extraction, the pure product was formed with isolated yield in the range of - %. it is noteworthy that the method was not effective when vinylaniline and -aminothiazole were used because of their radical sensitivity making them prone to either polymerization or decomposition in the reaction media, respectively. although no product was formed when secondary aliphatic amines were used, moderate yields of up to % were obtained from primary or tertiary aliphatic amines, while a complex mixture of products was formed when an aromatic h-p was used. [ ] . the crude products were purified by column chromatography (on silica) with isolated yields of % and % for the use of benzylamine and -chloroaniline, respectively. other amines were not investigated. although the method uses an inexpensive catalyst and oxidant, the applicability of the process was not explored using diverse h-p and amines. scheme . oxidative cross-coupling reaction of amines and h-phosphonates to form phosphoramidates in the presence of lii/tbhp. [ ] . the crude products were purified by column chromatography (on silica) with isolated yields of % and % for the use of benzylamine and -chloroaniline, respectively. other amines were not investigated. although the method uses an inexpensive catalyst and oxidant, the applicability of the process was not explored using diverse h-p and amines. lithium iodide-tert-butyl hydroperoxide (lii/tbhp) was used for the synthesis of p-n and other compounds by reddy et al. (scheme ) [ ] . the crude products were purified by column chromatography (on silica) with isolated yields of % and % for the use of benzylamine and -chloroaniline, respectively. other amines were not investigated. although the method uses an inexpensive catalyst and oxidant, the applicability of the process was not explored using diverse h-p and amines. scheme . oxidative cross-coupling reaction of amines and h-phosphonates to form phosphoramidates in the presence of lii/tbhp. many conventional routes to p-n synthesis as discussed above have used h-p (ro) p(o)h (r = alkyl, bn, ph) or (ro) p(o)cl (dacp; r = alkyl, bn, ph) and amine as the p-and n-sources, respectively. as a result, derivatives of p-n compounds synthesized through these routes are limited mainly to substituents on the amine starting materials. due to increased applicability, it has become necessary to synthesize p-n compounds with diverse functionalities. in order to achieve this, nitrene insertion from organic azides is becoming a popular one because the route uses phosphoryl azide, which acts a dual source of p and n. in this case, it is possible to add (or insert) diverse molecules on the p-n moiety through c-n bond formation. a route to p-n formation through c-n bond formation was explored by chang, kim, and coworkers (scheme a) [ ] . the p-n was synthesized in the presence of an ir(iii)-based catalyst using an amide or a ketone and a phosphoryl azide. the product was purified by column chromatography (on silica) with isolated yields in the range - %. the scope of the synthesis was explored using phosphoryl azide substituted with binaphthyl, aryl, and alkyl groups, while the phenone had substituents such as phenyl, methyl, and isobutane groups. furthermore, -methoxy, -cf , and tert-butyl were some of the substituents on the benzamide. biologically active phosphoramidates ( and ) were synthesized through this method (figure ). oxidative cross-coupling reaction of amines and h-phosphonates to form phosphoramidates in the presence of lii/tbhp. many conventional routes to p-n synthesis as discussed above have used h-p (ro) p(o)h (r = alkyl, bn, ph) or (ro) p(o)cl (dacp; r = alkyl, bn, ph) and amine as the p-and n-sources, respectively. as a result, derivatives of p-n compounds synthesized through these routes are limited mainly to substituents on the amine starting materials. due to increased applicability, it has become necessary to synthesize p-n compounds with diverse functionalities. in order to achieve this, nitrene insertion from organic azides is becoming a popular one because the route uses phosphoryl azide, which acts a dual source of p and n. in this case, it is possible to add (or insert) diverse molecules on the p-n moiety through c-n bond formation. a route to p-n formation through c-n bond formation was explored by chang, kim, and coworkers (scheme a) [ ] . the p-n was synthesized in the presence of an ir(iii)-based catalyst using an amide or a ketone and a phosphoryl azide. the product was purified by column chromatography (on silica) with isolated yields in the range - %. the scope of the synthesis was explored using phosphoryl azide substituted with binaphthyl, aryl, and alkyl groups, while the phenone had substituents such as phenyl, methyl, and isobutane groups. furthermore, -methoxy, -cf , and tert-butyl were some of the substituents on the benzamide. biologically active phosphoramidates ( and ) were synthesized through this method (figure ). che and coworkers later introduced a ru iv -porphyrin-catalyst to synthesize n-acylphosphoramidates (acp-n) from aldehydes and phosphoryl azide through nitrene insertion (scheme b) [ ] . the product was isolated by column chromatography (on silica). the observed product conversions were in the range - %. aromatic and aliphatic aldehydes such as -naphthaldehyde, benzo[d] [ , ] dioxole- -carbaldehyde, , -dihydro- h-pyran- -carbaldehyde and -methylbut- -enal were used in the reaction along with aromatic and aliphatic phosphoryl azide such as -nitrophenyl ( -nitrophenyl) phosphorazidate, bis( , , -trichloroethyl) phosphorazidate, and diethyl phosphorazidate. however, lower yields were obtained from aliphatic aldehydes when compared with the aromatic aldehydes, suggesting that the synthetic procedure favors electron-rich aromatic aldehydes. che and coworkers later introduced a ru iv -porphyrin-catalyst to synthesize n-acylphosphoramidates (acp-n) from aldehydes and phosphoryl azide through nitrene insertion (scheme b) [ ] . the product was isolated by column chromatography (on silica). the observed product conversions were in the range - %. aromatic and aliphatic aldehydes such as -naphthaldehyde, benzo[d] [ , ] dioxole- -carbaldehyde, , -dihydro- h-pyran- -carbaldehyde and -methylbut- -enal were used in the reaction along with aromatic and aliphatic phosphoryl azide such as -nitrophenyl ( -nitrophenyl) phosphorazidate, bis( , , -trichloroethyl) phosphorazidate, and diethyl phosphorazidate. however, lower yields were obtained from aliphatic aldehydes when compared with the aromatic aldehydes, suggesting that the synthetic procedure favors electron-rich aromatic aldehydes. a nitrene insertion route in the presence of ir(iii)-based catalyst was also used to synthesize p-ns by zhu et al. (scheme c) [ ] . the p-ns were synthesized under an inert environment and the isolated yield of the products was in the range of - %. the scope of the synthesis was investigated using diethyl phosphorazidate and diphenyl phosphorazidate along with -phenylpyridine or pyrazole derivatives with substituents such as me, ome, cf , cl, f, ph, and coome at ortho-, meta-, and para-positions as well as benzo[h]quinoline. although nitrene insertion provides a viable route to p-n synthesis and generates n gas as the only stoichiometric by-product, the process uses expensive catalysts in addition to many chemical additives. moreover, apart from the hazards associated with the handling of phosphoryl azide, the route requires a long reaction time, high temperature, and does not favor electron-deficient starting materials. an organic azide was generated in situ from organic halides and was coupled with (ro) p (r = alkyl) for the synthesis of p-n by sangwan et al. (scheme ) the crude product was purified by column chromatography (on silica) to afford isolated products in the range of - % yield. a broad scope of the reaction was tested; -bromoprop- -yne, -bromoprop- -ene, -bromopropane alkyl or (un)substituted aromatic halide such as (bromomethyl)benzene, -(bromomethyl)- -nitrobenzene, -(chloromethyl)- -nitrobenzene, (chloromethyl)benzene, -(bromomethyl)- , -dimethoxybenzene, or -( -(bromomethyl)- h- , , -triazol- -yl)benzyl acetate were reacted with dimethyl or diethyl h-p. -(chloromethyl)- -nitrobenzene, (chloromethyl)benzene, -(bromomethyl)- , -dimethoxybenzene, or -( -(bromomethyl)- h- , , -triazol- -yl)benzyl acetate were reacted with dimethyl or diethyl h-p. a similar method of p-n synthesis via in situ azide generation from organic halides was reported by dar [ ] , following the procedure previously reported by sangwan et al. (scheme ) [ ] . the yield range of the isolated p-ns, in this case, was - %. phosphoramidate-derived bioactive compound ( ) was synthesized using the proposed method ( figure ). scheme . phosphoramidate synthesis from organic azide and (ro) p. figure . structure of ( r, s, r)- -isopropyl- -methylcyclohexyl (dimethoxyphosphoryl)glycinate ( ) synthesized via in situ azide generation. in , koziara reported a two-step p-n synthetic route via organic azide generation using a [n-bu n]br catalyst (scheme ) [ ] . the crude product was purified by crystallization or vacuum distillation with - % isolated yield. the organic halides used were ( -bromopropyl)benzene, ( -bromoethyl)benzene, -bromohexane, bromocyclohexane, -bromobutane, or bromocyclopentane. routes via organic azide generation use readily available, less toxic organic halides and (ro) p (r = alkyl) relative to other routes and generate n as stoichiometric waste. however, the method scheme . phosphoramidate synthesis from organic azide and (ro) p. a similar method of p-n synthesis via in situ azide generation from organic halides was reported by dar [ ] , following the procedure previously reported by sangwan et al. (scheme ) [ ] . the yield range of the isolated p-ns, in this case, was - %. phosphoramidate-derived bioactive compound ( ) was synthesized using the proposed method ( figure ). column chromatography (on silica) to afford isolated products in the range of - % yield. a broad scope of the reaction was tested; -bromoprop- -yne, -bromoprop- -ene, -bromopropane alkyl or (un)substituted aromatic halide such as (bromomethyl)benzene, -(bromomethyl)- -nitrobenzene, -(chloromethyl)- -nitrobenzene, (chloromethyl)benzene, -(bromomethyl)- , -dimethoxybenzene, or -( -(bromomethyl)- h- , , -triazol- -yl)benzyl acetate were reacted with dimethyl or diethyl h-p. a similar method of p-n synthesis via in situ azide generation from organic halides was reported by dar [ ] , following the procedure previously reported by sangwan et al. (scheme ) [ ] . the yield range of the isolated p-ns, in this case, was - %. phosphoramidate-derived bioactive compound ( ) was synthesized using the proposed method ( figure ). scheme . phosphoramidate synthesis from organic azide and (ro) p. figure . structure of ( r, s, r)- -isopropyl- -methylcyclohexyl (dimethoxyphosphoryl)glycinate ( ) synthesized via in situ azide generation. in , koziara reported a two-step p-n synthetic route via organic azide generation using a [n-bu n]br catalyst (scheme ) [ ] . the crude product was purified by crystallization or vacuum distillation with - % isolated yield. the organic halides used were ( -bromopropyl)benzene, ( -bromoethyl)benzene, -bromohexane, bromocyclohexane, -bromobutane, or bromocyclopentane. routes via organic azide generation use readily available, less toxic organic halides and (ro) p (r = alkyl) relative to other routes and generate n as stoichiometric waste. however, the method figure . structure of ( r, s, r)- -isopropyl- -methylcyclohexyl (dimethoxyphosphoryl)glycinate ( ) synthesized via in situ azide generation. in , koziara reported a two-step p-n synthetic route via organic azide generation using a [n-bu n]br catalyst (scheme ) [ ] . the crude product was purified by crystallization or vacuum distillation with - % isolated yield. the organic halides used were ( -bromopropyl)benzene, ( -bromoethyl)benzene, -bromohexane, bromocyclohexane, -bromobutane, or bromocyclopentane. relies on the pre-functionalization and preparation of hazardous azide precursors in multi-step processes. phosphoramidate synthesis via two-step organic azide generation. transition metal-free synthesis of p-n from phosphoryl azide and amines was accomplished by chen et al. [ ] . the synthetic process uses the nitrogen atom available from the amine rather than from the azide (scheme ). the residue was purified by column chromatography (on silica) to afford the isolated product in - % yield. the scope of the reaction was examined by using diphenyl and diisopropyl phosphoryl azide along with primary amines (e.g., isopropylamine and t-butylamine), substituted primary amines (e.g., thiophen- -ylmethylamine and -chlorobenzylamine), and heterocyclic amines (e.g., pyrrolidine and morpholine). although the method circumvents the use of metal catalysts in the synthesis, the use of a hazardous azide and high temperature are its limitations. scheme . phosphoramidate synthesis from organic azide and amine. phosphoramidate synthesis via two-step organic azide generation. routes via organic azide generation use readily available, less toxic organic halides and (ro) p (r = alkyl) relative to other routes and generate n as stoichiometric waste. however, the method relies on the pre-functionalization and preparation of hazardous azide precursors in multi-step processes. transition metal-free synthesis of p-n from phosphoryl azide and amines was accomplished by chen et al. [ ] . the synthetic process uses the nitrogen atom available from the amine rather than from the azide (scheme ). the residue was purified by column chromatography (on silica) to afford the isolated product in - % yield. the scope of the reaction was examined by using diphenyl and diisopropyl phosphoryl azide along with primary amines (e.g., isopropylamine and t-butylamine), substituted primary amines (e.g., thiophen- -ylmethylamine and -chlorobenzylamine), and heterocyclic amines (e.g., pyrrolidine and morpholine). although the method circumvents the use of metal catalysts in the synthesis, the use of a hazardous azide and high temperature are its limitations. from the azide (scheme ). the residue was purified by column chromatography (on silica) to afford the isolated product in - % yield. the scope of the reaction was examined by using diphenyl and diisopropyl phosphoryl azide along with primary amines (e.g., isopropylamine and t-butylamine), substituted primary amines (e.g., thiophen- -ylmethylamine and -chlorobenzylamine), and heterocyclic amines (e.g., pyrrolidine and morpholine). although the method circumvents the use of metal catalysts in the synthesis, the use of a hazardous azide and high temperature are its limitations. scheme . phosphoramidate synthesis from organic azide and amine. beifuss et al. reported a microwave-assisted synthesis of n-arylphosphoramidates from nitrobenzene and (ro) p (r = alkyl) (scheme ) [ ] . the crude residue product was purified by column chromatography (on silica) resulting in pure p-ns with - % isolated yield. the scope of the synthesis was investigated using trimethyl or triethyl phosphite along with nitrobenzene, and mono-or di-substituted nitrobenzenes with substitution such as me, ome, cl, br, or cn groups. this method is attractive because it provides a different route to p-ns using nitro compounds instead of the conventional amines. nonetheless, the high temperature and amount of energy required during the synthesis are the obvious limitations. beifuss et al. reported a microwave-assisted synthesis of n-arylphosphoramidates from nitrobenzene and (ro) p (r = alkyl) (scheme ) [ ] . the crude residue product was purified by column chromatography (on silica) resulting in pure p-ns with - % isolated yield. the scope of the synthesis was investigated using trimethyl or triethyl phosphite along with nitrobenzene, and mono-or di-substituted nitrobenzenes with substitution such as me, ome, cl, br, or cn groups. this method is attractive because it provides a different route to p-ns using nitro compounds instead of the conventional amines. nonetheless, the high temperature and amount of energy required during the synthesis are the obvious limitations. afford the isolated product in - % yield. the scope of the reaction was examined by using diphenyl and diisopropyl phosphoryl azide along with primary amines (e.g., isopropylamine and t-butylamine), substituted primary amines (e.g., thiophen- -ylmethylamine and -chlorobenzylamine), and heterocyclic amines (e.g., pyrrolidine and morpholine). although the method circumvents the use of metal catalysts in the synthesis, the use of a hazardous azide and high temperature are its limitations. scheme . phosphoramidate synthesis from organic azide and amine. beifuss et al. reported a microwave-assisted synthesis of n-arylphosphoramidates from nitrobenzene and (ro) p (r = alkyl) (scheme ) [ ] . the crude residue product was purified by column chromatography (on silica) resulting in pure p-ns with - % isolated yield. the scope of the synthesis was investigated using trimethyl or triethyl phosphite along with nitrobenzene, and mono-or di-substituted nitrobenzenes with substitution such as me, ome, cl, br, or cn groups. this method is attractive because it provides a different route to p-ns using nitro compounds instead of the conventional amines. nonetheless, the high temperature and amount of energy required during the synthesis are the obvious limitations. hackenberger et al. synthesized p-n compounds from organic azide derivatives and (ro) p (r = alkyl) after lewis acid-catalyzed rearrangement (scheme ) [ ] . the crude product was concentrated under reduced pressure, and the desired product was isolated ( - % yield) through column chromatography (on silica). triethyl or trimethyl phosphite were used as the p-source along with organic azide derivatives such as (azidomethyl)benzene, azidocyclohexane, ( -azidopropan- -yl)benzene, azidobenzene, ethyl -azidoacetate, -azidodecane, (azidomethylene)dibenzene, and ( -azidoprop- -en- -yl)benzene. [ ] . the crude product was concentrated under reduced pressure, and the desired product was isolated ( - % yield) through column chromatography (on silica). triethyl or trimethyl phosphite were used as the p-source along with organic azide derivatives such as (azidomethyl)benzene, azidocyclohexane, ( -azidopropan- -yl)benzene, azidobenzene, ethyl -azidoacetate, -azidodecane, (azidomethylene)dibenzene, and ( -azidoprop- -en- -yl)benzene. zimin et al. reported the , -hydrophosphinylation of alkyl nitriles (r'-cn, r' = alkyl, ph, bn) using sodium dialkyl phosphite ((ro) pona, r = et, pr) to achieve the formation of a p-n featuring a p-c-n-p linkage in which the dialkyl phosphite group of (ro) pona was added to the carbon and nitrogen atoms of the nitriles (scheme ) [ ] . zimin et al. reported the , -hydrophosphinylation of alkyl nitriles (r'-cn, r' = alkyl, ph, bn) using sodium dialkyl phosphite ((ro) pona, r = et, pr) to achieve the formation of a p-n featuring a p-c-n-p linkage in which the dialkyl phosphite group of (ro) pona was added to the carbon and nitrogen atoms of the nitriles (scheme ) [ ] . zimin et al. reported the , -hydrophosphinylation of alkyl nitriles (r'-cn, r' = alkyl, ph, bn) using sodium dialkyl phosphite ((ro) pona, r = et, pr) to achieve the formation of a p-n featuring a p-c-n-p linkage in which the dialkyl phosphite group of (ro) pona was added to the carbon and nitrogen atoms of the nitriles (scheme ) [ ] . scheme . a , -hydrophosphinylation of nitriles to form phosphoramidates. chabour et al. reported a diastereoselective p-n synthesis via the pad process using a tsoh acid catalyst and a dienophile, maleimide (scheme ) [ ] . flash chromatography was used to isolate the desired product in - % yield. the scope of the synthesis was explored with maleimide derivatives with substituents such as -fc h ch , c h ch , c h coch , -brc h , phenyl, and methyl. the unsaturated aldehyde derivatives that were used were , -dimethylocta- , -dienal, -methylbut- -enal, pent- -enal, and hex- -enal. this newest route for p-n synthesis uses inexpensive catalysts, generates only h o as a by-product in stoichiometric amounts, and demonstrates an important route to the synthesis of polyfunctionalized p-n compounds. chabour et al. reported a diastereoselective p-n synthesis via the pad process using a tsoh acid catalyst and a dienophile, maleimide (scheme ) [ ] . flash chromatography was used to isolate the desired product in - % yield. the scope of the synthesis was explored with maleimide derivatives with substituents such as -fc h ch , c h ch , c h coch , -brc h , phenyl, and methyl. the unsaturated aldehyde derivatives that were used were , -dimethylocta- , -dienal, -methylbut- -enal, pent- -enal, and hex- -enal. this newest route for p-n synthesis uses inexpensive catalysts, generates only h o as a by-product in stoichiometric amounts, and demonstrates an important route to the synthesis of polyfunctionalized p-n compounds. zimin et al. reported the , -hydrophosphinylation of alkyl nitriles (r'-cn, r' = alkyl, ph, bn) using sodium dialkyl phosphite ((ro) pona, r = et, pr) to achieve the formation of a p-n featuring a p-c-n-p linkage in which the dialkyl phosphite group of (ro) pona was added to the carbon and nitrogen atoms of the nitriles (scheme ) [ ] . scheme . a , -hydrophosphinylation of nitriles to form phosphoramidates. chabour et al. reported a diastereoselective p-n synthesis via the pad process using a tsoh acid catalyst and a dienophile, maleimide (scheme ) [ ] . flash chromatography was used to isolate the desired product in - % yield. the scope of the synthesis was explored with maleimide derivatives with substituents such as -fc h ch , c h ch , c h coch , -brc h , phenyl, and methyl. the unsaturated aldehyde derivatives that were used were , -dimethylocta- , -dienal, -methylbut- -enal, pent- -enal, and hex- -enal. this newest route for p-n synthesis uses inexpensive catalysts, generates only h o as a by-product in stoichiometric amounts, and demonstrates an important route to the synthesis of polyfunctionalized p-n compounds. a wealth of information on the mechanisms for the synthetic routes to p-ns is available. for each of the main categories highlighted above, researchers have provided mechanistic insight into the reaction by determining the main sources of oxidation as well as electronic and steric factors provided from the substituents that influence the reaction rate. several examples of postulated catalytic cycles will be provided below, illustrating the key steps, similarities, and differences between them. atherton, openshaw and todd suggested that the two-step mechanistic process for the salt elimination could involve the formation of dialkyl or dibenzyl (trichloromethyl)phosphonate in the first step, from a reaction of (ro) p(o)h (h-p; r = alkyl) and ccl in the presence of et n [ ] . the desired product is formed in the second step when the (trichloromethyl)phosphonate reacts with amine, forming also chcl in the process (scheme ). although this mechanism is plausible, evidence by nmr spectroscopy suggests that in the first step, a reaction of the base and ccl results in salt formation [et ncl] + [ccl ] − [ ] . the h-p is deprotonated by [ccl ] − to form chcl and anionic phosphite, which reacts further with [et ncl] + to form (ro) p(o)cl (dacp; r = alkyl). in the second step, dacp reacts with amine to give the product. desired product is formed in the second step when the (trichloromethyl)phosphonate reacts with amine, forming also chcl in the process (scheme ). although this mechanism is plausible, evidence by nmr spectroscopy suggests that in the first step, a reaction of the base and ccl results in salt formation [et ncl] + [ccl ] − [ ] . the h-p is deprotonated by [ccl ] − to form chcl and anionic phosphite, which reacts further with [et ncl] + to form (ro) p(o)cl (dacp; r = alkyl). in the second step, dacp reacts with amine to give the product. scheme . atherton-todd mechanism proposed for the formation of phosphoramidates. for the modified atherton-todd reaction, the two-step mechanistic process involves the deprotonation of h-p by dbu, and the radical formed is autoxidized by o before it abstracts chlorine from chcl to form dacp (scheme ) [ ] . the nucleophilic substitution of dacp by the amine affords the p-n product. in the plausible mechanism for the direct conversion of diethyl hydrogen phosphate, hexamethyltriaminophosphine p(nme ) is oxidized to hexamethyltriaminodibromophosphorane ((me n) pbr ) which is ionized in solution (scheme ) [ ] . subsequently, the ionized (me n) pbr and diethyl hydrogen phosphate undergo ligand exchange in the presence of et n to form the scheme . atherton-todd mechanism proposed for the formation of phosphoramidates. for the modified atherton-todd reaction, the two-step mechanistic process involves the deprotonation of h-p by dbu, and the radical formed is autoxidized by o before it abstracts chlorine from chcl to form dacp (scheme ) [ ] . the nucleophilic substitution of dacp by the amine affords the p-n product. first step, from a reaction of (ro) p(o)h (h-p; r = alkyl) and ccl in the presence of et n [ ] . the desired product is formed in the second step when the (trichloromethyl)phosphonate reacts with amine, forming also chcl in the process (scheme ). although this mechanism is plausible, evidence by nmr spectroscopy suggests that in the first step, a reaction of the base and ccl results in salt formation [et ncl] + [ccl ] − [ ] . the h-p is deprotonated by [ccl ] − to form chcl and anionic phosphite, which reacts further with [et ncl] + to form (ro) p(o)cl (dacp; r = alkyl). in the second step, dacp reacts with amine to give the product. scheme . atherton-todd mechanism proposed for the formation of phosphoramidates. for the modified atherton-todd reaction, the two-step mechanistic process involves the deprotonation of h-p by dbu, and the radical formed is autoxidized by o before it abstracts chlorine from chcl to form dacp (scheme ) [ ] . the nucleophilic substitution of dacp by the amine affords the p-n product. in the plausible mechanism for the direct conversion of diethyl hydrogen phosphate, hexamethyltriaminophosphine p(nme ) is oxidized to hexamethyltriaminodibromophosphorane ((me n) pbr ) which is ionized in solution (scheme ) [ ] . subsequently, the ionized (me n) pbr and diethyl hydrogen phosphate undergo ligand exchange in the presence of et n to form the scheme . modified atherton-todd mechanism proposed in the formation of phosphoramidates. in the plausible mechanism for the direct conversion of diethyl hydrogen phosphate, hexamethyltriaminophosphine p(nme ) is oxidized to hexamethyltriaminodibromophosphorane ((me n) pbr ) which is ionized in solution (scheme ) [ ] . subsequently, the ionized (me n) pbr and diethyl hydrogen phosphate undergo ligand exchange in the presence of et n to form the intermediate, diethyl (phosphaneyloxy)phosphonate amino-hydrazine-bromide salt complex. nucleophilic attack from the amine onto the positively charged p results in the diethyl phosphoramidate bromide salt, which reacts with et n to give the desired product with a concomitant elimination of triethylammonium bromide. apart from the stoichiometric et n salt waste generated, the synthetic process is prone to the formation of significant quantities of pyrophosphate or phosphanetetraamine bromide salt side products, resulting in a low yield. nucleophilic attack from the amine onto the positively charged p results in the diethyl phosphoramidate bromide salt, which reacts with et n to give the desired product with a concomitant elimination of triethylammonium bromide. apart from the stoichiometric et n salt waste generated, the synthetic process is prone to the formation of significant quantities of pyrophosphate or phosphanetetraamine bromide salt side products, resulting in a low yield. the mechanistic steps for the synthesis of p-n via a salt elimination route were not proposed by zwierzak et al. [ ] ; however, based on the roles of khco , k co , and [n-bu n]br as outlined in the paper and a recently proposed mechanism for an atherton-todd type reaction, a probable mechanism can be postulated (scheme ) [ ] . in the reacting system, the hco − migrates to the the mechanistic steps for the synthesis of p-n via a salt elimination route were not proposed by zwierzak et al. [ ] ; however, based on the roles of khco , k co , and [n-bu n]br as outlined in the paper and a recently proposed mechanism for an atherton-todd type reaction, a probable mechanism can be postulated (scheme ) [ ] . in the reacting system, the hco − migrates to the organic phase in the presence of [n-bu n]br as the catalyst, where it is protonated and reacts with ccl to form anionic hco clh − and cationic cl c + . the dialkyl h-phosphonate ((ro) p(o)h, r = alkyl) is deprotonated by the cationic cl c + to yield chcl and anionic phosphate, (ro) p(o) − . next, the anionic phosphate reacts with hco clh − to form (ro) p(o)cl (dacp; r = alkyl) and h co . in the following step, a nucleophilic substitution between the amine and dacp results in the formation of the desired product. meanwhile, k co serves as the dehydrating agent in the reacting system. the plausible mechanism is also similar when naoh and the mechanistic steps for the synthesis of p-n via a salt elimination route were not proposed by zwierzak et al. [ ] ; however, based on the roles of khco , k co , and [n-bu n]br as outlined in the paper and a recently proposed mechanism for an atherton-todd type reaction, a probable mechanism can be postulated (scheme ) [ ] . in the reacting system, the hco − migrates to the organic phase in the presence of [n-bu n]br as the catalyst, where it is protonated and reacts with ccl to form anionic hco clh − and cationic cl c + . the dialkyl h-phosphonate ((ro) p(o)h, r = alkyl) is deprotonated by the cationic cl c + to yield chcl and anionic phosphate, (ro) p(o) − . next, the anionic phosphate reacts with hco clh − to form (ro) p(o)cl (dacp; r = alkyl) and h co . in the following step, a nucleophilic substitution between the amine and dacp results in the formation of the desired product. meanwhile, k co serves as the dehydrating agent in the reacting system. the plausible mechanism is also similar when naoh and [bnet n]cl are used in place of khco and [n-bu n]br, respectively. scheme . potential mechanism for the formation of phosphoramidates using a salt elimination route. [ ] . then, the dacp reacts with n-phenylformamide or the -chloro-n-phenylacetamide derivative to form the product. alternatively, the mechanism could proceed via a dialkyl (acetyl)(phenyl)phosphoramidate intermediate, which subsequently undergoes hydrolysis to afford the product. the mechanisms of p-n formation from gupta et al. [ ] and kaboudin et al. [ ] suggest that the dialkyl h-phosphonate ((ro) p(o)h, r = alkyl) reacts with trichloroisocyanuric acid (tcic-a) to form the intermediate (ro) p(o)cl (dacp; r = alkyl) and , , -triazinane- , , -trione (tato) (scheme ). following, a nucleophilic substitution between the dacp and the amine result in the p-n with hcl as the major by-product. the mechanisms of p-n formation from gupta et al. [ ] and kaboudin et al. [ ] suggest that the dialkyl h-phosphonate ((ro) p(o)h, r = alkyl) reacts with trichloroisocyanuric acid (tcic-a) to form the intermediate (ro) p(o)cl (dacp; r = alkyl) and , , -triazinane- , , -trione (tato) (scheme ). following, a nucleophilic substitution between the dacp and the amine result in the p-n with hcl as the major by-product. proposed mechanism for the base-free synthesis of phosphoramidates using a chlorinating agent. the mechanism of the reaction by jang et al. suggests that diphenyl phosphoric acid (dppa) undergoes chlorination in the presence of a base, which is subsequently attacked by the amine to afford the p-n product [ ] . although the authors did not propose any mechanism, they suggested that nucleophilic attack on the p atom was consistent with aliphatic amines resulting in a higher yield of products than the aromatic amines because aliphatic amines are more nucleophilic. the mechanistic step suggested by appel and einig involves the formation of oxo-triphenyl-diphosphoxanium salt intermediate from a deprotonation of phosphoric acid by ccl in the presence of phosphine (scheme ) [ ] . nucleophilic substitution from amine results in the desired product and triphenylphosphate as the by-product. scheme . proposed mechanism for the synthesis of phosphoramidates from phosphoric acid and amine in the presence of phosphine and ccl . in the suggested reaction mechanism by [ ] , iodine reacts with (ro) p (r = alkyl) to form trialkoxyiodophosphonium intermediate, which is further attacked by iodide ion to form dialkyl phosphoriodidate (dappi) (arbuzov reaction, scheme ). then, the dappi is attacked by the amine to give the desired p-n with an elimination of hi. n-phosphoryl amino acid esters and p-n with a free amino group were also synthesized using the iodine-mediated synthesis method. the challenges with this iodine-mediated p-n formation is that many side products are formed, and the proposed mechanism for the base-free synthesis of phosphoramidates using a chlorinating agent. the mechanism of the reaction by jang et al. suggests that diphenyl phosphoric acid (dppa) undergoes chlorination in the presence of a base, which is subsequently attacked by the amine to afford the p-n product [ ] . although the authors did not propose any mechanism, they suggested that nucleophilic attack on the p atom was consistent with aliphatic amines resulting in a higher yield of products than the aromatic amines because aliphatic amines are more nucleophilic. the mechanistic step suggested by appel and einig involves the formation of oxo-triphenyl-diphosphoxanium salt intermediate from a deprotonation of phosphoric acid by ccl in the presence of phosphine (scheme ) [ ] . nucleophilic substitution from amine results in the desired product and triphenylphosphate as the by-product. the mechanisms of p-n formation from gupta et al. [ ] and kaboudin et al. [ ] suggest that the dialkyl h-phosphonate ((ro) p(o)h, r = alkyl) reacts with trichloroisocyanuric acid (tcic-a) to form the intermediate (ro) p(o)cl (dacp; r = alkyl) and , , -triazinane- , , -trione (tato) (scheme ). following, a nucleophilic substitution between the dacp and the amine result in the p-n with hcl as the major by-product. proposed mechanism for the base-free synthesis of phosphoramidates using a chlorinating agent. the mechanism of the reaction by jang et al. suggests that diphenyl phosphoric acid (dppa) undergoes chlorination in the presence of a base, which is subsequently attacked by the amine to afford the p-n product [ ] . although the authors did not propose any mechanism, they suggested that nucleophilic attack on the p atom was consistent with aliphatic amines resulting in a higher yield of products than the aromatic amines because aliphatic amines are more nucleophilic. the mechanistic step suggested by appel and einig involves the formation of oxo-triphenyl-diphosphoxanium salt intermediate from a deprotonation of phosphoric acid by ccl in the presence of phosphine (scheme ) [ ] . nucleophilic substitution from amine results in the desired product and triphenylphosphate as the by-product. scheme . proposed mechanism for the synthesis of phosphoramidates from phosphoric acid and amine in the presence of phosphine and ccl . in the suggested reaction mechanism by [ ] , iodine reacts with (ro) p (r = alkyl) to form trialkoxyiodophosphonium intermediate, which is further attacked by iodide ion to form dialkyl phosphoriodidate (dappi) (arbuzov reaction, scheme ). then, the dappi is attacked by the amine to give the desired p-n with an elimination of hi. n-phosphoryl amino acid esters and p-n with a free amino group were also synthesized using the iodine-mediated synthesis method. the challenges with this iodine-mediated p-n formation is that many side products are formed, and the scheme . proposed mechanism for the synthesis of phosphoramidates from phosphoric acid and amine in the presence of phosphine and ccl . in the suggested reaction mechanism by [ ] , iodine reacts with (ro) p (r = alkyl) to form trialkoxyiodophosphonium intermediate, which is further attacked by iodide ion to form dialkyl phosphoriodidate (dappi) (arbuzov reaction, scheme ). then, the dappi is attacked by the amine to give the desired p-n with an elimination of hi. n-phosphoryl amino acid esters and p-n with a free amino group were also synthesized using the iodine-mediated synthesis method. the challenges with this iodine-mediated p-n formation is that many side products are formed, and the reaction procedure does not generate the desired product when n-iodosuccinimide (nis) is used as the iodine source. in addition, the reaction process is ineffective with aromatic h-phosphonates because the reaction is affected by steric effects on the substituents. in a mechanism of the i -mediated p-n formation, according to singh et al., h-p reacts with i to form an intermediate, which undergoes nucleophilic attack from the amine to generate a hydroxylated-dialkyl phenylphosphoramidate complex with an elimination of i (scheme ) [ ] . the phosphoramidate complex further undergoes dehydrogenation to eliminate h in form of h o and afford the desired products. the suggested mechanism of a similar reaction reported by prabhu et al. instead involves the nucleophilic substitution of phosphoriodate intermediate after the electrophilic iodination of the h-p starting material (scheme ) [ ] . then, the iodide ion is oxidized to active catalyst, hypoiodous acid, in the presence of h o . molecules , , x for peer review of reaction procedure does not generate the desired product when n-iodosuccinimide (nis) is used as the iodine source. in addition, the reaction process is ineffective with aromatic h-phosphonates because the reaction is affected by steric effects on the substituents. proposed mechanism for the iodine-mediated synthesis of phosphoramidates. in a mechanism of the i -mediated p-n formation, according to singh et al., h-p reacts with i to form an intermediate, which undergoes nucleophilic attack from the amine to generate a hydroxylated-dialkyl phenylphosphoramidate complex with an elimination of i (scheme ) [ ] . the phosphoramidate complex further undergoes dehydrogenation to eliminate h in form of h o and afford the desired products. [ ] . then, the iodide ion is oxidized to active catalyst, hypoiodous acid, in the presence of h o . scheme . proposed mechanism for the hypoiodous acid-mediated synthesis of phosphoramidates. in a copper-catalyzed oxidative cross-coupling reaction, hayes et al. proposed a reaction mechanism involving single-electron transfer oxidation of the copper catalyst, which favored the formation of the cu(ii)-amine complex (scheme ) [ ] . the cu(ii)-amine complex is further scheme . proposed mechanism for the iodine-mediated synthesis of phosphoramidates. because the reaction is affected by steric effects on the substituents. scheme . proposed mechanism for the iodine-mediated synthesis of phosphoramidates. in a mechanism of the i -mediated p-n formation, according to singh et al., h-p reacts with i to form an intermediate, which undergoes nucleophilic attack from the amine to generate a hydroxylated-dialkyl phenylphosphoramidate complex with an elimination of i (scheme ) [ ] . the phosphoramidate complex further undergoes dehydrogenation to eliminate h in form of h o and afford the desired products. [ ] . then, the iodide ion is oxidized to active catalyst, hypoiodous acid, in the presence of h o . proposed mechanism for the hypoiodous acid-mediated synthesis of phosphoramidates. in a copper-catalyzed oxidative cross-coupling reaction, hayes et al. proposed a reaction mechanism involving single-electron transfer oxidation of the copper catalyst, which favored the formation of the cu(ii)-amine complex (scheme ) [ ] . the cu(ii)-amine complex is further scheme . proposed mechanism for the iodine-mediated synthesis of phosphoramidates. scheme . proposed mechanism for the iodine-mediated synthesis of phosphoramidates. in a mechanism of the i -mediated p-n formation, according to singh et al., h-p reacts with i to form an intermediate, which undergoes nucleophilic attack from the amine to generate a hydroxylated-dialkyl phenylphosphoramidate complex with an elimination of i (scheme ) [ ] . the phosphoramidate complex further undergoes dehydrogenation to eliminate h in form of h o and afford the desired products. [ ] . then, the iodide ion is oxidized to active catalyst, hypoiodous acid, in the presence of h o . scheme . proposed mechanism for the hypoiodous acid-mediated synthesis of phosphoramidates. in a copper-catalyzed oxidative cross-coupling reaction, hayes et al. proposed a reaction mechanism involving single-electron transfer oxidation of the copper catalyst, which favored the formation of the cu(ii)-amine complex (scheme ) [ ] . the cu(ii)-amine complex is further scheme . proposed mechanism for the hypoiodous acid-mediated synthesis of phosphoramidates. in a copper-catalyzed oxidative cross-coupling reaction, hayes et al. proposed a reaction mechanism involving single-electron transfer oxidation of the copper catalyst, which favored the formation of the cu(ii)-amine complex (scheme ) [ ] . the cu(ii)-amine complex is further oxidized to form the cu(iii)-amine-phosphonate complex, which undergoes reductive elimination of the copper catalyst to yield the desired p-n. although this mechanism seems plausible, it could not easily explain the formation of the side products. molecules , , x for peer review of oxidized to form the cu(iii)-amine-phosphonate complex, which undergoes reductive elimination of the copper catalyst to yield the desired p-n. although this mechanism seems plausible, it could not easily explain the formation of the side products. according the plausible two-step mechanism for the reaction by purohit et al., (ro) p(o)cl (dacp; r = alkyl) is first generated from the electrophilic displacement of hydrogen on the dialkyl h-phosphonate ((or) p(o)h, r = alkyl) upon reacting with cucl (scheme a) [ ] . the phosphoramidate is formed in the second step by coupling dacp and amine. however, this reported mechanism could not properly explain the role of cs co in the coupling of alkyl chlorophosphate and the amine to give the p-n. this is because the coupling reaction was expected to eliminate other products along with cscl such as a highly reactive csh and a carbonate. however, it is to be understood that the use of cs co as the inorganic base was based on the poorly understood "caesium effect" in which the highly soluble caesium base selectively favors the formation of one functional group in preference to another [ , ] . molecules , , x for peer review of phosphoramidate is formed in the second step by coupling dacp and amine. however, this reported mechanism could not properly explain the role of cs co in the coupling of alkyl chlorophosphate and the amine to give the p-n. this is because the coupling reaction was expected to eliminate other products along with cscl such as a highly reactive csh and a carbonate. however, it is to be understood that the use of cs co as the inorganic base was based on the poorly understood "caesium effect" in which the highly soluble caesium base selectively favors the formation of one functional group in preference to another [ , ] . proposed mechanism for the cu-mediated synthesis of phosphoramidates. mizuno et al. proposed a mechanism for the reaction in which k co first deprotonates the amide, affording unpaired electrons on a cu-amide complex [ ] . furthermore, it was suggested that it is possible that k co deprotonates h-p or provides a medium for the h-p tautomer to further complex with the cu-amide complex. the cu-amide-phosphite intermediate undergoes reductive elimination to afford the desired product, while o re-oxidizes the reduced cu species to regenerate the active catalyst. although the oxidation state of cu in the reaction mechanism was not explained, the authors suggested that it could be + , and the fact that the reaction produced only stoichiometric amounts of the desired product (relative to the cu catalyst) under an inert atmosphere was a support to the re-oxidation of the cu species in air. from their experimental observations, zhou et al. strongly suggested that the oxidative dehydrocoupling reaction for p-n synthesis using cu-catalysts proceeds stereospecifically through an inversion of the configuration at p [ ] . this stereospecific inversion chemistry was used as an argument in the proposed mechanism to support a formation of halogen-phosphate intermediate when cu catalyst is reacted with h-phosphonate (h-p). the amine (as a nucleophile) subsequently attacks the intermediate from behind to give the product, while the cu(i) catalyst is then reoxidized by o to cu(ii) in the presence of hydrogen halide formed from the process. water is also formed as a stoichiometric waste (scheme b). the mechanism as reported by dangroo et al. involved transmetalation to form an azidocopper(ii) species in the first step (scheme ) [ ] . similar transmetalation reactions involving the phenylboronic acid-based substrates and the azidocopper(ii) species in the second scheme . proposed mechanism for the cu-mediated synthesis of phosphoramidates. mizuno et al. proposed a mechanism for the reaction in which k co first deprotonates the amide, affording unpaired electrons on a cu-amide complex [ ] . furthermore, it was suggested that it is possible that k co deprotonates h-p or provides a medium for the h-p tautomer to further complex with the cu-amide complex. the cu-amide-phosphite intermediate undergoes reductive elimination to afford the desired product, while o re-oxidizes the reduced cu species to regenerate the active catalyst. although the oxidation state of cu in the reaction mechanism was not explained, the authors suggested that it could be + , and the fact that the reaction produced only stoichiometric amounts of the desired product (relative to the cu catalyst) under an inert atmosphere was a support to the re-oxidation of the cu species in air. from their experimental observations, zhou et al. strongly suggested that the oxidative dehydrocoupling reaction for p-n synthesis using cu-catalysts proceeds stereospecifically through an inversion of the configuration at p [ ] . this stereospecific inversion chemistry was used as an argument in the proposed mechanism to support a formation of halogen-phosphate intermediate when cu catalyst is reacted with h-phosphonate (h-p). the amine (as a nucleophile) subsequently attacks the intermediate from behind to give the product, while the cu(i) catalyst is then reoxidized by o to cu(ii) in the presence of hydrogen halide formed from the process. water is also formed as a stoichiometric waste (scheme b). the mechanism as reported by dangroo et al. involved transmetalation to form an azidocopper(ii) species in the first step (scheme ) [ ] . similar transmetalation reactions involving the phenylboronic acid-based substrates and the azidocopper(ii) species in the second step result in the formation of a phenyl-azidocopper(ii) complex. the complex is oxidized in the presence of cu(ii) to a phenyl-azidocopper(iii) complex, which results in the formation of the organic azide and forms cu(i) after reductive elimination. from this, a reaction between the (ro) p (r = alkyl) and the organic azide gives phosphorimidate (with loss of n ), which undergoes hydrolysis to afford the desired product, while the cu(i) is reoxidized to cu(ii) in the catalytic cycle. although the mechanism satisfies much of the observed results, the proposed second step, transmetalation, would have resulted in borazidic acid (i.e., n -b(oh) ) and cu(ii)-phenyl species or cu(oh) species when the azidocopper(ii) species reacted with phenylboronic acid-based substrates, based on the exchange of ligands as in the first step. molecules , , x for peer review of scheme . proposed mechanism of the cu-catalyzed synthesis of phosphoramidates from phenylboronic acid/ester-based substrates and trialkyl phosphite. in a suggested mechanism for the organophotocatalytic synthesis of p-n, a photon from the visible light source is absorbed by the organic dye to become an excited radical (scheme ) [ ] . by a single-electron transfer process, the excited dye-radical abstracts an electron from the n atom of the amine to form an anionic dye radical and cationic amine radical. in the presence of o , the anionic dye radical is reoxidized to the organic dye, while the anionic superoxide that is formed in the process reacts with diethyl h-phosphonate (h-p) to generate anionic hydrogen peroxide and a diethyl phosphoryl radical. then, the radical reacts with the cationic-amine radical to form a protonated p-n, which is then deprotonated by the anionic hydrogen peroxide. the authors reported that after an initial % isolated product, the organic dye catalyst was recycled a second time using an acid-base extraction, and % isolated product was obtained the second time. scheme . proposed mechanism of the cu-catalyzed synthesis of phosphoramidates from phenylboronic acid/ester-based substrates and trialkyl phosphite. in a suggested mechanism for the organophotocatalytic synthesis of p-n, a photon from the visible light source is absorbed by the organic dye to become an excited radical (scheme ) [ ] . by a single-electron transfer process, the excited dye-radical abstracts an electron from the n atom of the amine to form an anionic dye radical and cationic amine radical. in the presence of o , the anionic dye radical is reoxidized to the organic dye, while the anionic superoxide that is formed in the process reacts with diethyl h-phosphonate (h-p) to generate anionic hydrogen peroxide and a diethyl phosphoryl radical. then, the radical reacts with the cationic-amine radical to form a protonated p-n, which is then deprotonated by the anionic hydrogen peroxide. the authors reported that after an initial % isolated product, the organic dye catalyst was recycled a second time using an acid-base extraction, and % isolated product was obtained the second time. the amine to form an anionic dye radical and cationic amine radical. in the presence of o , the anionic dye radical is reoxidized to the organic dye, while the anionic superoxide that is formed in the process reacts with diethyl h-phosphonate (h-p) to generate anionic hydrogen peroxide and a diethyl phosphoryl radical. then, the radical reacts with the cationic-amine radical to form a protonated p-n, which is then deprotonated by the anionic hydrogen peroxide. the authors reported that after an initial % isolated product, the organic dye catalyst was recycled a second time using an acid-base extraction, and % isolated product was obtained the second time. proposed mechanism for the visible light and organic dye-catalyzed synthesis of phosphoramidates. proposed mechanism for the visible light and organic dye-catalyzed synthesis of phosphoramidates. in the overall mechanism suggested for the alkali-metal catalyzed oxidative cross-coupling reaction, a phosphoryl iodide intermediate and lioh are generated in the first step from a reaction of lii and the h-phosphonate (h-p) in the presence of aq. tbhp (scheme ) [ ] . the second step involves the removal of a proton from the amine by the lioh to form lithium amide and h o, and the desired product is formed in the final step from the reaction of phosphoryl iodide and lithium amide. in the overall mechanism suggested for the alkali-metal catalyzed oxidative cross-coupling reaction, a phosphoryl iodide intermediate and lioh are generated in the first step from a reaction of lii and the h-phosphonate (h-p) in the presence of aq. tbhp (scheme ) [ ] . the second step involves the removal of a proton from the amine by the lioh to form lithium amide and h o, and the desired product is formed in the final step from the reaction of phosphoryl iodide and lithium amide. scheme . proposed mechanism for the synthesis of phosphoramidates using alkali-metal catalyst. the mechanism of the reaction of nitrene insertion according to chang, kim, and coworkers follows activation of the c-h bond cleavage on the amide or ketone substrate by the ir (iii) -based catalyst to generate an iridacyclic intermediate, which then reacts with the phosphoryl azide to produce a coordinated phosphoryl azide-iridacyclic adduct (scheme ) [ ] . by intramolecular migratory insertion of c-n into the ir-c bond, with removal of n , or by nucleophilic attack from the azide group on the ir-c bond to form the c-n bond with the elimination of n , a complex of ir-n-c bond is formed. proto-demetallation of the ir (iii) -based catalyst affords the desired product while scheme . proposed mechanism for the synthesis of phosphoramidates using alkali-metal catalyst. the mechanism of the reaction of nitrene insertion according to chang, kim, and coworkers follows activation of the c-h bond cleavage on the amide or ketone substrate by the ir (iii) -based catalyst to generate an iridacyclic intermediate, which then reacts with the phosphoryl azide to produce a coordinated phosphoryl azide-iridacyclic adduct (scheme ) [ ] . by intramolecular migratory insertion of c-n into the ir-c bond, with removal of n , or by nucleophilic attack from the azide group on the ir-c bond to form the c-n bond with the elimination of n , a complex of ir-n-c bond is formed. proto-demetallation of the ir (iii) -based catalyst affords the desired product while regenerating the catalyst. according to zhu et al., the mechanistic steps involve activation of the ir (iii) -based catalyst in the presence of agoac and agsbf , which abstracts an h atom from the -phenylpyridine derivative to cyclo-( -phenylpyridine)iridium(iii) complex [ ] . the complex further coordinates with phosphoryl azide to form a cyclo-( -phenylpyridine)iridium(iii)-phosphoryl azide complex, which undergoes migratory insertion to give a complex containing a c-n-ir (iii) bond, releasing n . the ir (iii) -based catalyst is released through proto-demetallation to generate the desired product. for the nitrene insertion using ru iv -porphyrin-based catalyst, che and coworkers proposed that a reactive intermediate ru iv -porphyrin-phosphorylimide species is first formed from the interaction of phosphoryl azide and the ru iv -porphyrin catalyst [ ] . then, the intermediate reacts with the aldehyde c-h bond through nitrene insertion to form a c-n bonded ru iv -porphyrin-phosphorylimide-aldehyde complex with the removal of n . the desired n-acylphosphoramidates (acp-n) product is formed after proto-demetallation of the ru iv -porphyrin catalyst, and the catalyst is regenerated in the process. molecules , for the nitrene insertion using ru iv -porphyrin-based catalyst, che and coworkers proposed that a reactive intermediate ru iv -porphyrin-phosphorylimide species is first formed from the interaction of phosphoryl azide and the ru iv -porphyrin catalyst [ ] . then, the intermediate reacts with the aldehyde c-h bond through nitrene insertion to form a c-n bonded ru iv -porphyrin-phosphorylimide-aldehyde complex with the removal of n . the desired scheme . proposed mechanism for the ir-catalyzed synthesis of phosphoramidates. in the reported mechanisms of the reaction for p-n synthesis via azide generation, organic azide is formed in the first step from nucleophilic substitution of the organic halide with the elimination of n gas. in the second step, the organic azide reacts with (ro) p (r = alkyl) to form the p-n compound after rearrangement or hydrolysis (scheme ) [ , , ] . interaction of phosphoryl azide and the ru iv -porphyrin catalyst [ ] . then, the intermediate reacts with the aldehyde c-h bond through nitrene insertion to form a c-n bonded ru iv -porphyrin-phosphorylimide-aldehyde complex with the removal of n . the desired n-acylphosphoramidates (acp-n) product is formed after proto-demetallation of the ru iv -porphyrin catalyst, and the catalyst is regenerated in the process. in the reported mechanisms of the reaction for p-n synthesis via azide generation, organic azide is formed in the first step from nucleophilic substitution of the organic halide with the elimination of n gas. in the second step, the organic azide reacts with (ro) p (r = alkyl) to form the p-n compound after rearrangement or hydrolysis (scheme ) [ , , ] . scheme . proposed mechanism to phosphoramidates via azide generation. according to the mechanism suggested by chen et al., the amine reacts with phosphoryl azide through a nucleophilic substitution reaction to generate an azido (hydroxy)-phosphanamine complex intermediate in the first step [ ] . in the second step, the azide anion departs as the leaving group to give a stable p-n product (scheme ). although the mechanism looks highly plausible, it is noteworthy that n is highly nucleophilic because in its canonical structure, one of the nitrogen atoms carries a double negative charge (n≡n + − n − ) [ ] , which makes it stable and a poor leaving group as n in the presence of amine [ ] . in addition, the mechanism does account for the potentially explosive hn in a closed flask at °c. however, it is possible that in the reaction process, the azide is first reduced or decomposed to a nitrene phosphonate radical ((ro) p(o)n; r = scheme . proposed mechanism to phosphoramidates via azide generation. according to the mechanism suggested by chen et al., the amine reacts with phosphoryl azide through a nucleophilic substitution reaction to generate an azido (hydroxy)-phosphanamine complex intermediate in the first step [ ] . in the second step, the azide anion departs as the leaving group to give a stable p-n product (scheme ). although the mechanism looks highly plausible, it is noteworthy that n is highly nucleophilic because in its canonical structure, one of the nitrogen atoms carries a double negative charge (n≡n + − n − ) [ ] , which makes it stable and a poor leaving group as n in the presence of amine [ ] . in addition, the mechanism does account for the potentially explosive hn in a closed flask at • c. however, it is possible that in the reaction process, the azide is first reduced or decomposed to a nitrene phosphonate radical ((ro) p(o)n; r = alkyl, ph), while n is liberated. in the second step, the electrophilic nitrene phosphonate radical reacts with or is substituted by the nucleophile amine to give the product. molecules , , x for peer review of alkyl, ph), while n is liberated. in the second step, the electrophilic nitrene phosphonate radical reacts with or is substituted by the nucleophile amine to give the product. proposed mechanism for an organic azide route to phosphoramidates. the proposed mechanism of the reaction for the nitro-group reduction route involves the reduction of nitrobenzene in the presence of (ro) p (r = alkyl) to give nitrosobenzene with a phosphate side product (scheme ) [ ] . in the second step, the nitrosobenzene is reduced further in the presence of another equivalent of (ro) p, generating phenylnitride with another phosphate side product. a further reaction of (ro) p (r = alkyl) with the basic phenylnitride generates a trialkyl phenylphosphorimidate intermediate, which undergoes hydrolysis to give the desired n-arylphosphoramidate product. experiments to verify the proposed mechanism using aniline in the presence of excess triethyl phosphite or triethyl phosphate did not yield the desired product under the optimized conditions, suggesting that the reaction proceeds via reduction of the nitro group to n − and that the product is not formed from phosphate. scheme . proposed mechanism for reduction of the nitro-group route to phosphoramidates. the plausible two-step mechanistic process suggested by hackenberger's group for the scheme . proposed mechanism for an organic azide route to phosphoramidates. the proposed mechanism of the reaction for the nitro-group reduction route involves the reduction of nitrobenzene in the presence of (ro) p (r = alkyl) to give nitrosobenzene with a phosphate side product (scheme ) [ ] . in the second step, the nitrosobenzene is reduced further in the presence of another equivalent of (ro) p, generating phenylnitride with another phosphate side product. a further reaction of (ro) p (r = alkyl) with the basic phenylnitride generates a trialkyl phenylphosphorimidate intermediate, which undergoes hydrolysis to give the desired n-arylphosphoramidate product. experiments to verify the proposed mechanism using aniline in the presence of excess triethyl phosphite or triethyl phosphate did not yield the desired product under the optimized conditions, suggesting that the reaction proceeds via reduction of the nitro group to n − and that the product is not formed from phosphate. in the presence of another equivalent of (ro) p, generating phenylnitride with another phosphate side product. a further reaction of (ro) p (r = alkyl) with the basic phenylnitride generates a trialkyl phenylphosphorimidate intermediate, which undergoes hydrolysis to give the desired n-arylphosphoramidate product. experiments to verify the proposed mechanism using aniline in the presence of excess triethyl phosphite or triethyl phosphate did not yield the desired product under the optimized conditions, suggesting that the reaction proceeds via reduction of the nitro group to n − and that the product is not formed from phosphate. scheme . proposed mechanism for reduction of the nitro-group route to phosphoramidates. the plausible two-step mechanistic process suggested by hackenberger's group for the synthesis involves the formation of phosphorimidate from the reduction of azide in the presence of (ro) p (r = alkyl), eliminating n , in the first step (scheme ) [ ] . in the second step, bf activates the nitrogen site of the phosphorimidate by forming a coordinated complex. electrophilic attack by a neighbouring alkyl group of phosphorimidate on the negatively charged n atom of the phosphorimidate-bf complex results in an ((alkyl)amino)trialkoxyphosphonium complex, cleaving off bf . nucleophilic attack on the trialkoxyphosphonium complex from a neighboring phosphorimidate completes the catalytic cycle, affording a p=o bond of the desired p-n product. scheme . proposed mechanism for reduction of the nitro-group route to phosphoramidates. the plausible two-step mechanistic process suggested by hackenberger's group for the synthesis involves the formation of phosphorimidate from the reduction of azide in the presence of (ro) p (r = alkyl), eliminating n , in the first step (scheme ) [ ] . in the second step, bf activates the nitrogen site of the phosphorimidate by forming a coordinated complex. electrophilic attack by a neighbouring alkyl group of phosphorimidate on the negatively charged n atom of the phosphorimidate-bf complex results in an ((alkyl)amino)trialkoxyphosphonium complex, cleaving off bf . nucleophilic attack on the trialkoxyphosphonium complex from a neighboring phosphorimidate completes the catalytic cycle, affording a p=o bond of the desired p-n product. molecules , , x for peer review of scheme . proposed mechanism for the catalyst-free staudinger reduction and lewis-acid catalyzed rearrangement route to phosphoramidates. for the hydrophosphinylation route to p-ns, zimin et al. proposed that in the first step, sodium dialkyl phosphite ((ro) pona, r = et, ph) reacts with alkyl nitrile to form an imine (c=n), which reacts further with a second equivalent of (ro) pona to form the , -addition product, sodium ( , -bis(dialkoxyphosphoryl)alkyl)amide (scheme ) [ ] . in the second step, sodium ( , -bis(dialkyoxyphosphoryl)alkyl)amide undergoes hydrolysis and isomerization to give the , -addition phosphoramidate product containing a p-c-n-p bond, eliminating a sodium cation in the process. scheme . proposed mechanism for the catalyst-free staudinger reduction and lewis-acid catalyzed rearrangement route to phosphoramidates. for the hydrophosphinylation route to p-ns, zimin et al. proposed that in the first step, sodium dialkyl phosphite ((ro) pona, r = et, ph) reacts with alkyl nitrile to form an imine (c=n), which reacts further with a second equivalent of (ro) pona to form the , -addition product, sodium ( , -bis(dialkoxyphosphoryl)alkyl)amide (scheme ) [ ] . in the second step, sodium ( , -bis(dialkyoxyphosphoryl)alkyl)amide undergoes hydrolysis and isomerization to give the , -addition phosphoramidate product containing a p-c-n-p bond, eliminating a sodium cation in the process. for the hydrophosphinylation route to p-ns, zimin et al. proposed that in the first step, sodium dialkyl phosphite ((ro) pona, r = et, ph) reacts with alkyl nitrile to form an imine (c=n), which reacts further with a second equivalent of (ro) pona to form the , -addition product, sodium ( , -bis(dialkoxyphosphoryl)alkyl)amide (scheme ) [ ] . in the second step, sodium ( , -bis(dialkyoxyphosphoryl)alkyl)amide undergoes hydrolysis and isomerization to give the , -addition phosphoramidate product containing a p-c-n-p bond, eliminating a sodium cation in the process. scheme . proposed mechanism for hydrophosphinylation route to phosphoramidates. chabour et al. proposed a mechanism of the reaction in which (eto) p(o)nh reacts with (or is activated by) acetic anhydride to form diethyl acetylphosphoramidate and acetic acid (scheme ) [ ] . meanwhile, the aldehyde protonated in the presence of an acid catalyst, tsoh, reacts with the diethyl acetylphosphoramidate complex to give the cationic diethyl (alkene-acetamido)phosphonate complex intermediate. the unstable cationic complex undergoes rearrangement with a hydride shift and deprotonation in presence of tso − to give a more stable diethyl acetyl(alk- , -diene)phosphoramidate. the subsequent direct addition of the maleimide derivative results in a p-n adduct intermediate, which is deacetylated in the presence of acetic acid to afford the desired product. scheme . proposed mechanism for hydrophosphinylation route to phosphoramidates. chabour et al. proposed a mechanism of the reaction in which (eto) p(o)nh reacts with (or is activated by) acetic anhydride to form diethyl acetylphosphoramidate and acetic acid (scheme ) [ ] . meanwhile, the aldehyde protonated in the presence of an acid catalyst, tsoh, reacts with the diethyl acetylphosphoramidate complex to give the cationic diethyl (alkene-acetamido)phosphonate complex intermediate. the unstable cationic complex undergoes rearrangement with a hydride shift and deprotonation in presence of tso − to give a more stable diethyl acetyl(alk- , -diene)phosphoramidate. the subsequent direct addition of the maleimide derivative results in a p-n adduct intermediate, which is deacetylated in the presence of acetic acid to afford the desired product. in this review, the applications, syntheses, and corresponding mechanisms of phosphoramidates have been discussed in depth. as demonstrated from the many categories for the uses of synthetic p-ns, it is clear that the p-n bond motif has structural importance in diverse fields. early synthetic routes to p-ns are limited due to harsh reaction conditions, air and scheme . proposed mechanism for the synthesis of phosphoramidates via the phosphoamidatealdehyde-dienophile (pad) route. in this review, the applications, syntheses, and corresponding mechanisms of phosphoramidates have been discussed in depth. as demonstrated from the many categories for the uses of synthetic p-ns, it is clear that the p-n bond motif has structural importance in diverse fields. early synthetic routes to p-ns are limited due to harsh reaction conditions, air and moisture-sensitive reagents, multi-step synthesis, and undesirable side reactions. the separation and purification of p-ns is also a challenge. in particular, most of the synthetic strategies involve extraction and/or column chromatography, which not only requires large amounts of solvent, but also takes a significant amount of time. therefore, unsurprisingly, extensive studies have been undertaken (with more to come in the future) for more sustainable, efficient, atom economic, selective, and milder synthetic procedures toward p-ns. in addition, the discussed mechanisms require a deeper understanding of the associated phosphorus chemistry in order to have better synthetic control and to optimize the yield and selectivity toward p-n products. to overcome these challenges, it is predicted that better catalysts, improved reaction conditions, and new solvent solutions for separating the product from the by-products and potentially improving selectivity are in the forecast. for example, there is a sharp rise into synthetic research under solvent-free conditions or using ionic liquids (ils) and deep eutectic solvents (des). these can be environmentally benign, highly versatile, selective, and recoverable solvents for use in catalysis. diastereoselective multicomponent phosphoramidate-aldehydedienophile (pad) process for the synthesis of polysubstituted cyclohex- -enyl-amine derivatives total synthesis of agrocin and phosmidosine as naturally occurring nucleotidic antibiotics having p-n bond linkages natural products containing 'rare' organophosphorus functional groups chemical structure and translation inhibition studies of the antibiotic microcin c properties of a toxic phospholipid in the northern blenny roe focus on phosphoarginine and phospholysine encyclopedia of metalloproteins chapter -thermolysin and related bacillus metallopeptidases synthesis of nucleotide antibiotics having n-acyl phosphoramidate linkages structure of the antifungal nucleotide antibiotic phosmidosine the diverse pharmacology and medicinal chemistry of phosphoramidates-a review remdesivir for severe acute respiratory syndrome coronavirus causing covid- : an evaluation of the evidence n-phosphorylation labeling for analysis of twenty natural amino acids and small peptides by using matrix-assisted laser desorption/ionization time-of-flight mass spectrometry structural effect of phosphoramidate derivatives on the thermal and flame retardant behaviors of treated cotton cellulose electrochemical determination of the organophosphate compound fenamiphos and its main metabolite, fenamiphos sulfoxide new o-substituted diphenylphosphinic amide ligands: synthesis, characterization and complexation with zn + , cu + and y + significant enhanced uranyl ions extraction efficiency with phosphoramidate-functionalized ionic liquids via synergistic effect of coordination and hydrogen bond the tribochemical study of novel phosphorous-nitrogen (p-n) type phosphoramidate additives in water the organophosphate pesticides synthesis and insecticidal activity of new phosphoramidates organophosphorus pesticides as food chain contaminants and type diabetes: a review current issues in organophosphate toxicology saligenin cyclic phosphoramidates and phosphoramidothionates as pesticides synthesis and insecticidal activity of new -benzylfuran- -yl n,n,n ,n -tetraethyldiamidophosphate derivatives negatively correlated cross-resistance and synergism between phosphoramidates and phosphorothiolates in their fungicidal actions on rice blast fungi a minireview on what we have learned about urease inhibitors of agricultural interest since mid- s design, synthesis, and biological evaluation of phosphoramide derivatives as urease inhibitors recent advances in design of new urease inhibitors: a review a review on the development of urease inhibitors as antimicrobial agents against pathogenic bacteria the tribological study of novel phosphorous-nitrogen type phosphoramidate additives in rapeseed oil halogenated flame retardants: do the fire safety benefits justify the risks? thermal degradation of cellulose acetate in presence of bis-phosphoramidates multiparameter toxicity assessment of novel dopo-derived organophosphorus flame retardants phosphoramidate-containing flame-retardant flexible polyurethane foams recent advances for flame retardancy of textiles based on phosphorus chemistry microwave assisted preparation of self-extinguishing cotton fabrics by small molecules containing phosphorous and nitrogen molecular firefighting-how modern phosphorus chemistry can help solve the challenge of flame retardancy el-shafei, a. preparation, polymerization, and performance evaluation of halogen-free radiation curable flame retardant monomers for cotton substrates systematically controlled decomposition mechanism in phosphorus flame retardants by precise molecular architecture: p-o vs p-n characterization of chars obtained from cellulose treated with phosphoramidate flame retardants novel intumescent flame retardants: synthesis and application in polycarbonate a method for the analysis of low-mass molecules by maldi-tof mass spectrometry suppression of matrix ions by n-phosphorylation labeling using matrix-assisted laser desorption-ionization time-of-flight mass spectrometry selective liquid-liquid extraction of uranium by phosphoramidate-group bearing ionic liquid selective micellar extraction of ultratrace levels of uranium in aqueous samples by task specific ionic liquid followed by its detection employing total reflection x-ray fluorescence spectrometry c(sp )-h bond activation induced by monohydroborane coordination at an iridium(iii)-phosphoramidate complex toward anti-markovnikov -alkyne o-phosphoramidation: exploiting metal-ligand cooperativity in a , -n,o-chelated cp*ir(iii) complex , -n,o-complexes of late transition metals. ligands with flexible bonding modes and reaction profiles phosphoramidate tantalum complexes for room-temperature c-h functionalization: hydroaminoalkylation catalysis copper-catalyzed synthesis of medium-and large-sized nitrogen heterocycles via n-arylation of phosphoramidates and carbamates palladium-catalyzed c-h arylation using phosphoramidate as a directing group at room temperature -(benzo[d]thiazol- -yl) phenyl- -nitrophenyl alkyl/aryl substituted phosphoramidates: synthesis, characterization and antimicrobial activity evaluation synthesis and biological evaluation of novel phosphoramidate derivatives of coumarin as chitin synthase inhibitors and antifungal agents convenient one-pot synthesis and biological evaluation of phosphoramidates and phosphonates containing heterocycles synthesis, spectral characterization, and proand antioxidant activity of phosphorylated derivatives of cis-tramadol. phosphorus sulfur silicon relat anti-cancer protides: tuning the activity of bvdu phosphoramidates related to thymectacin synthesis and evaluation of phosphoramidate and phosphorothioamidate analogues of amiprophos methyl as potential antimalarial agents phosphoramidates and phosphonamidates (protides) with antiviral activity anchimerically activatable antiviral protides new and efficient approach to aryl phosphoramidate derivatives of azt/d t as anti-hiv prodrugs aryloxy phosphoramidate triesters: a technology for delivering monophosphorylated nucleosides and sugars into cells the protide prodrug technology: from the concept to the clinic current knowledge about the antivirals remdesivir (gs- ) and gs- as therapeutic options for coronaviruses. one health polyphosphoramidate gene carriers: effect of charge group on gene transfer efficiency ternary complexes comprising polyphosphoramidate gene carriers with different types of charge groups improve transfection efficiency discovery and synthesis of a phosphoramidate prodrug of a pyrrolo gs- ) for the treatment of ebola and emerging viruses structure-reactivity correlation for the hydrolysis of phosphoramidate monoanions phosphoramidate solvolysis tunable ph-sensitive linker for controlled release second-generation tunable ph-sensitive phosphoramidate-based linkers for controlled release phosphoramidate derivates as controlled-release prodrugs of l-dopa phosphoramidate derivatives of acyclovir: synthesis and antiviral activity in hiv- and hsv- models in vitro the nature of the "inorganic phosphate" in voluntary muscle phosphorus compounds of muscle and liver the aquo ammono phosphoric acids. i. preparation of phenyl esters of amidoand diamidophosphoric acids the aquo ammono phosphoric acids. ii. preparation of n-substituted derivatives of the phenyl esters of amido-and diamidophosphoric acids . studies on phosphorylation. part ii. the reaction of dialkyl phosphites with polyhalogen compounds in presence of bases. a new method for the phosphorylation of amines . studies on phosphorylation. part iii. further observations on the reaction of phosphites with polyhalogen compounds in presence of bases and its application to the phosphorylation of alcohols . studies on phosphorylation. part iv. further studies on the use of dibenzyl chlorophosphonate and the examination of certain alternative phosphorylation methods the reaction of phosphorus-containing enzyme inhibitors with amines and amino acid derivatives the formation of p-n and p-n-p bonds by elimination of phenol in a basic condensation deoxygenation of nitro groups by trivalent phosphorus. indoles from o-nitrostyrenes the reactivity of organophosphorus compounds. part xx . p=o nucleophilicity in phosphonates and phosphoramidates reduction of nitro-and nitroso-compounds by tervalent phosphorus reagents. part v. reduction of alkyl-and methoxy-nitrobenzenes, and nitrobenzene by trialkyl phosphites phase-transfer-catalysed phosphorylation of amines in an aqueous system selective n-alkylation of diethyl phosphoramidate: tetrabutylaminium bromide as catalyst for nucleophilic substitution in a homogeneous medium a convenient method for the protection of amino groups as diethyl phosphoramidates direct conversion of diethyl hydrogen phosphate into diethyl phosphoramides zur umsetzung des zweikomponentensystems trialkylphosphit/tetrachlorkohlenstoff mit wasserstoffhaltigen nucleophilen . die reaktion mit ammoniak und aminen directed arbuzov-type reactions of -cyano- , -dimethylethyl deoxynucleoside phosphites a facile one-pot transformation of alkyl bromides into diethyl n-alkylphosphoroamidates preparation of sterically hindered phosphoramidates a phosphoryl radical-initiated atherton-todd-type reaction under open air a new approach to the atherton-todd reaction efficient and convenient one-pot synthesis of phosphoramidates and phosphates trichloroisocyanuric acid as an efficient reagent for the synthesis of phosphoroamidates via atherton-todd reaction under base-free conditions efficient amidation and esterification of phosphoric acid using cl ccn/ph p neues verfahren zur darstellung von phosphorsäureesteramiden, phosphinsäureamiden, phosphinsäureestern und gemischt substituierten phosphorsäuretriestern reinvestigation of the iodine-mediated phosphoramidation reaction of amines and p(or) and its synthetic applications iodine catalyzed solvent-free cross-dehydrogenative coupling of arylamines and h-phosphonates for the synthesis of n-arylphosphoramidates under atmospheric conditions cross-hetero-dehydrogenative coupling reaction of phosphites: a catalytic metal-free phosphorylation of amines and alcohols phosphoramidate synthesis via copper-catalysed aerobic oxidative coupling of amines and h-phosphonates copper-catalyzed aerobic oxidative cross-coupling of arylamines and dialkylphosphites leading to n-arylphosphoramidates fe o @mgo nanoparticles as an efficient recyclable catalyst for the synthesis of phosphoroamidates via the atherton-todd reaction a single-step one pot synthesis of o,o -dialkyl n,n-dialkylphosphoramidates from dialkylphosphites copper-catalyzed oxidative cross-coupling of h-phosphonates and amides to n-acylphosphoramidates stereospecific aerobic oxidative dehydrocoupling of p(o)-h bonds with amines catalyzed by copper copper catalyzed tandem chan-lam type c-n and staudinger-phosphite n-p coupling for the synthesis of n-arylphosphoramidates organophotocatalytic synthesis of phosphoramidates lii/tbhp mediated oxidative cross-coupling of p(o)-h compounds with phenols and various nucleophiles: direct access to the synthesis of organophosphates synthesis of phosphoramidates: a facile approach based on the c-n bond formation via ir-catalyzed direct c-h amidation ruthenium(iv) porphyrin catalyzed phosphoramidation of aldehydes with phosphoryl azides as a nitrene source iridium-catalyzed phosphoramidation of arene c-h bonds with phosphoryl azide catalyst free, one pot synthesis of phosphoramidates under environment friendly conditions an efficient synthesis of phosphoramidates from halides in aqueous ethanol transition-metal-free amination phosphoryl azide for the synthesis of phosphoramidates practical and reliable synthesis of dialkyl n-arylphosphoramidates with nitroarenes as substrates synthesis of n,n-disubstituted phosphoramidatesvia a lewis acid-catalyzed phosphorimidate rearrangement reaction of sodium salts of dialkylphosphorous acids with carboxylic acid nitriles study on the atherton-todd reaction mechanism cesium effect: high chemoselectivity in direct n-alkylation of amines improved cs co promoted o-alkylation of acids abnormally high nucleophilicity of micellar-bound azide ion one-pot procedure for diazo transfer and azide−alkyne cycloaddition: triazole linkages from amines we gratefully acknowledge ebonyi state university abakaliki and tertiary education trust fund (tetfund) nigeria for e.j.i's phd scholarship and the macdiarmid institute for advanced materials and nanotechnology, wellington, new zealand for financial support. the authors declare no conflict of interest. dialkyl chlorophosphate (dacp), dialkyl phosphoriodidate (dappi), h-phosphonate (h-p), phosphoramidate (p-n), trichloroisocyanuric acid (tcic-a), , , -triazinane- , , -trione (tato), n-acylphosphoramidates (acp-n), diphenylphosphine oxide (dppo), phosphonic acid (pp-a), n-bromosuccinimide (nbs), diphenyl phosphoric acid (dppa), triphenylphosphine (tpp), diisopropylchlorophosphate (dprcp), tetraethylpyrophosphate (tepyp), triphenylphosphate (tppo), , , , , , , , -octahydropyrimido[ , -a]azepine (dbu), matrix-assisted laser desorption-ionization time-of-flight mass spectrometry (maldi-tof ms), task specific ionic liquid (tsil), phosphorodiamidate (n-p-n), phenyl dichlorophosphate (pdcp), diphenylchlorophosphate (dpcp), tert-butyl hydroperoxide (tbhp). there were no suggested mechanisms for the reaction in the report by wang et al. [ ] . however, the results of the reagent screening for the reaction had suggested that other transition metal catalysts (e.g., cobr ) and other cu catalysts (e.g., cu(oac) ·h o) were inactive or inefficient for the catalysis. it was further suggested that although a cucl catalyst enabled reaction, it was not as effective as cubr and conducting the reaction under anaerobic conditions (e.g., under n ) did not lead to the desired product.in the fe o @mgo nanoparticle-mediated synthesis of p-ns as per the procedure reported by habibi et al. [ ] , the mechanism involves the formation of the (ro) p(o)cl (dacp; r = alkyl) intermediate from the reaction of dialkyl h-phosphonate ((or) p(o)h, r = alkyl) with ccl (scheme ). following, the amine in the presence of fe o @mgo nanoparticles couples with the dacp by nucleophilic substitution to give the p-n product, eliminating hcl. scheme . proposed mechanism for the fe o @mgo nanoparticle-mediated synthesis of phosphoramidates. proposed mechanism for the copper catalyzed oxidative cross-coupling of disubstituted h-phosphonates and amines to produce phosphoramidates.there were no suggested mechanisms for the reaction in the report by wang et al. [ ] . however, the results of the reagent screening for the reaction had suggested that other transition metal catalysts (e.g., cobr ) and other cu catalysts (e.g., cu(oac) ·h o) were inactive or inefficient for the catalysis. it was further suggested that although a cucl catalyst enabled reaction, it was not as effective as cubr and conducting the reaction under anaerobic conditions (e.g., under n ) did not lead to the desired product.in the fe o @mgo nanoparticle-mediated synthesis of p-ns as per the procedure reported by habibi et al. [ ] , the mechanism involves the formation of the (ro) oxidized to form the cu(iii)-amine-phosphonate complex, which undergoes reductive elimination of the copper catalyst to yield the desired p-n. although this mechanism seems plausible, it could not easily explain the formation of the side products. proposed mechanism for the copper catalyzed oxidative cross-coupling of disubstituted h-phosphonates and amines to produce phosphoramidates.there were no suggested mechanisms for the reaction in the report by wang et al. [ ] . however, the results of the reagent screening for the reaction had suggested that other transition metal catalysts (e.g., cobr ) and other cu catalysts (e.g., cu(oac) ·h o) were inactive or inefficient for the catalysis. it was further suggested that although a cucl catalyst enabled reaction, it was not as effective as cubr and conducting the reaction under anaerobic conditions (e.g., under n ) did not lead to the desired product.in the fe o @mgo nanoparticle-mediated synthesis of p-ns as per the procedure reported by habibi et al. [ ] , the mechanism involves the formation of the key: cord- -dh abx t authors: akkouh, ouafae; ng, tzi bun; singh, senjam sunil; yin, cuiming; dan, xiuli; chan, yau sang; pan, wenliang; cheung, randy chi fai title: lectins with anti-hiv activity: a review date: - - journal: molecules doi: . /molecules sha: doc_id: cord_uid: dh abx t lectins including flowering plant lectins, algal lectins, cyanobacterial lectins, actinomycete lectin, worm lectins, and the nonpeptidic lectin mimics pradimicins and benanomicins, exhibit anti-hiv activity. the anti-hiv plant lectins include artocarpus heterophyllus (jacalin) lectin, concanavalin a, galanthus nivalis (snowdrop) agglutinin-related lectins, musa acuminata (banana) lectin, myrianthus holstii lectin, narcissus pseudonarcissus lectin, and urtica diocia agglutinin. the anti-hiv algal lectins comprise boodlea coacta lectin, griffithsin, oscillatoria agardhii agglutinin. the anti-hiv cyanobacterial lectins are cyanovirin-n, scytovirin, microcystis viridis lectin, and microvirin. actinohivin is an anti-hiv actinomycete lectin. the anti-hiv worm lectins include chaetopterus variopedatus polychaete marine worm lectin, serpula vermicularis sea worm lectin, and c-type lectin mermaid from nematode (laxus oneistus). the anti-hiv nonpeptidic lectin mimics comprise pradimicins and benanomicins. their anti-hiv mechanisms are discussed. in the last few decades, africa remains the continent which has been afflicted to the most serious extent by the hiv/aids pandemic, although the number of infections elsewhere, for instance in asia, has also been on the rise [ ] . many children die from relatively preventable causes, especially in places where there is a high incidence of hiv infections/aids [ ] . the search for plant extracts with anti-hiv activity is continuing [ ] . polysaccharides [ ] and other compounds [ ] have been proposed or used for treatment. lectins have been extensively studied because they possess a variety of potentially exploitable activities. the purpose of this article is to review lectins with anti-hiv activity. lectins are carbohydrate-binding proteins of non-immunoglobulin nature. they are capable of recognition of and reversible binding to complicated glycoconjugates moieties without altering the covalent structure of any of the recognized glycosyl ligands [ ] . in nature, lectins are distributed across a wide variety of organisms like algae, fungi, sea corals, higher plants, prokaryotes, invertebrates and vertebrates. they are involved in many biological processes like recognition and binding of carbohydrates, host-pathogen interactions, cell targeting, cell-cell communication and induction of apoptosis, cancer metastasis and differentiation [ ] . the antiviral, antifungal and antibacterial activities of lectins have been reported [ , ] . the intent of this article is to review lectins with anti-hiv activity. the human immunodeficiency virus (hiv) type is an enveloped virus that causes the acquired immunodeficiency syndrome (aids). in humans the condition is manifested by progressive failure of the immune system [ , ] . it infects viral cells such as t-helper cells, macrophages and dendritic cells [ ] . this infection leads to low levels of cd + through a number of mechanisms. also, apoptosis of uninfected cells, direct viral killing of infected cells and killing of infected cd + cells by cytotoxic lymphocytes occur. the envelope protein complex determines viral tropism and facilitates the membrane fusion process that allows invasion of the viral genome [ ] . gp is a precursor env gene code that is later processed by a host cell protease to form the cleavage products gp and gp . the envelope protein complex and the gp are extensively glycosylated and proteolytically cleaved. when new virus particles come to the host cell, gp and gp will lie on opposite sides of the virus membrane. gp will sit on the outside of the virus particle, forming the virus's spikes and gp will sit on the inside of the membrane. each gp will be anchored to a gp through the membrane [ , ] . gp binds to cd receptor on any target cell that has such a receptor and will present to t-lymphocytes and macrophages. this cd is associated with spontaneous mutation in the env gene. the presence of cxcr is enough for this mutant strain to infect human cells. these strains have seven mutations in the sequence and coding for gp . it is also proposed that the mutations induce conformational changes in gp that allow the virus to directly interact with cxcr [ ] . prior to binding the host cell, gp remains effectively hidden from antibodies. gp is buried in the protein and shielded by sugars, which makes neutralization by antibodies very difficult. it is only exposed when in close proximity to a host cell and the space between the viral and host cell membranes is small enough to hinder the binding of antibodies. this is one of the reasons why there is not a vaccine available to hiv [ ] . gp is non-covalently bound to gp because it is buried within the viral envelope. when gp binds to cd , it will change its conformation and gp will become exposed, where it can assist in fusion with the host cell [ , ] . some lectins exhibit significant activity against human immunodeficiency virus (hiv) and other viruses with an envelope. this makes them particularly attractive targets for the development as novel antiviral drugs. cyanovirin-n and griffithsin are examples of lectins that can inhibit hiv and other viruses [ , [ ] [ ] [ ] . virally encoded glycoproteins cover the surfaces of retroviruses such as hiv and many other viruses with an envelope. gp and gp present on the envelope of hiv, heavily glycosylated with glycans, are estimated to contribute almost % of the molecular weight of gp [ ] [ ] [ ] . agents that strongly and specifically interact with the glycans may disturb the interaction between the cells of the host and the proteins of the viral envelope [ , ] . on the viral surface, sugar-binding proteins can crosslink with glycans and prevent other interactions with the co-receptors. antiviral lectins can prevent penetration of the host cells by the viruses. they prevent infection by binding to the sugars that adorn the surface of the envelope of hiv [ ] . the majority of other current antiviral therapeutics act through inhibition of the viral life cycle. antiviral lectins are suitable for topical applications and can exhibit lower toxicity than others used for antiviral therapy. the antiviral proteins are often resistant to low ph and high temperatures. also they are odorless, which are properties favorable for potential drugs [ ] . examples of lectins that exhibit antiviral activity and bind high-mannose carbohydrates are jacalin [ ] , concanavalin a [ ] , urtica diocia agglutinin [ ] , myrianthus holstii lectin, p. tetragonolobus lectin [ ] and narcissus pseudonarcissus lectin [ ] . lectins are also derived from marine organisms. the natural antiviral products that exhibit the highest activity among the lectins are cyanovirin-n, scytovirin, microcystis virdis lectin and griffithsin [ , , , ] . jacalin is the major protein from the jackfruit artocarpus heterophyllus seeds. artocarpus heterophyllus in the moraceae family is an evergreen tree that is grown in various tropical countries in south and southeast asia. the seed is large and oblong with a brown tegmen and a slim membranous testa. chatterjee et al. [ ] , roque-barreira et al. [ ] and kondoh et al. [ ] , were among the first who identified jacalin around . jacalin had the property of binding human iga with specificity for the iga subclass [ ] [ ] [ ] . in the recent years various studies have been carried out on jacalin, revealing its importance as a lectin of diverse applications ranging from the isolation of human iga to hiv research. jacalin has been reported to have a molecular mass of - kda [ ] . the seeds of this fruit contain a minor mannose-binding lectin [ ] . it is a two-chain lectin consisting of an α-chain of a amino acid residues non-covalently bound to a β-chain of amino acid residues [ ] . the glycosylated α-chain has a molecular mass of . kda [ ] . sugars which interact with the lectinic-site of jacalin suppress the binding of jacalin to cd . these findings give some insight into the mechanism by which jacalin inhibits hiv- infection of cd + cells [ ] . concanavalin a is a carbohydrate-binding protein isolated from canavalia ensiformis or jack bean. it is a member of the legume lectin family and is used for human nutrition or animal fodder [ ] . the beans of this plant are mildly toxic and copious consumption should be avoided. concanavalin a was the first lectin that was available on a commercial basis and is widely used in biology and biochemistry to characterize glycoproteins and sugar-containing entities on the surface of various cells [ ] . the plant contains large quantities of the enzyme urease. this is a reason that the plant cannot be used in fodder mixtures that contains urea. it will liberate harmful ammonia from urea. because of this, the plant has been investigated as a potential source of the urease enzyme [ ] . concanavalin a is a homotetramer with a molecular mass of . kda [ ] . it has amino acid residues and binds specifically to certain structures that are found in several glycoproteins, sugars and glycolipids, especially internal and non-reducing terminal α-d-mannosyl and α-d-glucosyl groups [ ] . con a binds to envelope glycoprotein gp of hiv- and hiv- [ ] . however, hiv- resistant strains have been reported [ ] . the detailed mechanism of lectin-mediated neutralization has not been completely elucidated, but there is a minimum of two mechanisms, a direct one and an indirect mechanism to account the efficacy of removal of only a few glycans to render hiv resistant. the direct mechanism is that the mutated n-glycosylation sites harbor the "key target glycans" for lectin-mediated neutralization and that these are removed by the mutations. binding of lectins to these glycans either prevent hiv binding to receptors on the cells or affect alterations of gp following binding. the indirect mechanism suggests that the removal of one or more n-glycosylation sites renders n-glycans elsewhere more exposed to carbohydrases in the golgi body and the endoplasmic reticulum. local changes in utilization of glycosylation site can affect the glycosylation setup and protein folding. thus, lectin-binding high-mannose glycans on the wild type are probably more highly processed in the lectin-selected strains, leading to a loss of their affinity for the [ ] . gna-related lectins are nearly related in their carbohydrate-binding activities [ ] [ ] [ ] and exhibit significant anti-hiv, anti-hsv and antitumor [ ] actions. the most gna-related lectins exhibit mannose-binding activity, while polygonatum cyrtonema lectin [ ] and ophiopogon japonicas lectin [ ] also display carbohydrate affinity to sialic acid. some agents that interact with viral-envelope glycans may compromise the efficient entry of the virus into its susceptible target cells. they do not interfere with the glycosylation enzymes from the cell, but rather act by directly binding to the intact glycans on the envelope. this kind of carbohydrate-binding agents force the virus to delete a part of its glycan shield so that it can escape drug pressure. the result of this can be the initiation of an immune response against uncovered immunogenic envelope epitopes. the mechanisms of antiviral action of the carbohydrate-binding agents are, firstly, direct antiviral activity by binding to the glycans of the viral envelope and blocking entry of the virus and secondly, an indirect antiviral action. this will result from the progressive creation of deletions in the glycan shield of the envelope. the immune system will be triggered to act against previously hidden immunogenic epitopes of the viral envelope [ ] . agglutinins from hippeastrum hybrid and galanthus nivalis inhibited cell-cell fusion between clone trevenv cells induced to express the viral envelope proteins, gp /gp (env), and highly cd -positive supt cells [ ] . the mannose-specific plant lectins hippeastrum hybrid agglutinin, galanthus nivalis agglutinin, narcissus pseudonarcissus agglutinin; cymbidium agglutinin; cyanovirin-n; and n-acetylglucosamine specific urtica dioica agglutinin efficiently abrogate dendritic cell-specific intercellular adhesion molecule- -grabbing nonintegrin (dc-sign)-directed hiv- capture and subsequent transmission to t lymphocytes [ ] . the mannose-specific plant lectins from galanthus, hippeastrum, narcissus, epipactis helleborine, and listera ovata, and the n-acetylglucosamine-specific urtica dioica lectin would primarily be targeted at the virus-cell fusion process [ ] . banana lectin binds to the glycosylated viral envelope and inhibits cellular entry and thus suppresses hiv- infection. its anti-hiv activity is comparable to snowdrop lectin and griffithsin, and to the anti-hiv drugs t- and maraviroc [ ] . myrianthus holstii is a tree with a large of branches then can grow from to meters in height. it is of the urticaceae family and often has stilt roots up to cm high and a short bole. the plant is harvested from the wild as local source of food and wood. this tree is grown in middle africa, in rainforest, montane forests, at edges or in regrowth and along rivers [ ] . myrianthus holstii lectin obtained from the roots by lh- chromatography has a molecular mass of kda. this lectin consists of two major constituents with a molecular mass of and da, respectively. myrianthus holstii lectin protected cem-ss cells from the cytopathic effects of hiv with an ec value of . μg/ml ( nm). this one is similar to the urtica diocia agglutinin (ec = nm). it inhibited syncytium formation with an ec value of . μg/ml indicating that this lectin reversibly inhibits hiv infection [ ] . narcissus pseudonarcissus is also known as wild daffodil or lent lily and belongs to the amaryllidaceae family. this plant produces seeds and is native to western europe. it grows in woods, grassland and rocky ground. it contains a dimeric protein with a molecular mass of kda. it is composed of two -kda subunits. it has specificity toward α-linked mannose and is used in characterization of early stages of apoptosis and has mitogenic activity toward human lymphocytes [ ] . this lectin agglutinates rabbit erythrocytes but is non-reactive with human erythrocytes [ ] . mannose-binding agglutinins from other narcissus species also manifest hiv- infection inhibitory activity like that of narcissus pseudonarcissus agglutinin. their hiv- infection inhibitory activity does not correlate with their hemagglutinating activity [ ] . comparative analyses revealed that the dimer-based super-structure may be important to the anti-hiv property of polygonatum cyrtonema lectin, a novel anti-hiv mannose-binding lectin from galanthus nivalis agglutinin-related lectin family [ ] . urtica diocia is a herbaceous perennial flowering plant and is often called stinging nettle or common nettle. it grows in europe, north america, africa and asia and will not grow more than meters in height. it has a long history of use as a source of fiber, food and medicine. urtica diocia agglutinin is a special plant lectin that differs from other plant lectins. the cytopathic effect of hiv has a rate of ec = nm [ ] and has a small molecular mass of . kda. this is a t cell mitogen distinguishable from classical t cell lectin mitogens. it is able to discriminate a particular population of cd + and cd + cells, and can induce an original pattern of t cell cytokine production as well as activation. plant lectin mitogens have been successfully and extensively used in the analysis of events that occur during lymphocyte activation and proliferation [ , ] . the lectin suppressed entry of hiv- into host cells with a half-maximal effective concentration at . nm. surface plasmon resonance analysis disclosed a high association constant of the lectin with gp . the lectin binds to viral envelope hemagglutinin against various strains, including a clinical isolate of pandemic h n - virus, indicating that it is potentially useful for antiviral therapy [ ] . griffithsin is a -kda protein with amino acids isolated from the red alga griffithsia. this protein is a highly potent hiv entry inhibitor [ ] . it blocks cd -dependent gp binding to the receptor expressing cells. there it will bind to other gp's like gp , and . this will happen in a glycosylation-dependent manner. it inhibits gp binding of monoclonal antibody (mab) g , which recognizes a carbohydrate-dependent motif, and (mab) d, which binds to cd -induced epitope. griffithsin also moderately interfered with the binding of gp to scd . the binding of griffithsin to soluble gp was not inhibited by fucose, xylose, galactose, n-acetylgalactosamine or sialic acid containing glycoproteins but it was inhibited by mannose, glucose and n-acetylglucosamine. griffithsin binds the terminal mannose residues of surface n-linked glycans on hiv- , hiv- , ebola virus, hepatitis c virus, and severe acute respiratory syndrome coronavirus. it means that this is a type of lectin that binds to several viral glycoproteins in a monosaccharide-dependent manner. griffithsin interacts with gp leading to exposure of the cd -binding site cd bs through binding of the glycan at position [ ] . unlike cyanovirin and galanthus nivalis agglutinin, the interaction between griffithsin and gp relies on the specific trimeric "sugar tower" including n and n . this was supported by findings from griffithsin env binding experiments [ ] . griffithsin does not have mitogenic activity toward human t-cells and does not elicit secretion of proinflammatory cytokines in treated human cell lines. subutaneous injections of griffithsin induce no toxicity other than some splenomegaly and hepatomegaly. serum samples of griffithsin-treated animals display activity against hiv- -enveloped pseudoviruses in a cell-based neutralization assay. griffithsin suppresses hiv gp binding to dc-sign and dc-sign-mediated transfer of hiv- to cd + t-lymphocytes. it drives gp away from the gp -dc-sign complex. functionally intact carbohydrate binding sites are essential to its optimal action. its dimeric form is paramount to its hiv inhibitory action. griffithsin synergizes with the antiviral drugs enfuvirtide, maraviroc and tenofovir against hiv- clade b and clade c isolates in primary peripheral blood mononuclear cells and in cd + mt- cells. dissimilarity in glycosylation patterns on the viral envelope of clade b and clade c gp does not affect the synergism [ ] . however, a discrepancy exists between hiv- gp binding activity and hiv inhibitory activity of the griffithsin variants d a, d a, and d a, suggesting the presence of an anti-hiv mechanism unrelated to simple gp binding [ ] . griffithsin has three nearly-equivalent mannose binding sites all essential for anti-hiv activity. griffithsin mutants d a, d a, and d a, each with mutation of one of the three sites, demonstrated only a - fold decrement in binding to immobilized gp but -or more fold decline in hiv inhibiting ability in single-round hiv infection assays, hiv cytopathicity assays and co-cultivation assays. d a exhibited superior hiv inhibitory activity despite reduced gp binding activity compared with d a and d a in some strains, furnishing evidence for absence of a direct correlation between binding to hiv gp and hiv inhibition. hence gp binding does not necessarily ensue in an antiviral effect; the anti-hiv mechanism of griffithsin action involves not only binding and sterically hindering the activity of gp , but more importantly to alter the gp structure or its oligomeric state as a consequence of the binding of g mannose residues, and cross-link gp glycans, to render hiv uninfective [ ] . griffithsin interacts with gp leading to exposure of the cd -binding site cd bs through binding of the glycan at position [ ] . unlike cyanovirin and galanthus nivalis agglutinin, the interaction between griffithsin and gp relies on the specific trimeric "sugar tower" including n and n . this was supported by findings from griffithsin env binding experiments [ ] . however, a discrepancy exists between hiv- gp binding activity and hiv inhibitory activity of the griffithsin variants d a, d a, and d a, suggesting the presence of an anti-hiv mechanism unrelated to simple gp binding [ ] . griffithsin synergizes with other carbohydrate-binding agents in inhibitory action against hiv- , hiv- , and even against hiv- strains demonstrating resistance to some carbohydrate-binding agents. the carbohydrate-binding agents tested have different binding patterns on the gp envelope and hence no steric hindrance. the observation opens up the possibility of employing combinations of carbohydrate binding agents in topical microbicidal agents to counter hiv transmission [ ] . a mannose-binding lectin from the green alga oscillatoria agardhii (oaa) with selectivity for the cluster of á - -mannose, its homologue oscillatoria agardhii agglutinin homologue (oaah), and a designed hybrid oaah (opa), inhibit hiv replication, syncytium formation between hiv- -infected and uninfected t cells, dc-sign-mediated hiv- capture and transmission to cd + target t cells, infectivity of a variety of hiv- and hiv- clinical isolates. surface plasmon resonance analysis and flow cytometry revealed that both are competitive inhibitors of the binding of the manα( - )manspecific g monoclonal antibody. the hiv- nl . ( g res), nl . (mvnres) and iiib (grftres) strains were inhibited as well as the wild-type hiv- strains. oaa and opa synergize with hippeastrum hybrid agglutinin, and griffithsin [ ] . combinations of grft and other cbas showed synergistic activity against hiv- , hiv- , and even against certain cba-resistant hiv- strains. the cbas tested appear to have distinct binding patterns on the gp envelope and therefore do not necessarily compete with each other's glycan binding sites on gp . as a result, there might be no steric hindrance between two different cbas in their competition for glycan binding (except for the hha/gna combination). these data are encouraging for the use of paired cba combinations in topical microbicide applications (e.g., creams, gels, or intravaginal rings) to prevent hiv transmission [ ] . combined nmr and crystallographic results provide structural insights into the mechanism by which oaa specifically recognizes the branched man- core, distinctly different from the recognition of the d and d arms at the non-reducing end of high-mannose carbohydrates by other antiviral lectins [ ] . cyanovirin-n is a bacterial protein that is produced by the cyanobacterium nostoc ellipsosporum that shows virucidal activity against several viruses [ , ] . it is an entry inhibitor of hiv [ ] . it was discovered during a screening program in the search for naturally occurring virucidal agents that may be developed into anti-hiv microbicides. this protein consists of a single amino acid chain, which exhibits significant internal sequence duplication. it reveals conservative amino acid changes as well as direct homology between amino acid residues. the molecular mass is approximately kda [ ] . it also consists of four cysteines, which can form two intrachain disulfide bonds. the intact disulfide bonds appear to be required for anti-hiv activity. when cleavage of these bonds happens due to β-mercaptoethanol treatment its hiv inhibitory effects were abolished. when stored in solution phase, reduced cyanovirin-n could reform the required disulfide bonds, accompanied by recovery of anti-hiv activity [ ] . cyanovirin-n inhibited the in vitro cytopathicity of laboratory strains and several clinical isolates of hiv type , and simian immunodeficiency virus. it also effectively prevented cell-to-cell fusion and transmission of hiv from infected cells to uninfected cells. hiv virions that have undergone pretreatment with cyanovirin-n neutralized virus infectivity, but are nontoxic to host cells. the unique virucidal effects arise from its association with the viral envelope glycoprotein . cyanovirin-n interferes with critical interactions between viral gp and cell surface receptors, which are required for successful virus fusion and entry into cells [ ] . linker-cyanovirin-n (a cyanovirin-n derivative with a flexible and hydrophilic linker (gly ser) at the n-terminus), and k peg-ald-lcvn (n-terminal α-amine of n-terminal α-amine of linkercyanovirin-n pegylated to create k peg-aldehyde-linker-cyanovirin-n), are characterized by the specificity and affinity of cyanovirin-n for high mannose n-glycans. linker-cyanovirin-n displayed anti-hiv- activity with reduscd cytotoxicity on cat keratinocytes and mt- t lymphocytes. k peg-ald-lcvn inactivated hiv- with markedly decreased cytotoxicity and pronounced cell-to-cell fusion inhibitory activity in vitro. the linker-extended cyanovirin-n and the mono-pegylated derivative are thus promising candidates for the development of an anti-hiv- agent [ ] . humic acids potentiate the anti-hiv activity of azt, griffithsin, and cyanovirin [ ] . cvn specifically recognizes with nanomolar affinity man( )glcnac( ) and the d d isomer of man( )glcnac( ) [ ] . scytovirin is an antiviral protein isolated from the cyanobacterium scytonema varium. this protein consists of a single -amino acid chain with a molecular mass of . kda and significant internal sequence duplication. it has cysteines forming five intrachain disulfide bonds [ ] . [ , ] . cvn specifically recognizes with nanomolar affinity man( )glcnac( ) and the d d isomer of man( )glcnac( ) [ ] [ ] [ ] . microcystis viridis is a unicellular freshwater bloom-forming cyanobacterium. it showed transient hemagglutinating activity in laboratory culture during stationary phase under nonaeration conditions. this lectin is composed of a single -kda polypeptide with amino acid residues and two tandemly repeated homologous domains of amino residues. microcystis viridis lectin binds oligomannosides with sub-micromolar affinities and that two novel carbohydrate recognition domains composed of four non-contiguous regions. the residues make numerous intermolecular contacts with their carbohydrate ligands. this lectin inhibits hiv type envelope-mediated cell fusion with an ic [ ] . the cyanobacterial lectin microvirin is potentially exploitable since it potently inhibits hiv- with minimal toxicity. synchronized infectivity assays reveal different kinetics of hiv- entry inhibition by microvirin and cyanovirin. synergism demonstrated by combinations of the inhibitors in spite of common specificity for maná( - )man terminals indicate recognition of distinctly different glycans and/or glycan conformations on gp . entry assays utilizing amphotropic viruses disclose the inactivity of microvirin, in contrast to the potent inhibitory activity of cyanovirin. in view of the similarity in the carbohydrate-binding site common to microvirin and cyanovirin, it appears that gp is a clustered glycan epitope and multivalent-protein interactions applicable to cyanovirin but not to microvirin are needed for inhibition of certain viruses [ ] . actinohivin, an actinomycete lectin, is composed of amino acid residues and contains three segments, all of which display high specific affinity to gp due to multivalent interaction of the three sugar-binding pockets with three high-mannose type glycans hmtgs of gp via the "cluster effect" of lectin. it exhibits potent in vitro anti-hiv activity [ ] . actinohivin suppresses entry of hiv- to susceptible cells. specific and potent anti-hiv activity is produced by cooperative binding of three segments of actinohivin to three high mannose-type glycans (hmtgs) of hiv- gp . the anti-hiv activity of actinohivin is enhanced after dimerization due to a rise in the number of hmtg-binding pockets [ ] . the -kda â-galactose-specific lectin inhibited the cytopathic effects brought about by hiv- and the production of viral p antigen. its effect at the early stage of viral replication was disclosed by time-of-addition analysis of its anti-hiv- activity indicated. the lectin inhibited cell-to-cell fusion of hiv infected and uninfected cells. its suppression of hiv- entry into host cells was shown by employing fluorescence-based real-time quantify pcr [ ] . the lectin inhibited the production of viral p antigen and cytopathic effect induced by hiv with ec values of . and . μg/ml, respectively [ ] . dendritic cells (dcs) play a vital role in hiv- transmission; dcs capture assaulting hiv- via interaction of the c-type lectin dc-sign with gp oligosaccharides and translocate to lymphoid tissues in which transmission of hiv- to t cells occurs. it is reasonable to target gp for inhibiting interactions with dcs and transmission of hiv- . the nematode (laxus oneistus) ca + -dependent c-type lectin mermaid, a structural homologue of dc-sign devoid of cytotoxicity, shares the glycan specificity with dc-sign, interacts with high mannose structures on gp and prevents hiv- binding to dc-sign on dcs. mermaid inhibits dc-sign-mediated hiv- transmission from dc to t cells. thus the lectin has potential for development into an anti-hiv- agent [ ] . pradimicins and benanomicins are non-peptidic lectin mimics which recognize d-mannopyranoside in the presence of calcium ions. they exert antifungal and anti-hiv activities through binding to mannopyranoside-containing glycans of pathogens. pradimicin a, an original member of pradimicins, binds to terminal d-mannopyranoside residues at the non-reducing end of glycans, as well as to internal -substituted mannopyranoside residues [ ] . mannose binding lectin triggers the complement pathway culminating in pathogen opsonization and phagocytosis. mannose binding lectin deficiency is associated with to hiv transmission and progression of aids. expression of the lectin has been detected in astrocytes, microglia, neurons, and oligodendrocytes of the hiv- -infected frontal cortex. in hiv encephalitis, monomeric and trimeric mannose binding lectin expression and mcp- expression are elevated in the neuronal axons. differential mbl expression was not observed in healthy hiv-negative subjects and normal or gp transgenic mice. the findings indicate that mannose binding lectin could cause neuroinflammation and neuronal injury through activating the complement pathway [ ] . hiv- goes into the brain at the early stages of infection and leads to damage and impairment of the central nervous system mannose binding lectin binds to the n-linked mannose residues on gp , a neurotoxin, and is instrumental in intravesicular packaging of gp in neuronal subcellular organelles and subcellular trafficking of gp through the endoplasmic reticulum and golgi vesicles.the vesicular complexes were translocated along the microtubule network a functional carbohydrate recognition domain necessary for the actions of mannose binding lectin [ ] . in summary, the use of lectins extracted from various sources is a great promise and potential for use in future hiv therapy. latest research has found potent results towards the anti-hiv effects of various lectins as shown in this review. there is still no vaccine against hiv. the n-linked glycans of gp v protects the v region of oligomeric gp from potential neutralizing antibodies [ ] . because of the inventive immunological escape mechanisms of this virus, additional research should be funded in order to understand these mechanisms. also for the lectins, research should be promoted and funded and aid in the transition to clinical application. the mannose binding lectins actinohivin (ah), cyanovirin-n (cv-n), griffithsin (grft), microcystis viridis lectin (mvl), oscillatoria agardhii agglutinin (oaa), scytovirin (svn), from cyanobacteria and algae display high anti-hiv potencies with nanomolar-picomolar ic values. they differ in tertiary and quaternary structures, but contain internal repeats within their primary structures. the number of sequence repeats often corresponds to the number of domains and binding sites in each lectin. there is pronounced amino acid sequence homology and also structural resemblance between the domains. each of the lectins has distinctive characteristics. some are multimeric and/or manifest domain swapping, whereas others are monomeric. monomeric cv-n, oaa, and svn have two sugar binding sites; ah and engineered monomeric grft possess three sites; domain-swapped cv-n and mvl display four sites; and domain-swapped grft has six sugar binding sites. the lectins recognize different epitopes on high mannoses. the number of binding sites on the protein and/or the number of epitopes recognized on man- / correlate with its antiviral activity. they target mannose on gp of hiv, inhibiting the conformational change into the active state. the most straightforward delivery mode is introduction into the rectum or vagina, or in multifunctional contraceptive gels or rings, or in situ expression by modified commensal bacteria which inhabit the gut or vagina [ ] . engineered vaginal lactobacillus strain has been proposed for mucosal delivery of the human immunodeficiency virus inhibitor cyanovirin-n to prevent sexual transmission of the virus [ ] . grft could be obtained in multigram quantities after extraction from nicotiana benthamiana plants transducted with a tobacco mosaic virus vector expressing the lectin. the employment of lactobacillus jensenii expressing an anti-hiv lectin can curtail the costs of the development of a user-friendly microbicide [ ] . the anti-hiv activities of various lectins are summarized in tables and . hiv/aids-related mortality in africa and asia: evidence from indepth health and demographic surveillance system sites cause-specific childhood mortality in africa and asia: evidence from indepth health and demographic surveillance system sites in vitro anti-hiv activity of five selected south african medicinal plant extracts polymeric anti-hiv therapeutics hiv/aids epidemiology, pathogenesis, prevention, and treatment lectins as cell recognition molecules discovery of cyanovirin-n, a novel human immunodeficiency virus-inactivating protein that binds viral surface envelope glycoprotein gp : potential applications to microbicide development purification and characterization of a mucin specific mycelial lectin from aspergillus gorakhpuresis: application for mitogenic and antimicrobiol activity how does hiv cause aids manipulation of dendritic cell function by viruses robbins basic pathology hiv entry and its inhibition the hiv- envelope glycoproteins: fusogens, antigens, and immunogens spontaneous mutations in the env gene of the human immunodeficiency virus type ndk isolate are associated with a cd -independent entry phenotype access of antibody molecules to the conserved coreceptor binding site on glycoprotein gp is sterically restricted on primary human immunodeficiency virus type cyanovirin-n binds to gp to interfere with cd -dependent human immunodeficiency virus type virion binding, fusion, and infectivity but does not affect the cd binding site on gp or soluble cd -induced conformational changes in gp cyanovirin-n inhibits hepatitis c virus entry by binding to envelope protein glycans isolation and characterization of griffithsin, a novel hiv-inactivating protein from the red alga griffithsia sp structural studies of algal lectins with anti-hiv activity improved procedure for the construction of neoglycolipids having antigenic and lectin-binding activities, from reducing oligosaccharides a novel in vitro system to generate and study latently hiv-infected long-lived normal cd + t-lymphocytes inhibition of hiv entry by carbohydrate-binding proteins proteins that bind high-mannose sugars of the hiv envelope jacalin, a lectin interacting with o-linked sugars and mediating protection of cd + cells against hiv- , binds to the external envelope glycoprotein gp inhibition of human immunodeficiency virus infection by the lectin jacalin and by a derived peptide showing a sequence similarity with gp effects of succinylated concanavalin a on infectivity and syncytial formation of human immunodeficiency virus the mannose-specific plant lectins from cymbidium hybrid and epipactis helleborine and the (n-acetylglucosamine)-specific plant lectin from urtica dioica are potent and selective inhibitors of human immunodeficiency virus and cytomegalovirus replication in vitro isolation and characterization of myrianthus holstii lectin, a potent hiv- inhibitory protein from the plant myrianthus holstii inhibition of herpes simplex, rabies and rubella viruses by lectins with different specificities a potent novel anti-hiv protein from the cultured cyanobacterium scytonema varium the antiviral lectin cyanovirin-n: probing multivalency and glycan recognition through experimental and computational approaches comparative studies of new marker lectins for alkali-labile and alkali-stable carbohydrate chains in glycoproteins jacalin: an iga-binding lectin jacalin, a jackfruit lectin, precipitates iga but not iga subclass on gel diffusion reaction preparation and x-ray characterization of four new crystal forms of jacalin, a lectin from artocarpus integrifolia activation of t and b cells by a crude extract of artocarpus integrifolia is mediated by a lectin distinct from jacalin a jackfruit (artocarpus heterophyllus) seed-derived lectin of versatile applications in immunobiological research structural and electron-microscopic studies of jacalin from jackfruit (artocarpus integrifolia) show that this lectin is a kda tetramer jacalin, a lectin with anti-hiv- properties, and hiv- gp envelope protein interact with distinct regions of the cd molecule in silico analysis of molecular mechanisms of legume lectin-induced apoptosis in cancer cells ultrastructural visualization of surface carbohydrate structures on mycoplasma membranes by concanavalin a canatoxin, a toxic protein from jack beans (canavalia ensiformis), is a variant form of urease (ec . . . ): biological effects of urease independent of its ureloytic activity non-glycosylated recombinant pro-concanavalin a is active without polypeptide cleavage isolation, physicochemical characterization and carbohydrate-binding specificity of lectins correlation between carbohydrate structures on the envelope glycoprotein gp of hiv- and hiv- and syncytium inhibition with lectins resistance of human immunodeficiency virus type to the high-mannose binding agents cyanovirin n and concanavalin a structural analysis and binding properties of isoforms of tarin, the gna-related lectin from colocasia esculenta induction of apoptosis by polygonatum odoratum lectin and its molecular mechanisms in murine fibrosarcoma l cells polygonatum cyrtonema lectin induces apoptosis and autophagy in human melanoma a cells through a mitochondria-mediated ros-p -p pathway molecular modeling, docking and dynamics stimulations of gna-related lectins for potential prevention of influenza virus (h n ) molecular mechanisms of polygonatum cyrtonema lectin-induced apoptosis and autophagy in cancer cells galanthus nivalis agglutinin (gna)-related lectins: traditional proteins, burgeoning drugs? targeting the glycans of glycoproteins: a novel paradigm for antiviral therapy inhibition of hiv- env-mediated cell-cell fusion by lectins, peptide t- , and neutralizing antibodies carbohydrate-binding agents efficiently prevent dendritic cell-specific intercellular adhesion molecule- -grabbing nonintegrin (dc-sign)-directed hiv- transmission to t lymphocytes current lead natural products for the chemotherapy of human immunodeficiency virus (hiv) infection a lectin isolated from bananas is a potent inhibitor of hiv replication edible wild plants of tanzania; regional land management unit the potentially insecticidal narcissus pseudonarcissus lectin demonstrates age-related mitogenecity related mannose-specific lectins from different species of the family amaryllidaceae anti-human immunodeficiency virus type (hiv- ) activity of lectins from narcissus species crystal structures of a novel anti-hiv mannose-binding lectrin from polygonatum cyrtonema hua with unique ligand-binding property and super-structure urtica dioica agglutinin: a superantigenic lectin from stinging nettle rhizome early biochemical events in lymphocyte t activation by mitogens boodlea coacta is a potent entry inhibitor of hiv- and influenza viruses binding of the mannose-specific lectin, griffithsin, to hiv- gp exposes the cd -binding site removal of two high-mannose n-linked glycans on gp renders human immunodeficiency virus largely resistant to the carbohydrate-binding agent griffithsin synergistic activity profile of griffithsin in combination with tenofovir, maraviroc and enfuvirtide against hiv- clade c the role of individual carbohydrate-binding sites in the function of the potent anti-hiv lectin griffithsin development of vaginal microbicides for the prevention of heterosexual transmission of hiv. hiv/aids in women: an expanding epidemic broad anti-hiv activity of the oscillatoria agardhii agglutinin homologue lectin family combinations of griffithsin with other carbohydrate-binding agents demonstrate superior activity against hiv type , hiv type , and selected carbohydrate-binding agent-resistant hiv type strains structural basis of the anti-hiv activity of the cyanobacterial oscillatoria agardhii agglutinin primary sequence determination, and disulfide bond structure of cyanovirin-n, an anti-hiv (human immunodeficiency virus) protein from the cyanobacterium nostoc ellipsosporum pegylation of cyanovirin-n, an entry inhibitor of hiv linker-extended native cyanovirin-n facilitates pegylation and potently inhibits hiv- by targeting the glycan ligand humic acids (ha) strongly potentiate anti-hiv effects of azt, griffithsin, and cyanovirin the potent anti-hiv protein cyanovirin-n contains two novel carbohydrate binding sites that selectively bind to man d d and man with nanomolar affinity: implications for binding to the hiv envelope protein gp the novel fold of scytovirin reveals a new twist for antiviral entry inhibitors mechanisms of hiv- subtype c resistance to grft, cv-n and svn the cyanobacterial lectin scytovirin displays potent in vitro and in vivo activity against zaire ebola virus differential inhibitory effects of cyanovirin-n, griffithsin, and scytovirin on entry mediated by envelopes of gammaretroviruses and deltaretroviruses isolation and characterization of a mannan-binding lectin from the freshwater cyanobacterium (blue-green algae) microcystis viridis solution structure of the monovalent lectin microvirin in complex with man(α)( - )man provides a basis for anti-hiv activity with low toxicity mechanism by which the lectin actinohivin blocks hiv infection of target cells the high mannose-type glycan binding lectin actinohivin: dimerization greatly improves anti-hiv activity a β-galactose-specific lectin isolated from the marine worm chaetopterus variopedatus possesses anti-hiv- activity a new lectin from the sea worm serpula vermicularis: isolation, characterization and anti-hiv activity c-type lectin mermaid inhibits dendritic cell mediated hiv- transmission to cd +t cells mannose-binding geometry of pradimicin a expression of mannose binding lectin in hiv- -infected brain: implications for hiv-related neuronal damage and neuroaids intracellular mannose binding lectin mediates subcellular trafficking of hiv- gp in neurons protection of neutralization epitopes in the v loop of oligomeric human immunodeficiency virus type glycoprotein by n-linked oligosaccharides in the v region engineered vaginal lactobacillus strain for mucosal delivery of the human immunodeficiency virus inhibitor cyanovirin-n antiviral lectins as potential hiv microbicides algal lectins as potential hiv microbicide candidates the antiviral protein cyanovirin-n: the current state of its production and applications mutational pathways, resistance profile, and side effects of cyanovirin relative to human immunodeficiency virus type strains with n-glycan deletions in their gp envelopes , )-mannose-specific lectin isolated from microcystis aeruginosa, has anti-hiv- activity comparable with that of cyanovirin-n but a much higher safety profile subtype c gp and sensitivity to the lectins, griffithsin, cyanovirin-n and scytovirin new carbohydrate specificity and hiv- fusion blocking activity of the cyanobacterial protein mvl: nmr, itc and sedimentation equilibrium studies overexpression and purification of scytovirin, a potent, novel anti-hiv protein from the cultured cyanobacterium scytonema varium primary structure and carbohydrate binding specificity of a potent anti-hiv lectin isolated from the filamentous cyanobacterium oscillatoria agardhii griffithsin, a potent hiv entry inhibitor, is an excellent candidate for anti-hiv microbicide investigation of griffithsin's interactions with human cells confirms its outstanding safety and efficacy profile as a microbicide candidate scaleable manufacture of hiv- entry inhibitor griffithsin and validation of its safety and efficacy as a topical microbicide component potential anti-hiv agents from marine resources: an overview carbohydrate-binding proteins of marine invertebrates we gratefully acknowledge the award of an hmrf research grant (reference no. ) from food and health bureau, the government of hong kong special administrative region. o.a. and t.b.n. were responsible for writing the review and did the final editing of the manuscript. s.s.s., y.s.c. and w.p. prepared the tables. r.c.f.c. assisted in providing references for the manuscript and proofread the manuscript. x.d. and c.y. helped in compilation of references. the authors have declared that no competing interests exist. key: cord- - nalst authors: boobphahom, siraprapa; nguyet ly, mai; soum, veasna; pyun, nayoon; kwon, oh-sun; rodthongkum, nadnudda; shin, kwanwoo title: recent advances in microfluidic paper-based analytical devices toward high-throughput screening date: - - journal: molecules doi: . /molecules sha: doc_id: cord_uid: nalst microfluidic paper-based analytical devices (µpads) have become promising tools offering various analytical applications for chemical and biological assays at the point-of-care (poc). compared to traditional microfluidic devices, µpads offer notable advantages; they are cost-effective, easily fabricated, disposable, and portable. because of our better understanding and advanced engineering of µpads, multistep assays, high detection sensitivity, and rapid result readout have become possible, and recently developed µpads have gained extensive interest in parallel analyses to detect biomarkers of interest. in this review, we focus on recent developments in order to achieve µpads with high-throughput capability. we discuss existing fabrication techniques and designs, and we introduce and discuss current detection methods and their applications to multiplexed detection assays in relation to clinical diagnosis, drug analysis and screening, environmental monitoring, and food and beverage quality control. a summary with future perspectives for µpads is also presented. microfluidics is the science of fluid manipulation in a microscale network and deals with controlling fluid samples with low volumes ( − to − l) inside micrometer-scale channels [ , ] . though the first known microfluidic device was documented in [ ] , it mostly dealt with materials like silicon, glass, and polymers such as polymethylmethacrylate (pmma), polystyrene (ps), polycarbonate (pc), and polydimethylsiloxane (pdms) [ ] . miniaturization of experimental apparatuses has led to lab-on-chip environments in which sample and reagent consumption is low, reactions are faster, and mixing or separation is more efficient, thereby enabling quick, modern, high-technology research [ ] . hence, microfluidics has garnered significant attention in analytical chemistry. after a few decades of development, microfluidics started to blossom with the utilization of papers (chromatography, filter, and nitrocellulose paper); this was triggered by whitesides' publication in [ ] . these inexpensive and ubiquitous materials allow researchers to use simple and feasible methods with minimal accessories to create affordable microfluidic paper-based analytical [ ] . copyright , american chemical society. (b) the electrochemical (e)-µpads for multiplexed analytical detections of paracetamol, caffeine, and ascorbic acid in drugs. reproduced with permission from the authors of [ ] . copyright , elsevier b.v. (c) lateral flow assay (lfa) that can be used for detecting four biomarkers (hepatitis b virus (hbv), hepatitis c virus (hcv), human immunodeficiency virus (hiv), and treponema pallidum (tp)), simultaneously. reproduced with permission from the authors of [ ] . copyright under the terms and conditions of the creative commons attribution. we describe fabrication strategies highlighting novel techniques and on how different research groups have successfully oriented their devices toward high-throughput screening, including rapid fabrication, mass production (with high reproducibility), rapid prototyping, and compatibility with automated processes. we summarize the fabrication methods of p-cmf devices, integration of functionality and reproducibility, and mass production automation fabrication processes. from the very beginning, µpads have been dominantly fabricated by using simple, rapid methods that facilitate prototyping and minimize cost while ensuring high-quality testing. this coincides with the trend to fabricate general microfluidic devices based on the cost of materials and related equipment and tools, faster turnaround time, and increased functionality [ ] . table shows a collection of reported methods used for the quick fabrication of µpads. for an explanation of each fabrication method and for the procedures used in each method, readers are redirected to excellent review papers [ ] [ ] [ ] [ ] . [ ] . copyright , american chemical society. (b) the electrochemical (e)-µpads for multiplexed analytical detections of paracetamol, caffeine, and ascorbic acid in drugs. reproduced with permission from the authors of [ ] . copyright , elsevier b.v. (c) lateral flow assay (lfa) that can be used for detecting four biomarkers (hepatitis b virus (hbv), hepatitis c virus (hcv), human immunodeficiency virus (hiv), and treponema pallidum (tp)), simultaneously. reproduced with permission from the authors of [ ] . copyright under the terms and conditions of the creative commons attribution. we describe fabrication strategies highlighting novel techniques and on how different research groups have successfully oriented their devices toward high-throughput screening, including rapid fabrication, mass production (with high reproducibility), rapid prototyping, and compatibility with automated processes. we summarize the fabrication methods of p-cmf devices, integration of functionality and reproducibility, and mass production automation fabrication processes. from the very beginning, µpads have been dominantly fabricated by using simple, rapid methods that facilitate prototyping and minimize cost while ensuring high-quality testing. this coincides with the trend to fabricate general microfluidic devices based on the cost of materials and related equipment and tools, faster turnaround time, and increased functionality [ ] . table shows a collection of reported methods used for the quick fabrication of µpads. for an explanation of each fabrication method and for the procedures used in each method, readers are redirected to excellent review papers [ ] [ ] [ ] [ ] . table . recent fabrication of µpads including paper-based continuous-flow microfluidic (p-cmf) and paper-based dmf (p-dmf) devices. patterning electrodes/creating dielectric layer agnp; cnt/parylene-c-teflon inkjet printer/vapor deposition chamber; spin coater [ , ] patterning electrodes/creating dielectric layer agnp/plastic wrap-silicon oil conductive ballpoint pen; plotter [ ] screen printing/taping patterning electrodes/creating dielectric carbon; silver/adhesive tape-nevosil screen print [ , ] patterning electrodes/creating dielectric layer agnp/pdmssilicon oil microsyringe dispenser/spin coater [ ] spraying/taping patterning electrodes/creating dielectric layer graphite/tape-nevosil spray [ ] molecules , , of the need to apply research to solve real-life issues has led to the need to add more functions to µpads to fulfill the testing requirements, those requirements ranging from d-guided channels for fluid samples transportation, sample preparation, programming fluid transportation, and sensing materials incorporation for the readout of results. herein, we pay more attention to d-µpads. as proposed by whitesides' group, d-µpads allow transport of fluids both laterally and vertically, enabling distribution, combination, and/or filtration of fluid to perform a function or to add a new function to the system [ ] (figure a ). they are especially suitable for high-throughput screening in terms of our definition. compared to the channels in d-µpads, those in d-µpads offer higher flow speed and more capability to perform multistep chemical reactions orderly or multiple assays simultaneously [ ] . for instance, to the best of our knowledge, a d origami-based µpad, which had been designed to extract nucleic acids from viscous samples, was first reported by govindarajan et al. in [ ] . a year later, ge et al. reported a multifunction-integrated immunodevice based on a similar method; that device contained (i) a test tab surrounded by (ii) a filter tab to obtain serum from treated whole blood samples by separating red blood cells, (iii) two waste tabs for washing off nonspecifically bound proteins, and (iv) a reagent tab for the distribution of chemiluminescence reagents to obtain a detectable signal. when these tabs were folded correctly, multiplex immunoassays could be completed within min [ ] . figure b shows a representative of a d-µpad fabricated by using an origami method and d-µpad was designed specifically for assay detection of dnas [ ] . molecules , , of the need to apply research to solve real-life issues has led to the need to add more functions to µpads to fulfill the testing requirements, those requirements ranging from d-guided channels for fluid samples transportation, sample preparation, programming fluid transportation, and sensing materials incorporation for the readout of results. herein, we pay more attention to d-µpads. as proposed by whitesides' group, d-µpads allow transport of fluids both laterally and vertically, enabling distribution, combination, and/or filtration of fluid to perform a function or to add a new function to the system [ ] (figure a) . they are especially suitable for high-throughput screening in terms of our definition. compared to the channels in d-µpads, those in d-µpads offer higher flow speed and more capability to perform multistep chemical reactions orderly or multiple assays simultaneously [ ] . for instance, to the best of our knowledge, a d origami-based µpad, which had been designed to extract nucleic acids from viscous samples, was first reported by govindarajan et al. in [ ] . a year later, ge et al. reported a multifunction-integrated immunodevice based on a similar method; that device contained (i) a test tab surrounded by (ii) a filter tab to obtain serum from treated whole blood samples by separating red blood cells, (iii) two waste tabs for washing off nonspecifically bound proteins, and (iv) a reagent tab for the distribution of chemiluminescence reagents to obtain a detectable signal. when these tabs were folded correctly, multiplex immunoassays could be completed within min [ ] . figure b shows a representative of a d-µpad fabricated by using an origami method and d-µpad was designed specifically for assay detection of dnas [ ] . representative methods for the fabrication of d-µpads. (a) d-µpad was fabricated by stacking layers of paper channels and adhesive tapes to form d channels. reproduced with permission from the authors of [ ] . copyright , the national academy of sciences of the usa. (b) d-µpad was fabricated by using an origami method to form a d channel. the channels were folded that allowed fluid samples to flow in a vertical direction. reproduced with permission from the authors of [ ] . copyright under the terms and conditions of the creative commons attribution. sample preparations including separation and preconcentration of analytes and the mixing of reagents are very important steps in analytical assay protocols. in µpads, preconcentrating can be done by using the electrophoretic separation technique where mainly two conductive electrodes (anode and cathode) are integrated into a p-cmf channel. the electrodes located at the beginning and the end of d or d (origami-based) paper-based channels are applied with a certain electric field to separate the target analytes from the mixture based on the interaction between the analytes and electrophoretic force [ ] [ ] [ ] . when the electrophoretic separation technique is integrated into µpads, it enables users to analyze a sample that has relatively low concentration and to improve the limit of detection (lod) [ , , ] . this technique was applied successfully in µpads to preconcentrate various biomarkers including amino acid [ ] , bovine serum albumin (bsa) [ , ] , dna [ ] , and microvesicle/exosome [ ] . electrophoretic separation technique has been used in µpads as a sample preparation step in various analytical detection methods such as ion-transmission representative methods for the fabrication of d-µpads. (a) d-µpad was fabricated by stacking layers of paper channels and adhesive tapes to form d channels. reproduced with permission from the authors of [ ] . copyright , the national academy of sciences of the usa. (b) d-µpad was fabricated by using an origami method to form a d channel. the channels were folded that allowed fluid samples to flow in a vertical direction. reproduced with permission from the authors of [ ] . copyright under the terms and conditions of the creative commons attribution. sample preparations including separation and preconcentration of analytes and the mixing of reagents are very important steps in analytical assay protocols. in µpads, preconcentrating can be done by using the electrophoretic separation technique where mainly two conductive electrodes (anode and cathode) are integrated into a p-cmf channel. the electrodes located at the beginning and the end of d or d (origami-based) paper-based channels are applied with a certain electric field to separate the target analytes from the mixture based on the interaction between the analytes and electrophoretic force [ ] [ ] [ ] . when the electrophoretic separation technique is integrated into µpads, it enables users to analyze a sample that has relatively low concentration and to improve the limit of detection (lod) [ , , ] . this technique was applied successfully in µpads to preconcentrate various biomarkers including amino acid [ ] , bovine serum albumin (bsa) [ , ] , dna [ ] , and microvesicle/exosome [ ] . electrophoretic separation technique has been used in µpads as a sample preparation step in various analytical detection methods such as ion-transmission mass spectrometry (ms) for analysis of amino acids [ ] , matrix-assisted laser desorption ionization time-of-flight mass spectrometry (maldi-tof-ms) for detection of albumin and glycated hemoglobin (hba c) [ ] , and lateral flow immunoassay (lfa) for the detection of igg [ ] typically, fluid control and programmability are vital to automate and to minimize the processing in the fabrication and the operation, but maximize the use of µpads. by controlling the fluid flow in the µpad, researchers have been able to achieve high-sensitivity detection and automated programmable assays [ , ] . because fluid transport in the paper-based channel is induced by the capillary action generated by the interfacial interaction between the fluid and the fibrous network of the paper channel, the flow of fluid can be controlled by changing the surface energy, permeability, dimensions, and geometry of the paper channel [ ] [ ] [ ] [ ] [ ] [ ] . commonly, those paper channels are made of chromatography paper and filter papers. for the first time, soum et al. introduced programmable photo-paper-based microfluidic devices in which the fluid flow could be quantitatively controlled. fluid flow in the photo-paper-based channel can be controlled by using hydrophobic and hydrophilic patterns to decrease and increase the speed, respectively. depending on the shapes and width of the patterns modified in the channel, the fluid transportation can be controlled quantitatively ( figure ) [ ] . recent review papers have summarized progress in the development of flow control tools and the integration of those tools for paper microfluidics [ , , , ] . molecules , , of mass spectrometry (ms) for analysis of amino acids [ ] , matrix-assisted laser desorption ionization time-of-flight mass spectrometry (maldi-tof-ms) for detection of albumin and glycated hemoglobin (hba c) [ ] , and lateral flow immunoassay (lfa) for the detection of igg [ ] typically, fluid control and programmability are vital to automate and to minimize the processing in the fabrication and the operation, but maximize the use of µpads. by controlling the fluid flow in the µpad, researchers have been able to achieve high-sensitivity detection and automated programmable assays [ , ] . because fluid transport in the paper-based channel is induced by the capillary action generated by the interfacial interaction between the fluid and the fibrous network of the paper channel, the flow of fluid can be controlled by changing the surface energy, permeability, dimensions, and geometry of the paper channel [ ] [ ] [ ] [ ] [ ] [ ] . commonly, those paper channels are made of chromatography paper and filter papers. for the first time, soum et al. introduced programmable photo-paper-based microfluidic devices in which the fluid flow could be quantitatively controlled. fluid flow in the photo-paper-based channel can be controlled by using hydrophobic and hydrophilic patterns to decrease and increase the speed, respectively. depending on the shapes and width of the patterns modified in the channel, the fluid transportation can be controlled quantitatively ( figure ) [ ] . recent review papers have summarized progress in the development of flow control tools and the integration of those tools for paper microfluidics [ , , , ] . to detect the target analytes, the paper-based microfluidic platforms must be integrated with sensing materials such as reagents (chemicals, enzymes, antibodies) or electrochemical sensors to provide detectable signals such as color and electrical current. these sensing materials can be stored directly by physical absorption in the matrix of the paper and chemically immobilization on the fibrous paper while electrochemical sensors can be patterned on the surface of the paper. being able to store reagents, µpads make processes of analytical assays become faster and easier with low reagent consumption because less equipment and steps are involved. however, the stability of the reagents is lower compared to those reagents stored in the standard condition in the laboratories. to provide a longer lifetime of the reagents stored in the µpads, researchers need to incorporate with functional materials for stabilization purposes and identify optimum storage conditions such as temperature, humidity, and light [ ] . electrochemical sensors can be printed directly on the surface of paper being used for µpads by using screen printing, inkjet printing, and ballpoint pen printing to detect the target analytes, the paper-based microfluidic platforms must be integrated with sensing materials such as reagents (chemicals, enzymes, antibodies) or electrochemical sensors to provide detectable signals such as color and electrical current. these sensing materials can be stored directly by physical absorption in the matrix of the paper and chemically immobilization on the fibrous paper while electrochemical sensors can be patterned on the surface of the paper. being able to store reagents, µpads make processes of analytical assays become faster and easier with low reagent consumption because less equipment and steps are involved. however, the stability of the reagents is lower compared to those reagents stored in the standard condition in the laboratories. to provide a longer lifetime of the reagents stored in the µpads, researchers need to incorporate with functional materials for stabilization purposes and identify optimum storage conditions such as temperature, humidity, and light [ ] . electrochemical sensors can be printed directly on the surface of paper being used for µpads by using screen printing, inkjet printing, and ballpoint pen printing methods. screen printing is the most suitable method for printing electrochemical sensors on chromatography or filter paper because the method can print high-viscosity ( - , cp) conductive inks that generate conductive electrodes within a single printing time [ ] . the inkjet printing may require several or single printing time(s) to pattern good conductive electrodes because it can only print low viscosity ink ( - cp), which depends on the type of paper and inkjet printer used for the fabrication [ , ] . lastly, the ballpoint pen printing method was reported that it could print conductive electrodes on photo paper within a single printing time [ , ] . a recent review paper by noviana et al. has summarized methods for the fabrication of electrochemical paper-based devices [ ] . reproducibility, which we limit to the reproducibility of results, is defined as the ability to perform and confirm a previously reported finding by using procedures that are the same or closely matched to those in the original setting [ ] . it is an important criterion in assessing the fabrication and the performance of a device. for robust results, during prototyping, device components and experiment parameters must be optimized if an efficient µpad is to be produced. for this, a simple method will facilitate the process. garcia et al. presented a handheld stamping process that produced a µpad in seconds independently of the user's technical skills [ ] . we considered the fabrication methods that involve digitally control (e.g., wax printing, inkjet printing, and digital cutter) provides high reproducibility compared to the manual control methods (e.g., handwriting) [ , , , , , , , , ] . when addressing fabrication, one must consider the mass production and the abilities to automate and/or program a device; these factors have a closed relationship and affect one another. mass production uses a standardized process for creating interchangeable parts in large quantities at a low cost [ ] . normally, for a product to be produced in large quantities, (i) it should have a useful proven application and an economic value, (ii) it can be manufactured with minimal, cost-effective equipment, tools and materials, and (iii) it should be fabricated or handled via automation with less power consumption. akyazi et al. provided a critical review that analyzed the strengths and weaknesses of each fabrication method and the potential to apply those methods commercially [ ] . due to isotropic wicking and multidirectional transport of fluids in the paper, the authors strongly emphasized that for successful commercialization of µpads, an integrated flow control method was needed. we appreciate this work albeit the fact that objectively, no perfect fabrication method exists, particularly in this new exploding field. in summary, the cost and the value of the final product must be considered when selecting and optimizing µpads for use in specific applications. wax screen printing, as an example, is reported to be a possible fabrication method for mass production [ ] ; however, it requires a heating step, and wax materials cannot be used with any organic solvents. sameenoi et al., therefore, proposed a one-step polymer screen printing method using polystyrene, which allowed mass production of flexible µpads in a roll-to-roll format [ ] . the demonstration showed their devices had good analysis capability for both inorganic (hydrogen peroxide-h o ) and organic ( , -diphenyl- -picrylhydrazyl-dpph) solvents. another example is the use of a commercial inkjet printer and an xyz dispensing platform to mass-produce barcoded µpads with high precision [ ] . the fabricated devices showed good performance for rapid multiplexed immunoassays and nucleic acid detection. based on our own perspective, mass production of paper-based microfluidic devices can be achieved when a fabrication method for µpads is transferred to a manufacturing process after it is well optimized in the laboratory. however, the cost of material for the fabrication should be a primary concern before shifting to mass production. in p-dmf devices, the movement of a fluid droplet is induced by using an electric field on the electrode arrays that are coated by a hydrophobic dielectric layer. the droplet can be actuated by electrowetting force created at the interface between the hydrophobic dielectric layer and conductive droplet. as the applied voltage increases, the contact angle of the droplet becomes smaller as the wettability is increased [ ] . this property can be explained by the young-lippmann equation: where θ l (v) and θ y ( ) are the contact angle of the droplet when a voltage (v) and no voltage are applied, respectively. c and v are the capacitance, and applied voltage, respectively. γ lv is the interfacial tension of the droplet. two main components for the fabrication of a p-dmf device are conductive electrode arrays and hydrophobic dielectric layers that have the function to deliver applied voltage to actuate electrodes and to suppress electrical current from leaking, respectively. moreover, an electrical switching system (operation system) to activate each of the individual electrodes is required for the basic droplet manipulations of p-dmf devices. the inkjet method, a familiar printing method frequently used for handling fluids through ewod, allows electrodes to be printed on paper in a very accessible way by using personal computers and printers ( figure a ) [ ] . on the printed electrode, as mentioned above, is a dielectric layer for preventing electrolysis; a parylene-c coating fabricated using chemical vapor deposition (cvd) is commonly coated with a hydrophobic layer that reduces the contact angle and increases the initial contact angle. these surface modification steps cause the fluid to move smoothly. to efficiently program the chip design and to reduce its complexity, researchers have proposed that chips be constructed from component libraries and verified through simulations before fabrication [ ] . by decomposing the system into modules, which are sets of components with customized functions, one can reduce the system's complexity. the system can be controlled by using computer-based software and a smartphone app (figure c ,d) [ ] . even though µpads provide extraordinary advantages, including simple fabrication and mass- fobel et al. printed five reservoir electrodes and drive electrodes on photo paper by using inkjet printing with silver nanoparticles [ ] . through automation with an open-source dropbot drop controller, they performed the steps ( dispense, mix, and sprite steps, measurements) required for a homogenous chemiluminescence assay with higher reproducibility in less than one hour with only the initial three pipettings. meanwhile, yafia et al. used a mask to squeeze a cnt ink onto paper to print the electrode by using screen printing [ ] . in this case, complete contact between the screen and the paper substrate is important for resolution. instead of materials such as parylene-c, polydimethylsiloxane (pdms), and cytop ™ amorphous fluoropolymer, which are mainly used as dielectric layers [ ] , applying a stretched parafilm, they were able to avoid the need for additional equipment. they also realized the automation of fluid routing by using an arduino mega unit and a card edge connector ( - - , te connectivity). another very simple method for printing electrodes is the ballpoint pen printing method [ ] . by mounting a conductive ballpoint pen with a digital plotter, printing can be done quickly and very easily without expensive equipment. for the dielectric layer, the authors of ref. [ ] used a simple process of wrapping the electrodes with commercial premade linear-low-density polyethylene (lldpe) plastic ( -ul thick), which is inexpensive and easy to obtain. silicon oil was applied to the top and the bottom of the plastic wrap to avoid bubbles between the electrodes and the wrap and to reduce friction between the fluid and the chip. this significantly reduced the contact angle to facilitate fluid movement. their control system for the chip consisted of a hardware prototype and a smartphone, and control was accomplished by running a custom-developed app that could send commands via bluetooth. as the chip can be manufactured using a variety of easy ways and can be automated using electricity, bioassays and diagnoses can be performed quickly and conveniently with minimal manpower. a summary of methods for the fabrication of p-dmf devices is shown in table . even though µpads provide extraordinary advantages, including simple fabrication and mass-scalability at an affordable cost, if an advanced paper-based analytical device providing high performance and sensitivity is to be achieved, suitable detection mechanisms are necessary for the µpads. the most common detection techniques in µpads are colorimetric, electrochemical, chemiluminescent, and electrochemiluminescent techniques. herein, we briefly summarize and compare such detection techniques. colorimetric assays have been widely used in µpads for the simultaneous detection of target analytes, which is usually related to a biological reaction or color-change chemical assay, and have provided qualitative or semiquantitative results [ ] . the results can be visualized qualitatively and semiquantitatively by a naked eye due to a biological or chemical interaction ( figure ). the first µpad was demonstrated for the colorimetric detection of glucose and proteins and was based on a change in color when the sample was introduced into the reaction zone [ ] . however, the detection sensitivity in conventional paper-based devices is relatively low because they suffer from limitations on the physical flow behavior and the time delay of the reaction. the use of metal and metal oxide nanoparticles [ , ] , biological catalysts [ ] , and chitosan [ ] to modify the paper's surface has been proposed to enhance the detection signals from µpads used for automated and multiplexed colorimetric analyses. in addition, the design of µpads as multilayers and origami platforms can significantly improve the colorimetric signal for highly sensitive detection. because the sample solution added onto multilayered µpads can be automatically pretreated in the microfluidic channel before carrying out the colorimetric reactions at the detection zones, the color intensity and reproducibility are greatly increased [ , , , ] . integrating colorimetric µpads with cell culture microarrays has been developed for high-throughput drug screening. a µpad containing a drug-concentration gradient was designed and fabricated for high-throughput testing of the cell's response to drugs. this drug screening platform can be applied for drug discovery and for diagnostic studies with simultaneous and parallel tests of drugs under various concentration gradients [ ] . several conventional µpads used for colorimetric detection provide low sensitivity of detection compared to uv-visible spectroscopy and absorption spectroscopy. they are mainly limited by the interpretation of color intensity via the naked eye, which is different for each individual, by the nature of the ambient light, and by the properties of the paper substrate [ ] . colorimetric µpads require additional devices, such as a camera, scanner, and smartphone-based reporting system, to increase their accuracy, detection sensitivity, and high-throughput ability. the intensity of a color signal can be interpreted using imaging software, where a pixel value is related to the concentration of the analyte. the data can be analyzed by using such a system [ ] [ ] [ ] [ ] . remarkably, the p-dmf chip, which is governed by the same basic principle as other dmf devices involved with fluid manipulation by using ewod. the p-dmf device is able to manipulate small-volume droplets individually without the channels, pumps, and microvalves required by other devices; hence, it is proper for multistep, as well as for other single-step, assay procedures [ , ] . not only the p-dmf devices can transport fluids and delay fluid delivery, they also efficiently mix several samples with reagents for analysis and for tunable dilutions. moreover, p-dmf devices are cost-effective, portable, and disposable, which makes them more suitable for point-of-care testing than other dmf devices on glass or polymer substrates. recently, p-dmf devices have emerged as microfluidic platforms for colorimetric detection. abadian et al., with the goal of expanding microfluidic capabilities for poct, developed a new p-dmf device. in the detection, the device performed preprocessing steps of controlling the flow of the liquid sample and transferring it to a paper-based channel where the reagent was immobilized for glucose detection [ ] . the detection performance of this device indicated that it could be applied as a poct device in multistep microfluidics assays, such as a biosensor in enzyme-linked immunosorbent assays (elisa). jafry et al. demonstrated a novel p-dmf chip based on double-sided electrohydrodynamic jet printing of circuitry and the dispensing of high-viscosity silver nanoparticles as a two-dimensional array of electrodes on a single paper sheet. the proposed p-dmf device effectively performed as a low-cost, disposable poct platform for pesticide detection ( figure ) [ ] . the p-dmf platform could be operated with a low operating voltage to move the droplet toward an actuated electrode; such a low operating voltage would not be harmful to bioassays and would be appropriate for portable poct applications. atabakhsh et al. introduced thermocapillary several conventional µpads used for colorimetric detection provide low sensitivity of detection compared to uv-visible spectroscopy and absorption spectroscopy. they are mainly limited by the interpretation of color intensity via the naked eye, which is different for each individual, by the nature of the ambient light, and by the properties of the paper substrate [ ] . colorimetric µpads require additional devices, such as a camera, scanner, and smartphone-based reporting system, to increase their accuracy, detection sensitivity, and high-throughput ability. the intensity of a color signal can be interpreted using imaging software, where a pixel value is related to the concentration of the analyte. the data can be analyzed by using such a system [ ] [ ] [ ] [ ] . remarkably, the p-dmf chip, which is governed by the same basic principle as other dmf devices involved with fluid manipulation by using ewod. the p-dmf device is able to manipulate small-volume droplets individually without the channels, pumps, and microvalves required by other devices; hence, it is proper for multistep, as well as for other single-step, assay procedures [ , ] . not only the p-dmf devices can transport fluids and delay fluid delivery, they also efficiently mix several samples with reagents for analysis and for tunable dilutions. moreover, p-dmf devices are cost-effective, portable, and disposable, which makes them more suitable for point-of-care testing than other dmf devices on glass or polymer substrates. recently, p-dmf devices have emerged as microfluidic platforms for colorimetric detection. abadian et al., with the goal of expanding microfluidic capabilities for poct, developed a new p-dmf device. in the detection, the device performed preprocessing steps of controlling the flow of the liquid sample and transferring it to a paper-based channel where the reagent was immobilized for glucose detection [ ] . the detection performance of this device indicated that it could be applied as a poct device in multistep microfluidics assays, such as a biosensor in enzyme-linked immunosorbent assays (elisa). jafry et al. demonstrated a novel p-dmf chip based on double-sided electrohydrodynamic jet printing of circuitry and the dispensing of high-viscosity silver nanoparticles as a two-dimensional array of electrodes on a single paper sheet. the proposed p-dmf device effectively performed as a low-cost, disposable poct platform for pesticide detection ( figure ) [ ] . the p-dmf platform could be operated with a low operating voltage to move the droplet toward an actuated electrode; such a low operating voltage would not be harmful to bioassays and would be appropriate for portable poct applications. atabakhsh et al. introduced thermocapillary actuation working with low-voltage batteries to manipulate, transport, and confine the droplets on a p-dmf platform. this proposed device could enhance the controllability of water-droplet transport and decrease the droplet's surface tension; it was applied for a biochemical glucose colorimetric detection assay [ ] . molecules , , of longer detection time. in addition, additional tools, such as cameras or scanners, must be used to amplify the colorimetric signal, leading to more operational steps and higher analysis costs. µpads using electrochemical detection have emerged as a promising alternative to high-sensitivity quantitative measurements, as will be discussed in detail below. an electrochemical technique has become one of the most promising methods of analytical detection for µpads because of the availability of relatively low-cost potentiostats, the ease of their use, and their easy miniaturization for on-site measurements. in contrast to colorimetric detection, this technique can provide both qualitative and quantitative information in the nanomolar range with high sensitivity and selectivity, enabling low detection limits and fast responses. moreover, various material-modified electrodes on µpads have led to increased surface areas and increased conductivities, which significantly improve the response signal of µpads used for electrochemical detection. therefore, the results from a number of research programs involving the development of electrochemical µpads (e-µpads) using metallic nanoparticles (au, ag, pt nps), carbon-based nanomaterials (graphene, cnts), and chemical/biocatalysts (enzymes, antibodies) for analytical measurements of target analytes have been reported. currently, e-µpads exhibit the possibility of performing multianalyses, which sets the stage for the development of various promising applications, especially in poct and biomedical diagnosis. wu et al. fabricated a multiplexed e-µpad by using wax printing and screen printing for the simultaneous detection of multiple tumor biomarkers in a clinical sample. this design provided wide linearity and a low detection limit with a good correlation to the data obtained using standard equipment [ ] . the design of e-µpad platforms without a wax printer has been introduced as an alternative for simultaneous multiple-analyte determination. fava et al. revealed the fabrication of a disposable e-µpad using an adhesive mask containing - channels that could provide a multiplexing capability with a low detection limit of × − mol l − for glucose detection in urine (figure ) [ ] . additionally, fava et al. utilized working electrodes on one e-µpad for the multiplexed analyses of four biomarkers in real urine samples [ ] . colorimetric detection has been widely employed in many µpad designs because of the simplicity, ease of operation, handheld capability, and practical applications of µpads. nevertheless, colorimetric detection still suffers from the main drawbacks of low selectivity, low sensitivity, and longer detection time. in addition, additional tools, such as cameras or scanners, must be used to amplify the colorimetric signal, leading to more operational steps and higher analysis costs. µpads using electrochemical detection have emerged as a promising alternative to high-sensitivity quantitative measurements, as will be discussed in detail below. an electrochemical technique has become one of the most promising methods of analytical detection for µpads because of the availability of relatively low-cost potentiostats, the ease of their use, and their easy miniaturization for on-site measurements. in contrast to colorimetric detection, this technique can provide both qualitative and quantitative information in the nanomolar range with high sensitivity and selectivity, enabling low detection limits and fast responses. moreover, various material-modified electrodes on µpads have led to increased surface areas and increased conductivities, which significantly improve the response signal of µpads used for electrochemical detection. therefore, the results from a number of research programs involving the development of electrochemical µpads (e-µpads) using metallic nanoparticles (au, ag, pt nps), carbon-based nanomaterials (graphene, cnts), and chemical/biocatalysts (enzymes, antibodies) for analytical measurements of target analytes have been reported. currently, e-µpads exhibit the possibility of performing multianalyses, which sets the stage for the development of various promising applications, especially in poct and biomedical diagnosis. wu et al. fabricated a multiplexed e-µpad by using wax printing and screen printing for the simultaneous detection of multiple tumor biomarkers in a clinical sample. this design provided wide linearity and a low detection limit with a good correlation to the data obtained using standard equipment [ ] . the design of e-µpad platforms without a wax printer has been introduced as an alternative for simultaneous multiple-analyte determination. fava et al. revealed the fabrication of a disposable e-µpad using an adhesive mask containing - channels that could provide a multiplexing capability with a low detection limit of × − mol l − for glucose detection in urine (figure ) [ ] . additionally, fava et al. utilized working electrodes on one e-µpad for the multiplexed analyses of four biomarkers in real urine samples [ ] . additionally, the simultaneous detection of multiple targeted analytes from a single sample by using electroanalytical techniques is challenging due to poor selectivity and sensitivity for other species in the mixture caused by changes in the assay conditions towards a target analyte. to overcome this issue, henry's group reported janus e-µpads as a new approach for high-performance multiplexed electrochemical sensing under different solution conditions on a single sample at the same time by using a single potentiostat. the janus e-µpad was utilized for the simultaneous detection of multiple neurotransmitters with the capability for in situ ph control and optimized electrochemical experiments. this device showed great potential as a low-cost platform that could provide multiplexed analysis by using simple steps (figure ) [ ] . the janus e-µpad was also applied for high-throughput determination of the concentrations of different metals, including cd, pb, cu, fe, and ni metals, from a single sample of airborne particulate matter, and the results were in agreement with those obtained using a traditional method ( % confidence) [ ] . additionally, the simultaneous detection of multiple targeted analytes from a single sample by using electroanalytical techniques is challenging due to poor selectivity and sensitivity for other species in the mixture caused by changes in the assay conditions towards a target analyte. to overcome this issue, henry's group reported janus e-µpads as a new approach for high-performance multiplexed electrochemical sensing under different solution conditions on a single sample at the same time by using a single potentiostat. the janus e-µpad was utilized for the simultaneous detection of multiple neurotransmitters with the capability for in situ ph control and optimized electrochemical experiments. this device showed great potential as a low-cost platform that could provide multiplexed analysis by using simple steps (figure ) [ ] . the janus e-µpad was also applied for high-throughput determination of the concentrations of different metals, including cd, pb, cu, fe, and ni metals, from a single sample of airborne particulate matter, and the results were in agreement with those obtained using a traditional method ( % confidence) [ ] . moreover, electrochemical detection seems well suited for integration with dmf technology because the processing steps required to form electrodes for droplet operation in the dmf devices are similar to those required to form electrodes for electrochemical detection. this combination represents a versatile system with high sensitivity, minimal reagent consumption, and automation capabilities for microscale analysis of many targeted analytes [ , ] . dmfs on paper substrates using electrochemical detection have become subjects of interest lately because the processes used to fabricate these devices are simple and incur lower costs. ruecha et al. developed the first working electrochemical p-dmf sensors for multiplexed human healthcare monitoring by using printing and modular fabrication; these devices were operated using a portable, wireless control system. an electrochemical sensor and the electrodes on the device platform for electrowetting actuation of digital drops were fabricated using affordable printing techniques (figure ). the working electrode was modified with reduced graphene and au nps to enhance the sensitivity of the sensor. this hybridized open-close chip format demonstrated excellent multiplexed detection, which provided a wide linear range of low-level detection for three biological molecules in human serum [ ] . during the fabrication step, a challenge for the design of the p-dmf device is the interference between the moving droplets and the voltages on the control lines because the electrode arrays and the control lines are printed on the same side of a paper sheet. to resolve this issue, researchers proposed control-fluidic codesign methodologies, which simultaneously adjusted the control line routing and the arrangement of the fluid droplets. the proposed method successfully eliminated the interferences during operation of the p-dmf device and achieved an optimal solution [ , ] . the improved droplet and electrode-oriented flows might increase the potential for p-dmf devices to be used as electrochemical detectors. all the publications reviews revealed the high potential for the use of electrochemical detection techniques with µpads in the simultaneous detection of multiple analytes. electrochemical techniques are open to further miniaturization, increased portability, and higher throughput ability for in-field use. moreover, electrochemical detection seems well suited for integration with dmf technology because the processing steps required to form electrodes for droplet operation in the dmf devices are similar to those required to form electrodes for electrochemical detection. this combination droplet and electrode-oriented flows might increase the potential for p-dmf devices to be used as electrochemical detectors. all the publications reviews revealed the high potential for the use of electrochemical detection techniques with µpads in the simultaneous detection of multiple analytes. electrochemical techniques are open to further miniaturization, increased portability, and higher throughput ability for in-field use. chemiluminescence is an efficient analytical technique that has been successfully performed with µpads by taking advantage of their simplicity and high sensitivity. for detection, this technique can reduce background noise without the need for an external light source, but a portable chemiluminescence reader must be operated in the dark, which makes device fabrication more complex. this detection method was used with µpads for biomarker detection [ ] , immunoassay [ ] , biomolecule detection [ ] , environmental monitoring [ ] , and food safety control [ ] . in recent years, chemiluminescence sensing has become popular for clinical biomarker detection and immunoassays due to its wide dynamic range and complete automation. yang et al. demonstrated a novel paper-based chemiluminescence analytical device based on immunocomplex formation for the simultaneous detection of heart disease biomarkers. this device could be applied for simple, sensitive, and selective detection of three biomarkers in human serum and had low detection limits in the picogram range [ ] . guo et al. fabricated a µpad by using a dipping method based on a chemiluminescence immunoreaction system for multiplexed detection of three cancer biomarkers in human serum samples [ ] . han et al. fabricated a novel paper-based immunoassay device for the chemiluminescence detection of allergen-specific immunoglobulin e (sige) in patients with allergyrelated diseases. this strategy has led to a novel platform for use in clinical diagnosis [ ] . even though paper-based microfluidic concepts with chemiluminescence detection show great potential for the development of analytical devices with a variety of applications, these devices still chemiluminescence is an efficient analytical technique that has been successfully performed with µpads by taking advantage of their simplicity and high sensitivity. for detection, this technique can reduce background noise without the need for an external light source, but a portable chemiluminescence reader must be operated in the dark, which makes device fabrication more complex. this detection method was used with µpads for biomarker detection [ ] , immunoassay [ ] , biomolecule detection [ ] , environmental monitoring [ ] , and food safety control [ ] . in recent years, chemiluminescence sensing has become popular for clinical biomarker detection and immunoassays due to its wide dynamic range and complete automation. yang et al. demonstrated a novel paper-based chemiluminescence analytical device based on immunocomplex formation for the simultaneous detection of heart disease biomarkers. this device could be applied for simple, sensitive, and selective detection of three biomarkers in human serum and had low detection limits in the picogram range [ ] . guo et al. fabricated a µpad by using a dipping method based on a chemiluminescence immunoreaction system for multiplexed detection of three cancer biomarkers in human serum samples [ ] . han et al. fabricated a novel paper-based immunoassay device for the chemiluminescence detection of allergen-specific immunoglobulin e (sige) in patients with allergy-related diseases. this strategy has led to a novel platform for use in clinical diagnosis [ ] . even though paper-based microfluidic concepts with chemiluminescence detection show great potential for the development of analytical devices with a variety of applications, these devices still have some drawbacks. in a paper-based format, assays, in particular elisa, using such devices have low sensitivity and limited throughput capability. therefore, p-dmf devices using chemiluminescence detection have been introduced to effect fully automated, complex, multistep assay protocols. fobel et al. demonstrated for the first time a sandwich elisa using chemiluminescence detection ( figure ). the p-dmf device with more than one-hundred working parts was constructed by printing arrays of silver electrodes and reservoirs connected to contact pads onto a paper substrate. the proposed device was inexpensive, easy to use, and fast; one device cost less than $ . and could be fabricated in approximately min. the device was applied for homogeneous, chemiluminescence elisa of rubella igg, which required discrete steps for each concentration evaluated and showed a detection limit of . iu/ml [ ] . this indicates that p-dmf devices based on inexpensive platforms can provide multiplexed performance and high detection sensitivity for chemiluminescence immunoassays. overall, the chemiluminescence detection technique is providing new abilities for µpads; however, the utility of chemiluminescence detection will depend on advances in cost reduction and increased simplicity of device fabrication. figure ). the p-dmf device with more than one-hundred working parts was constructed by printing arrays of silver electrodes and reservoirs connected to contact pads onto a paper substrate. the proposed device was inexpensive, easy to use, and fast; one device cost less than $ . and could be fabricated in approximately min. the device was applied for homogeneous, chemiluminescence elisa of rubella igg, which required discrete steps for each concentration evaluated and showed a detection limit of . iu/ml [ ] . this indicates that p-dmf devices based on inexpensive platforms can provide multiplexed performance and high detection sensitivity for chemiluminescence immunoassays. overall, the chemiluminescence detection technique is providing new abilities for µpads; however, the utility of chemiluminescence detection will depend on advances in cost reduction and increased simplicity of device fabrication. the electrochemiluminescence technique is based on electro-generated luminescence due to optical emission from an excited state of an electrochemiluminescent luminophore produced at the electrode's surface by electrochemical reactions. the integration of electrochemical and spectroscopic methods provides better selectivity, increased dynamic concentration range, and multiplexed bioassay properties. compared to chemiluminescence alone, this integrated technique stands out for its unique properties, such as lower background and greater control of the emission location and the reaction time. this facilitates the detection of multiple analytes and improves the reproducibility of the electrochemiluminescence technique is based on electro-generated luminescence due to optical emission from an excited state of an electrochemiluminescent luminophore produced at the electrode's surface by electrochemical reactions. the integration of electrochemical and spectroscopic methods provides better selectivity, increased dynamic concentration range, and multiplexed bioassay properties. compared to chemiluminescence alone, this integrated technique stands out for its unique properties, such as lower background and greater control of the emission location and the reaction time. this facilitates the detection of multiple analytes and improves the reproducibility of an analytical process. such µpads have been used for multiplexed detection of electrochemiluminescence from biomarkers [ , ] , immunoassays [ , ] , biomolecules [ ] , and drugs [ ] . to perform an electrochemiluminescence immunoassay with a µpad for sensitive and multiplexed analysis, ge et al. constructed a three-dimensional electrochemiluminescence µpad based on wax-patterned technology and screen-printed electrodes for the detection of four tumor markers in serum samples ( figure ) [ ] . electrochemiluminescence µpads with bipolar electrodes have been used to promote both reduction and oxidation reactions without the need for an external power supply [ , , ] . zhang et al. developed a bipolar electrochemiluminescence paper-based platform consisting of three electrodes integrated into parallel into a single device for rapid, high-throughput detection of different biomarkers [ ] . liu et al. fabricated bipolar electrochemiluminescence µpads in which multiple electrodes were situated in two parallel channels to achieve multiplexed detection of glucose in four complex samples [ ] . an external power supply [ , , ] . zhang et al. developed a bipolar electrochemiluminescence paper-based platform consisting of three electrodes integrated into parallel into a single device for rapid, high-throughput detection of different biomarkers [ ] . liu et al. fabricated bipolar electrochemiluminescence µpads in which multiple electrodes were situated in two parallel channels to achieve multiplexed detection of glucose in four complex samples [ ] . furthermore, the electrochemiluminescence technique has been integrated with dmf technology to achieve multiplexed assays with higher throughput. a dmf with electrochemiluminescence was demonstrated for the first time in ; that platform could move, dispense, mix, and split the droplets on arrays of insulated electrodes and was compatible with a wide range of analytical detection approaches. this novel platform was applied with a nucleic acid hybridization assay for the detection of mirna and exhibited an analytical performance with a . -fm detection limit for small sample volumes of . µl [ ] . to our knowledge, no p-dmf devices using electrochemiluminescence detection have been reported so far. nonetheless, the integration of electrochemiluminescence with dmfs has opened opportunities for p-dmf platforms, which offer lower costs, faster in-place fabrication with printers and ink, and better disposability. in the near future, fluid manipulation using dmf technology may be combined with capillary wetting features to create paper "hybrid" devices that take advantage of the significant capabilities of both formats for high-throughput electrochemiluminescence detection. as highlighted in the previous sections, µpads are promising platforms that can be applied in various fields owing to their advantages of low cost, disposability, and ease of use when compared to traditional microfluidic devices [ , , ] . the current applications of µpads are summarized in table . due to the urge and importance of medical testing, µpads development has focused more on medical diagnosis with high-throughput screening. research has been conducted using real samples, such as human serum, blood, and urine, for the detection of multiple analytes. the common biomolecules studied in clinical diagnosis, including glucose [ ] , lactate [ ] , and uric acid [ ] , have been successfully identified by using µpads with colorimetric sensing as the main technique furthermore, the electrochemiluminescence technique has been integrated with dmf technology to achieve multiplexed assays with higher throughput. a dmf with electrochemiluminescence was demonstrated for the first time in ; that platform could move, dispense, mix, and split the droplets on arrays of insulated electrodes and was compatible with a wide range of analytical detection approaches. this novel platform was applied with a nucleic acid hybridization assay for the detection of mirna and exhibited an analytical performance with a . -fm detection limit for small sample volumes of . µl [ ] . to our knowledge, no p-dmf devices using electrochemiluminescence detection have been reported so far. nonetheless, the integration of electrochemiluminescence with dmfs has opened opportunities for p-dmf platforms, which offer lower costs, faster in-place fabrication with printers and ink, and better disposability. in the near future, fluid manipulation using dmf technology may be combined with capillary wetting features to create paper "hybrid" devices that take advantage of the significant capabilities of both formats for high-throughput electrochemiluminescence detection. as highlighted in the previous sections, µpads are promising platforms that can be applied in various fields owing to their advantages of low cost, disposability, and ease of use when compared to traditional microfluidic devices [ , , ] . the current applications of µpads are summarized in table . due to the urge and importance of medical testing, µpads development has focused more on medical diagnosis with high-throughput screening. research has been conducted using real samples, such as human serum, blood, and urine, for the detection of multiple analytes. the common biomolecules studied in clinical diagnosis, including glucose [ ] , lactate [ ] , and uric acid [ ] , have been successfully identified by using µpads with colorimetric sensing as the main technique for detection. to improve the throughput and the sensitivity of µpads, researchers have developed and constructed p-dmf devices for use as diagnostic platforms for highly sensitive detection of multiple biomolecules [ , , ] . nucleic acids, including deoxyribonucleic acid (dna) and ribonucleic acid (rna), have long been interesting study subjects because of their importance in genetics, molecular analysis, detection, diagnosis, and monitoring. nevertheless, nucleic acids exist in very small amounts in living cells: for example, only hundreds of nanograms of dna can be extracted from one microliter of blood [ , ] ). for that reason, the pcr and its modifications (quantitative pcr, reverse-transcriptase pcr, nested pcr, and multiplex pcr) are conventional methods used to amplify nucleic acids. although these methods are very efficient, they often require special equipment and large amounts of laboratory space, consume significant amounts of power, and need to be applied by skilled, trained technicians. since the explosion of microfluidic analytical devices, researchers around the world have directed their attention to the development and application of pcr chips as very promising goals. various approaches based on distinct platforms from continuous-flow pcr chips to digital droplet pcr devices have been proposed, but at the time of this review, no work, to the best of our knowledge, has been reported for "paper-based pcrs". this is reasonable because thermal cycles, especially at high temperatures, and the time-dependent continuous-flow of fluid are usually not recommended for paper or paper-related materials. this roadblock, however, has paved the way for the successful application of isothermal amplification techniques in µpads for poct. these techniques include, but are not limited to, nucleic acid sequence-based amplification (nasba), transcription-mediated amplification (tma), self-sustained sequence replication ( sr), helicase-dependent amplification (hda), rolling-circle amplification (rca), signal-mediated amplification of rna technology (smart), strand-displacement amplification (sda), recombinase polymerase amplification (rpa), and loop-mediated isothermal amplification (lamp), which is probably the most well-known of these techniques [ , ] . generally, the above-mentioned techniques target specific and unique sequences of dna [ , ] or rna [ , ] and in many cases, microrna (mirna) [ ] [ ] [ ] . paper-based nucleic acid amplification and detection are useful in diseases diagnosis [ , ] , forensic analysis [ ] , food [ ] and beverage [ ] control, and environmental testing [ ] . recently, several reviews on nucleic acid testing (nat) using paper-based devices have been reported [ ] [ ] [ ] , so in this section, we only select and discuss those notable accomplishments that we believe have potential for, or may contribute significantly to, high-throughput screening. as we discussed in the fabrication section of this review, µpads for nat should have integrated functions, as well as the capability to yield reproducible data, even though automation and mass production might still be limited. in this manner, choi et al., for the first time, reported a paper-based biosensor that incorporated nucleic acid extraction, amplification, and visual detection or quantification by using a smartphone to detect escherichia coli and streptococcus pneumonia [ ] . their device consisted of four layers, including from top to bottom, (i) a lateral flow layer for visual detection, (ii) a glass fiber layer for immersion of nucleic acid, (iii) an fta card for paper-based nucleic acid extraction, and (iv) a bottom layer for sample purification and washing. to control fluid flow between connecting layers, the authors used hydrophobic polyvinyl-chloride (pvc) backing pads as "valves". the lamp reaction was optimized at • c for min by using a handheld battery-powered heating device. next, denaturation of double-strand dna was performed at • c for s so that the product could be hybridized with the probe-labeled gold nanoparticles in the top layer. finally, a lateral flow assay occurred in a disposable centrifuge tube. the entire "sample-to-answer" assay could be completed within h. this system, however, suffered from the lack of both reproducibility and the capability to perform multiplex detection; furthermore, it was still complicated due to different temperatures that had to be applied. another effort came from rodriguez et al., who provided a platform for molecular diagnosis of cervical cancer by combining nucleic acid isolation, isothermal amplification, and lateral flow assay for visual detection of human papillomavirus (hpv) dna [ ] . the device contained (i) an adhesive base made from laminating sheets, (ii) a polyethersulfone (pes) filter paper disk for sample loading and nucleic acid extraction, (iii) an absorbent pad made from cellulose blotting paper for washing, and (iv) a lateral flow immunoassay (lfia) detection strip, all assembled via folding and alignment. for testing, the clinical cervical specimen was initially resuspended in a lysis buffer, after which it was pipetted onto the sample port of the chip, where it wicked through the absorbent pad. after several washings, the absorbent pad was removed. next, a lamp reaction mix was added to the sample port, and another folding step was taken to seal and protect the sample from evaporation. the chip was placed on a • c heating block or hot plate for min for amplification. after incubation, part of the device was peeled off to expose the pes membrane to the lfia. once eluted by water, the amplified products could be introduced to the test strip, and results could be obtained within two minutes. though innovative, this platform required many steps of complicated folding and fluid manipulation, which limited its usefulness for poct. later in , the same group reported a similar, but simpler platform with the addition of internal amplification control for the detection of sexually transmitted infections caused by neisseria gonorrhoeae [ ] . to address multiplex nat, in , cooper's group published a notable study regarding the use of a paper-based origami device to diagnose malaria by using whole blood from finger prick [ ] . their device consisted of five panels fabricated by wax printing one folded onto another and a plastic cover for lamp incubation. lamp reagents were predeposited onto four different spots for amplification of different species; including spots for internal control, plasmodium pan, plasmodium falciparum, and plasmodium vivax. the sample was added onto the folded panels, followed by the addition of lysis and a washing buffer for dna extraction and purification. next, the folded panel was turned over, and elution started to deliver purified dna to the final panel for amplification and detection. lamp reactions were carried out at • c for min on a simple hotplate, after which results could be simply readout using a handheld uv lamp. later in , the same group upgraded their device to a more user-friendly platform and applied it to poct in low-resource rural communities in uganda [ ] . with the embedded finger-activated pumps for fluid transportation and the inclusion of lateral flow assay, the results could be easily read without the need for an additional readout device. sixty-seven patients were tested, and the device provided an impressive sensitivity of % for plasmodium pan and plasmodium falciparum. considerable improvements in nat using µpads have been achieved. to avoid the relatively high temperature and long reaction time of lamp, margo et al. used reverse transcription-rpa for multiplexed detection of different rna sequences of the ebola virus [ ] , and ahn et al. utilized rpa for simultaneous detection of multiple foodborne pathogens [ ] . these isothermal reactions were complete min after the injection of isolated rna or dna samples at temperatures ranging from to • c. integrating this with mirna extraction, deng et al. employed the exponential amplification reaction (expar), which allowed amplification at • c in min by using a portable heating block for detection of mir- and mir- , two potential biomarkers associated with lung cancer [ ] . to increase the sensitivity of fluorimetric detection of mirna in µpad, liang et al. proposed a method of growing an interconnected, dense flower-like silver (fls) layer on the surface of a cellulose fiber in the fluorescence detection zone [ ] . this fls-µpad rendered not only a reduced background fluorescence signal but also a sensitive metal-enhanced fluorescence resonance-energy transfer (fret) efficiency. on the other hand, for colorimetric detection, teengam et al. promoted the use of a cationic pyrrolidinyl peptide nucleic acid (pna) probe as a replacement for the dna and the rna probes [ ] . the anionic silver nanoparticles (agnps) interacted with the cationic pna probe and aggregated. if a complementary target sequence was present, it outcompeted and disaggregated the agnps, leading to a change of color in the detection zone. albeit promising, if a convenient, all-in-one device is to be had, these tracks need to incorporate more functions. to adapt to the new trend toward simpler, faster, but effective detection in nat, bender et al. presented a µpad that engaged isotachophoresis (itp) and rpa to simultaneously extract and amplify target nucleic acids [ ] . the device consisted of a plasma separation membrane placed on top of a porous glass fiber strip, which was located between two liquid buffer reservoirs and was housed within a sealed petri dish; two electrodes were embedded in the plastic lid for this system. a whole blood sample was pipetted onto the membrane where blood cells were held as the filtered plasma wicked into the proteinase pretreated sample pad region of the glass fiber strip. next, a mixture of leading electrolyte (le) and rpa reagents was added to the wet region from the glass fiber strip to the le reservoir. reservoirs were then filled with le and trailing electrolyte (te) solutions. next, the chip was sealed by closing the lid. once an electric field had been applied, nucleic acids were separated from the whole blood sample, and together with the rpa reagents, they gathered within an itp plug where amplification took place; results could be observed with a fluorescence microscope. interestingly, without the use of any specific heating device, the ipt-rpa still occurred by leveraging the joule heating effect when a current was used to attain an appropriate temperature for rpa within the itp plug. when the temperature was controlled using a hot plate, the total processing time was less than min, including min for filtration and digestion and min for separation and amplification. another creative idea came from kaarj et al., who separated zika virus rna from different samples just by capillary action in a single-layer printed wax-paper channel [ ] . bulky biomolecules remained at the beginning of the channel while virus rna could pass through and be focused at the end of the chip, where it was cut out and amplified by using an rt-lamp reaction coupled with a ph indicator-based colorimetric assay. although these systems still need further improvement, they have encouraged researchers to explore all possible fabrication and detection methods for nat using a µpad. we finish by discussing a published work from phillips et al. for a poc microfluidic rapid and autonomous analysis device (microraad) for the detection of the human immunodeficiency virus (hiv) [ ] . the apparatus was built using a laminated and replaceable µpad, a kapton tape with printed resistive heating elements, and a temperature control circuit that could connect to a power source like a cell phone or portable battery, all assembled inside a plastic case as shown in figure a . the main component, the µpad, was composed of a glass fiber membrane for wash buffer distribution, a blood separator membrane (mf ), a pes filter membrane sandwiched between two dried polyethylene terephthalate (pef) films preloaded with rt-lamp reagents, wax valve strips for fluidic manipulation control, and an lfia strip for visual detection. a whole blood sample was dropped into the sample inlet, followed by the addition of rt-lamp rehydrating mixture, with a washing buffer being added into the buffer inlet. the two inlets were sealed with adhesive tape to avoid evaporation. here, the wax valves acted as barriers to constrain flows from the buffer zone to the amplification zone and from the amplification zone to the lfia. fluid movements were illustrated in figure b through three main steps: (i) once powered, the amplification zone was heated first, reaching • c within seconds and remaining at that temperature for min. the pre-set temperature control circuit then automatically terminated heating in this zone and initiated heating of the two wax valves at the same time. (ii) upon heating, the wax valves opened, and washing buffer was passed through the amplification zone, carrying amplicons, if any, to the lfia. hence, result bands could be observed to min after the valves had been opened (iii). in fact, to the best of our knowledge, this is the most complete system reported to date, yet it was not equipped with internal control and multiplex testing; furthermore, the performance time was also longer than those for other systems. nevertheless, undoubtedly, these weaknesses can be overcome by careful adjustment and modification. this example, once again, points to a wide application of µpads in nat for poct in the very near future. in the diagnosis of diseases of interest, the use of sensitive, disposable µpads will allow early diagnosis and greatly increase the chances for successful treatment. several researchers have focused on the development of µpads for multiplexed detection of cancer biomarkers and tumor cells based on the immunoassay method. these µpads were mainly integrated with chemiluminescence [ ] and electrochemiluminescence techniques [ ] . for the immunoassay, some researchers have developed µpads using the colorimetric method, but the use of those devices was often limited by insufficient sensitivity and a higher detection limit in comparison to other spectrometric methods. thus, a p-dmf device using chemiluminescence detection has been proposed to enhance the detection sensitivity and the multiplexing capability of immunoassays [ ] . moreover, µpads have been demonstrated to have considerable potential for poct to help in the diagnosis of diseases caused by bacteria. infectious bacterial diseases have always been problematic worldwide; infections arising from common bacteria, such as acinetobacter baumannii, escherichia coli, and staphylococcus aureu, have increased drastically in recent years. hence, the demand for a simple device with a fast response for the simultaneous screening of multiple bacteria is high. an aptamer µpad for multiplex analysis of whole-cell bacteria based on aptamers bound to nitrocellulose membranes integrated into a chip has been developed. the detection involves a change in the color intensity as a bacterial cell attaches itself to specific aptamers labeled with biotin [ ] . the capability of µpads has been further expanded to drug analysis and screening in a highthroughput manner. a simple µpad designed with a barcode feature has been utilized in the colorimetric detection of illegal drugs based on a flow lateral immunoassay system labeled with aunps [ ] . a facile e-µpad has been fabricated based on gold-paper screen-printed electrodes and the principle of kirigami and origami. this µpad was successfully used to electrochemically detect cancer cells and to screen in situ anticancer drugs in a high-throughput system with a low detection limit and wide linear range [ ] . in the development of conventional µpads for high-throughput in the diagnosis of diseases of interest, the use of sensitive, disposable µpads will allow early diagnosis and greatly increase the chances for successful treatment. several researchers have focused on the development of µpads for multiplexed detection of cancer biomarkers and tumor cells based on the immunoassay method. these µpads were mainly integrated with chemiluminescence [ ] and electrochemiluminescence techniques [ ] . for the immunoassay, some researchers have developed µpads using the colorimetric method, but the use of those devices was often limited by insufficient sensitivity and a higher detection limit in comparison to other spectrometric methods. thus, a p-dmf device using chemiluminescence detection has been proposed to enhance the detection sensitivity and the multiplexing capability of immunoassays [ ] . moreover, µpads have been demonstrated to have considerable potential for poct to help in the diagnosis of diseases caused by bacteria. infectious bacterial diseases have always been problematic worldwide; infections arising from common bacteria, such as acinetobacter baumannii, escherichia coli, and staphylococcus aureu, have increased drastically in recent years. hence, the demand for a simple device with a fast response for the simultaneous screening of multiple bacteria is high. an aptamer µpad for multiplex analysis of whole-cell bacteria based on aptamers bound to nitrocellulose membranes integrated into a chip has been developed. the detection involves a change in the color intensity as a bacterial cell attaches itself to specific aptamers labeled with biotin [ ] . the capability of µpads has been further expanded to drug analysis and screening in a high-throughput manner. a simple µpad designed with a barcode feature has been utilized in the colorimetric detection of illegal drugs based on a flow lateral immunoassay system labeled with aunps [ ] . a facile e-µpad has been fabricated based on gold-paper screen-printed electrodes and the principle of kirigami and origami. this µpad was successfully used to electrochemically detect cancer cells and to screen in situ anticancer drugs in a high-throughput system with a low detection limit and wide linear range [ ] . in the development of conventional µpads for high-throughput drug analysis and screening, the devices need to be designed as multilayers by folding sheets of paper, which complicates the fabrication process. the use of p-dmf devices is an alternative simple and inexpensive approach to performing highly sensitive multiplexed drug analysis and screening. together with the explosion and development of science and technology, activities of human beings, especially in manufacturing, construction, transportation, and agriculture, have caused global impacts on nature, which leads to retroacting on human beings. daily, a large number of pollutants are released into the air, soil, and water, bringing about air pollution, food contamination, health problems and imbalance of ecosystem, etc. accordingly, the application of µpad in environmental analysis also receives a lot of attention. in this manner, readers are redirected to well-organized reviews by meredith et al. [ ] and kung et al. [ ] . compared to other testing methods, environmental analysis seems not to be limited by a low amount of sample; however, it requires a dedicated study design and a careful sampling plan to collect representative samples from a vast heterogeneous area and preserve in proper condition to avoid sample degradation [ ] . different from bulky analysis systems, µpad lightens the workload but still fulfills requirements of environmental analysis and monitoring. for example, heavy metals and pesticides should be given more consideration for priority control because of their high toxicity, relative stability, and easy accumulation in living organisms. thus, devices that allow on-site detection and accurate monitoring of environmental conditions are needed. µpads have been developed to improve the accuracy and performance for multiplexed colorimetric detection of pesticides [ ] and heavy metals in the air [ ] and rivers [ , ] . the devices developed thus far are portable and can be used as rapid on-site monitoring platforms for the detection of multiple heavy-metal ions; they have a wide linear range and a low detection limit down to the microgram level. [ ] β agonist swine hair chemiluminescence . nm [ ] to date, significant progress in the development of µpads for high-throughput screening has been made, various platforms for a short time and multiplex assays can be used for multiple samples from different individuals. as for traditional p-cmf devices, they are limited by low sensitivity and by difficulty in enabling multistep analysis with automatic processes because p-cmf devices suffer from passive capillary wicking. to circumvent, researchers re-engineered those devices by incorporating methods to control the fluid transport in paper channels such as layer assembly, wax valve usage, or patterning hydrophobic and hydrophilic substrates inside channels. when fluid transport is programmed, complicated assay protocols can be accomplished automatically without the need for an external control system. however, the fabrication of controllable p-cmfs still needs further improvements and new breakthroughs because the devices are being applied more frequently for analytical applications. meanwhile, p-dmf devices may become a promising tool for screening multiple analytes in a variety of applications. because the p-dmf device is based on the ewod technique, which can be programmed to manage the fluid flow better, multiple target analytes can be detected with high sensitivity by using a single-or multistep assay protocol. although p-dmf devices have unique advantages in terms of fluid flow control, they still require a portable electrical switching system and a controlled software interface for the assay protocols, which make the p-dmf devices more expensive than conventional p-cmf devices. therefore, the usefulness of p-dmf devices will depend on advances in reducing the costs of detection assays and fabrication processes. overall, the development of µpads with a lower cost of fabrication and controllable protocol is strongly recommended to explore this novel approach to high-throughput screening of any potential target analyte. it is worth noting that high-throughput screening can only be obtained when the whole system is equipped with high-throughput detection and readout tools. for that reason, researchers have utilized and combined different methods to increase sensitivity, accuracy, and reproducibility of the results. to make poct and on-field monitoring more affordable, interestingly, supporting devices gradually have been scaled down to minimal but still in well-functioned forms such as printed heater [ ] , portable fluorometer [ ] , mobile power supply [ ] , handheld raman spectrometer [ ] , etc. this is indeed a very dramatic transformation that triggers the competition between manufacturers and allows small enterprises to enter the market, thus opening up the opportunity of commercialization of µpads. either in the form of accessories modifications or functional integration, the application of µpads in high-throughput screening has been extending in various fields. the more widely µpads are being used, the more innovations are to be made, which bring more benefits to the society, especially in low-income or under-developed countries. as we have shown in the previous part, µpads are most suited to on-site health screening with high-throughput capability. this plays an important role in early diagnosis, (isolation, if it is a must) and treatment to eliminate the spread of infectious or deadly diseases throughout the community, particularly when epidemic or pandemic is happening. notably, biologists have alerted that harmful microbes have the ability to accumulate mutations and evolve to resist antibiotics and drugs. to detect a new type of microbe, new µpads need to be developed. therefore, it is necessary to optimize and set up a clear procedure for the fast prototyping of new µpads. even in normal conditions, quick and regular health screening also helps to build up personal medical records, which can be used for precision medicine. when readout methods like barcodes [ ] or qr codes [ ] are being used, results can be converted to digital information. in the vision of smart e-government, if a well-mannered procedure and collection can be established, such digital information can be encrypted and stored in a centralized cloud computing system managed by the government. whenever necessary, medical experts can access the database and withdraw essential information (without personal information) to predict and evaluate the risk of a new disease in the population. to sum up, the basic foundation of µpads has been well set up for more than a decade, and the potential usage of µpads is updated frequently. by targeting different applications, ideally, researchers can use various fabrication methods to alter different compartments and ultimately build up their platforms that meet the requirements of high-throughput screening and commercialization. for sample-to-answer purposes, the future of µpad will be a fully integrated system with delicate design and user-friendly interface, providing a trustable result for poct, all coming with a low cost and ready to be used in even resource-limited regions. it might not solely contain paper, but it should be either totally disposable or partially reusable. very optimistically, the emerging and expanding of µpad would play an important role not only in the field of analytical chemistry or microfluidic study but also in generally any side of life, via a process of detecting and solving, improving quality of life. the authors declare no conflict of interest. reference module in chemistry chemical analysis of food: techniques and applications a gas chromatographic air analyzer fabricated on a silicon wafer introduction: the origin, current status, and future of microfluidics fernández-la-villa, a. integrated microfluidic electrochemical sensors to enhance automated flow analysis systems patterned paper as a platform for inexpensive novel paper-based cholesterol biosensor using graphene/polyvinylpyrrolidone/polyaniline nanocomposite label-free paper-based electrochemical impedance immunosensor for human interferon gamma detection a copper oxide-ionic liquid/reduced graphene oxide composite sensor enabled by digital dispensing: non-enzymatic paper-based microfluidic determination of creatinine in human blood serum electrochemical immunosensor based on gold-labeled monoclonal anti-lipl for leptospirosis diagnosis sensitive electrochemical sensor using a graphene-polyaniline nanocomposite for simultaneous detection of zn (ii), cd (ii), and pb (ii) acid-base titrations using microfluidic paper-based analytical devices chelate titrations of ca + and mg + using microfluidic paper-based analytical devices a low-cost and simple paper-based microfluidic device for simultaneous multiplex determination of different types of chemical contaminants in food multiplexed paper microfluidics for titration and detection of ingredients in beverages advances in microfluidic paper-based analytical devices for food and water analysis low voltage electrowetting-on-dielectric active digital microfluidic paper chips with inkjet-printed patterned electrodes paper microfluidics goes digital toward practical application of paper-based microfluidics for medical diagnostics: state-of-the-art and challenges electrochemical paper-based devices: sensing approaches and progress toward practical applications a fully integrated paperfluidic molecular diagnostic chip for the extraction, amplification, and detection of nucleic acids from clinical samples a paperfluidic platform to detect neisseria gonorrhoeae in clinical samples colorimetric paper bioassay for the detection of phenolic compounds fast and flexible strategy to produce electrochemical paper-based analytical devices using a craft cutter printer to create wax barrier and screen-printed electrodes skiving stacked sheets of paper into test paper for rapid and multiplexed assay a review of current methods in microfluidic device fabrication and future commercialization prospects recent developments in paper-based microfluidic devices fabrication techniques for microfluidic paper-based analytical devices and their applications for biological testing: a review fabrication, flow control, and applications of microfluidic paper-based analytical devices programmable paper-based microfluidic devices for biomarker detections viscosity measurements utilizing a fast-flow microfluidic paper-based device a review on wax printed microfluidic paper-based devices for international health colored wax-printed timers for two-dimensional and three-dimensional assays on paper-based devices fabrication and characterization of paper-based microfluidics prepared in nitrocellulose membrane by wax printing inkjet-printed barcodes for a rapid and multiplexed paper-based assay compatible with mobile devices fully inkjet-printed distance-based paper microfluidic devices for colorimetric calcium determination using ion-selective optodes drop-slip" bulk sample flow on fully inkjet-printed microfluidic paper-based analytical device development of paper-based microfluidic analytical device for iron assay using photomask printed with d printer for fabrication of hydrophilic and hydrophobic zones on paper by photolithography electrochemical detection for paper-based microfluidics flexographically printed fluidic structures in paper a low-cost paper-based platform for fast and reliable screening of cellular interactions with materials paper-based microfluidic devices by plasma treatment patterning and modeling three-dimensional microfluidic devices fabricated on a single sheet of paper fabrication of fully enclosed paper microfluidic devices using plasma deposition and etching. lab a chip engineering fluidic delays in paper-based devices using laser direct-writing. lab a chip femtosecond-laser-structured nitrocellulose membranes for multi parameter point-of-care tests a paper-based in vitro model for on-chip investigation of the human respiratory system a simple paper-based sensor fabricated by selective wet etching of silanized filter paper using a paper mask one-step polymer screen-printing for microfluidic paper-based analytical device (µpad) fabrication glucose biosensor based on disposable electrochemical paper-based transducers fully fabricated by screen-printing a simple method to produce d and d microfluidic paper-based analytical devices for clinical analysis fabrication of low-cost paper-based microfluidic devices by embossing or cut-and-stack methods flexible time-temperature indicator: a versatile platform for laminated paper-based analytical devices direct spraying method for fabrication of paper-based microfluidic devices a chemically patterned microfluidic paper-based analytical device (c-µpad) for point-of-care diagnostics a portable smartphone-based sensing system using a d-printed chip for on-site biochemical assays three-dimensional microfluidic devices fabricated in layered paper and tape d origami-based multifunction-integrated immunodevice: low-cost and multiplexed sandwich chemiluminescence immunoassay on microfluidic paper-based analytical device a three-dimensional origami microfluidic device for paper chromatography: application to quantification of tartrazine and indigo carmine in food samples d vertical-flow paper-based device for simultaneous detection of multiple cancer biomarkers by fluorescent immunoassay three-dimensional paper-based microfluidic chip device for multiplexed fluorescence detection of cu + and hg + ions based on ion imprinting technology three-dimensional paper-based microfluidic electrochemical integrated devices ( d-pmed) for wearable electrochemical glucose detection three-dimensional paper-based microfluidic device for assays of protein and glucose in urine double-sided d printing on paper towards mass production of three-dimensional paper-based microfluidic analytical devices ( d-µpads) single step and mask-free d wax printing of microfluidic paper-based analytical devices for glucose and nitrite assays d printed paper-based microfluidic analytical devices d printed lego ® -like modular microfluidic devices based on capillary driving from microfluidic paper-based analytical devices to paper-based biofluidics with integrated continuous perfusion novel d printed microfluidic paper-based analytical device with integrated screen-printed electrodes for automated viscosity measurements flow controllable three-dimensional paper-based microfluidic analytical devices fabricated by d printing technology instrument-free fabrication of microfluidic paper-based analytical devices through d pen drawing fabricating paper based devices using correction pens low-cost fabrication of paper-based microfluidic devices by one-step plotting low-cost printing of poly(dimethylsiloxane) barriers to define microchannels in paper a novel highly flexible, simple, rapid and low-cost fabrication tool for paper-based microfluidic devices (µpads) using technical drawing pens and in-house formulated aqueous inks high-throughput rapid-prototyping of low-cost paper-based microfluidics paper-based digital microfluidic chip for multiple electrochemical assay operated by a wireless portable control system affordable fabrication of conductive electrodes and dielectric films for a paper-based digital microfluidic chip hybrid paper-based microfluidics: combination of paper-based analytical device (µpad) and digital microfluidics (dmf) on a single substrate fabrication of digital microfluidic devices on flexible paper-based and rigid substrates via screen printing double-sided electrohydrodynamic jet printing of two-dimensional electrode array in paper-based digital microfluidics paper-based digital microfluidics fabrication of paper-based microfluidic analysis devices: a review microfluidic origami for point-of-care extraction of nucleic acids from viscous samples paper-based microfluidics for dna diagnostics of malaria in low resource underserved rural communities electrophoresis of proteins on filter paper electrophoretic separations on paper: past, present, and future-a review a new apparatus for electrophoretic analysis of colloidal mixtures two orders of magnitude improvement in detection limit of lateral flow assays using isotachophoresis -fold sample focusing on paper-based microfluidic devices electrophoretic separation in a microfluidic paper-based analytical device with an on-column wireless electrogenerated chemiluminescence detector a fully disposable paper-based electrophoresis microchip with integrated pencil-drawn electrodes for contactless conductivity detection low-voltage origami-paper-based electrophoretic device for rapid protein separation low-voltage paper isotachophoresis device for dna focusing origami-paper-based device for microvesicle/exosome preconcentration and isolation situ ion-transmission mass spectrometry for paper-based analytical devices simultaneous pre-concentration and separation on simple paper-based analytical device for protein analysis quantitatively controllable fluid flows with ballpoint-pen-printed patterns for programmable photo-paper-based microfluidic devices enhancing the sensitivity of colorimetric lateral flow assay (clfa) through signal amplification techniques enhancing capillary-driven flow for paper-based microfluidic channels hollow-channel paper analytical devices improving sensitivity of gold nanoparticle-based lateral flow assays by using wax-printed pillars as delay barriers of microfluidics programming fluid transport in paper-based microfluidic devices using razor-crafted open channels rapid flow in multilayer microfluidic paper-based analytical devices tunable-delay shunts for paper microfluidic devices progress in the development and integration of fluid flow control tools in paper microfluidics recent advances of fluid manipulation technologies in microfluidic paper-based analytical devices (µpads) toward multi-step assays fully inkjet-printed paper-based potentiometric ion-sensing devices a paper-based electrochemical sensor using inkjet-printed carbon nanotube electrodes d paper-based microfluidic device: a novel dual-detection platform of bisphenol a programmable contact printing using ballpoint pens with a digital plotter for patterning electrodes on paper a handheld stamping process to fabricate microfluidic paper-based analytical devices with chemically modified surface for clinical assays pen-on-paper strategies for point-of-care testing of human health chapter -types of production situations review on microfluidic paper-based analytical devices towards commercialisation a low-cost, simple, and rapid fabrication method for paper-based microfluidics using wax screen-printing droplet actuation by electrowetting-on-dielectric (ewod): a review a correct-by-construction design and programming approach for open paper-based digital microfluidics dielectric materials for electrowetting-on-dielectric actuation recent advances in paper-based sensors pcr-free paper-based nanobiosensing platform for visual detection of telomerase activity via gold enhancement mimicking peroxidase-like activity of co o -ceo nanosheets integrated paper-based analytical devices for detection of glucose with smartphone double-layered microfluidic paper-based device with multiple colorimetric indicators for multiplexed detection of biomolecules colorimetric point-of-care detection of cholesterol using chitosan nanofibers three-dimensional microfluidic paper-based device for multiplexed colorimetric detection of six metal ions combined with use of a smartphone a concentration gradient generator on a paper-based microfluidic chip coupled with cell culture microarray for high-throughput drug screening signal amplified gold nanoparticles for cancer diagnosis on paper-based analytical devices a paper-based optical probe for chromium by using gold nanoparticles modified with , -thiodiacetic acid and smartphone camera readout determination of norfloxacin residues in foods by exploiting the coffee-ring effect and paper-based microfluidics device coupling with smartphone-based detection smartphone-imaged multilayered paper-based analytical device for colorimetric analysis of carcinoembryonic antigen a review of digital microfluidics as portable platforms for lab application of paper ewod (electrowetting-on-dielectrics) chip: protein tryptic digestion and its detection using maldi-tof mass spectrometry thermal actuation and confinement of water droplets on paper-based digital microfluidics devices label-free microfluidic paper-based electrochemical aptasensor for ultrasensitive and simultaneous multiplexed detection of cancer biomarkers electrochemical paper-based microfluidic device for high throughput multiplexed analysis new disposable electrochemical paper-based microfluidic device with multiplexed electrodes for biomarkers determination in urine sample janus electrochemistry: simultaneous electrochemical detection at multiple working conditions in a paper-based analytical device janus electrochemical paper-based analytical devices for metals detection in aerosol samples integrated digital microfluidic platform for voltammetric analysis electrochemical detection on electrowetting-on-dielectric digital microfluidic chip control-fluidic codesign for paper-based digital microfluidic biochips integrated control-fluidic codesign methodology for paper-based digital microfluidic biochips chemiluminescence immunoassays for simultaneous detection of three heart disease biomarkers using magnetic carbon composites and three-dimensional microfluidic paper-based device a paper-based chemiluminescence immunoassay device for rapid and high-throughput detection of allergen-specific ige high-resolution temporally resolved chemiluminescence based on double-layered d microfluidic paper-based device for multiplexed analysis a miniaturized chemiluminescence detection system for a microfluidic paper-based analytical device and its application to the determination of chromium (iii) fabrication of paper-based microfluidic device by recycling foamed plastic and the application for multiplexed measurement of biomarkers a microfluidic electrochemiluminescent device for detecting cancer biomarker proteins three-dimensional paper-based electrochemiluminescence immunodevice for multiplexed measurement of biomarkers and point-of-care testing rotational paper-based electrochemiluminescence immunodevices for sensitive and multiplexed detection of cancer biomarkers graphite paper-based bipolar electrode electrochemiluminescence sensing platform a novel paperfluidic closed bipolar electrode-electrochemiluminescence sensing platform: potential for multiplex detection at crossing-channel closed bipolar electrodes a paper-based electrochemiluminescence electrode as an aptamer-based cytosensor using ptni@ carbon dots as nanolabels for detection of cancer cells and for in situ screening of anticancer drugs disposable paper-based bipolar electrode array for multiplexed electrochemiluminescence detection of pathogenic dnas electrochemiluminescence on digital microfluidics for microrna analysis multiplex quantification of metals in airborne particulate matter via smartphone and paper-based microfluidics simultaneous quantification of multiple biomarkers on a self-calibrating microfluidic paper-based analytic device electrochromic sensor for multiplex detection of metabolites enabled by closed bipolar electrode coupling method validation for extraction of nucleic acids from peripheral whole blood a comparative evaluation of four dna extraction protocols from whole blood sample isothermal amplification of nucleic acids a fully disposable and integrated paper-based device for nucleic acid extraction, amplification and detection a novel mutation tolerant padlock probe design for multiplexed detection of hypervariable rna viruses equipment-free nucleic acid extraction and amplification on a simple paper disc for point-of-care diagnosis of rotavirus a paperfluidic chip device for small rna extraction, amplification, and multiplexed analysis metal-enhanced fluorescence/visual bimodal platform for multiplexed ultrasensitive detection of microrna with reusable paper analytical devices ultrasensitive microfluidic paper-based electrochemical/visual biosensor based on spherical-like cerium dioxide catalyst for mir- detection visual detection of dna on paper chips single-step recombinase polymerase amplification assay based on a paper chip for simultaneous detection of multiple foodborne pathogens an integrated paper-based sample-to-answer biosensor for nucleic acid testing at the point of care paper-based nucleic acid amplification tests for point-of-care diagnostics paper-based microfluidics for rapid diagnostics and drug delivery shaping up field-deployable nucleic acid testing using microfluidic paper-based analytical devices paper-origami-based multiplexed malaria diagnostics from whole blood paper-based rna detection and multiplexed analysis for ebola virus diagnostics multiplex paper-based colorimetric dna sensor using pyrrolidinyl peptide nucleic acid-induced agnps aggregation for detecting mers-cov, mtb, and hpv oligonucleotides semiquantitative nucleic acid test with simultaneous isotachophoretic extraction and amplification simpler, faster, and sensitive zika virus assay using smartphone detection of loop-mediated isothermal amplification on paper microfluidic chips microfluidic rapid and autonomous analytical device (microraad) to detect hiv from whole blood samples. lab a chip a paper-based device with an adjustable time controller for the rapid determination of tumor biomarkers screening of highly-specific aptamers and their applications in paper-based microfluidic chips for rapid diagnosis of multiple bacteria based electrochemical cyto-device for sensitive detection of cancer cells and in situ anticancer drug screening paper-based analytical devices for environmental analysis microfluidic paper-based analytical devices for environmental analysis of soil, air, ecology and river water environmental chemical analysis improved assessment of accuracy and performance using a rotational paper-based device for multiplexed detection of heavy metals simultaneous colorimetric detection of metallic salts contained in low explosives residue using a microfluidic paper-based analytical device (µpad). forensic chem multiplex paper-based designs for point-of-care (poc) diagnostics a paper-based device for performing loop-mediated isothermal amplification with real-time simultaneous detection of multiple dna targets andersson-svahn, h. a vertical flow paper-microarray assay with isothermal dna amplification for detection of neisseria meningitidis a novel microfluidic paper-based analytical device based on chemiluminescence for determination of β-agonists in swine hair znse quantum dot based ion imprinting technology for fluorescence detecting cadmium and lead ions on a three-dimensional rotary paper-based microfluidic chip paper-based bipolar electrode-electrochemiluminescence (bpe-ecl) device with battery energy supply and smartphone read-out: a handheld ecl system for biochemical analysis at the point-of-care level detection of cardiovascular disease associated mir- a using paper-based microfluidics and surface enhanced raman scattering printable qr code paper microfluidic colorimetric assay for screening volatile biomarkers key: cord- - kt xw i authors: dasgupta, tumpa; ferdous, shomita; tse-dinh, yuk-ching title: mechanism of type ia topoisomerases date: - - journal: molecules doi: . /molecules sha: doc_id: cord_uid: kt xw i topoisomerases in the type ia subfamily can catalyze change in topology for both dna and rna substrates. a type ia topoisomerase may have been present in a last universal common ancestor (luca) with an rna genome. type ia topoisomerases have since evolved to catalyze the resolution of topological barriers encountered by genomes that require the passing of nucleic acid strand(s) through a break on a single dna or rna strand. here, based on available structural and biochemical data, we discuss how a type ia topoisomerase may recognize and bind single-stranded dna or rna to initiate its required catalytic function. active site residues assist in the nucleophilic attack of a phosphodiester bond between two nucleotides to form a covalent intermediate with a ′-phosphotyrosine linkage to the cleaved nucleic acid. a divalent ion interaction helps to position the ′-hydroxyl group at the precise location required for the cleaved phosphodiester bond to be rejoined following the passage of another nucleic acid strand through the break. in addition to type ia topoisomerase structures observed by x-ray crystallography, we now have evidence from biophysical studies for the dynamic conformations that are required for type ia topoisomerases to catalyze the change in the topology of the nucleic acid substrates. normal cell growth requires replication of the genome and regulated transcription. certain enzymes play crucial roles in these vital cellular processes. for example, dna polymerase enzymes are essential for dna replication [ ] and rna polymerases are required for transcription [ ] . because cellular genomes exist as very long double-stranded dna, topoisomerases are needed for their unique role as master regulators of dna topology during dna replication, transcription, recombination, chromosome remodeling and many other genomic processes [ ] [ ] [ ] [ ] [ ] . topoisomerases catalyze the interconversion of different topological forms of dna by creating transient breaks on one or both strands of the dna duplex [ , ] . the twin supercoiling domain model proposed by liu and wang described the topological problems encountered during transcription that would require topoisomerase action ahead and behind the elongating rna polymerase complex [ , ] . in bacteria, topoisomerase i belonging to the type ia subfamily relaxes the negative supercoiling generated by transcription behind the rna polymerase complex to prevent the formation of dna-rna hybrids/r-loops that in turn can inhibit the transcription process [ , , [ ] [ ] [ ] . in addition to the relaxation of negatively supercoiled dna and preventing hypernegative supercoiling, type ia topoisomerases can also catalyze the decatenation of replication intermediates [ ] [ ] [ ] [ ] [ ] and the knotting or unknotting of single-stranded dna circles or nicked duplex dna [ , ] . every living organism has at least one type ia topoisomerase that can resolve topological barriers, including replication and recombination intermediates or other entangled dna structures, by passing dna through a transient break in a single strand of dna [ ] . the ability to unknot single-stranded rna circles was first observed for escherichia coli topoisomerase iii [ ] . this rna topoisomerase activity might have been related to the similarity between e. coli topoisomerase iii (top ) and an ancestral type ia topoisomerase present in the rna world. the interest in the physiological significance of rna topoisomerase activity was greatly heightened by the findings that human topoisomerase iiiβ (top b) has both dna and rna topoisomerase activities [ , ] . furthermore, mutations in human top b are linked to disorders in neurodevelopment and mental health [ ] [ ] [ ] [ ] [ ] . many other type ia topoisomerases in all three domains of life have been found to possess rna topoisomerase activity [ ] [ ] [ ] . while top b is likely to participate in the regulation of transcription-associated supercoiling and r-loops within the nucleus [ ] [ ] [ ] [ ] , top b has been shown to bind to mrna within the cytoplasm [ ] [ ] [ ] and may play a role in translation. potential roles of top b in unlinking molecules of mrna or overcoming torsional stress of mrna have been proposed [ ] , but the exact cellular functions of the rna topoisomerase activity remain to be fully elucidated. remarkably, top b has recently been demonstrated to be a host factor hijacked for the efficient viral replication of positive-sense single-stranded rna viruses that include flaviviruses and coronaviruses [ ] . figure shows some of the potential topological barriers occurring during the life cycle of a positive-sense single-stranded rna virus that may require the rna topoisomerase activity of human top b. tdrd plays an important role in the stabilization and activation of the dna and rna topoisomerase activities of human and drosophila top b [ , , ] . crispr/cas -based deletion that targeted the top b-tdrd complex did not affect flavivirus translation and replication, but was found to diminish the late-stage production of infectious virus particles [ ] . it is not known yet if the top b-tdrd complex is involved similarly in the different stages of the coronavirus life cycle. molecules , , x for peer review of replication and recombination intermediates or other entangled dna structures, by passing dna through a transient break in a single strand of dna [ ] . the ability to unknot single-stranded rna circles was first observed for escherichia coli topoisomerase iii [ ] . this rna topoisomerase activity might have been related to the similarity between e. coli topoisomerase iii (top ) and an ancestral type ia topoisomerase present in the rna world. the interest in the physiological significance of rna topoisomerase activity was greatly heightened by the findings that human topoisomerase iiiβ (top b) has both dna and rna topoisomerase activities [ , ] . furthermore, mutations in human top b are linked to disorders in neurodevelopment and mental health [ ] [ ] [ ] [ ] [ ] . many other type ia topoisomerases in all three domains of life have been found to possess rna topoisomerase activity [ ] [ ] [ ] . while top b is likely to participate in the regulation of transcription-associated supercoiling and r-loops within the nucleus [ ] [ ] [ ] [ ] , top b has been shown to bind to mrna within the cytoplasm [ ] [ ] [ ] and may play a role in translation. potential roles of top b in unlinking molecules of mrna or overcoming torsional stress of mrna have been proposed [ ] , but the exact cellular functions of the rna topoisomerase activity remain to be fully elucidated. remarkably, top b has recently been demonstrated to be a host factor hijacked for the efficient viral replication of positivesense single-stranded rna viruses that include flaviviruses and coronaviruses [ ] . figure shows some of the potential topological barriers occurring during the life cycle of a positive-sense singlestranded rna virus that may require the rna topoisomerase activity of human top b. tdrd plays an important role in the stabilization and activation of the dna and rna topoisomerase activities of human and drosophila top b [ , , ] . crispr/cas -based deletion that targeted the top b-tdrd complex did not affect flavivirus translation and replication, but was found to diminish the late-stage production of infectious virus particles [ ] . it is not known yet if the top b-tdrd complex is involved similarly in the different stages of the coronavirus life cycle. figure . some of the potential topological problems during the life cycle of a positive-sense singlestranded rna virus that may require the rna topoisomerase activity of human top b. genome circularization can be mediated by rna-rna interactions and proteins binding to the ′ and ′ ends. decatenation of the catenated circular viral genome by top b is required to remove blocks of (a) viral protein translation, (b) viral rna transport and packaging. (c) when a translating ribosome or a helicase unwinds a duplex region in a viral rna hairpin, and if the hairpin is bound to an immobile rnp or cellular matrix, the helical torsion will need to be relaxed by top b. the figure is modified from a published version for potential top b action on mrna in [ ] . two recent reviews on type ia topoisomerases have discussed the essential functions of bacterial type ia topoisomerase [ ] and the many versatile collaborations engaged by eukaryotic top to carry out different topological transactions [ ] . this review on the mechanism of type ia topoisomerases will focus more on the results from recent structural, biophysical, biochemical and figure . some of the potential topological problems during the life cycle of a positive-sense singlestranded rna virus that may require the rna topoisomerase activity of human top b. genome circularization can be mediated by rna-rna interactions and proteins binding to the and ends. decatenation of the catenated circular viral genome by top b is required to remove blocks of (a) viral protein translation, (b) viral rna transport and packaging. (c) when a translating ribosome or a helicase unwinds a duplex region in a viral rna hairpin, and if the hairpin is bound to an immobile rnp or cellular matrix, the helical torsion will need to be relaxed by top b. the figure is modified from a published version for potential top b action on mrna in [ ] . two recent reviews on type ia topoisomerases have discussed the essential functions of bacterial type ia topoisomerase [ ] and the many versatile collaborations engaged by eukaryotic top to carry out different topological transactions [ ] . this review on the mechanism of type ia topoisomerases will focus more on the results from recent structural, biophysical, biochemical and genetic studies that help us gain a better understanding of how type ia topoisomerases can act as magicians to manipulate the topology of both dna and rna, in addition to key remaining questions on the catalytic mechanism. topoisomerases are classified into two major types, type i and type ii topoisomerases, based on their ability to cleave one or both dna strands, respectively, during the catalytic process. according to structural homologies and reaction mechanisms, type ii topoisomerases can be subdivided into two subgroups: type iia (including gyrase and topoisomerase iv found in prokaryotes; topoisomerase iiα and topoisomerase iiβ in humans) and type iib (topoisomerase vi and viii found in archaea and bacteria) [ ] [ ] [ ] . type i topoisomerases can be divided into type ia and type ib, ic subgroups [ ] . type ia topoisomerases, ubiquitous in bacteria, archaea and eukarya [ ] , can relax negatively supercoiled dna but not positive supercoils because of the requirement of single-stranded dna for binding [ ] . type ia topoisomerase introduces a transient cleavage of the single dna strand and forms a covalent linkage to the -terminal phosphate of the cleaved dna, followed by the passing of an intact strand (t-strand) of dna through the dna break in the cleaved g-strand and the subsequent religation of the nicked g-strand of dna. this process is referred to as the enzyme-bridged or enzyme-gated mechanism [ , , ] . type ib and type ic topoisomerases form a covalent linkage to the -terminal phosphate during catalysis that involves a • rotation of the cleaved free end of dna around the intact strand to relax both positive and negative supercoils with the controlled swivel or controlled rotation mechanism [ , , ] . type ic differs from type ib or any other known topoisomerase in sequence or structure, with a unique fold in its n-terminal domain [ ] . the first information of type ia topoisomerase structure came from the crystal structure of the kda n-terminal fragment of e. coli topoisomerase i (pdb ecl) [ ] . we can see in this structure a type ia core domain structure formed by domains d -d that is present in the n-terminal region of all type ia topoisomerase i and topoisomerase iii ( figure ) determined subsequently for e. coli topoisomerase iii (pdb d m) [ ] , thermotoga maritima topoisomerase i (pdb gai) [ ] , mycobacterium tuberculosis topoisomerase i (pdb d h) [ ] , streptococcus mutans topoisomerase i (pdb ozw) [ ] , mycobacterium smegmatis topoisomerase i (pdb pcm) [ ] , human topoisomerase iii alpha (pdb cgy) [ ] and iii beta (pdb gvc) [ ] . in the structure of reverse gyrases from archaeoglobus fulgidus (pdb gku) [ ] and t. maritima (pdb ddu) [ ] , type ia core domains are found in the c-terminal region preceded by the helicase-like domain [ ] . the elucidation of the crystal structure of e. coli topoisomerase i core domains [ ] provided support and further detail for the previously proposed enzyme-gated mechanism of catalysis that should be applicable for the other members of the type ia subfamily of topoisomerases. the interior cavity of the torus structure observed in this study was noted to be big enough to hold single-and double-stranded dna substrates to accomplish the relaxation of supercoiled dna or catenation/decatenation of nicked double-stranded dna. the g-strand of dna fits into a binding groove in domain d , and follows the path of one strand of a b form dna, as observed in the subsequently obtained cocrystals of e. coli topoisomerase iii (pdb i d) [ ] and topoisomerase i (pdb mw ) [ ] . the active site tyrosine is part of domain d and situated at the interface of domains d and d and responsible for the transient breakage of the g-strand of dna along with the subsequent formation of the phosphoryl-tyrosyl covalent complex ( figure ). the d n mutation of e. coli topoisomerase i was found to be extremely lethal because the resulting deficiency in dna religation leads to the accumulation of the covalent intermediate [ ] . this mutation was utilized to obtain the crystal structure of the covalent complex formed between e. coli topoisomerase i core domains and cleaved dna (pdb px ) [ ] . while the conformation of the individual n-terminal domains remained largely the same in this covalent complex and in the full-length e. coli topoisomerase i structure with the c-terminal domains present (pdb rul) [ ] , there are conformational changes in the n-terminal core domains including the relative orientations of the individual domains that allow type ia topoisomerases to bind the g-strand and form the active site ( figure a ) as observed for e. coli topoisomerase i, e. coli topoisomerase iii and m. tuberculosis topoisomerase i [ , , , ] . a comparison between the structures of the topoisomerase-dna complex and apoenzyme showed that conformational change in domain d created the binding groove for the g-strand of dna ( figure b ). this is brought about by the movement of an alpha helix that follows a strictly conserved glycine residue (gly in e. coli topoisomerase i, figure d ). the flexibility of this glycine may facilitate this conformational change required for g-strand binding [ ] . polar and positively charged residues in this alpha helix, including strictly conserved arg and gln in e. coli topoisomerase i and arg and gln of m. tuberculosis topoisomerase i, have been noted to interact with the g-strand phosphodiester backbone [ , , ] . site-directed mutagenesis of e. coli topoisomerase at gly , arg and gln confirmed that mutations at these residues reduced the relaxation activity significantly [ , ] . an additional strictly conserved arginine residue in domain d (corresponding to arg in e. coli topoisomerase i, figure d ) also interacts with a phosphate of the g-strand [ , ] . the deoxyribose and bases of the g-strand interact with other polar, positively charged and aromatic residues that extend from the interface of core domains d and d into d ( figure b ). arg and asp in e. coli topoisomerase i are strictly conserved in type ia topoisomerases and have been shown with site-directed mutagenesis to be required for the relaxation and g-strand dna cleavage [ , ] . the r c substitution in e. coli topoisomerase i was found recently in genetic studies to increase the rate of sequence deletion and duplication events, resulting in a mutator phenotype [ ] . another e. coli topoisomerase i mutation, r p, was associated with an increase in short-sequence deletions in the same study. it is not known if the genetic instability is due to the effect of the reduction in topoisomerase i relaxation activity on dna supercoiling, or the disruption of the enzyme catalytic cycle by the mutation [ ] . present (pdb rul) [ ] , there are conformational changes in the n-terminal core domains including the relative orientations of the individual domains that allow type ia topoisomerases to bind the gstrand and form the active site ( figure a ) as observed for e. coli topoisomerase i, e. coli topoisomerase iii and m. tuberculosis topoisomerase i [ , , , ] . a comparison between the structures of the topoisomerase-dna complex and apoenzyme showed that conformational change in domain d created the binding groove for the g-strand of dna ( figure b ). this is brought about by the movement of an alpha helix that follows a strictly conserved glycine residue (gly in e. coli topoisomerase i, figure d ). the flexibility of this glycine may facilitate this conformational change required for g-strand binding [ ] . polar and positively charged residues in this alpha helix, including strictly conserved arg and gln in e. coli topoisomerase i and arg and gln of m. tuberculosis topoisomerase i, have been noted to interact with the g-strand phosphodiester backbone [ , , ] . site-directed mutagenesis of e. coli topoisomerase at gly , arg and gln confirmed that mutations at these residues reduced the relaxation activity significantly [ , ] . an additional strictly conserved arginine residue in domain d (corresponding to arg in e. coli topoisomerase i, figure d ) also interacts with a phosphate of the g-strand [ , ] . the deoxyribose and bases of the g-strand interact with other polar, positively charged and aromatic residues that extend from the interface of core domains d and d into d ( figure b ). arg and asp in e. coli topoisomerase i are strictly conserved in type ia topoisomerases and have been shown with site-directed mutagenesis to be required for the relaxation and g-strand dna cleavage [ , ] . the r c substitution in e. coli topoisomerase i was found recently in genetic studies to increase the rate of sequence deletion and duplication events, resulting in a mutator phenotype [ ] . another e. coli topoisomerase i mutation, r p, was associated with an increase in short-sequence deletions in the same study. it is not known if the genetic instability is due to the effect of the reduction in topoisomerase i relaxation activity on dna supercoiling, or the disruption of the enzyme catalytic cycle by the mutation [ ] . topoisomerase i and reverse gyrase enzymes in the type ia topoisomerase family exhibit a preference for a cytosine base at four nucleotides upstream (− ) of the dna cleavage sites [ ] [ ] [ ] . in the structure of the e. coli topoisomerase i covalent complex and m. tuberculosis topoisomerase i noncovalent complex representing the pretransition state (pdb cq ), this cytosine base fits into a sterically restrictive cavity formed by residues that are conserved in the topoisomerase i and reverse gyrase sequences (highlighted in blue in figure d ). in these crystal structures, a tyrosine side chain (tyr in e. coli topoisomerase i) wedges between the − and − dna bases and creates a kink in the g-strand. the substitution of tyr with serine to abolish the base-stacking interaction resulted in the complete loss of dna cleavage and relaxation activity [ ] . phenylalanine at this position ( figure d ) likely plays the same role in reverse gyrases. alanine substitution at arg switched the dna cleavage sequence site preference to having an adenine at the − position instead of cytosine and reduced the relaxation activity by -fold, while substitution at arg reduced dna cleavage and relaxation activity less severely, and did not change the cytosine preference [ ] . the specific topoisomerase i and reverse gyrase enzymes in the type ia topoisomerase family exhibit a preference for a cytosine base at four nucleotides upstream (− ) of the dna cleavage sites [ ] [ ] [ ] . in the structure of the e. coli topoisomerase i covalent complex and m. tuberculosis topoisomerase i noncovalent complex representing the pretransition state (pdb cq ), this cytosine base fits into a sterically restrictive cavity formed by residues that are conserved in the topoisomerase i and reverse gyrase sequences (highlighted in blue in figure d ). in these crystal structures, a tyrosine side chain (tyr in e. coli topoisomerase i) wedges between the − and − dna bases and creates a kink in the g-strand. the substitution of tyr with serine to abolish the base-stacking interaction resulted in the complete loss of dna cleavage and relaxation activity [ ] . phenylalanine at this position ( figure d ) likely plays the same role in reverse gyrases. alanine substitution at arg switched the dna cleavage sequence site preference to having an adenine at the − position instead of cytosine and reduced the relaxation activity by -fold, while substitution at arg reduced dna cleavage and relaxation activity less severely, and did not change the cytosine preference [ ] . the specific binding of the cytosine positions the phosphate four nucleotides downstream at the position for nucleophilic attack by the active site tyrosine. topoisomerase iii sequences have different amino acid residues present at positions corresponding to arg , arg and tyr in the alignment ( figure d ), and may have a different mechanistic basis for selectivity in cleavage sites. for rna topoisomerase activity, the g-strand is expected to be accommodated in the same binding groove in type ia topoisomerase core domains. the helix in d that follows the flexible glycine may play a similar role in movement to facilitate g-strand binding and interact with the phosphodiester backbone of the g-strand rna. structural and sequence variation in the binding groove may influence the relative molecules , , of efficiency of dna versus rna topoisomerase activity. sequence selectivity of rna cleavage by type ia topoisomerases have not been analyzed. alanine substitution at arg of e. coli topoisomerase i was found to have a more severe effect on the enzyme's rna unknotting activity than its dna unknotting activity, suggesting that the cytosine may need to be bound selectively at the − position for the rna g-strand cleavage by e. coli topoisomerase i [ ] . in addition to the preference of a cytosine at the − position, there may also be structural features that are relevant for the binding and cleavage of rna as the g-strand due to the presence of the -hydroxyl groups on the rna ribose rings. type ia topoisomerases achieve the relaxation of supercoiled dna through a transesterification reaction that includes two sequential nucleophilic attacks involving the active site tyrosine residue situated in d and positioned at the junction of domains d and d [ ] . in type ia topoisomerases, the hydroxyl group in the side chain of the active site tyrosine residue is responsible for the first nucleophilic attack on the scissile phosphate of a single-stranded dna resulting in the cleavage of the g-strand and the formation of the transient -phospho-tyrosyl covalent linkage [ ] . while the cleaved g-strand segment with the phosphate is covalently linked to the enzyme, the cleaved g-strand segment with the leaving hydroxyl is bound by multiple noncovalent interactions [ ] . the covalent and noncovalent interactions on both sides of the g-strand cleavage site not only facilitate the enzyme-bridged t-strand passage through the break, but also prevent any inadvertent release of the cleaved dna that can harm the chromosome integrity. after the intact t-strand passes through the nick, rejoining of the g-dna backbone occurs through the second nucleophilic attack by the -oh group of the cleaved g-strand on the phosphotyrosine linkage to release the enzyme from the covalent complex and allow the enzyme to release the substrate and be ready for the next catalytic cycle [ , ] . a number of conserved amino acids with acidic and basic side chains are present in close proximity of active site tyrosine in type ia topoisomerases to play a role in g-strand cleavage and religation [ , ] . a water molecule has been postulated to be acting as the general base for deprotonation of the tyrosine hydroxyl nucleophile for dna cleavage [ , ] . the deprotonation of the nucleophile by water is likely to be in concert with the formation of the transition state at the scissile phosphate, similar to the nucleotide transfer mechanism proposed for dna polymerases [ ] . the positively charged side chain of the strictly conserved arginine residue separated by one residue from the active site tyrosine (arg in e. coli topoisomerase i, figure c ) plays a critical role in stabilizing the negative charge on the hydroxyl nucleophile and the transition state. the positive charge of divalent ions can also activate the nucleophile and stabilize the transition state for both g-strand cleavage and religation even though divalent ions are absolutely required only for g-strand religation, and can be absent for cleavage of the g-strand by type ia topoisomerases. however, when arg in e. coli topoisomerase i or the corresponding arg in yersinia pestis topoisomerase i was substituted with an aromatic residue, dna cleavage could still take place but became divalent ion dependent [ ] . moreover, the presence of divalent ions cannot compensate for the loss of this arginine residue in g-strand religation, resulting in a dominant lethal cell killing because of the accumulation of topoisomerase i-mediated dna breaks [ ] . a r w mutation introduced at the corresponding arginine residue in human topoisomerase iiiβ has been shown to facilitate trapping of the intracellular covalent complex with both dna and rna [ ] , confirming a similar role for this arginine residue in the cleavage and rejoining of the rna g-strand. type ia and type iia topoisomerases require divalent ions for catalytic activity. at the active sites of these topoisomerases, divalent ions are coordinated by the toprim motifs (with a conserved glutamate and two conserved aspartates dxd) also seen in many nucleotidyl transferases/hydrolases [ ] . in the crystal structure of m. tuberculosis topoisomerase i [ ] , one mg + ion is coordinated with the negatively charged carboxylate side chain of glu (corresponding to glu in e. coli topoisomerase i, figure a ) and glu directly, and to glu indirectly through a water molecule, in addition to the scissile phosphate. the positive charge of mg + can help to position the -oh of the cleaved g-strand for a nucleophilic attack on the phosphotyrosine linkage and stabilize the transition state for dna rejoining. the dna cleavage also became mg + dependent when the first aspartate of the dxd toprim motif was mutated to asparagine without the negatively charged side chain [ , ] . overexpression of the d n mutant topoisomerase i is toxic to the cells because of deficiency in dna religation resulting in the accumulation of the cleavage complex on chromosomal dna. the d n mutation in the e. coli topoisomerase i n-terminal core domain fragment enabled the isolation of the covalent complex formed with cleaved oligonucleotide for structural determination [ ] . other mutations that affect mg + binding at the active site of bacterial topoisomerase i also resulted in bacterial cell death when the mutant topoisomerase i was overexpressed because of the inhibition of dna religation and accumulation of dna breaks [ ] [ ] [ ] [ ] . the requirement for dna religation is more stringent than for dna cleavage because both the phosphotyrosine linkage and -hydroxyl nucleophile have to be placed exactly at positions required for dna rejoining to take place. in the crystal structures of human topoisomerase iiiα [ ] and human topoisomerase iiiβ [ ] , dna substrate is not present. a single mg + is bound directly to the glu side chain of the toprim motif and through a water molecule to the first asp of the toprim dxd. it is expected that upon binding of the g-strand at the active site, the scissile phosphate will displace the water molecules seen in the crystal structure as ligands for the mg + . it cannot be ruled out that a transient interaction with additional mg + not currently observed in the crystal structures of type ia topoisomerases can take place during the course of dna cleavage and rejoining. in addition to acting as a ligand for mg + , the toprim glutamate side chain has been proposed to interact directly during dna cleavage as a general acid [ ] with the g-strand -hydroxyl leaving group to provide a proton from a nearby positively charged histidine (his in e. coli topoisomerase i) side chain via proton relay through the d side chain [ , ] . reversal of the proton relay may take place during religation for the glutamate to act as a general base in the activation of the -hydroxyl nucleophile [ ] . mutation of the toprim glutamate to alanine or glutamine abolished the dna cleavage and relaxation activity [ ] but did not affect the rna cleavage activity observed for m. smegmatis topoisomerase i, suggesting that the -oh of rna could potentially participate in the proton relay for the rna cleavage [ ] . though the type ia topoisomerase core domain that forms the characteristic torus structure contains all the highly conserved motifs responsible for g-strand binding and cleavage religation, the c-terminal domains of bacterial topoisomerase i have been shown to be required for removing negative supercoils from dna rapidly in a processive mechanism [ , , [ ] [ ] [ ] [ ] . results from these studies indicated that the c-terminal domain of type ia topoisomerases possesses a significant affinity to substrate dna. unlike the n-terminal core domains, the c-terminal domains among type ia topoisomerases greatly varied in size and sequence. two distinct types of structural motifs ( figure a ) have been observed in the crystal structures of bacterial topoisomerase i [ , , , ] . tetracysteine motifs that form the zinc ribbon fold [ ] can be found in the topoisomerase i gene of a majority of bacterial species. the zinc ribbon fold comprises a four-stranded antiparallel β-sheet with a zn(ii)-binding site on the top of the motif ( figure a ). the crystal structure of full-length e. coli topoisomerase i [ ] showed three such topo_c_znrpt zinc ribbon domains (d -d ) followed by two zinc ribbon-like domains (d , d ) that did not bind to zn(ii) due to the absence of cysteines [ ] . the four cysteines in the single zinc ribbon domain (d ) in t. maritima topoisomerase i did not bind to the zn(ii) ion in the crystal structure but formed two disulfide bonds to stabilize the zinc ribbon fold [ ] . while the deletion of the c-terminal domains in e coli topoisomerase i resulted in the complete loss of relaxation activity [ ] , t. maritima topoisomerase i retained some activity upon deletion of the c-terminal domain [ ] . in certain species of bacteria in the actinobacteria phylum including members of the mycobacterium and streptomyces genera, the c-terminal domains are organized with repeated topo_c_rpt domains first predicted based on the m. tuberculosis topoisomerase i crystal structure [ ] that do not have zn(ii)-binding cysteines, ending with a tail rich in positively charged lysines and arginines. the topo_c_rpt fold has an antiparallel four-stranded β-sheet flanked by a cterminal helix on one side ( figure a ). [ ] ). conserved aromatic residues from each c-terminal domain form π-π stackings with the nucleotide bases. in the crystal structures of the topoisomerase-dna complex, π-π stacking interactions between tyrosine and phenylalanine amino acids at specific positions of the topo_c_znrpt or topo_c_rpt motifs and nucleotide bases are utilized for dna binding by the c-terminal domains of e. coli and m. smegmatis topoisomerase i ( figure b ). additional interactions with the ssdna include hydrogen bonds and cation-π interactions. flexible loops connect between these c-terminal domains and to the n-terminal core domains. topoisomerase iiiα and iiiβ in higher eukaryotes have large numbers of cysteines in their c-terminal domains that could potentially form multiple zn(ii)-binding motifs that should be similar to the topo_c_znrpt motifs in e. coli topoisomerase i. structural studies are needed to determine the exact folding of these c-terminal motifs in eukaryotic topoisomerase iii enzymes. in actinobacteria such as mycobacteria and streptomyces, a lysine-rich c-terminal tail follows the topo_c_rpt motifs to also participate in dna binding [ , ] . biochemical analysis demonstrated that these elements in type ia topoisomerases contribute to the interaction with the single-stranded dna region in the substrate and are important for the processivity of enzyme activity [ , , [ ] [ ] [ ] . there is further evidence that the bacterial topoisomerase i c-terminal domains have a specific role for interacting with the t-strand in strand passage for the efficient recognition and relaxation of negatively supercoiled dna [ , , , ] . figure illustrates a model of relaxation of supercoiled dna by bacterial topoisomerase i that adds the binding of t-strand by c-terminal domains to the first step of previously proposed topoisomerase i catalytic mechanism [ ] . an rgg-box rich in arginines and glycines follows the zn(ii)-binding motifs in the c-terminal domains of topoisomerase iiiβ found in higher eukaryotes [ , ] . a similar rgg-box is found in [ ] ). conserved aromatic residues from each c-terminal domain form π-π stackings with the nucleotide bases. in certain species of bacteria in the actinobacteria phylum including members of the mycobacterium and streptomyces genera, the c-terminal domains are organized with repeated topo_c_rpt domains first predicted based on the m. tuberculosis topoisomerase i crystal structure [ ] that do not have zn(ii)-binding cysteines, ending with a tail rich in positively charged lysines and arginines. the topo_c_rpt fold has an antiparallel four-stranded β-sheet flanked by a c-terminal helix on one side ( figure a ). in the crystal structures of the topoisomerase-dna complex, π-π stacking interactions between tyrosine and phenylalanine amino acids at specific positions of the topo_c_znrpt or topo_c_rpt motifs and nucleotide bases are utilized for dna binding by the c-terminal domains of e. coli and m. smegmatis topoisomerase i ( figure b ). additional interactions with the ssdna include hydrogen bonds and cation-π interactions. flexible loops connect between these c-terminal domains and to the n-terminal core domains. topoisomerase iiiα and iiiβ in higher eukaryotes have large numbers of cysteines in their c-terminal domains that could potentially form multiple zn(ii)-binding motifs that should be similar to the topo_c_znrpt motifs in e. coli topoisomerase i. structural studies are needed to determine the exact folding of these c-terminal motifs in eukaryotic topoisomerase iii enzymes. in actinobacteria such as mycobacteria and streptomyces, a lysine-rich c-terminal tail follows the topo_c_rpt motifs to also participate in dna binding [ , ] . biochemical analysis demonstrated that these elements in type ia topoisomerases contribute to the interaction with the single-stranded dna region in the substrate and are important for the processivity of enzyme activity [ , , [ ] [ ] [ ] . there is further evidence that the bacterial topoisomerase i c-terminal domains have a specific role for interacting with the t-strand in strand passage for the efficient recognition and relaxation of negatively supercoiled dna [ , , , ] . figure illustrates a model of relaxation of supercoiled dna by bacterial topoisomerase i that adds the binding of t-strand by c-terminal domains to the first step of previously proposed topoisomerase i catalytic mechanism [ ] . terminal domains and basic amino acid residues. the protein-protein interactions with other cellular proteins are relevant for the physiological functions and regulation of type ia topoisomerase activities [ ] . basic residues in the c-terminal domains of e. coli topoisomerase i [ ] and the cterminal tail of m. smegmatis topoisomerase i [ ] have been proposed to interact directly with the β′ subunit of rna polymerase for the function of topoisomerase i in transcription elongation [ , , ] to suppress hypernegative supercoiling and r-loop accumulation [ , , ] . a recent report of an additional active site in the c-terminal domains of helicobacter pylori topoisomerase i [ ] further illustrates the diverse properties of type ia topoisomerase c-terminal domains. there are eight tyrosine residues present in the c-terminal domains of h. pylori an rgg-box rich in arginines and glycines follows the zn(ii)-binding motifs in the c-terminal domains of topoisomerase iiiβ found in higher eukaryotes [ , ] . a similar rgg-box is found in many proteins involved in mrna processing [ ] . the deletion of the rgg-box from topoisomerase iiiβ reduced both the dna and rna topoisomerase activities [ ] . the rgg-box in topoisomerase iiiβ may contribute to both dna and rna binding. post-translational methylation of specific arginine residues in the topoisomerase iiiβ rgg-box enhances the dna and rna topoisomerase activities, and also the interaction between topoisomerase iiiβ and its auxiliary factor tdrd [ ] . the acetylation of lysine residues can also influence the topoisomerase activity of e. coli topoisomerase i [ , ] . it can be postulated that the last universal common ancestor (luca) of type ia topoisomerase may resemble the bacterial and archaeal topoisomerase iii enzymes consisting of the n-terminal core domains and relatively short c-terminal domain. the c-terminal domains observed in the topoisomerase i of bacteria and the topoisomerase iii of higher eukaryotes have evolved to enhance the interactions with nucleic acid substrates and protein-protein interactions using the repeated c-terminal domains and basic amino acid residues. the protein-protein interactions with other cellular proteins are relevant for the physiological functions and regulation of type ia topoisomerase activities [ ] . basic residues in the c-terminal domains of e. coli topoisomerase i [ ] and the c-terminal tail of m. smegmatis topoisomerase i [ ] have been proposed to interact directly with the β subunit of rna polymerase for the function of topoisomerase i in transcription elongation [ , , ] to suppress hypernegative supercoiling and r-loop accumulation [ , , ] . a recent report of an additional active site in the c-terminal domains of helicobacter pylori topoisomerase i [ ] further illustrates the diverse properties of type ia topoisomerase c-terminal domains. there are eight tyrosine residues present in the c-terminal domains of h. pylori topoisomerase. it is not known which of these tyrosine residues may act as an alternative active site nucleophile for relaxation of negatively supercoiled dna. in the enzyme-bridged mechanism [ ] , the two ends of the cleaved g-strand in the covalent intermediate formed by type ia topoisomerases need to be separated by a significant distance through an extensive conformational change of the covalent complex to create the opening at the gate for strand passage. significant conformational changes of bacterial topoisomerase i have been detected by bulk fluorescence measurements [ , ] . trapped ion mobility spectrometry-mass spectrometry (tims-ms) also demonstrated microheterogeneity of e. coli topoisomerase i conformational states [ ] . the extent of gate opening was measured experimentally for e. coli topoisomerase i and topoisomerase iii in single-molecule assays [ ] . the distance between the cleaved ends of single-stranded dna bound covalently to the topoisomerases was found to increase by as much as nm. such a distance was estimated by molecular dynamics simulation to be required for the passage of a double-stranded dna through the break for a catenation/decatenation reaction involving double-stranded circular dna with a nick or single-stranded gap [ ] . a significant opening of the g-strand gate would also be needed for the passage of a single strand of dna through the break to catalyze the relaxation of negatively supercoiled dna. according to the model for type ia topoisomerase catalysis proposed when the crystal structure of the core domains first showed [ ] a toroid enclosed by interactions of d with d and d , d must move away from d and d for gate opening. following the entry of dna into the interior of the toroid hole, d needs to be brought back by a conformational change to close the g-strand gate and religate the break in the g-strand ( figure ). opening of the toroid hole also needs to occur at the end of the catalytic cycle for the release of dna from the interior. a decatenation loop present in e. coli topoisomerase iii but not topoisomerase i has been proposed to keep the gate open to assist the catalysis of decatenation [ ] . in contrast, the rapid closing of the dna gate formed by e. coli topoisomerase i observed in the single-molecule assays would allow a fast rate of relaxation of negatively supercoiled dna [ ] . the hinge region between domains d and d could play an important role in the gate opening and closing. combined techniques of magnetic tweezers and total internal reflection fluorescence microscopy also detected e. coli topoisomerase i conformational change that is necessary for strand passage, but an alternative model of sliding the domains past each other to create a gate for capturing the t-strand of dna into the interior of the toroid was proposed [ ] . the mechanism of the g-strand gate opening and t-strand transport by type ia topoisomerases remains to be fully elucidated. communication between the n-terminal core domains and the c-terminal domains bound to the t-strand may be required for coordinating the opening and closing of the g-gate with strand passage. the guiding of the t-strand in and out of the toroid hole is also not well understood. direct evidence and characterization of additional transient intermediates that are part of the proposed catalytic cycle would further our understanding of the mechanism at the molecular level. the recent discovery of the rna topoisomerase activity for type ia topoisomerases expanded the scope of potential cellular functions for this ubiquitous class of topoisomerases. even though the interaction between topoisomerase iiiβ and mrna in vivo has been linked to synaptic functions [ ] [ ] [ ] , there is no direct experimental evidence so far for a change in cellular rna topology catalyzed by rna topoisomerase activity. separation of function mutations or inhibitors that can be identified for topoisomerase iiiβ would be valuable research tools for investigating the cellular activity and function of topoisomerase iiiβ. eukaryotic dna polymerases in dna replication and dna repair rna polymerase ii transcription initiation: a structural view dna topoisomerases: structure, function, and mechanism dna topoisomerases: harnessing and constraining energy to govern chromosome topology all tangled up: how cells direct, manage and exploit topoisomerase function cellular roles of dna topoisomerases: a molecular perspective new mechanistic and functional insights into dna topoisomerases type ii dna topoisomerases: enzymes that can unknot a topologically knotted dna molecule via a reversible double-strand break supercoiling of the dna template during transcription dna supercoiling during transcription transcription generates positively and negatively supercoiled domains in the template new modulators of genome dynamics and function replication and the functions of bacterial type a topoisomerases topoisomerase iii can serve as the cellular decatenase inescherichia coli topa, the sulfolobus solfataricus topoisomerase iii, is a decatenase topoisomerase α is required for decatenation and segregation of human mtdna genome-wide mapping of topoisomerase i activity sites reveal its role in chromosome segregation topoisomerase iii acts at the replication fork to remove precatenanes knotted single-stranded dna rings: a novel topological isomer of circular single-stranded dna formed by treatment with escherichia coli ω protein catenation and knotting of duplex dna by type topoisomerases: a mechanistic parallel with type topoisomerases an rna topoisomerase deletion of top β, a component of fmrp-containing mrnps, contributes to neurodevelopmental disorders top β is an rna topoisomerase that works with fragile x syndrome protein to promote synapse formation topoisomerase β is the major topoisomerase for mrnas and linked to neurodevelopment and mental dysfunction deletion of top b is associated with cognitive impairment and facial dysmorphism top b: a novel candidate gene in juvenile myoclonic epilepsy? cytogenet rna topoisomerase is prevalent in all domains of life and associates with polyribosomes in animals type ia topoisomerases can be "magicians" for both dna and rna in all domains of life a type ia dna/rna topoisomerase with rna hydrolysis activity participates in ribosomal rna processing arginine methylation facilitates the recruitment of top b to chromatin to prevent r loop accumulation arginine methylation of the c-terminus rgg motif promotes top b topoisomerase activity and stress granule localization loss of top b leads to increased r-loop formation and genome instability topoisomerase β interacts with rnai machinery to promote heterochromatin formation and transcriptional silencing in drosophila topoisomerase iii-ss is required for efficient replication of positive-sense rna viruses dna and rna topoisomerase activities of top β are promoted by mediator protein tudor domain-containing protein role of rna-binding proteins during the late stages of flavivirus replication cycle the many lives of type ia topoisomerases purification of a dna topoisomerase ii from the hyperthermophilic archaeon sulfolobus shibatae. a thermostable enzyme with both bacterial and eucaryal features a novel subfamily of type iib topoisomerases encoded by free or integrated plasmids in archaea and bacteria phylogenomics of dna topoisomerases: their origin and putative roles in the emergence of modern organisms bacterial dna topoisomerase i can relax positively supercoiled dna containing a single-stranded loop the mechanism of type ia topoisomerases three-dimensional structure of the k n-terminal fragment of e. coli dna topoisomerase i topoisomerase v relaxes supercoiled dna by a constrained swiveling mechanism a model for the mechanism of human topoisomerase i structural studies of type i topoisomerases the structure of escherichia coli dna topoisomerase iii crystal structure of full length topoisomerase i from thermotoga maritima insights from the structure of mycobacterium tuberculosis topoisomerase i with a novel protein fold crystal structure of the -kilodalton amino-terminal fragment of dna topoisomerase i from the gram-positive model organism streptococcus mutans mechanistic insights from structure of mycobacterium smegmatis topoisomerase i with ssdna bound to both n-and c-terminal domains structural and mechanistic insight into holliday-junction dissolution by topoisomerase iiiα and rmi structural basis of the interaction between topoisomerase iiiβ and the tdrd auxiliary factor crystal structure of reverse gyrase: insights into the positive supercoiling of dna crystal structures of thermotoga maritima reverse gyrase: inferences for the mechanism of positive dna supercoiling crystal structure of a complex of a type ia dna topoisomerase with a single-stranded dna molecule structure of a complex between e. coli dna topoisomerase i and single-stranded dna asp-to-asn substitution at the first position of the dxd toprim motif of recombinant bacterial topoisomerase i is extremely lethal to e. coli crystal structure of a covalent intermediate in dna cleavage and rejoining by escherichia coli dna topoisomerase i structural basis for suppression of hypernegative dna supercoiling by e. coli topoisomerase i investigating mycobacterial topoisomerase i mechanism from the analysis of metal and dna substrate interactions at the active site flexibility at gly- is required for dna cleavage and relaxation activity ofescherichia colidna topoisomerase i site-directed mutagenesis of residues involved in g strand dna binding byescherichia colidna topoisomerase i point mutations in topoisomerase i alter the mutation spectrum in e. coli and impact the emergence of drug resistance genotypes covalent bonds between protein and dna. formation of phosphotyrosine linkage between certain dna topoisomerases and dna archaebacterial reverse gyrase cleavage-site specificity is similar to that of eubacterial dna topoisomerases i analysis of dna relaxation and cleavage activities of recombinant mycobacterium tuberculosis dna topoisomerase i from a new expression and purification protocol residues of e. coli topoisomerase i conserved for interaction with a specific cytosine base to facilitate dna cleavage synthesizing topological structures containing rna type ia topoisomerases: a simple puzzle? studies of bacterial topoisomerases i and iii at the single-molecule level site-directed mutagenesis of conserved aspartates, glutamates and arginines in the active site region ofescherichia colidna topoisomerase i identification of active site residues inescherichia colidna topoisomerase i extensive free-energy simulations identify water as the base in nucleotide addition by dna polymerase the strictly conserved arg- residue in the active site of escherichia coli topoisomerase i plays a critical role in dna rejoining topoisomerase b (top b) dna and rna cleavage complexes and pathway to repair top b-linked rna and dna breaks toprim-a conserved catalytic domain in type ia and ii topoisomerases, dnag-type primases, old family nucleases and recr proteins deciphering the distinct role for the metal coordination motif in the catalytic activity of mycobacterium smegmatis topoisomerase i mutation adjacent to the active site tyrosine can enhance dna cleavage and cell killing by the toprim gly to ser mutant of bacterial topoisomerase i bacterial cell killing mediated by topoisomerase i dna cleavage activity the dna relaxation activity and covalent complex accumulation of mycobacterium tuberculosis topoisomerase i can be assayed in escherichia coli: application for identification of potential fret-dye labeling sites biochemical characterization of an invariant histidine involved inescherichia colidna topoisomerase i catalysis c-terminal lysine repeats in streptomyces topoisomerase i stabilize the enzyme-dna complex and confer high enzyme processivity a highly processive topoisomerase i: studies at the single-molecule level carboxyl terminal domain basic amino acids of mycobacterial topoisomerase i bind dna to promote strand passage the carboxyl terminal domain ofescherichia coli dna topoisomerase i confers higher affinity to dna c-terminal domains of escherichia coli topoisomerase i belong to the zinc-ribbon superfamily solution structure of the c-terminal single-stranded dna-binding domain of escherichia coli topoisomerase i probing the structural domains and function in vivo of escherichia coli dna topoisomerase i by mutagenesis thermotoga maritima-escherichia coli chimeric topoisomerases. answers about involvement of the carboxyl-terminal domain in dna topoisomerase i-mediated catalysis the zn(ii) binding motifs of e. coli dna topoisomerase i is part of a high-affinity dna binding domain expression and dna-binding properties of the k carboxyl terminal fragment of escherichia coli dna topoisomerase i the topoisomerase α zinc-finger domain t of arabidopsis thaliana is required for targeting the enzyme activity to holliday junction-like dna repair intermediates the role of the zn(ii) binding domain in the mechanism of e. coli dna topoisomerase i cloning and characterization of drosophila topoisomerase iiibeta. relaxation of hypernegatively supercoiled dna how arginine-rich domains coordinate mrna maturation events deacetylation of topoisomerase i is an important physiological function of e. coli cobb biochemical basis of e. coli topoisomerase i relaxation activity reduction by nonenzymatic lysine acetylation characterization of molecular interactions between escherichia coli rna polymerase and topoisomerase i by molecular simulations distinct mechanism evolved for mycobacterial rna polymerase and topoisomerase i protein-protein interaction direct interaction between escherichia coli rna polymerase and the zinc ribbon domains of dna topoisomerase i transcription facilitated genome-wide recruitment of topoisomerase i and dna gyrase growth inhibition mediated by excess negative supercoiling: the interplay between transcription elongation, r-loop formation and dna topology escherichia coli dna topoisomerase i inhibits r-loop formation by relaxing transcription-induced negative supercoiling molecular dissection of helicobacter pylori topoisomerase i reveals an additional active site in the carboxyl terminus of the enzyme effect of mg(ii) binding on the structure and activity ofescherichia colidna topoisomerase i type ia topoisomerase inhibition by clamp closure microheterogeneity of topoisomerase ia/ib and their dna-bound states direct observation of topoisomerase ia gate dynamics identification of a unique domain essential for escherichia coli dna topoisomerase iii-catalysed decatenation of replication intermediates an orthogonal single-molecule experiment reveals multiple-attempt dynamics of type ia topoisomerases funding: this work was supported by nih grant r -gm (to y.t.). the authors declare no conflict of interest. key: cord- - fvkx f authors: singh, rajveer; gautam, anupam; chandel, shivani; ghosh, arijit; dey, dhritiman; roy, syamal; ravichandiran, velayutham; ghosh, dipanjan title: protease inhibitory effect of natural polyphenolic compounds on sars-cov- : an in silico study date: - - journal: molecules doi: . /molecules sha: doc_id: cord_uid: fvkx f the current pandemic, caused by sars-cov- virus, is a severe challenge for human health and the world economy. there is an urgent need for development of drugs that can manage this pandemic, as it has already infected million people and led to the death of around , people worldwide. at this time, in-silico studies are providing lots of preliminary data about potential drugs, which can be a great help in further in-vitro and in-vivo studies. here, we have selected three polyphenolic compounds, mangiferin, glucogallin, and phlorizin. these compounds are isolated from different natural sources but share structural similarities and have been reported for their antiviral activity. the objective of this study is to analyze and predict the anti-protease activity of these compounds on sars-cov- main protease (mpro) and tmprss protein. both the viral protein and the host protein play an important role in the viral life cycle, such as post-translational modification and viral spike protein priming. this study has been performed by molecular docking of the compounds using pyrx with autodock vina on the two aforementioned targets chosen for this study, i.e., sars-cov- mpro and tmprss . the compounds showed good binding affinity and are further analyzed by (molecular dynamic) md and molecular mechanics poisson-boltzmann surface area mm-pbsa study. the md-simulation study has predicted that these natural compounds will have a great impact on the stabilization of the binding cavity of the mpro of sars-cov- . the predicted pharmacokinetic parameters also show that these compounds are expected to have good solubility and absorption properties. further predictions for these compounds also showed no involvement in drug-drug interaction and no toxicity. the current covid- pandemic is the most serious global outbreak to have occurred in recent times. researchers from different fields are trying to understand it by different approaches [ ] . however, until now, no medication or any established therapy has been established. natural compounds always offer a great promise in the cure of critical diseases. polyphenolic compounds are naturally occurring phenolic compounds that contain more than one phenol group. polyphenols are found largely in the fruits, vegetables, cereals. it has been observed that fruits such as apples, pears, grapes, and cherries swiss-model server has developed three different models of tmprss , out of which we have selected the best model for molecular docking based upon the qmeans score. the homology model is considered as good and reliable if the target sequence was found to be more than %. sequence alignment for the tmprss and human hepsin tmprss ( ce . .a) shows a similarity of . % (figure ). the models were built depending on the template-target alignment utilizing the promod . the geometry of the model was regularized by utilizing the force field. the quality was assessed by the qmean scoring function. qmeans value [ , ] and generalized quantum master equations (gmqe) value of the model was found to be − . and . , respectively. we further analyzed the homology model of tmprss ( amino acid residues) with the help of rampage software and noticed that . % of the total residues were in favored regions, . % of the total residues were in allowed regions and only . % of the total residues were in outlier region (supplementary material figure s ). molecules , , x for peer review of the help of rampage software and noticed that . % of the total residues were in favored regions, . % of the total residues were in allowed regions and only . % of the total residues were in outlier region (supplementary material figure s ). we have selected three natural compounds from different natural source for molecular docking against the main proteases of sars-cov- (mpro) and tmprss . these compounds have previous reports of anti-viral properties and structural similarities. the binding affinities of the compounds are listed in the tables for the mpro of sars-cov- and tmprss protein. mangiferin showed the highest binding affinity for mpro, which is − . kcal/mol, while the binding affinity of glucogallin and phlorizin was − kcal/mol − . kcal/mol, respectively. the binding and mode of interactions of these compounds with mpro are shown in figure . . we have also compared the binding affinity of our compounds with three different reference compounds remdesivir, x , and n for mpro with previously reported docking scores of − . kcal/mol, − . kcal/mol, and − . kcal/mol, respectively, which is near to our obtained docking scores of − . kcal/mol, − . kcal/mol, and− . kcal/mol [ ] [ ] [ ] . remdesivir showed five we have selected three natural compounds from different natural source for molecular docking against the main proteases of sars-cov- (mpro) and tmprss . these compounds have previous reports of anti-viral properties and structural similarities. the binding affinities of the compounds are listed in the tables for the mpro of sars-cov- and tmprss protein. mangiferin showed the highest binding affinity for mpro, which is − . kcal/mol, while the binding affinity of glucogallin and phlorizin was − kcal/mol − . kcal/mol, respectively. the binding and mode of interactions of these compounds with mpro are shown in figure . from the interactions, it has been shown that primarily there are three types of interaction (h bonding, electrostatic, van der waals forces). . we have also compared the binding affinity of our compounds with three different reference compounds remdesivir, x , and n for mpro with previously reported docking scores of − . kcal/mol, − . kcal/mol, and − . kcal/mol, respectively, which is near to our obtained docking scores of − . kcal/mol, − . kcal/mol, and− . kcal/mol [ ] [ ] [ ] (table ) . (table ). for tmprss protein, phlorizin showed the highest binding affinity with a binding energy of − . kcal/mol. the binding energy of both glucogallin and mangiferin was found to be − . kcal/mol. the binding residues and binding modes of these compounds are shown in figure . mangiferin showed the conventional h bonding interaction with residues ala (a) and glu (a). van der waals interaction of mangiferin was found to be with the residues trp (a), asn table ) . we have used dockthor server [ ] to cross check the results of our docking study. the docking score from this server showed docking scores near to pyrx autodock vina (tables s and s ). after analyzing the docking scores of the reference compounds obtained from both of the servers with the docking scores that are already published, we observed that the published docking scores of remdesivir and n are near to the docking scores that are obtained from the pyrx autodock vina software. whereas, the published docking score of x are near to the docking score obtained from the dockthor server. the absorption, distribution, metabolism, and excretion (adme) parameters have been analyzed by the pkcsm database, which gives the result after submission of small molecules in smiles format or simply providing the linear structure. the molecular properties of the compounds have been mentioned in the supplementary data. the pharmacokinetic parameter of the three compounds showed less absorption from the git tract, no bbb permeability, and no effect on the cyp d , cyp a , cyp a , cyp c , cyp c , cyp d , cyp a cytochrome enzymes (tables s -s ). the prediction of targets was analyzed by the online swiss target prediction server, which gives the top results in the form of pie-chart ( figure ). in the case of mangiferin the pie chart showed probable protein target type as . % enzyme, . kinases, . % proteases, . % lyase, . % family a g-protein-coupled receptor, % membrane receptor. the pie chart of glucogallin showed . % enzyme, . % phosphatases, . % hydrolases, . % proteases, . % primary active transporter, and family a g protein-coupled receptor. the pie chart of phlorizin showed that . % proteases, . % of electrochemical transporter, . % cytochrome p , . % of family a g-protein-coupled receptor, . % of oxidoreductase, and other cytosolic protein ( figure ) . these were the possible target sites to which the compounds may bind which were predicted by the server. the toxicity study along with the reference compounds was performed with the pkcsm database, which predicted that the selected natural compounds do not have any ames toxicity, hepatotoxicity, and skin sensitivity. whereas all the reference compounds may possess hepatotoxicity, and x and n may possess ames toxicity as predicted by the server. the natural compounds also may not have the hergi and hergii inhibition activity, but all the reference compounds may have hergii inhibition activity (table ). the toxicity study along with the reference compounds was performed with the pkcsm database, which predicted that the selected natural compounds do not have any ames toxicity, hepatotoxicity, and skin sensitivity. whereas all the reference compounds may possess hepatotoxicity, and x and n may possess ames toxicity as predicted by the server. the natural compounds also may not have the hergi and hergii inhibition activity, but all the reference compounds may have hergii inhibition activity (table ) . molecular dynamic simulation (mds) offers a great insight into the proper binding of ligand candidates with the protein under analysis. a workflow combined with molecular docking, molecular dynamic, and free energy calculation has been engaged in understanding the properties of individual natural products in solvation state. in this study, a ns mds was performed to calculate the binding affinity and conformational stability of natural products to the mpro and tmprss . reference compounds such as remdesivir, n , and x for mpro and camostat mesylate for tmprss were selected along with the polyphenols, mangiferin, glucogallin, and phlorizin for the mds study. the mds trajectories were screened out on the following parameters-root mean square deviation (rmsd), root mean square fluctuation (rmsf), solvent-accessible surface area (sasa), rg, and binding free energies. rmsd plot of phlorizin-mpro complex achieves the conformational stability at . ns with rmsd of . nm and stayed at the same conformation up to . ns. the glucogallin-mpro protein complex achieved optimum confirmation of . nm at around . ns and remained unchanged until ns. rmsd plot of the mangiferin-mpro showed very stable conformation around . nm, starting from ns to ns. the rmsd plots of all the three ligand-protein complexes showed very stable conformation throughout the simulation study, which demonstrates that it has a huge impact on the mpro target ( figure s a ) as the reference compound n . in case of tmprss , the phlorizin-tmprss complex became stable after . ns and stays stable up to ns with rmsd of less than . nm. the rmsd plot of glucogallin-tmprss complex started above . nm and remained stable from ns to ns. the mangiferin-tmprss complex achieved the conformation stability after . ns having the rmsd value more than . nm. all the ligand-tmprss complexes having low rmsd value with little fluctuation showed that the ligands have a huge impact on the protein structures ( figure s b ). to further analyze the effect of these compounds on the active sites of the targets, the root means square fluctuations (rmsf) study has been performed. rmsf study of the three natural compounds with sars-cov- mpro showed very less fluctuations, and the value was found to be less than . nm, which indicates that the ligands bind properly with the active sites of the protein such as the reference compounds ( figure s a ). in the case of tmprss , ligands-tmprss complexes showed little fluctuation upto amino acid , but after that, it became stable. the rmsf values of all the natural products are less than . nm ( figure b ). furthermore, we analyzed the solvent-accessible surface area (sasa), to screen hydrophilic and hydrophobic residues of the target-protein and docked ligand-protein complexes. the sasa plots of the natural compounds docked with mpro and tmprss showed less value compared to the un-docked protein targets ( figure s ). the radius of gyration (rg) plot, which indicates the compactness of protein structure, was also performed. the selected natural compounds showed the rg value between . to . nm for the mpro. in the case of tmprss , the phlorizin showed the plateau up to ns w.r.t. tmprss to further analyze the effect of these compounds on the active sites of the targets, the root means square fluctuations (rmsf) study has been performed. rmsf study of the three natural compounds with sars-cov- mpro showed very less fluctuations, and the value was found to be less than . nm, which indicates that the ligands bind properly with the active sites of the protein such as the reference compounds ( figure s a ). in the case of tmprss , ligands-tmprss complexes showed little fluctuation upto amino acid , but after that, it became stable. the rmsf values of all the natural products are less than . nm ( figure b ). furthermore, we analyzed the solvent-accessible surface area (sasa), to screen hydrophilic and hydrophobic residues of the target-protein and docked ligand-protein complexes. the sasa plots of the natural compounds docked with mpro and tmprss showed less value compared to the un-docked protein targets ( figure s ). the radius of gyration (rg) plot, which indicates the compactness of protein structure, was also performed. the selected natural compounds showed the rg value between . to . nm for the mpro. in the case of tmprss , the phlorizin showed the plateau up to ns w.r.t. tmprss protein and then regained the plateau phase nearly at ns. the rg value was found to be around . ± . nm. the second compound glucogallin showed plateau up to . ns and regains the same phase after . ns till the end of mds with a rg value of . ± . nm. the mangiferin showed the plateau phase with tmprss undocked protein from ns to ns with the rg value of . ± . nm (figure ) . mm-pbsa analysis has been carried out on all the three target-ligand complexes to analyze the affinity of the natural compounds towards the protein molecules. the binding free energy calculation was carried out for , ps based on the mds trajectories. the energy calculations provide solvation energy, binding energy, electrostatic energy, van der waals energy, and sasa energy after the md simulation process. in case of mpro-ligand complexes, mpro-phlorizin complex showed the highest binding energy of - . ± . kcal/mol followed by glucogallin-mpro and mangiferin-mpro complexes showing binding energy of − . ± . and − . ± . kcal/mol, respectively. whereas in the case of tmprss -ligand complexes, mangiferin-tmprss complex showed the highest binding energy of − . ± . kcal/mol followed by tmprss -phlorozin andglucogallin-tmprss complexes with binding energy of − . ± . kcal/mol and − . ± . kcal/mol, respectively. the resulted energies by mm-pbsa of the md trajectories (after ns) showed that the natural compounds still bind at active pockets of the protein. the energies of target-ligand complexes are listed in table . mm-pbsa analysis has been carried out on all the three target-ligand complexes to analyze the affinity of the natural compounds towards the protein molecules. the binding free energy calculation was carried out for , ps based on the mds trajectories. the energy calculations provide solvation energy, binding energy, electrostatic energy, van der waals energy, and sasa energy after the md simulation process. in case of mpro-ligand complexes, mpro-phlorizin complex showed the highest binding energy of - . ± . kcal/mol followed by glucogallin-mpro and mangiferin-mpro complexes showing binding energy of − . ± . and − . ± . kcal/mol, respectively. whereas in the case of tmprss -ligand complexes, mangiferin-tmprss complex showed the highest binding energy of − . ± . kcal/mol followed by tmprss -phlorozin andglucogallin-tmprss complexes with binding energy of − . ± . kcal/mol and − . ± . kcal/mol, respectively. the resulted energies by mm-pbsa of the md trajectories (after ns) showed that the natural compounds still bind at active pockets of the protein. the energies of target-ligand complexes are listed in table . polyphenolic compounds are one of the groups of phenolic compounds that are already popular for their multiple medicinal activities. in search for some potential polyphenolic compounds against sars-cov- , some in-silico studies have already showed that polyphenols from different natural sources such as black tea, green tea, mint, velvet bean, etc., were found to have inhibitory potentiality, and some flavonoid compounds were successfully repurposed for the management of the covid- [ ] [ ] [ ] . our current study is focused to screen three selected polyphenolic compounds present in fruits such as amla, apple, and mango as a potential inhibitor for sars-cov- . in summarized form, we did the molecular docking study of the three selected natural polyphenolic compounds against the main protease of sars-cov- and tmprss protease. all the three natural compounds showed good affinity towards the viral main protease and tmprss . as there is no d structure available for tmprss , we have built a homology model using its protein sequence and d structure of hepsin (tmprss ) on swiss model server. analyzing the three ligand-tmprss complexes, we observed that the compounds phlorizin and glucogallin interacts with the residues of the homology model that are common with the primary sequence of hepsin. this indicates the reliability of our homology model.however, interacting residues of mangiferin does not match with any common residues of the homologous sequence of hepsin. phlorizin showed the best affinity towards tmprss , compared to mangiferin and glucogallin. mangiferin showed the highest binding affinity of − . kcal/mol with sars-cov- main protease. mangiferin was previously reported as an immune and respiratory stimulant agent along with anti-viral properties [ ] [ ] [ ] [ ] . the rest of the two compounds also showed good binding affinity to the main protease. analyzing the molecular docking result of three compounds in comparison to the reference compounds, we observed that they possess some common interacting residues. this indicates that the compounds are binding in the same pocket of the target proteins as the reference compounds. glucogallin comes under the class of gallotannins, which have been reported to possess anti-viral property against herpes simplex virus type [ ] . to analyze the pharmacokinetics and toxicity parameter, we used the pkcsm database. the properties of these compounds show that they can transport through specific membranes, distribute in the system, and bind to the specific proteins. the absorption of the molecules mainly depends upon the lipophilicity and hydrophilicity factors. all compounds have sugar moieties, which increase the water solubility. these compounds do not have any inhibition effect on liver enzymes (such as cyp a etc.) that are involved in the metabolism of the drugs. thus, the metabolism of anticancer, anticonvulsant, and antimalarial drugs remain unaffected. no interference with cyp c and cyp d enzymes involved in the metabolism of cardiovascular drugs and nsaids, respectively, was also noted. these compounds are not involved in drug-drug interaction. whereas the server predicted that the reference compounds may have the inhibition activity on the liver enzymes that are involved in drug metabolism. the toxicity study of the polyphenolic compounds predicts that it is safe for animal and human consumption, but the reference compounds may have hepatotoxicity. the hepatoxicity of remdesivir have already been reported [ ] . the molecular dynamic simulation showed that the selected natural compounds bind and stabilized the active site of both the proteins such as mpro and tmprss . the resultof the mm-pbsa free energy calculations suggested that the phlorizin have the lowest binding free energy towards the mpro, followed by the glucogallin and mangiferin. for tmprss , mangiferin showed the lowest binding free energy, followed by phlorizin and glucogallin. for this study, we have selected three natural compounds from different natural sources that were previously reported to have antiviral activity. to study the ligand-protein interaction, we have downloaded the "sdf" file of ligands from the pubchem. the protein crystalline structure of covid- main protease (mpro) with ligand (pdb- lu ) was downloaded from the rcsb protein databank (www.rcsb.org). the protein structure of proteases was cleaned by removing the ligand, water molecule or hetatom by using biovia discovery studio and saved the protein in protein data bank (pdb) format. homology modeling is extensively used to generate a valid structure of protein utilizing the amino acid sequence [ ] . to date, the crystal structure of the tmprss enzyme was not established. we used the swiss-model server for the generation of the homology model of the tmprss . the protein sequence of tmprss was given in the swiss-model server, the human hepsin tmprss ( ce . .a). a shows a higher similarity of . %. after preparing the d structure of the protein by swiss-model server, we downloaded the pdb file, and it was used for the molecular docking [ , ] . the molecular docking was performed by using pyrx version . , together with autodock vina. both the protein and ligands were loaded to the pyrx virtual screening software. (https: //pyrx.sourceforge.io/) [ ] . the protease protein was put as fixed, but the ligands had rotatable torsions. the size of the box was designed around the center of protease protein, with an exhaustiveness parameter of for all docking. the best binding affinity ligands were selected for the analysis of the inter-residue interaction. to cross check the docking scores of the compounds obtained from pyrx autodock vina, we also used dockthor server [ ] . the ligand and protein interaction were generated by the discovery studio visualize (dsv) version , from biovia. absorption, distribution, metabolism, and excretion (adme) studies are important for better understanding of pharmacodynamic parameter of the selected molecules or drugs. for the analysis of the adme parameter, the pkcsm database [ ] was used. the user can select smiley string or smile file of the compounds. the database also provides the additional information about molecular properties (log p, rotatable bonds, acceptor, donors, and surface area), absorption (solubility, intestinal absorption, skin permeability, p-glycoprotein binding), distribution (volume distribution, protein binding, cns, and blood-brain barrier permeability), metabolism (cyp substrate and inhibitors), and excretion (total clearance and renal transport). this study has the importance in the identification of the possible targets, phenotypical side effects, or potential cross-reactivity of selected molecules [ ] . the swiss target prediction website has been used for the analysis of the possible target. the toxicity study is required for the analysis or prediction of the small molecules that are safe for human and animal uses. pkcsm online database has been used for the toxicity prediction. the molecules can be submitted in the form of smiles in the software. the database provides the ames toxicity, human maximum tolerated dose, hergi and ii, ld , hepato-toxicity, skin toxicity, t. pyriformis, and minnow toxicity of compounds [ ] . molecular dynamics (md) simulation was executed with the docked structure that have the minimum energy using gromacs (version . . ) [ ] with charmm -march [ ] force field using the tip p model [ ] . for the preparation of ligands, the charmm general force field server (https://cgenff.umaryland.edu/) was used. pbc was applied by generating a dodecahedron box. the system was neutralized by adding an adequate number of na + or cl − ions. energy minimization was followed by system equilibration for ps at k using isochoric-isothermal (nvt) equilibration keeping the fs time step. the isothermal-isobaric (npt) ensemble was performed for ps at k with fs time step. electrostatic and van der waals interactions cut-offs for both nvt and npt were kept at . nm. for calculation of long-range interactions, the smooth particle mesh ewald (pme) method was applied. further, , ps md-simulation was performed using the same cut-off. further ns trajectories were submitted to mm-pbsa for energy calculation with , frames [ , ] . from our data, we can conclude that the natural polyphenolic compounds, i.e., glucogallin, mangiferin, and phlorizin, may have the potential to be used as safe protease inhibitors, which may act by inhibiting tmprss and sars-cov- main protease, thus preventing sars-cov- spike protein priming and viral protein post-translational modification (figure ). other advantages of these compounds are that they possess good absorption, good solubility in water, no inhibitory effect on liver enzymes, no effect on the metabolism of essential drugs such as anticancer, anticonvulsant, and anti-coagulant drugs. the potentiality of these compounds can be further verified and may be confirmed by in vitro study. the following are available online, figure s : ramachandran plot of swiss-modelpredicted structure of tmprss , . % favored region. . % allowed region and . % outlier region. figure acknowledgments: anupam gautam will like to acknowledge debaleena bhowmik and kazi amirul hossain for discussion on gromacs and data communication by mega. the authors declare no conflict of interest. enhanced covid- data for improved prediction of survival enhancing the therapeutic effects of polyphenols with macromolecules polyphenols, inflammation, and cardiovascular disease polyphenols and their effects on diabetes management: a review the role of phenolic compounds in the fight against cancer-a review neuroprotectiverole of natural polyphenols antibacterial properties of polyphenols: characterization and qsar (quantitative structureactivity relationship) models characteristics and occurrence of phenolic phytochemicals an in silico approach for identification of novel inhibitors as potential therapeutics targeting covid- main protease sars-cov- cell entry depends on ace and tmprss and is blocked by a clinically proven protease inhibitor proteolytic activation of the influenza virus hemagglutinin efficient activation of the severe acute respiratory syndrome coronavirus spike protein by the transmembrane protease tmprss antiviral effect of mangiferin and isomangiferin on herpes simplex virus antiviral activity of mangiferin against herpes simplex virus type in vitro immunotherapeutic effects of mangiferin mediated by the inhibition of oxidative stress to activated lymphocytes, neutrophils and macrophages mangiferin prevents guinea pig tracheal contraction via activation of the nitric oxide-cyclic gmp pathway cardioprotective effect of ritonavir, an antiviral drug, in isoproterenol induced myocardial necrosis: a new therapeutic implication hydrolyzable tannins: gallotannins and ellagitannins qmeandisco-distance constraints applied on model quality estimation assessing the local structural quality of transmembrane protein models using statistical potentials (qmeanbrane) a search for medications to treat covid- via in silico molecular docking models of the sars-cov- spike glycoprotein and cl protease drug repurposing against sars-cov- using e-pharmacophore based virtual screening, molecular docking and molecular dynamics with main protease as the target screening of plant-based natural compounds as a potential covid- main protease inhibitor: an in silico docking and molecular dynamics simulation approach structure of mpro from sars-cov- and discovery of its inhibitors a taxonomically-driven approach to development of potent, broad-spectrum inhibitors of coronavirus main protease including sars-cov- (covid- ). be publ highly flexible ligand docking: benchmarking of the dockthorprogram on the leads-pep protein-peptide data set evaluation of green tea polyphenols as novel corona virus (sars cov- ) main protease (mpro) inhibitors-an in silico docking and molecular dynamics simulation study natural products' role against covid- potential of flavonoid-inspired phytomedicines against covid- drug-induced liver injury in a covid- patient: potential interaction of remdesivir with p-glycoprotein inhibitors homology modeling a fast tool for drug discovery: current perspectives swiss-model: homology modelling of protein structures and complexes automated comparative protein structure modeling with swiss-model and swiss-pdbviewer: a historical perspective small-molecule library screening by docking with pyrx predicting small-molecule pharmacokinetic and toxicity properties using graph-based signatures swiss target prediction: updated data and new features for efficient prediction of protein targets of small molecules gromacs: a message-passing parallel molecular dynamics implementation charmm united atom chain model for lipids and surfactants charmm tip p water model suppresses peptide folding by solvating the unfolded state open source drug discovery consortium g_mmpbsa-a gromacs tool for high-throughput mm-pbsa calculations this article is an open access article distributed under the terms and conditions of the creative commons attribution key: cord- - bthqj authors: huang, jian; ru, beibei; dai, ping title: bioinformatics resources and tools for phage display date: - - journal: molecules doi: . /molecules sha: doc_id: cord_uid: bthqj databases and computational tools for mimotopes have been an important part of phage display study. five special databases and eighteen algorithms, programs and web servers and their applications are reviewed in this paper. although these bioinformatics resources have been widely used to exclude target-unrelated peptides, characterize small molecules-protein interactions and map protein-protein interactions, a lot of problems are still waiting to be solved. with the improvement of these tools, they are expected to serve the phage display community better. phages, also known as bacteriophages, are viruses that infect bacterial cells. many phages such as m and fd are good expression vectors. in , george p. smith displayed foreign peptides on the virion surface by inserting the foreign dna fragments into the filamentous phage gene iii [ ] . it was demonstrated that foreign peptides in fusion proteins on the virion surface were in immunologically accessible form and specific fusion phage could be enriched by affinity selection [ ] . also in that paper, smith, considered the father of phage display technology, inferred that desired clones could be isolated from a phage library of random inserts in a fusion-phage vector by one or more rounds of selection [ ] . since the pioneering work described above, phage display technology has further been developed and improved by scientists from various fields, and its applications has extended from epitope mapping to antibody engineering and organ targeting [ ] [ ] [ ] [ ] . so far, phage display technology has been widely open access used in basic research such as studying the sites and the networks of protein-protein interactions [ ] [ ] [ ] [ ] , and in applied research such as developing new diagnostics, therapeutics and vaccines [ ] [ ] [ ] [ ] [ ] . the most common protocol of phage display technology can be summarized as: ( ) preparing phage library e.g. random library and cdna library; ( ) immobilizing the target; ( ) incubating the phage library with the immobilized target; ( ) washing away the unbound phage with buffer; ( ) eluting the bound phage with template or stronger buffer; ( ) amplifying the eluted phage by infecting bacteria; ( ) repeating steps ( )-( ) for more rounds; ( ) amplifying the eluted phage and randomly picking up some clones for binding test and dna sequencing. the foreign peptides displayed on the surface of virion can then be derived from the dna sequences. in the foregoing description, the target refers to the substance used to screen the phage library, which is also known as bait or selector; the template refers the natural partner binding to the target. the whole process of affinity selection is usually called biopanning or just panning. the acquired peptides mimicking the binding site on the template and binding to the target are defined as mimotopes. the term mimotope was first introduced by geysen et al. to describe peptides able to bind to the antigen combining site of an antibody but different from the epitope inducing the antibody [ ] . in fact, it was hard to get mimotopes using geysen's method because all their peptides were synthesized chemically. this situation has changed since the coming of the phage display technology. mimotopes can now be obtained in a relatively cheap, efficient and convenient way, i.e. screening phage-displayed random peptide libraries with a given target. however, not all eluted phage clones are target-specific, because the target itself is only one component of the screening system [ ] . from time to time, clones binding to contaminants in the target sample or other components of the screening system such as the solid phase (e.g. plastic plates) and the capturing molecule (e.g. streptavidin and secondary antibody) rather than binding to the actual target, are recovered with those target-specific binders during panning. peptides displayed on these phage clones are called target-unrelated peptides (tups), a term coined recently by menendez and scott [ ] . since all the tups mentioned above are favored during the affinity selection process itself, they are categorized as the selection-related tups. there is also another category of target-unrelated peptides, called the propagation-related tups, which are favored because of increased infectivity or productivity during phage propagations [ ] [ ] [ ] . mimotopes obtained from phage display experiments are very valuable. first, these peptides are candidates for new diagnostics, therapeutics and vaccines. second, they can be used to predict the networks or sites of protein-protein interactions. therefore, bioinformatics studies on integrating all available mimotope data and developing more powerful tools for mimotope analysis are of great importance. in this review, we will summarize the special databases, algorithms, programs, web servers and their applications in the phage display area, focusing on the tools for mimotope-based epitope mapping. as a high-throughput technology, the amount of peptide sequence data derived from phage display has been accumulating rapidly. however, the data related to phage display experiments has been scattered in separate resources for a long period of time. for example, the structure data of mimotopes were store in the pdb database; the sequence data of phage vectors might be stored in the genbank; the mimotope data were stored in various reference databases with the published papers. special databases for mimotopes and other associate information are urgently needed since data integration plays a fundamental role in bioinformatics. available databases for mimotope data are listed in table . [ ] aspd is short for artificially selected proteins/peptides database. it is the first special database for mimotopes [ ] . this database became available at the beginning of the st century, collecting panning results from random libraries, mutant libraries and cdna libraries. at present, the aspd database contains data on selection experiments, which were described in original papers. for each experiment, the following information is given: target, template, links to the external databases such as swiss-prot and pdb, aligned sequences of peptides retrieved through in vitro evolution and relevant native or constructed sequences, rounds of selection, occurrences of clones with each sequence. for each paper, a full reference, a link to the medline database and the name of the corresponding author with his email address are recorded. all curated data are stored in two flat files. one is for panning experiment; another is for literature. aspd has a user-friendly interface and can be searched by means of the srs system. there is a blast search tool against the aspd database for looking directly for homologous sequences. regretfully, the aspd database has not been updated for years. the relic peptides is a relational database created with oracle i and can be accessed through a web interface coded with asp. it currently houses over , peptide sequences that have been selected with small molecule metabolites such as atp, gtp and glucose and drugs such as taxol and taxotere, as well as random clones from parent libraries. as part of the relic suite, this database is indispensible because many programs in relic are dependent on the data of relic peptides [ ] . the motif database contains , peptides obtained from the public domain and by competitive screening of phage display libraries using antisera raised against allergens and industrial enzymes [ ] . it also contains , binding motifs derived from the sequence alignments of these mimotopes. this database was integrated into an epitope mapping tool (emt), which can search motifs in the database against a given antigen structure. matches indicate all possible epitopic regions on the antigen. the pepbank database also includes some peptide data from phage display technology [ ] . at the time of writing, it contains a total of , individual peptide entries. the major source of peptide sequence data comes from text mining of medline abstracts with a program coded with perl. another component of the pepbank database is the peptide sequence data from aspd and uniprot. an additional, smaller part of the database is manually curated from sets of full text articles and text mining results. the mimodb database is the newest database for mimotopes, which is developed by our team very recently [ ] . this database is scheduled to be revised and updated quarterly, collecting mimotopes from random libraries. peptides with sequences longer than residues or selected from phage display cdna libraries (e.g. antibody phage display libraries) are not included. in the current release, it has , peptides manually curated from publications, which were grouped into , sets. these peptides are selected with different targets. the type of targets is quite diverse, varying from small compounds to nucleic acids, proteins, cells, tissues, organs and even entire organisms. nonetheless, proteins including antibodies and receptors from human and mouse are the most used targets. at present, there are known templates in mimodb. for most of the peptides, their templates are not determined. the database also stores solved structures for target-template complex, which are related to mimotope sets. there are five solved structures for target-mimotope complex, which are related to four mimotope sets. for structures of target-template or target-mimotope complex, contact residues making the interface can be viewed interactively with jmolapplet in mimodb. the mysql relational database management system is used to store and manage all the data described above. the mimodb database can be browsed and searched through a user-friendly web interface coded with php. all the databases described here are important resources for the phage display community. with the large amount of sequences in these databases, it is feasible to find out new target-unrelated peptides. for example, in our preliminary analysis, among the , peptides in mimodb, , peptides appear only once; peptides appear times [ ] . svsvgmkpsprp, haiyprh and lpltplp are most frequent, which are seen in , and sets of mimotopes selected with different targets. svsvgmkpsprp and haiyprh have been proved to be propagation-related tups [ , ] . experimental biologists can search the mimodb database to verify if their results have appeared in the database. if so, the match might mean that different research groups have isolated the same peptide with different targets. in this situation, the peptide may not be a true target binder. it is also convenient for computational biologists to derive benchmarks and customized data sets from these databases, which are useful for new algorithm development and tool evaluation. mimotopes are considered to be similar with the corresponding template in some way since they bind the target competitively. it is only natural that the first thought would be their sequence similarity. it is true in some cases. mimotope sequence might be very similar or even identical to one part of its template sequence, indicating the location of the protein-protein interaction site [ , ] . eyes and tools are often used to align mimotope sequence to its template. even if the template is not determined (e.g. panning against a small molecule drug with no priori knowledge about the corresponding drug target), a local alignment search against the non-redundant protein database with blast might help to find it out. however, most existing sequence alignment tools are developed and optimized for long protein sequences. they are not good at the analysis of short peptides deduced from phage display. moreover, it is more often that the mimotope has little sequence similarity with its template, though they do have similar physicochemical properties and spatial organization locally. therefore, many computational tools have been developed to interpret mimotope data reasonably onto the structure of its template. to improve the performance of these tools, algorithms and programs for excluding tups have also been developed. all these algorithms, programs and web servers are listed in table . with these tools, phage display technology has shown its power in exploring the interactions between proteins, peptides and small molecule ligands. generally, all methods described in this section can be divided into two categories, i.e. the sequence-sequence alignment category and the sequence-structure alignment category. findmap, epimap and the mimalign method of the mimop belong to the first category. all methods left can be included in the sequence-structure alignment category, which requires mimotopes and the structure of template as input. in this category, there are motif-based methods (such as dex, peptide, mimox, mimop and etc.), pairs-based methods (such as mapitope and denisova method), patch-based methods (such as sitelight and episearch), graph-based methods (such pep- d-search and pepsurf) and all kinds of hybrid methods (such as mimopro). though different from each other, most mentioned methods mainly address on deciphering the protein-protein interactions, especially epitope mapping. although specific peptides can be selected from the phage-displayed random peptide libraries with various targets ranging from small compounds to whole organisms, the most frequently used targets are proteins, especially antibodies. actually, phage display technology was used for epitope mapping from its infancy [ ] . powered by special computational tools developed for phage display technology, not only linear epitope but also conformational epitope formed by discontinuous residues brought into spatial proximity by protein folding can be mapped reasonably. the tramontano lab from italy might be the first team that tried to map discontinuous epitope computationally based on mimotopes and the antigen structure [ ] . their method includes four steps. first, the solvent accessible area of each residue of the antigen is calculated with the program what if and then surface residues were determined. second, a program called peptide, which they developed, is used to create a file in pir format containing all the peptide sequences of a given length that can mimic a preselected number of side chains exposed on the surface of antigen structure. third, the commercial package such as gcg is used to search the pir file with the consensus sequence obtained from a set of mimotopes. last, energy minimizations and molecular dynamic simulations are performed with the commercial software packages such as insight and discover. as the commercial programs or the rd party software were expensive and not integrated well enough, this method were not widely used by the phage display community. in , the jensen-jarolim group from austria proposed a -dimensional epitope search method and applied it to localize the major ige epitope on bet v , the major birch pollen allergen [ ] . in brief, the -dimensional coarse-grained epitope search was based on the x-ray structure of bet v . each amino acid in the structure was localized using the coordinates of its cβ atom (for glycine, the cα atom). based on this cβ grid, neighboring amino acids were found in a distance smaller than . Å. fitting of only two amino acids consecutively induced a broader attempt, which allowed gaps. the model was further simplified by classifying all amino acids into four groups: polar, lipophilic, acidic and basic amino acids. all hits were statistically evaluated. using this method internally, they found two regions on bet v surface significantly similar with mimotope, though aligning the mimotope with bet v sequence using the gcg package failed to find any similarities. in , mumey et al. described the program findmap [ ] . this program aligns the mimotope sequence to its template sequence, allowing any permutations and local rearrangements (e.g. inversion) of the mimotope sequence. it is therefore different from traditional sequence alignment and has proved to be np-complete. a branch-and-bound algorithm was used to solve this alignment problem in practice and implemented with c++ originally. findmap is unique because it is only based on sequences. the sequences of mimotope and its template are enough; the structure of template is not needed. with this advantage, it has been widely used to explore the epitope and topology of membrane proteins, which lack solved structures due to difficulties in crystallization [ ] [ ] [ ] [ ] [ ] . recently, an improved version with the name epimap has been proposed and coded with java [ ] . while findmap can deal with only one mimotope or a consensus sequence, epimap is capable of: ( ) aligning each mimotope to the template and producing a set of the top-scoring alignments; ( ) selecting the most mutually compatible alignments and filtering out spurious alignments with the program epifilter. the program sitelight developed by halperin et al. might be the first patch-based method [ ] . briefly, the template surface is divided into overlapping patches based on geodesic distances between two cα atoms of amino acids; each mimotope is compared with each patch and a bipartite graph is created for each potential match scored by a similarity matrix; the best alignment of a mimotope and a patch represented in a bipartite graph is found by the maximal bipartite matching algorithm. the mapitope algorithm was originally proposed by enshell-seijffers et al. [ ] . the core idea of mapitope is that: ( ) the simplest meaningful fragment of an epitope is an amino acid pair; ( ) amino acid pairs on the template surface can be simulated by amino acid pairs in the mimotope sequence. two amino acids on the template surface can be considered as a pair when the distance between their cα atoms is less than a threshold, for example Å. to predict the epitope with a set of mimotopes, at first, each peptide is converted to overlapping sequence pairs. all amino acid pairs derived from the set of mimotopes are then pooled, and the frequency of each type is calculated and it is determined whether its representation in the pool is higher than the random expectation. once the most significant amino acid pairs of the pool are identified, the algorithm seeks the match pairs on the template surface and attempts to link them into clusters. the mapitope program was implemented with c++ and has been applied in predicting epitope recognized by monoclonal antibodies against viruses [ , , ] . as the above method typically predicts only a fraction of the epitope, denisova et al. added a filling step to improve the performance of mapitope [ ] . in the filling step, an additional set of amino acid pairs (separated by distance no longer than Å) for every predicted cluster based on the mimotope sequences, is created. this new dataset is then compared to the total amino acid pair composition of the mimotopes. new pairs that were present in the original dataset but were not selected initially because they did not meet the statistical threshold are indentified. the cluster analysis is then repeated using the new pairs and the largest clusters are predicted to be the epitope. the improved method was applied to the prediction of epitopes for five monoclonal antibodies against the west nile virus e protein. in the case of e monoclonal antibody, only three contact residues were uncovered by the original algorithm, while additional nine contact residues were found by the improved algorithm. in our opinion, there is quite a lot information loss while converting mimotopes to overlapping sequence pairs linked with a peptidyl bond [ ] . other pairs, perhaps all pairs, should be considered. according to our observation, other conformations such as helix, turn, hairpin and even globular shape can be seen in the structures of free and bound mimotopes besides the extended conformation. this means that two residues far away in a mimotope sequence may fold together to form a space pair. furthermore, based on our computations on representative structures in the pdb database, the cα distances between two residues separated by one amino acid in all types of conformations are below Å. this indicates that at least pairs separated by one residue in the mimotope sequence are space pairs and should not be ignored [ ] . the same fact might have also been noticed by other groups. in a recent method based on pattern recognition theory, all possible space pairs (for example, pairs separated by one residue, two residues, three residues, and so on) in mimotope sequences are taken into account [ , ] . this new method can be regarded as a derivative of mapitope. however, it is specially designed for elucidating epitope specificity within antiserum. the method consists of two phases: learning and identification. during the learning phase, a large set of mimotopes is collected through panning against specific monoclonal antibodies. the mimotopes are analyzed to identify epitope specific pairs. during the identification phase, mimotopes selected using patient antiserum are interrogated for the presence of the epitope specific pairs. in , the tool dex (short for d-epitope-explorer) was developed by schreiber et al. [ ] . to localize a mimotope on the structure of its template, dex maps each amino acid in the mimotope into a table, containing all the same amino acids on the template surface. the residues in each table are then connected one by one if their cα or cβ distances below the predefined threshold. gaps are allowed in the connecting process. in , a php program named mimop was developed by moreau et al. [ ] . it includes two methods. mimalign, the first method, combines results from four multiple sequence alignments of the template and its mimotopes. for each position, a frequency and a score are calculated. convergent positions are then selected and clustered based on their topology. the clusters obtained are considered as potential epitopic regions, then scored and ranked. the second method named mimcons, evaluates the similarity of the mimotopes and clusters them accordingly. consensus patterns are identified from mimotope sequences of each cluster. the template surface is scanned to look for all possible exposed consensus patterns. the two methods can be run independently or their results combined. mimox might be the first freely accessible web tool for mimotope-based epitope mapping [ ] . it was coded with perl using modules from the bioperl project. mimox has two sections. in the first section, it provides a simple interface for clustalw to align a set of mimotopes and a consensus sequence is derived from the alignments. in the second section, mimox can map a single mimotope or a consensus sequence, or part of them, onto the corresponding antigen structure and search for all of the clusters of residues that could represent the native epitope. naccess is used to evaluate the surface accessibility of the candidate clusters; and jmol is embedded to view them interactively in their d context. mimox is an interactive rather than an automatic tool at present. the default parameters of the program are optimized to decrease the load of the server. thus, the users often need to adjust the parameters many times to get a reasonable result. mimox has been applied by immunologists to characterizing both monoclonal antibody and antiserum [ , ] . in , perschinka et al. proposed a structural alignment method to identify conformational epitopes on heat shock protein associated with atherosclerosis [ ] . in their method, each mimotope is divided into a set of overlapping -mer peptides. these peptides are then superimposed onto the template surface according to cβ atoms. an alignment score is calculated for every superimposition based on similarity of the superimposed amino acids and the distance between the superimposed cβ atoms. for each -mer peptide, the calculated structural alignment score of each surface exposed amino acid is plotted. a control plot can be done with a random peptide. peaks in the plot for mimotope with high scores can easily be observed and taken as the positive hits. the program was written in matlab . and the source code is available on request from the authors. mayrose et al. described a graph-based tool pepsurf for mapping a set of mimotopes onto the solved structure of the template [ ] . in pepsurf, the problem is converted into the task of aligning a set of query peptides to a graph representing the template surface. the best match of each mimotope is found by aligning it against virtually all possible paths in the graph. a clustering step then combines the most significant matches and a predicted epitope is inferred. the program was written in c++ and can be used directly through the pepitope web server [ ] . the mapitope and a combination of pepsurf and mapitope algorithm are also implemented in the pepitope. the pepsurf algorithm and the pepitope web server have been widely used in predicting epitopes on toxins, allergens and receptors recognized by monoclonal or polyclonal antibodies [ ] [ ] [ ] [ ] . in , huang et al. proposed another graph-based tool pep- d-search [ ] . in this method, a surface graph of all exposed residues on the template is created at first. then, the algorithm can be employed in two modes. the first mode is the mimotope mode, which searched for matching paths on the template surface with each query mimotope by the ant colony optimization (aco) algorithm. all paths were scored to the corresponding mimotope according to an amino-acid substitution matrix. putative candidate epitopes were then picked out by the p-value calculation algorithm and the depth-first search algorithm. the second mode is the motif mode, which directly mapped the motif onto the template surface using the aco algorithm and took the top-scoring paths as epitope candidates. all source code is in visual basic and can be downloaded freely. in , negi et al proposed another patch-based method called episearch [ ] . with a set of mimotopes (up to peptide sequences) and corresponding template structure as input, the algorithm first divides the surface of template into overlapping surface patches around each solvent accessible amino acid residue with a radius of Å. then it ranks all surface exposed patches according to the frequency distribution of similar residues in each mimotope and in each patch. episearch is fully automated and has shown an impressive performance in the reported test cases. the web server mimopro has been available on line very recently. it is a mixture of the patch-based method and the graph-based method coded with java. firstly, the template surface is divided into overlapping surface patches centered at the cα atom of each surface residue with a Å radius. then, the surface patches are converted into graphs by specifying two amino acids as neighbor amino acids using a fluctuating distance threshold guided by the compactness factor. for each patch, a complete search method is conducted to find the best alignment for each mimotope sequence. dynamic programming and branch-bound methods are adopted to avoid repeating search and narrow the search space. the floating distance threshold used in mimopro is quite new as all tools mentioned previously use a fixed distance threshold. different from the tools described above, the relic server was particularly designed for the study of the interaction of small molecules with proteins [ ] . by analyzing the sequence of a protein and the sequences of small molecule affinity-selected, phage-displayed peptides, relic can predict proteins or some residues on the protein that bind to drugs, drug candidates and small metabolites. relic is not a single program but rather a suite of computational tools. it currently includes programs: dna pro, aafreq, popdiv, aadiv, info, divaa, motif , motif , closecon, heteroalign, distsim, match, fastacon and fastaskan (see table ). the dna pro is designed to get mimotope sequence from the ph.d.- and ph.d.-c c phage libraries of new england biolabs. any other library of interest is supported if provided with the start and end dna sequences of the vector. aafreq, popdiv, aadiv, info and divaa are designed to analyze the statistical properties of a peptide population. these data are particularly valuable when calculated in conjunction with randomly chosen members of the unselected library, as some propagation-related tups can be identified and subtracted. motif and motif are designed to identify weak sequence motifs within short peptide sequence populations. closecon, heteroalign and distsim use pdb file of the template as the basis for analysis of protein-ligand interactions. if the structure of the template is not available, match, fastacon and fastaskan can be used to do optimal sequence alignments between mimotopes and its template sequence. all programs in the relic suite were developed in fortran with a dos based, command-line user interface. the dos based applications were then converted into web based applications by creating com+ wrappers around the legacy code. as complicated software package online, relic, especially its tools for population analysis, motif identification and sequence alignment are extensively used by the phage display community [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] . as discussed previously, the results from phage display technology are noisy. besides mimotopes, target-unrelated peptides often creep into and even dominate the biopanning results [ ] [ ] [ ] [ ] . although strict control and subtractive experiment might help to decrease tups, either selection-related tups or propagation-related tups can not be eradicated. undoubtedly, taking tups as mimotopes would make the experimental and computational conclusions misleading. to improve the accuracy of programs for mimotope-based analysis, procedures or special tools for excluding tups have been developed. when peptide population of both affinity-selected library and unselected library are analyzed by the program info in the relic suite, the propagation-related tups can be identified and theoretically subtracted from the affinity-selected library. the method is based on the theory of information by shannon. this virtual subtraction process is used not only by info, but is also an option in the relic programs heteroalign, match, and fastaskan to reduce the noise in affinity-selected peptide sequences and amplify the signal, i.e. mimotope [ ] . noise filtering procedure has also been implemented at the level of amino acid pairs. in the algorithm proposed by denisova et al. recently, three groups of peptides are used [ , ] . group a is a collection of mimotopes isolated by affinity selection using a series of specific monoclonal antibody. group b is the set of peptides obtained by antiserum, which needs characterizing. group c consists of irrelevant peptides to be used as a negative control. at the learning phase, the entire collection of group a peptides is used to filter amino acid pairs that are common to more than one antibody. we have developed a free web tool called sarotup, which can be used to scan, exclude and report possible target-unrelated peptides from mimotopes [ ] . at present, a set of tup motifs collected from literature are compiled in the program. among them, one motif indicates propagationrelated tups; motifs indicate selection-related tups, including motifs specific for the capturing agents, five motifs specific for the constant region of antibody, three motifs specific for the screening solid phase and two motifs specific for the contaminants in the target sample. these motifs are converted to regular expressions and then used to check input peptide sequence one by one. however, there are a lot of target-unrelated peptides bearing no known motifs [ ] . as these tups are not embedded in sarotup at present, it is possible that a true tup cannot be detected by sarotup. one way to reduce such false negatives is to search the mimodb database. as stated previously, analysis on peptides in the mimodb database can also revealed new tups. the existing bioinformatics resources and tools reviewed in this paper have benefited the phage display community. however, there are still a lot of problems need to be solved. for example, known tups, especially propagation-related tups are very limited. is there any pattern exists in propagationrelated tups? most tools ignore tups and only a few tools have integrated a filtering procedure. no tools can map epitope formed by two or more chains. the performances of available tools are far from satisfying, and etc. we believe all the mentioned problems will be solved in the coming years. with the advances of bioinformatics tools, the powerful phage display technology will become even more powerful. we can expect that some tools can also be used to other similar surface display technology such as ribosome display, yeast display and bacterial display, producing broader influence. filamentous fusion phage: novel expression vectors that display cloned antigens on the virion surface searching for peptide ligands with an epitope library random peptide libraries: a source of specific protein binding molecules phage antibodies: filamentous phage displaying antibody variable domains organ targeting in vivo using phage display peptide libraries phage display affinity selection from biological libraries a combined experimental and computational strategy to define protein interaction networks for peptide recognition modules probing a protein-protein interaction by in vitro evolution small peptides as potent mimetics of the protein hormone erythropoietin peptide agonist of the thrombopoietin receptor as potent as the natural cytokine display of heterologous proteins on the surface of microorganisms: from the screening of combinatorial libraries to live recombinant vaccines towards a solution for hepatitis c virus hypervariability: mimotopes of the hypervariable region can induce antibodies cross-reacting with a large number of viral variants a peptide-based erythropoietin-receptor agonist for pure red-cell aplasia a priori delineation of a peptide which mimics a discontinuous antigenic determinant the nature of target-unrelated peptides recovered in the screening of phage-displayed random peptide libraries with antibodies selection by phage display of peptides targeting the hiv- tar element a target-unrelated peptide in an m phage display library traced to an advantageous mutation in the gene ii ribosome-binding site corruption of phage display libraries by target-unrelated clones: diagnosis and countermeasures aspd (artificially selected proteins/peptides database): a database of proteins and peptides evolved in vitro relic--a bioinformatics server for combinatorial peptide analysis and identification of protein-ligand interaction sites an in silico method using an epitope motif database for predicting the location of antigenic determinants on proteins in a structural context pepbank--a database of peptides based on sequence text mining and public peptide data sources mimodb: a new repository for mimotope data derived from phage display technology identification of biologically active peptides using random libraries displayed on phage mapping epitopes on protein surfaces allergen mimotopes for -dimensional epitope search and induction of antibodies inhibiting human ige a new method for mapping discontinuous antibody epitopes to reveal structural features of proteins sitelight: binding-site prediction using phage display libraries the mapping and reconstitution of a conformational discontinuous b-cell epitope of hiv- stepwise prediction of conformational discontinuous b-cell epitopes using the mapitope algorithm d-epitope-explorer ( dex): localization of conformational epitopes within three-dimensional structures of proteins mimox: a web tool for phage display based epitope mapping discontinuous epitope prediction based on mimotope analysis filtering epitope alignments to improve protein surface prediction epitope mapping using combinatorial phage-display libraries: a graph-based algorithm pepitope: epitope mapping from affinityselected peptides identification of atherosclerosis-associated conformational heat shock protein epitopes by phage display and structural alignment pep- d-search: a method for b-cell epitope prediction based on mimotope analysis a novel computer algorithm improves antibody epitope prediction using affinity-selected mimotopes: a case study using monoclonal antibodies against the west nile virus e protein deciphering epitope specificities within polyserum using affinity selection of random peptides and a novel algorithm based on pattern recognition theory applying bioinformatics for antibody epitope prediction using affinity-selected mimotopes -relevance for vaccine design automated detection of conformational epitopes using phage display peptide sequences sarotup: scanner and reporter of target-unrelated peptides constraints on the conformation of the cytoplasmic face of dark-adapted and light-excited rhodopsin inferred from antirhodopsin antibody imprints equilibrium between metarhodopsin-i and metarhodopsin-ii is dependent on the conformation of the third cytoplasmic loop new insights into the membrane topology of the phagocyte nadph oxidase: characterization of an anti-gp -phox conformational monoclonal antibody c-terminal tail phosphorylation of n-formyl peptide receptor: differential recognition of two neutrophil chemoattractant receptors by monoclonal antibodies nfpr and nfpr new p -phox monoclonal antibodies: identification of a conformational probe for cytochrome b computational prediction of the cross-reactive neutralizing epitope corresponding to the monclonal antibody b specific for hiv- gp mapping a neutralizing epitope on the sars coronavirus spike protein: computational prediction based on affinityselected peptides information loss and noise inclusion risk in mimotope based epitope mapping towards the definition of a chimpanzee and human conserved cd domain epitope recognized by t monoclonal antibody induction of immunity in sheep to fasciola hepatica with mimotopes of cathepsin l selected from a phage display library characterization of a key neutralizing epitope on pertussis toxin recognized by monoclonal antibody b peptide mimotopes recognized by antibodies cetuximab and matuzumab induce a functionally equivalent anti-egfr immune response characterization of ige epitopes of cuc m , the major melon allergen, and their role in crossreactivity with pollen profilins mapping protective epitopes in the tick and mosquito subolesin ortholog proteins phage display reveals multiple contact sites between fhua, an outer membrane receptor of escherichia coli, and tonb interactions between tonb from escherichia coli and the periplasmic protein fhud in vivo phage display selection yields atherosclerotic plaque targeted peptides for imaging t lytic phage-displayed peptide libraries exhibit less sequence bias than m filamentous phage-displayed peptide libraries trinucleotide cassettes increase diversity of t phage-displayed peptide library ifats collection: combinatorial peptides identify alpha beta integrin as a receptor for the matricellular protein sparc on adipose stromal cells efficient one-cycle affinity selection of binding proteins or peptides specific for a small-molecule using a t phage display pool identification of microtubule-binding domains on microtubule-associated proteins by major coat phage display technique identification of peptides with targeted adhesion to bone-like mineral via phage display and computational modeling the adsorption of preferential binding peptides to apatitebased materials a d monoclonal antibody recognizes a new linear epitope from sag a toxoplasma gondii tachyzoites, identified by phage display bioselection phage-displayed combinatorial peptide libraries in fusion to betalactamase as reporter for an accelerated clone screening: potential uses of selected enzyme-linked affinity reagents in downstream applications novel beta-lactamase-random peptide fusion libraries for phage display selection of cancer cell-targeting agents suitable for enzyme prodrug therapy from combinatorial peptide selection to drug prototype (i): targeting the vascular endothelial growth factor receptor pathway from combinatorial peptide selection to drug prototype (ii): targeting the epidermal growth factor receptor pathway sample availability: contact the authors this article is an open access article distributed under the terms and conditions of the creative commons attribution license the authors are grateful to the anonymous reviewers for their valuable comments, which have led to the improvement of this review. this work was supported by the national natural science foundation of china under the grant and the scientific research foundation of uestc for youth under the grant jx . key: cord- -lx vy authors: emwas, abdul-hamid; szczepski, kacper; poulson, benjamin gabriel; chandra, kousik; mckay, ryan t.; dhahri, manel; alahmari, fatimah; jaremko, lukasz; lachowicz, joanna izabela; jaremko, mariusz title: nmr as a “gold standard” method in drug design and discovery date: - - journal: molecules doi: . /molecules sha: doc_id: cord_uid: lx vy studying disease models at the molecular level is vital for drug development in order to improve treatment and prevent a wide range of human pathologies. microbial infections are still a major challenge because pathogens rapidly and continually evolve developing drug resistance. cancer cells also change genetically, and current therapeutic techniques may be (or may become) ineffective in many cases. the pathology of many neurological diseases remains an enigma, and the exact etiology and underlying mechanisms are still largely unknown. viral infections spread and develop much more quickly than does the corresponding research needed to prevent and combat these infections; the present and most relevant outbreak of sars-cov- , which originated in wuhan, china, illustrates the critical and immediate need to improve drug design and development techniques. modern day drug discovery is a time-consuming, expensive process. each new drug takes in excess of years to develop and costs on average more than a billion us dollars. this demonstrates the need of a complete redesign or novel strategies. nuclear magnetic resonance (nmr) has played a critical role in drug discovery ever since its introduction several decades ago. in just three decades, nmr has become a “gold standard” platform technology in medical and pharmacology studies. in this review, we present the major applications of nmr spectroscopy in medical drug discovery and development. the basic concepts, theories, and applications of the most commonly used nmr techniques are presented. we also summarize the advantages and limitations of the primary nmr methods in drug development. the unexpected sars-cov- /covid- outbreak, with over million confirmed cases globally (oct. ) and the struggle for survival in the absence of a proven and efficient treatments, emphasizes molecules , the critical need to develop effective, novel, and rapid drug discovery methodologies. even though the pharmaceutical industry works constantly to discover and develop novel drugs, the process is still slow and expensive. the cost of introducing a new drug has increased steadily, with current cost estimates predicting that a future drug will cost in excess of $ . billion. the typical development cost is usually spread out over the course of years [ ] [ ] [ ] , making investment even more difficult (i.e., cost recovery delay). this high investment barrier for drug development is a result of numerous testing phases (scheme ), with each phase requiring a statistically significant number of cases. although there are several other substantial costs to drug development, that discussion of experimental methods to reduce costs is beyond the scope of this review. emphasizes the critical need to develop effective, novel, and rapid drug discovery methodologies. even though the pharmaceutical industry works constantly to discover and develop novel drugs, the process is still slow and expensive. the cost of introducing a new drug has increased steadily, with current cost estimates predicting that a future drug will cost in excess of $ . billion. the typical development cost is usually spread out over the course of years [ ] [ ] [ ] , making investment even more difficult (i.e. cost recovery delay). this high investment barrier for drug development is a result of numerous testing phases (scheme ), with each phase requiring a statistically significant number of cases. although there are several other substantial costs to drug development, that discussion of experimental methods to reduce costs is beyond the scope of this review. the emergence of a pandemic and the emergencies it creates worldwide understandably drive and motivate the rapid development and/or optimization of drugs. however, patient safety and subsequent earned public trust is a primary requirement. drug redirecting/repurposing (scheme ) is an efficient short-cut method in disease treatment that utilizes existing tools, and combines artificial intelligence, machine learning algorithms, and experimental nmr techniques (i.e. "from bench to bedside"). this process must be relatively rapid and efficient to have any benefit to patients and the health-care system. scheme . schematic representation of drug discovery [ ] . numerical data were reported from meigs et al. [ ] . compared to mass spectrometry and high-performance liquid chromatography (hplc), nuclear magnetic resonance (nmr) is another powerful technique with several unique advantages [ ] [ ] [ ] [ ] . nmr is intrinsically quantitative, and it provides several different approaches that are routinely utilized to identify and structurally elucidate molecules of interest [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] . in contrast to mass spectrometry, nmr is non-destructive, non-invasive, has extremely high reproducibility permitting in addition to the advantages provided by nmr, it is often used with complementary methods such as x-ray crystallography, hplc, and mass spectrometry [ ] . an example of this is found in work by wyss et al. [ ] , where they combined x-ray crystallography with nmr fragment-based screening to create the first inhibitor candidate for bace- in alzheimer's disease. bace- is a membrane-anchored aspartic acid protease and is responsible for the production of amyloid beta peptides in neurons related to the progression of alzheimer's disease [ , ] . using nmr fragmentbased screening, wyss et al. identified isothiourea as binding to bace- and confirmed this observation with the x-ray crystal structure of the complex of a ligand-efficient isothiourea fragment. information obtained from these experiments aided in design optimization, resulting in the selection in addition to the advantages provided by nmr, it is often used with complementary methods such as x-ray crystallography, hplc, and mass spectrometry [ ] . an example of this is found in work by wyss et al. [ ] , where they combined x-ray crystallography with nmr fragment-based screening to create the first inhibitor candidate for bace- in alzheimer's disease. bace- is a membrane-anchored aspartic acid protease and is responsible for the production of amyloid beta peptides in neurons related to the progression of alzheimer's disease [ , ] . using nmr fragment-based screening, wyss et al. identified isothiourea as binding to bace- and confirmed this observation with the x-ray crystal structure of the complex of a ligand-efficient isothiourea fragment. information obtained from these experiments aided in design optimization, resulting in the selection of iminopyrimidinones as bace- inhibitors [ ] . this is a perfect example of using different complementary methods to maximize scientific outcome. however, in order to be efficient, one must know the advantages and disadvantages of each method. one of the major issues regarding nmr is the effective size restriction when measuring targets such as proteins above kda. recent progress has extended this mass limit; an example of this is the resolved structure of chaperone secb in complex with unstructured prophoa (pdb id jtl) with a total mass of kda using nmr [ ] . in this review, we present practical guideline to use nmr techniques in drug design studies and provide examples of the successful use of nmr in drug-design. nmr is a versatile tool for studying biomolecules of all kinds and is a unique regarding the biophysical analysis of drugs [ ] [ ] [ ] [ ] . the basic feature of nmr lies in the fact that it inductively detects the larmor precession of individual nuclei (i.e., spins) which vary because of different atomic, electronic, and chemical environments (i.e., structural atomic relationships). initially, the sample is placed in a strong, static, and homogeneous magnetic field. because spins contain angular momentum, they exhibit larmor precessions around this static magnetic field. a net magnetization builds up over time as the spin population (represented by different energy levels) is minutely differential in the presence of the magnetic field. these levels are dictated by the spin quantum number and can be roughly thought of as different orientations with respect to the static field. subsequently, induced electromagnetic fields at radiofrequencies (called rf pulses) are applied transverse to the plane of the static magnetic field, and the net or bulk magnetization undergoes an effective rotation. the bulk coherence moves into the transverse plane and the subsequent coherently precessing magnetization vectors induce a detectable alternating voltage in the nmr receiver coil. this tiny alternating voltage is amplified and converted from an analog time domain signal to a frequency reading via fourier transformation. these signals are recorded in response to the induced radio-wave pulses ( figure ) and are representative of the larmor frequencies that are converted into normalized values termed chemical shifts in order to be field independent. molecules , , x for peer review of of iminopyrimidinones as bace- inhibitors [ ] . this is a perfect example of using different complementary methods to maximize scientific outcome. however, in order to be efficient, one must know the advantages and disadvantages of each method. one of the major issues regarding nmr is the effective size restriction when measuring targets such as proteins above kda. recent progress has extended this mass limit; an example of this is the resolved structure of chaperone secb in complex with unstructured prophoa (pdb id jtl) with a total mass of kda using nmr [ ] . in this review, we present practical guideline to use nmr techniques in drug design studies and provide examples of the successful use of nmr in drug-design. nmr is a versatile tool for studying biomolecules of all kinds and is a unique regarding the biophysical analysis of drugs [ ] [ ] [ ] [ ] . the basic feature of nmr lies in the fact that it inductively detects the larmor precession of individual nuclei (i.e. spins) which vary because of different atomic, electronic, and chemical environments (i.e. structural atomic relationships). initially, the sample is placed in a strong, static, and homogeneous magnetic field. because spins contain angular momentum, they exhibit larmor precessions around this static magnetic field. a net magnetization builds up over time as the spin population (represented by different energy levels) is minutely differential in the presence of the magnetic field. these levels are dictated by the spin quantum number and can be roughly thought of as different orientations with respect to the static field. subsequently, induced electromagnetic fields at radiofrequencies (called rf pulses) are applied transverse to the plane of the static magnetic field, and the net or bulk magnetization undergoes an effective rotation. the bulk coherence moves into the transverse plane and the subsequent coherently precessing magnetization vectors induce a detectable alternating voltage in the nmr receiver coil. this tiny alternating voltage is amplified and converted from an analog time domain signal to a frequency reading via fourier transformation. these signals are recorded in response to the induced radio-wave pulses ( figure ) and are representative of the larmor frequencies that are converted into normalized values termed chemical shifts in order to be field independent. table . h chemical shift assignments is shown as an example. the chemical structure and simulated h-nmr spectrum were created using chemdraw . . this is the final, representative spectroscopic signature of the chemical and magnetic environment of the atom, and it provides detailed atomic resolution information about the molecular structure [ ] [ ] [ ] [ ] . a wealth of information can be derived from the nmr signal made up components such as the chemical shift position, signal linewidth, and observed couplings/multiplet structure. the table . h chemical shift assignments is shown as an example. the chemical structure and simulated h-nmr spectrum were created using chemdraw . . this is the final, representative spectroscopic signature of the chemical and magnetic environment of the atom, and it provides detailed atomic resolution information about the molecular structure [ ] [ ] [ ] [ ] . a wealth of information can be derived from the nmr signal made up components such as the chemical shift position, signal linewidth, and observed couplings/multiplet structure. the signal contains precise details about the chemical environment of the involved and interacting spins in the structure of the molecule, dynamics of the spins in various timescales, conformational exchange, etc. [ ] [ ] [ ] . any change in the environment of the associated spin can be observed. these changes include molecules , , of molecular binding, interactions, and/or exchange between different conformations [ , [ ] [ ] [ ] . thus, nmr has been used to study a wide range of functional molecules such as natural products [ ] [ ] [ ] , saccharides [ , ] , metabolites [ , ] , dna [ , ] , and proteins [ ] , and its use as an analytical tool in drug design research has increased immensely in recent years (see figure ). as nmr is non-destructive in nature, the same sample can be analyzed repeatedly. nmr can be performed first and then submitted to mass spectrometry (ms); however, the addition of common deuterated nmr solvents (such as d o) can perturb ms results and should be avoided (e.g., tube-in-tube or by using non-deuterated solvent and running the nmr unlocked). in fact, high-performance liquid chromatography (hplc), ion-trap ms and nmr have been combined to detect the effects of drugs demonstration in urine and blood serum samples [ , , ] . corcoran and spraul [ ] emphasize that liquid chromatography (lc), ms, and nmr utilized in parallel give comprehensive structural data on molecules of novel drugs in development. in the following subsections we briefly describe nmr methods that have been used in drug design, and then discuss how nmr principles are used in drug discovery research. the one-dimensional ( d) experiment is by far the most common nmr experiment used for drug studies. the d acquisition takes the least amount of time, has one of the simplest hardware requirements, and therefore, in most cases, d-nmr is more attractive for high throughput studies. one dimensional nmr spectroscopy normally incorporates a preparation period, some form of induced excitation to form coherence, and lastly, a signal "read" detection period. the preparation period can be modified according to the needs of the experiment or the specifics of the sample. simple d nmr is capable of rapidly producing high-quality spectra of drugs and their targets while revealing how the drugs and targets may interact at the atomic level. d h-nmr is extremely effective in drug design studies because it has a (relatively) high sensitivity, it is non-destructive, and because hydrogen atoms are extremely abundant in most molecules of interest. therefore the resulting spectra usually contains a large amount of relevant information and this wealth of data can be acquired in a relatively short period of time. the basic d h-nmr, along with d c-nmr, d n-nmr, and d p-nmr, and their respective uses in drug design/discovery are briefly discussed below. the h hydrogen isotope is nmr visible, has the highest gyromagnetic ratio (apart from tritium) of all of nmr active nuclei, and is combined with a vast natural abundance in organic chemical compounds. this makes the d- h-nmr experiment the most commonly applied nmr approach. moreover, many software databases [ ] [ ] [ ] [ ] are well established for h-nmr spectra therefore assisting with processing, analyzing, and identifying the detected molecules automatically. since almost all drug discovery and drug development studies are performed on samples dissolved in water, many different solvent suppression methods have been applied. the most common is presaturation [ , ] . the key point of this method is to use a low power induced field at the specific frequency of water. this effectively averages out any coherence of the water resonance. the experiment is simple for common hardware to perform and easy to set up; however, presaturation has a substantial disadvantage in that signals resonating close to the solvent signal will show decreased intensity [ , ] or may be lost entirely. this is due to the fact the even selective pulses or very low power pulses also excite some area around the water signal. also suppressed hydrogen from h o in solution can exchange with atoms of interest in the molecule and effectively bleed the suppressive spin state to any neighboring atoms. the water signal itself is usually broad, so a wider area of suppression is not necessarily undesirable but affects more of the molecule(s) of interest. more recent water suppression techniques have been developed such as those based on a scheme known as excitation sculpting [ , ] . the basic pulse sequence consists of a double pulsed field gradient echo (dpfge) in each of which a selective component pulse is flanked by two pulsed field gradients [ ] . the particular elements differ for different applications. in the case of water suppression known as water suppression by gradient tailored excitation (watergate), this involves an initial encoding gradient along with the middle element; a combination of two selective • rotations on the water along with a central non-selective • excitation of all resonances [ ] . this is predicated in that water experiences a • rotation (effectively nothing) while all other spins experience • rotation. the application of the second refocusing gradient does not rephase the water and therefore removes the signal. the reader is referred to the detailed literature [ , , ] for further information. in principle, a water suppression element (or many elements combined) can be incorporated in any existing pulse sequence to enhance the performance, and it has been implemented in various d, d, and triple resonance d/ d experiments. although h is the most sensitive nucleus for nmr yielding strong, sharp signals within a few minutes [ ] , chemical shift dispersion of h is quite narrow (only around ppm). this has prompted the consideration of other nuclei such as c, n, or p for resolution improvements. compared with h, c has a much higher chemical shift dispersion (~ ppm), however the natural abundance of c is low ( . %). additionally, the gyromagnetic ratio is~ times weaker than h and therefore c spectra are far more difficult to obtain especially for less concentrated samples. there are some polarization transfer techniques such as distortionless enhancement by polarization transfer (dept) or insensitive nuclei enhanced by polarization transfer (inept), which can enhance signal intensity by starting the magnetization on a higher sensitivity and abundance proton and then transferring magnetization to the less sensitive carbon nuclei for subsequent direct detection [ ] , but this requires additional hardware and acquisition times. the use of d c in drug design studies was illustrated by tsujimoto et al. [ ] . the goal of the study was to examine if a metabolomics approach based on h and c offers significant improvements when comparing potential drugs. the authors prepared a total of samples with five different citrus-type crude drugs (kijitsu, tohi, chimpi, kippi and seihi) and measured d h and d c for each sample. while h-nmr spectra allowed the identification of three compounds (naringin, sucrose, and β-glucose), using c-nmr allowed unambiguous identification of eight additional compounds (naringin, neohesperidin, αand β-glucose, sucrose, limonene, narirutin, and synephrine). the added signal resolution from c-nmr spectra allowed researchers to obtain better structural information about the compounds than from h-nmr spectra alone. in comparison to the previous example, n has a lower shift dispersion (~ ppm) than c, but higher than that of h. here, the situation is unfortunately severely limited due to an even lower natural abundance ( . %) and a gyromagnetic ratio~ times smaller than h. this means that n's combined sensitivity is around , times lower than h. as a result, isotopic enrichment of n combined with h-mediated enhancement via indirect detection is often needed in order to obtain a satisfactory d n spectra. similar to c, a few methods are available to overcome such low sensitivity. one of them focuses on tagging molecules with carboxyl groups using n-ethanolamine and later detecting the signal using a d heteronuclear correlation nmr experiment [ ] . currently, novel approaches such as "smart isotope labeling" have been developed [ ] . also, the sofast (band-selective optimized flip angle short transient) technique can help but results in substantial hardware considerations/drawbacks and often increased concentrations, and/or dramatically longer experiments are still required [ ] [ ] [ ] . promising methods are on the horizon. these methods include n heteronuclear signal enhancement via signal amplification by reversible exchange in shield enables alignment transfer to heteronuclei (sabre-sheath); however, more work and research are required before such methods can be applied for biomedical purposes [ ] . with a natural abundance of % and a gyromagnetic ratio of about . times smaller than h, one may think that phosphorus could be broadly used for nmr experiments regarding the drug discovery and development. however, the application of p is limited due to the fact that most of the molecules of interest simply do not contain a phosphorus atom. therefore p-nmr is usually applicable for studies related to energy, phospholipid metabolism (atp, nadp), and/or characterization of changes in dna [ , , ] . for example, overall et al. conducted an experiment in which they showed that p solid-state nmr can be used for quantitative analysis of dna dynamics within live bacteria [ ] . for that, the researchers first prepared untreated cultures of e. coli, and measured them using a hartmann-hahn h to p cross-polarization ( p cp) experiment. afterwards, they measured e. coli treated with ampicillin and maculatin . (mac . ) in a similar manner. spectra obtained from treated bacteria compared to those obtained from untreated bacteria showed alterations in the lineshape, reduced signal intensity at the spectrum's edges, and a shift in spectral density towards ppm which indicated the increased dynamics of the phosphorus from nucleic acids [ ] . over time, several innovations have been applied to expand the usage of p. like in c and n labeling of specific biological compounds, incorporation of p can also be used. in order to achieve that, -chloro- , , , -tetramethyldioxaphospholane (ctmdp) can be used for tagging lipids containing hydroxyl, aldehyde, and carboxyl groups that can later be detected with better resolution [ ] . another fairly recent method enables toxicological screening of p in living cells for several hours without affecting cell viability [ ] . this specific method can be used to observe the changes in energy metabolism in real-time while enabling the evaluation of the effects of administered drugs. nmr experiments are not limited to one-dimensional direct acquisition; they can be extended to multidimensional methods including d, d, d, and even higher dimensionality. the focus of this section is common d nmr experiments that have been used in drug design and drug development. a brief description of correlation spectroscopy (cosy), total correlation spectroscopy (tocsy), and heteronuclear multiple bond correlation (hmbc), along with their uses in drug design and discovery will be presented. cosy is one of the simplest and most frequently used d nmr experiment [ ] . it shows the homonuclear coupling of nuclei (i.e., h- h) separated by up to several covalent bonds. the pulse sequence consists of a • excitation pulse followed by a specific evolution time (t ), a second pulse, and finally a measurement period (t , not to be confused with relaxation rates or times). the second pulse can be • or • or • , depending upon the specific requirements, and respectively yield cosy [ ] , cosy- or cosy- functionality (see [ ] [ ] [ ] ). a two-dimensional fourier transform (ft) yields the final spectra and shows the frequencies for proton ( h) or carbon (in the case of carbon detection) along both axes. there are two types of peaks; (i) diagonal peaks, which represent the peaks of the conventional d spectra, and (ii) cross-peaks, which have different values in the two frequency axes and are therefore off the diagonal. these off diagonal cross-peaks are the most important pieces of information as they mark correlations between pairs of nuclei due to through bond magnetization transfer. this helps in identifying which atoms are connected [ ] , critical for structural elucidation of both known molecules and unknown molecules in solution [ ] . by implementing phase-cycling [ , ] , it is also possible to distinguish different types of coupling and yields further helpful information about the chemical structure of a molecule [ ] . as an example, the use of the cosy experiment was presented in the work of zheng et al. [ ] . the main goal of their work was to investigate potential biological differences and compare the pharmacological effects between danggui (an herbal drug used in traditional chinese medicine) and european danggui. the difference between the two experiments is that a tocsy experiment will show multiple cross-peaks including indirectly coupled nuclei (i.e., longer range via scalar coupling) throughout the j-coupled spin system of a chemical compound. the basic pulse sequence of the tocsy consists of excitation by a • pulse, followed by a free variable evolution period which encodes the indirect dimension. this is normally followed by an isotropic mixing sequence to transfer magnetization between spins via the strong scalar coupling. the mixing generates in-phase magnetization throughout a spin coupled network of the associated nuclei during the mixing time. lastly, a direct detection is performed. a major advantage of the tocsy experiment is that it detects in-phase magnetization (i.e., pure absorptive line-shape) which is far easier to analyze compared to the anti-phase information in the phase sensitivity cosy-type experiment. the isotropic mixing is usually performed using a composite pulse scheme such as waltz, mlev or dipsi [ , ] pulse train, and can be sandwiched between two z-filters [ ] where isotropic mixing is performed on the longitudinal magnetization. the most obvious advantage of tocsy is that all cross-peaks of the same spin system can be observed for whole spin system at once. this is useful for identifying the complete network of spins and reducing the ambiguity of any spectral overlap. the tocsy experiment can be produced as d with a relatively shorter time and easier analysis compared to d but lacks the benefit of multi-dimensional resolution. the d tocsy is usually done to resolve spectra overlap [ ] when first identifying molecules [ ] [ ] [ ] . for example, jiang et al. used this to predict the response to gemcitabine-carboplatin (gc) chemotherapy in patients with metastatic breast cancer who were previously exposed to treatment with both anthracyclines and taxanes [ ] . for that, researchers collected serum samples from patients prior to treatment and measured them using d h-nmr. additionally, they conducted d nmr experiments such as the h, h-cosy, , h-tocsy, h, c-hsqc, and h, c-hmbc to help assign serum metabolites. after receiving the treatment with gemcitabine-carboplatin, patients were divided into four groups based on the results from the computed tomography: complete response (cr), partial response (pr), stable disease (sd), or progressive disease (pd). after comparing nmr results prior to the treatment with the outcome of chemotherapy, the researchers observed lower baseline levels of serum format and acetate in breast cancer patients who progressed with the disease than in those who achieved a clinical benefit from therapy, indicating that those two biomarkers could be used to distinguish between patients who will benefit from gc treatment from those who do not [ ] . d-heteronuclear single quantum coherence (hsqc) experiments are commonly used to help resolve spectral overlap [ ] while providing c information without the inherent sensitivity losses involved in c direct detection (see below). hsqc shows the correlations between directly coupled nuclei [ ] , e.g., h- c or h- n [ ] . as such, an hsqc spectrum will show clean peaks for each unique proton directly connected to the heteronuclear nuclear atom of interest [ , ] . in h, c/ n hsqc experiments, the magnetization is transferred from the more sensitive nucleus (i: h) to the less sensitive nucleus (s: c/ n) [ ] [ ] [ ] (figure ). this is especially useful when applying nmr spectroscopy to drug design, as most drugs are organic (i.e., contain carbon atoms), and the relative abundance of c ( . %) is quite low [ ] . by transferring sensitivity from h to c, one can circumvent the long experimental time required for d c experiments [ ] . molecules , , x for peer review of abundance of c ( . %) is quite low [ ] . by transferring sensitivity from h to c, one can circumvent the long experimental time required for d c experiments [ ] . for example, de castro et al. [ ] studied ptac s and its related cytotoxicity to the cisplatinresistant epithelial ovarian carcinoma (eoc), skov- cells. in the study, they used nmr spectroscopy and multi-variate statistical analysis to observe how skov- cells reacted to treatment with ptac s. in particular, they used h, c-hsqc along with h-cosy and heteronuclear multiple bond correlation (hmbc), and the human metabolome database to assign the chemical shifts of the lipid metabolites present in the studied samples. interestingly, skov- cells treated with ptac s produced more pyruvate than skov- cells treated with cisplatin. the authors also noticed an unexpected difference in lipid metabolite expression levels between the cells treated with ptac s and those treated with cisplatin. these results provide a possible explanation for how ptac s is able to overcome cisplatin resistance in skov- cells [ ] . heteronuclear d experiments are useful for transferring magnetization from sensitive nuclei (i.e. h) to less sensitive nuclei (i.e. c) [ ] thereby reducing the time needed for the acquisition of spectra [ ] . heteronuclear single quantum coherence (hsqc) will only show one cross peak for each coupled pair [ , ] of nuclei. this makes hsqcs useful for assigning the backbone of proteins [ ] and in metabolites of complex biofluids [ ] , whose d h-nmr spectra can suffer from severe spectral overlap. the hmbc technique, while similar to hsqc, is an example of a heteronuclear d experiment that reveals correlations between nuclei separated by two or more chemical bonds while also suppressing one-bond correlations at the same time. this experiment combined with hsqc is often used to assign nmr spectra for studied molecules in drug design experiments [ , , , ] . as an example, hmbc was used in a recent study by xu et al. [ ] to investigate the changes in the metabolic profiles of rats treated with different dosages of the "renqingmangjue" pill, a traditional tibetan medicine. in this study, the rats were divided into four groups based on the amount of "renqingmangjue" administered: low dose group (ld)- . mg/kg/day, middle dose group (md)- . mg/kg/day, high dose group (hd)- . mg/kg/day and a control group (nc). after days of consecutive administration, half of the rat population was used to collect samples such as serum, kidney, and liver tissue, while the other half underwent an additional days of recovery before the same samples were acquired. the samples were measured using h-nmr cpmg (an experiment used to suppress signals from larger molecules, see below) [ ] [ ] [ ] along with h, h-cosy, h, c-hsqc, and h, c-hmbc used for molecules assignment. the obtained spectra showed that the "renqingmangjue" pill alters many metabolites, which are related to a variety of metabolic pathways including energy metabolism, amino acid metabolism, and lipid metabolism indicating potentially harmful effects on kidneys and liver. for example, de castro et al. [ ] studied ptac s and its related cytotoxicity to the cisplatin-resistant epithelial ovarian carcinoma (eoc), skov- cells. in the study, they used nmr spectroscopy and multi-variate statistical analysis to observe how skov- cells reacted to treatment with ptac s. in particular, they used h, c-hsqc along with h-cosy and heteronuclear multiple bond correlation (hmbc), and the human metabolome database to assign the chemical shifts of the lipid metabolites present in the studied samples. interestingly, skov- cells treated with ptac s produced more pyruvate than skov- cells treated with cisplatin. the authors also noticed an unexpected difference in lipid metabolite expression levels between the cells treated with ptac s and those treated with cisplatin. these results provide a possible explanation for how ptac s is able to overcome cisplatin resistance in skov- cells [ ] . heteronuclear d experiments are useful for transferring magnetization from sensitive nuclei (i.e., h) to less sensitive nuclei (i.e., c) [ ] thereby reducing the time needed for the acquisition of spectra [ ] . heteronuclear single quantum coherence (hsqc) will only show one cross peak for each coupled pair [ , ] of nuclei. this makes hsqcs useful for assigning the backbone of proteins [ ] and in metabolites of complex biofluids [ ] , whose d h-nmr spectra can suffer from severe spectral overlap. the hmbc technique, while similar to hsqc, is an example of a heteronuclear d experiment that reveals correlations between nuclei separated by two or more chemical bonds while also suppressing one-bond correlations at the same time. this experiment combined with hsqc is often used to assign nmr spectra for studied molecules in drug design experiments [ , , , ] . as an example, hmbc was used in a recent study by xu et al. [ ] to investigate the changes in the metabolic profiles of rats treated with different dosages of the "renqingmangjue" pill, a traditional tibetan medicine. in this study, the rats were divided into four groups based on the amount of "renqingmangjue" administered: low dose group (ld)- . mg/kg/day, middle dose group (md)- . mg/kg/day, high dose group (hd)- . mg/kg/day and a control group (nc). after days of consecutive administration, half of the rat population was used to collect samples such as serum, kidney, and liver tissue, while the other half underwent an additional days of recovery before the same samples were acquired. the samples were measured using h-nmr cpmg (an experiment used to suppress signals from larger molecules, see below) [ ] [ ] [ ] along with h, h-cosy, h, c-hsqc, and h, c-hmbc used for molecules assignment. the obtained spectra showed that the "renqingmangjue" pill alters many metabolites, which are related to a variety of metabolic pathways including energy metabolism, amino acid metabolism, and lipid metabolism indicating potentially harmful effects on kidneys and liver. relaxation in nmr is a phenomenon describing the time dependence involved in signal intensity after an induced rf (radiofrequency) pulse is applied [ ] . after application of a • rf pulse, the bulk magnetization will move to the transverse (xy) plane and will gradually return to its original equilibrium position along the longitudinal (z) axis [ ] . this process is described in figure , and is termed t relaxation. the details are beyond the scope of the manuscript and interested readers are directed to [ ] and references therein. relaxation times for nmr are even more complicated and exist in two categories: t and t . t refers to the rate of longitudinal (or spin-lattice) z-axis relaxation as the system returns to equilibrium. a second component also contributes, i.e., t relaxation and refers to the rate of transverse (or spin-spin) relaxation [ ] which occurs in the xy plane. t is independent of the longitudinal relaxation (t ) and represents the loss of coherence in the precessing spins. therefore nmr relaxation spectroscopy can be based on t and/or t [ ] , and is collectively referred to as "relaxation edited nmr" [ ] . molecules , , x for peer review of relaxation in nmr is a phenomenon describing the time dependence involved in signal intensity after an induced rf (radiofrequency) pulse is applied [ ] . after application of a ° rf pulse, the bulk magnetization will move to the transverse (xy) plane and will gradually return to its original equilibrium position along the longitudinal (z) axis [ ] . this process is described in figure , and is termed t relaxation. the details are beyond the scope of the manuscript and interested readers are directed to [ ] and references therein. relaxation times for nmr are even more complicated and exist in two categories: t and t . t refers to the rate of longitudinal (or spin-lattice) z-axis relaxation as the system returns to equilibrium. a second component also contributes, i.e. t relaxation and refers to the rate of transverse (or spin-spin) relaxation [ ] which occurs in the xy plane. t is independent of the longitudinal relaxation (t ) and represents the loss of coherence in the precessing spins. therefore nmr relaxation spectroscopy can be based on t and/or t [ ] , and is collectively referred to as "relaxation edited nmr" [ ] . over time (ms to seconds, and in extreme cases, minutes [ ] ), the bulk magnetization will decrease in the transverse plane, and increase in the longitudinal axis, returning to its original, equilibrium value. (a) represents t relaxation and (b) represents t relaxation. t -based methods typically measure and compare the t times of the free and bound ligands. a common way to measure the t value of a small molecule is the inversion recovery experiment [ , ] , although other experiments are also available such as ultrafast nmr t [ ] and saturation inversion recovery [ ] . in general, the shorter t the relaxation time the less intense the peak signal will be and the broader the signal linewidth [ ] . the t values of free and bound ligand will differ depending on how strongly the ligand binds because molecular interactions with the target will influence the ligand's molecular motion, and hence, its longitudinal relaxation [ ] . bound ligands will have smaller t values than in their free form because, overall, they will experience slower molecular motion upon interacting with a target [ ] therefore behaving like a much larger molecule. they can (depending on molecule size) also display a negative noe difference spectrum (transferred noe) [ ] , whereas non-binding ligands normally show small-positive noes [ ] . for binding ligands to display negative noes, their t values must be comparatively longer than the /koff value of the target [ ] . t -based methods typically measure and compare the t times of the free and bound ligands. a common way to measure the t value of a small molecule is the inversion recovery experiment [ , ] , although other experiments are also available such as ultrafast nmr t [ ] and saturation inversion recovery [ ] . in general, the shorter t the relaxation time the less intense the peak signal will be and the broader the signal linewidth [ ] . the t values of free and bound ligand will differ depending on how strongly the ligand binds because molecular interactions with the target will influence the ligand's molecular motion, and hence, its longitudinal relaxation [ ] . bound ligands will have smaller t values than in their free form because, overall, they will experience slower molecular motion upon interacting with a target [ ] therefore behaving like a much larger molecule. they can (depending on molecule size) also display a negative noe difference spectrum (transferred noe) [ ] , whereas non-binding ligands normally show small-positive noes [ ] . for binding ligands to display negative noes, their t values must be comparatively longer than the /k off value of the target [ ] . t relaxation times can be easily used to screen small molecules as ligands for dna [ ] and serve as a basis for hts experiments [ ] . an experiment related to drug design that utilized d and d relaxation edited nmr was done by hajduk et al. [ ] in which he and others used d and d relaxation edited nmr techniques to detect ligands that bind to fk binding protein and stromelysin. one year earlier, liu et al. [ ] used relaxation edited one-and two-dimensional h-nmr spectroscopy to characterize biological fluids. tang et al. [ ] extended this by applying relaxation edited nmr spectroscopy to improve the detection of metabolites in blood plasma. more recently, jaremko et al. commented on available models used to interpret n protein relaxation data [ ] , and even used deficient n relaxation data to rapidly calculate the dynamics of proteins [ ] . the t relaxation experiment relies on so-called carr-purcell-meiboom-gill (cpmg) building blocks ( figure ). molecules , , x for peer review of t relaxation times can be easily used to screen small molecules as ligands for dna [ ] and serve as a basis for hts experiments [ ] . an experiment related to drug design that utilized d and d relaxation edited nmr was done by hajduk et al. [ ] in which he and others used d and d relaxation edited nmr techniques to detect ligands that bind to fk binding protein and stromelysin. one year earlier, liu et al. [ ] used relaxation edited one-and two-dimensional h-nmr spectroscopy to characterize biological fluids. tang et al. [ ] extended this by applying relaxation edited nmr spectroscopy to improve the detection of metabolites in blood plasma. more recently, jaremko et al. commented on available models used to interpret n protein relaxation data [ ] , and even used deficient n relaxation data to rapidly calculate the dynamics of proteins [ ] . the t relaxation experiment relies on so-called carr-purcell-meiboom-gill (cpmg) building blocks ( figure ). figure . cpmg pulse sequence. first, a ° rf pulse is applied and results in transverse magnetization in the xy plane. then a °y pulse is applied to re-phase the magnetization vectors. after °y, the vectors who were faster during the dephasing are overtaken by the slower vectors, which results in re-phasing and generation of a spin-echo signal. this process is repeated several times. this pulse sequence is explained with the following steps: first, application of a ° rf pulse creates a transverse (xy plane) magnetization. second, a spin-echo period (delay- °-delay block) is responsible for mx/y magnetization decay. this period is repeated "n'' times (cpmg building blocks). it is essential to point out that every nmr experiment involving a large number of pulses (e. g. due to the repeating building blocks) is likely to be sensitive to hardware restrictions and small miscalibrations of the duration of the applied pulses. to attenuate the unwanted effects of miscalibrations, meiboom and gill modified the previously used carr-purcell sequence [ ] by changing the phase of the applied ° pulses from x to y [ ] . this procedure can be used to measure t relaxation times of any type of nuclei. for instance, in the case of c, all pulses and acquisitions are applied on c channel, while broadband proton decoupling is applied during all pulse sequences. it works analogically for different nmr-active nuclei [ ] . in a typical cpmg experiment, the effective transverse relaxation rate, r ,eff, is typically measured by fitting the signal decay as a function of a variable number of cpmg blocks [ ] . the experimental half-height linewidth (d) of a given resonance signal is directly related to t * (also called as 'effective' or "observed'') by the following equation: ( ) figure . cpmg pulse sequence. first, a • rf pulse is applied and results in transverse magnetization in the xy plane. then a • y pulse is applied to re-phase the magnetization vectors. after • y , the vectors who were faster during the dephasing are overtaken by the slower vectors, which results in re-phasing and generation of a spin-echo signal. this process is repeated several times. this pulse sequence is explained with the following steps: first, application of a • rf pulse creates a transverse (xy plane) magnetization. second, a spin-echo period (delay- • -delay block) is responsible for mx/y magnetization decay. this period is repeated "n" times (cpmg building blocks). it is essential to point out that every nmr experiment involving a large number of pulses (e. g. due to the repeating building blocks) is likely to be sensitive to hardware restrictions and small miscalibrations of the duration of the applied pulses. to attenuate the unwanted effects of miscalibrations, meiboom and gill modified the previously used carr-purcell sequence [ ] by changing the phase of the applied • pulses from x to y [ ] . this procedure can be used to measure t relaxation times of any type of nuclei. for instance, in the case of c, all pulses and acquisitions are applied on c channel, while broadband proton decoupling is applied during all pulse sequences. it works analogically for different nmr-active nuclei [ ] . in a typical cpmg experiment, the effective transverse relaxation rate, r ,eff , is typically measured by fitting the signal decay as a function of a variable number of cpmg blocks [ ] . the experimental half-height linewidth (d) of a given resonance signal is directly related to t * (also called as 'effective' or "observed") by the following equation: t represents the transverse relaxation times, and additional broadening comes from the magnetic field inhomogeneities (t inh ), which must be taken into account. t measurements of ligands are also useful for determining the binding nature of a small molecule. the t values of small molecules are quite large compared to those of bigger molecules (i.e., proteins) mostly because macromolecules have more spin-spin diffusion [ ] . bound ligands will, therefore, display shorter t values than non-binding ligands because they interact with the target (i.e., protein), adopting similar vibrational and rotational energies to the target [ ] . this interaction is represented by the resonance line broadening in the binding ligand's spectrum when a receptor is introduced into the sample [ ] . given the sizable difference of t values of binding and non-binding ligands, one can utilize d relaxation-edited experiments to distinguish the binding ligands from the non-binding ligands efficiently and effectively based on the differences in the t values [ ] . these and other related relaxation edited experiments prove useful in drug design. relaxation edited nmr spectroscopy takes advantage of an inherent atomic property (i.e., the return of bulk magnetization back to equilibrium [ ] ), so no molecular enrichment (e.g., n isotopic enrichment of protein targets) is required [ ] . furthermore, the slow time scale of nmr relaxation allows the user to manipulate the external conditions (i.e., length and power of pulse) to increase the resolution of targets and potential drugs [ ] in nmr drug design experiments. however, this slow timescale also sets the lower limit at which nmr drug design experiments can be performed [ ] , meaning that any external manipulations cannot decrease experimental time below a certain threshold. this varies based on the drugs and targets used in the experiment. low drug solubility is also a challenge, as the ligands must be at a sufficiently high concentration to allow detection via nmr, although the use of organic solvents has helped to attenuate this effect in relaxation edited nmr spectroscopy [ ] . for examples of experiments that use different nmr techniques mentioned above, see table . emodin can affect the immune response and interrupt energy metabolism (citric acid cycle) along with glutathione synthesis, which can lead to oxidative stress. dox decreases atp production and induces oxidative stress in h c cells. dex counteracts those changes, having a cardioprotective effect on h c cell lines. [ ] curcumin shows antihyperlipidemic effects on c bl/ slac mice by partially restoring metabolic defects induced by a high-fat diet. affected metabolic pathways include the citric acid cycle, glycolysis and gluconeogenesis, ketogenesis of bcaa, synthesis of ketone bodies and cholesterol, and choline and fatty acid metabolism. formosanin c shows the ability to inhibit synthesis and methylation of dna as well as reducing the activity of the citric acid cycle and energy metabolism in the mitochondria of hepg cells. melamine melamine disrupts metabolism of glucose, nitrogen, and protein in the liver of wistar rat. h-nmr cpmg [ ] aristolochic acid (aa) aristolochic acid causes renal lesions and a disorder in tubular reabsorption in wistar rats. evaluating response outcome for patients with rheumatoid arthritis, treated with rituximab. identification of metabolites changes between responders and non-responders such as succinate, taurine, lactate, pyruvate and aspartate. no distinction between metabolite profiles of serum from patients treated with levetiracetam, lamotrigine and topiramate. could not evaluate response of initial treatment of epilepsy. [ ] metallaprism mainly affects lipid metabolism in a (human ovarian cancer) and hek- (human embryonic kidney) cells, and increases gsh levels in all cell lines. in a cisr (cisplatin resistant a ) cells, lipid biogenesis and glycosylation are affected by treatment with metallaprism. extract from centella asiatica promotes glycolysis, boosts the citric acid cycle and decreases gluconeogenesis and lipid metabolism in t dm sprague-dawley rats. vr and vr improve glycerol metabolism, decrease betaine levels, and normalize the altered level of myoinositol in serum of dmh-induced crc (colorectal cancer) wistar rats. evaluating the efficacy (determined by the concentration of a drug able to reach the therapeutic site) of four drugs in cerebrospinal fluid of tuberculous meningitis patients. genipin can recover energy metabolism to normal levels, and regulate methylamine and amino acid metabolisms of diabetic sprague dawley rats. h-noesy- d [ ] adriamycin (adr) identification of seven biomarkers: trimethylamine oxide (tmao), taurine, trimethylamine (tma), hippurate, trigonelline, citrate and -oxoglutarate that can predict tumor's (gastric adenocarcinoma) response to adr treatment in balb/c-nu/nu mice. h-noesypresat- d [ ] danggui/european danggui fuzi causes a shift in energy metabolism (from aerobic respiration to anaerobic), induces membrane toxicity, and disrupts the balance of gut microbiota of wistar rats. administrating fuzi with gancao diminishes the toxic effects of fuzi. h-nmr spectra enabled the identification of three compounds (naringin, sucrose, and β-glucose), and c-nmr enabled the identification of eight compounds (naringin, neohesperidin, αand β-glucose, sucrose, limonene, narirutin, and synephrine). as stated, nmr spectroscopy can be fundamental in studying how drugs interact with their targets. this has been done mainly via the fragment based drug design (fbdd) approach, which has two sub-approaches: target-(i.e., protein) based, or ligand-(drug) based. target based screening monitors how the target responds to binding molecules in a method called structure activity relationship ("sar") by nmr. ligand (drug)-based screening methods provide ways to observe the binding/non-binding behavior of the drug in approaches such as saturation transfer difference (std) and other nuclear overhauser effect (noe) type methods, diffusion-based methods, relaxation-based methods (i.e., t and t ). target based screening, ligand (drug) based screening, and their respective methods, are discussed in detail below. nmr-based drug discovery can be broadly classified into two groups: chemical and biological (in-cell) categories. one of the principal methods of drug discovery using nmr spectroscopy is called fragment-based drug design (fbdd) [ ] . in-cell nmr (biological) based drug discovery techniques will be discussed later in this review. fbdd was first reported in [ ] and used throughout the late s as evidenced by the use of keywords related to fbdd in papers published during this time [ ] . the use of fbdd as a viable drug screening technique began to be widely adopted in the mid- s [ ] . high throughput screening (hts) is another technique widely used in drug discovery [ ] . hts analyzes molecules from a chemical library to see which ones are suitable leads [ ] [ ] [ ] [ ] (see figure ) . fbdd techniques will screen against a carefully designed fragment library composed of a few thousand molecules (for details on the choice of compounds and design of fragment libraries, see [ , ] ) and identified hits are further developed via fragment growing, fragment merging, or fragment linking [ ] . for examples of drugs derived from the fbdd approach that are currently in clinical trials, refer to table . at the time of writing, and to the best of our knowledge, there are three food and drug administration (fda)-approved drugs derived from the fbdd approach [ ] , and over are in clinical trials [ ] . the first marketed drug derived via the fbdd approach is vemurafenib [ ] . vemurafenib is also the first drug approved for treatment of braf-mutant cancer [ ] , and is reported to exhibit significant clinical benefit for patients with metastatic melanoma [ ] . venetoclax, a common drug used to treat patients with chronic lymphocytic leukemia [ ] , is considered the second drug to be discovered using the fbdd approach [ ] , and ribociclib, a cdk inhibitor, the third [ ] . the names, structures, targets/applications, and clinical status of vemurafenib, venetoclax, ribociclib, and other drugs are listed in table . hts has been productive in drug design [ , ] , but the method is time and resource intensive [ ] and expensive [ ] because of the numerous molecules to be examined (~ million) [ ] . furthermore, the success rate is only estimated to be at~ % [ , ] . unlike traditional hts, which can survey a large number of molecules ranging from a few hundred thousand to a few million [ ] , fbdd usually surveys a few thousand molecules (~ - ) from libraries with greater chemical diversity [ , ] . fbdd is a main-stream screening technique for drug discovery [ , , [ ] [ ] [ ] [ ] [ ] [ ] and nmr is standard for many fbdd studies [ ] . additional methods and techniques such as spr, x-ray crystallography [ , [ ] [ ] [ ] [ ] etc. have also been used in fbdd studies, accompanied or unaccompanied by nmr experiments. for examples of fbdd derived drugs using methods besides nmr, refer to table . at the time of writing, and to the best of our knowledge, there are three food and drug administration (fda)-approved drugs derived from the fbdd approach [ ] , and over are in clinical trials [ ] . the first marketed drug derived via the fbdd approach is vemurafenib [ ] . vemurafenib is also the first drug approved for treatment of braf-mutant cancer [ ] , and is reported to exhibit significant clinical benefit for patients with metastatic melanoma [ ] . venetoclax, a common drug used to treat patients with chronic lymphocytic leukemia [ ] , is considered the second drug to be discovered using the fbdd approach [ ] , and ribociclib, a cdk inhibitor, the third [ ] . the names, structures, targets/applications, and clinical status of vemurafenib, venetoclax, ribociclib, and other drugs are listed in table . nmr, x-ray crystallography, inhibition of cathepsin d as mentioned, nmr spectroscopy can be used in fbdd in two different ways: ( ) target (or receptor) based screening, and ( ) ligand-based screening. with the stated advantages and disadvantages, researchers must select based on their available compounds. target based screening typically utilizes the "sar by nmr" (structure-activity-relationship by nuclear magnetic resonance) approach [ ] . sar is primarily used to identify and develop extremely tight-binding ligands [ ] . the ligand to target binding is traditionally monitored via chemical shift changes [ ] using a correlation spectroscopy such as h- n hsqc starting with the target and no ligand present [ ] . multiple spectra for the target are recorded in the presence and absence of ligands. the binding ligand will cause chemical shift perturbations in the target, and these perturbations are often easily visualized by overlaying the two spectra [ ] . for example hajduk et al. investigated the binding interactions of -phenylimidazole with the fkbp protein as shown in figure [ ] . disadvantages, researchers must select based on their available compounds. target based screening typically utilizes the "sar by nmr" (structure-activity-relationship by nuclear magnetic resonance) approach [ ] . sar is primarily used to identify and develop extremely tight-binding ligands [ ] . the ligand to target binding is traditionally monitored via chemical shift changes [ ] using a correlation spectroscopy such as h- n hsqc starting with the target and no ligand present [ ] . multiple spectra for the target are recorded in the presence and absence of ligands. the binding ligand will cause chemical shift perturbations in the target, and these perturbations are often easily visualized by overlaying the two spectra [ ] . for example hajduk et al. investigated the binding interactions of -phenylimidazole with the fkbp protein as shown in figure [ ] . from the overlaid spectra, chemical shift changes are measured, and from the molecular location, extent, and rate of the chemical shift changes, the binding site and affinity of the ligand is calculated [ ] . then, by following a procedure completely analogous to that of fbdd (see figure ), a ligand developed from multiple fragments can be optimized for the binding site of interest, again by monitoring the changes in chemical shifts of the target. several examples of the successful applications of sar by nmr in drug design research are replete in the scientific literature [ , , ] . sar by nmr spectroscopy allows researchers to observe directly ligand binding [ ] in both solution state and solid-state spectra [ ] , increasing the method's versatility [ ] . it works particularly well for targeting proteins with adjacent "subpocket" binding sites [ ] . furthermore, sar by nmr is cost-effective when combined with hts (high throughput screening) [ ] . sar by nmr can also be used even when atomic peak assignments in spectra are unknown, though it is much more powerful when the resonance frequency of each atom is known [ ] . the main limitation of sar by nmr, however, is its inability to distinguish between multiple binding modes (i.e. cleavage from the overlaid spectra, chemical shift changes are measured, and from the molecular location, extent, and rate of the chemical shift changes, the binding site and affinity of the ligand is calculated [ ] . then, by following a procedure completely analogous to that of fbdd (see figure ), a ligand developed from multiple fragments can be optimized for the binding site of interest, again by monitoring the changes in chemical shifts of the target. several examples of the successful applications of sar by nmr in drug design research are replete in the scientific literature [ , , ] . sar by nmr spectroscopy allows researchers to observe directly ligand binding [ ] in both solution state and solid-state spectra [ ] , increasing the method's versatility [ ] . it works particularly well for targeting proteins with adjacent "subpocket" binding sites [ ] . furthermore, sar by nmr is cost-effective when combined with hts (high throughput screening) [ ] . sar by nmr can also be used even when atomic peak assignments in spectra are unknown, though it is much more powerful when the resonance frequency of each atom is known [ ] . the main limitation of sar by nmr, however, is its inability to distinguish between multiple binding modes (i.e., cleavage of covalent bonds or allosteric changes), and if multiple binding modes are present, it can be difficult to pinpoint the "true" binding site of the ligand solely using data obtained using sar by nmr [ ] . ligand-based screening, the second approach of nmr in fbdd, has three main categories: ) saturation transfer difference (std) and nuclear overhauser effect (noe) type methods, based on ) diffusion methods, or ) relaxation-based methods (i.e., t and t ). saturation transfer difference (std) nmr depends on the nuclear overhauser effect (noe), which is often used to enhance the sensitivity of less sensitive nuclei such as c and n [ , ] . this increase in sensitivity is possible because of dipolar coupling (i.e., through space interactions of separate nuclei) [ ] . the increase in sensitivity is actually brought about by applying a long, low power radiofrequency pulse that selectively saturates the magnetization [ ] of a specific chemical group (i.e., the methyl groups on a protein), which is then given time to transfer to another chemical group via the noe dipolar coupling within a few angstroms [ ] . the transfer in magnetization is easily visualized on a nmr spectrum that takes the differences in the signal intensities from before and after the irradiation. this new spectrum is called a "difference spectrum", and it reveals what chemical groups interact with the irradiated signal [ ] (see figure ). of covalent bonds or allosteric changes), and if multiple binding modes are present, it can be difficult to pinpoint the "true" binding site of the ligand solely using data obtained using sar by nmr [ ] . ligand-based screening, the second approach of nmr in fbdd, has three main categories: ) saturation transfer difference (std) and nuclear overhauser effect (noe) type methods, based on ) diffusion methods, or ) relaxation-based methods (i.e. t and t ). saturation transfer difference (std) nmr depends on the nuclear overhauser effect (noe), which is often used to enhance the sensitivity of less sensitive nuclei such as c and n [ , ] . this increase in sensitivity is possible because of dipolar coupling (i.e. through space interactions of separate nuclei) [ ] . the increase in sensitivity is actually brought about by applying a long, low power radiofrequency pulse that selectively saturates the magnetization [ ] of a specific chemical group (i.e. the methyl groups on a protein), which is then given time to transfer to another chemical group via the noe dipolar coupling within a few angstroms [ ] . the transfer in magnetization is easily visualized on a nmr spectrum that takes the differences in the signal intensities from before and after the irradiation. this new spectrum is called a "difference spectrum", and it reveals what chemical groups interact with the irradiated signal [ ] (see figure ). std nmr is an application of noe used to probe the binding of ligands to a specific site within the targeted proteins [ ] . a generic example of detecting ligand binding via std is presented in figure a . the std nmr method follows the same concepts as a normal noe experiment: a spectrum of the ligand in the free, non-binding form is recorded, the ligand is allowed to bind to the protein, which has a functional group of interest (i.e. methyls) with a saturated signal from a previous selective radiofrequency pulse. the saturated signal travels to the ligand, increasing the intensity of a signal on the ligand spectrum and finally a difference spectrum is used to determine precisely which sections of the ligands bind. the difference in peak intensities proves the presence of ligand binding [ ] . water-ligand observed through gradient spectroscopy (waterlogsy) is a second type of std (see figure b ). the main difference with normal std nmr is that water is the saturated signal [ ] , and instead of observing lower peak intensities, peak inversions indicate the presence of ligand binding [ ] . std nmr is an application of noe used to probe the binding of ligands to a specific site within the targeted proteins [ ] . a generic example of detecting ligand binding via std is presented in figure a . the std nmr method follows the same concepts as a normal noe experiment: a spectrum of the ligand in the free, non-binding form is recorded, the ligand is allowed to bind to the protein, which has a functional group of interest (i.e., methyls) with a saturated signal from a previous selective radiofrequency pulse. the saturated signal travels to the ligand, increasing the intensity of a signal on the ligand spectrum and finally a difference spectrum is used to determine precisely which sections of the ligands bind. the difference in peak intensities proves the presence of ligand binding [ ] . water-ligand observed through gradient spectroscopy (waterlogsy) is a second type of std (see figure b ). the main difference with normal std nmr is that water is the saturated signal [ ] , and instead of observing lower peak intensities, peak inversions indicate the presence of ligand binding [ ] . for std nmr to work properly, the ligand concentration must be in large excess (often - fold) over the receptor so that effective saturation transfer can take place [ ] . this means that for std nmr, and waterlogsy, only small amounts (µg) of protein are required to get results [ ] [ ] [ ] . this is advantageous for researchers, as they can perform std nmr on a protein of interest, and preserve the rest of the unused sample for future/other experiments. also, the same sample can be used for multiple nmr measurements. std nmr facilitates the differentiation of binding ligands from non-binding ligands because the change in signal (as determined by the difference spectrum) is easy to measure and observe, as shown in figure . waterlogsy has been extended to study ligand interactions with dna and rna [ ] . for std nmr to work properly, the ligand concentration must be in large excess (often - fold) over the receptor so that effective saturation transfer can take place [ ] . this means that for std nmr, and waterlogsy, only small amounts (µ g) of protein are required to get results [ ] [ ] [ ] . this is advantageous for researchers, as they can perform std nmr on a protein of interest, and preserve the rest of the unused sample for future/other experiments. also, the same sample can be used for multiple nmr measurements. std nmr facilitates the differentiation of binding ligands from non-binding ligands because the change in signal (as determined by the difference spectrum) is easy to measure and observe, as shown in figure . waterlogsy has been extended to study ligand interactions with dna and rna [ ] . there are additional noe-type experiments (trnoe, inpharma, salmon, etc.) used for drug design, and specific details regarding individual methods are found in the scientific literature [ ] . with the pressing search for new antiviral drugs, any techniques for identifying and characterizing novel leads has become increasingly important. benie et al. [ ] described the use of saturation transfer difference (std) nmr spectroscopy [ , [ ] [ ] [ ] [ ] [ ] [ ] to identify and characterize the binding of an antiviral compound to native human rhinovirus serotype (hrv ). the experiments demonstrated that it is possible to subject targets of the size and complexity of whole viruses (for a model of an hrv particle cut open, cf. the table of contents) to std nmr experiments. the principles of std nmr have been known for many years [ , ] but it was only recently that the potential of this technique for screening libraries for compounds with binding activity toward protein receptors has been realized [ , ] . the technique also permitted the analysis of epitopes of ligands bound to receptor proteins. previous nmr studies of virus-ligand interactions used chemical shift titrations, which required very large quantities of the virus. this approach was unworkable when studying pathogenic viruses. benie et al. [ ] demonstrated that solution state std methodology not only reduces the amount of virus required by approximately orders of magnitude, but also allows for the identification and characterization of virus-ligand interactions with atomic resolution [ ] . there are additional noe-type experiments (trnoe, inpharma, salmon, etc.) used for drug design, and specific details regarding individual methods are found in the scientific literature [ ] . with the pressing search for new antiviral drugs, any techniques for identifying and characterizing novel leads has become increasingly important. benie et al. [ ] described the use of saturation transfer difference (std) nmr spectroscopy [ , [ ] [ ] [ ] [ ] [ ] [ ] to identify and characterize the binding of an antiviral compound to native human rhinovirus serotype (hrv ). the experiments demonstrated that it is possible to subject targets of the size and complexity of whole viruses (for a model of an hrv particle cut open, cf. the table of contents) to std nmr experiments. the principles of std nmr have been known for many years [ , ] but it was only recently that the potential of this technique for screening libraries for compounds with binding activity toward protein receptors has been realized [ , ] . the technique also permitted the analysis of epitopes of ligands bound to receptor proteins. previous nmr studies of virus-ligand interactions used chemical shift titrations, which required very large quantities of the virus. this approach was unworkable when studying pathogenic viruses. benie et al. [ ] demonstrated that solution state std methodology not only reduces the amount of virus required by approximately orders of magnitude, but also allows for the identification and characterization of virus-ligand interactions with atomic resolution [ ] . the very large size of viruses makes them particularly attractive for studies by std nmr, as they inherently yield large line widths allowing for easy irradiation of the virus without affecting the ligand protons. furthermore, because of the larger correlation time of a virus in comparison to an average-sized protein, spin diffusion, and thus saturation transfer, is very efficient. the large line width has additional benefits not just for std-based nmr methods but also for transfer noesy spectra, as protons from the virus capsid are invisible in the nmr spectra (for an example of a transfer noesy spectrum, see [ ] ). moreover, competitive std titration experiments can be used to determine the k d value of a ligand [ ] . analysis of the std spectra using the group epitope mapping method [ ] allows for the determination of the binding epitope. std nmr methods can considerably speed up the determination of the binding epitope for potential antiviral lead compounds. simple std nmr experiments provide substantial information on the binding of ligands to native viruses and require very small amounts of the virus with measurement times in the range of tens of minutes. this allows for a high throughput of ligand samples without significant consumption of viral material because it remains unaffected by the experiments and is easily separated from the low molecular weight ligands by ultra-filtration subsequently. in addition to the detection of binding, a complete mapping of the ligand-binding epitope can be achieved [ ] . noroviruses (nv) are non-enveloped, single-stranded, positive-sense rna viruses that are the major cause of epidemic outbreaks of gastroenteritis worldwide [ ] [ ] [ ] . the viral coat consists of a single protein, vp , which assembles into a capsid with overall icosahedral symmetry [ ] [ ] [ ] . attachment of human noroviruses to histo-blood group antigens (hbgas) is thought to be critical for the infection process [ ] . the protruding domains of the vp proteins, called p-domains, harbor highly conserved binding sites for hbgas. std nmr-based epitope mapping was used [ , ] to identify structural features of different core types critical for the binding of synthetic a-and b-tetrasaccharides [ ] to virus-like particles (vlps) of a highly homologous gii. strain (ast ). std nmr experiments provide a robust and straightforward technique for obtaining ligand binding epitopes at atomic resolution. comparing binding epitopes of related ligands then delivers critical information about structural requirements for ligand recognition. conversely, comparison of binding epitopes of a given ligand binding to wild type, and to mutant proteins reveals the importance of individual amino acids for binding. std nmr experiments with l-fuc and b-trisaccharide in the presence of wild type and mutant vlps yield virtually identical binding epitopes and suggest that these two mutations do not significantly alter hbga recognition. the std nmr approach to characterize binding of hbga ligands to noroviruses has employed vlps as targets and thus taken advantage of the large size of vlps yielding excellent signal-to-noise ratios of the corresponding std nmr spectra, as demonstrated previously [ ] . the application of the transferred noe (tr-noe) effect was first demonstrated by bothner-by [ ] . the tr-noe is the nuclear overhauser effect between ligand spins, which are in chemical exchange between the bound and unbound form with the protein or receptor. ligands, which are a mixture of target molecules, are small in size (below - da). since they are usually low molecular weight molecules, they exhibit much shorter correlation times when compared to the receptor and have slow noe build-ups with no spin diffusion. this is the reason they show small positive noes in the free form. when binding to a protein receptor, the situation changes, where the ligand acquires large correlation times in the bound state with rapid noe build-up. then they show spin diffusion and a strong negative noe, which is termed the transferred noe. signals arising from the protein are usually not observed for large proteins as they are generally kept low in concentration, with ligands in a high excess concentration. in addition, most of the time protein signals are suppressed by their very short t period. it is worthwhile to mention that ligands that are in fast exchange between the bound and the free form (dissociation constants ranging from µm to mm) get enough bound time to transfer the negative noe from the protein complex to the population of the free molecules, yet usually retain the chemical shift of the free molecule along with the relaxation characteristics. in order to observe tr-noes, the following condition have to be fulfilled: where n and ∂ represent the number of molecules and the cross-relaxation rate, respectively. the subscript b and f represent the bound and free form, respectively. therefore, to observe the tr-noes, a high excess concentration of ligands over protein is maintained. on the other hand, if the ligand concentration is kept too high, the excess free ligand in solution will exhibit positive noe, which can result in a significant reduction of the tr-noesy enhancements due to negative noe developed by the very small concentration of bound ligand. hence, the preparation of the sample becomes tricky and an optimum ratio between - to is maintained while considering the dissociation constant values. the binding of a ligand to a receptor protein can easily be identified by observing the sign and size of the noes. there are some distinct experimental features for the discrimination between tr-noes from the bound state and noes of the ligand in free states like the build-up rate. for tr-noes, this is in the range of to ms, whereas for small ligands it is much longer. there have been various instances of experimental implementations to quickly determine the binding activity of ligand libraries. one example was to find the ligand molecule among a library of similar structure polysaccharides that is bioactive in binding with recombinant e-selectin [ ] . this is a protein present in an igg chimera with a molecular weight of about kda. in this case, two d noesy spectra were recorded. the noesy spectra for the ligand library was measured at several temperatures and it was found that most of the compounds exhibited the weak positive noes at k, which was then chosen to differentiate between trnoes showing large negative values. the trnoesy spectra of the ligand library in the presence of protein was recorded at different ratios, such as : , : , : , : , and : , at k. in all the ratios, trnoes were observed; however, the ratio of : represented the best-case scenario. the inpharma method (see figure ) was designed to determine the relative orientation between two competitive ligands in the receptor-binding pocket through the observation of inter-ligand noe between the two ligands. it is a tr-noe in nature as it is mediated by the bound conformation of the competing ligands and in exchange with the receptor protein. the first example was competitive binding and observation of inter-ligand noe between baccatin iii and epothilone a in the presence of tubulin, which acts as a receptor [ ] . since the observation is on the ligand site, it provides unique advantages. the detailed conformation of a ligand-protein complex can be addressed by conventional nmr. however, it is time-consuming and demands full solving of the structure and there is also a size limitation. from that aspect, ligand-based methods are more useful. the only limiting fact is that it should fulfill all the conditions of tr-noe explained previously in terms of dissociation constant (k d ), fast exchange regime, and proper ligand to protein ratio. then, information on the ligand structure can be derived from tr-noe build up as a function of mixing time. this can be readily explained using the originally proposed schematics [ ] . the noesy spectrum of a mixture of the two ligands a and b in the presence of the common receptor (t) is recorded. under the situation that each of a and b exhibit competitive binding in a fast exchange regime with the receptor t, intermolecular tr-noe peaks between the two ligands a and b can then be observed in the noesy spectrum due to extensive spin diffusion. during the noesy mixing time, the first proton of ligand a (h a ) binds to receptor t, which results in transfers of magnetization from h a to h t . subsequently, the complex at dissociates as they fulfill the dissociation constant range, which creates the opportunity for ligand b to bind to the receptor t at the same binding site. this results in the transfer of the magnetization of h t , which had been originally coming from h a , to h b . as a result, an inter-molecular correlation h a -h b can be seen, and this inter-molecular noe will be a function of mixing time as described above. the detailed analysis of such intermolecular noe peaks helps in assessing the relative orientation of each ligand in the binding pocket. diffusion is the random, translational motion of molecules in solution as a consequence of their thermal energy [ ] . this type of motion is often referred to as "brownian motion", a motion that describes molecular movement induced by random collisions between the molecules [ ] . in the presence of a concentration gradient, molecules will naturally move from places of higher concentration to places of lower concentration [ ] after a period of time, t, as shown in figure . fick's law can be used to model this type of movement [ ] . the distribution of the diffusing molecules is accurately represented by a gaussian curve, a normal distribution centered at a single point, which gradually "flattens" as t approaches infinity [ ] . the extent to which a molecule diffuses is directly related to its shape, size, and mass [ ] . in homogeneous isotropic solutions, the root mean square distance (zrms) traveled by a molecule is given by following equation [ , ] : where d is the diffusion coefficient of the molecule, and t is the diffusion time. making the assumption that the molecules are solid rigid spheres, the value of d can be calculated according to the famous einstein-stokes equation (equation ( )): where kb is the boltzmann's constant ( . × − j/k), t is the absolute temperature, η is the solution viscosity, and rs is the hydrodynamic radius of the molecule [ ] . equation ( ) and equation ( ), however, are not universally applicable; they only apply to molecules that are freely diffusing in isotropic, homogeneous solutions, and importantly that can be accurately described as hard, rigid spheres [ ] . different molecular geometries and additional modes of diffusion (i.e. restricted and anisotropic) require more advanced mathematics and theory [ , ] , but the essential concepts of diffusion remain the same. diffusion is the random, translational motion of molecules in solution as a consequence of their thermal energy [ ] . this type of motion is often referred to as "brownian motion", a motion that describes molecular movement induced by random collisions between the molecules [ ] . in the presence of a concentration gradient, molecules will naturally move from places of higher concentration to places of lower concentration [ ] after a period of time, t, as shown in figure . fick's law can be used to model this type of movement [ ] . the distribution of the diffusing molecules is accurately represented by a gaussian curve, a normal distribution centered at a single point, which gradually "flattens" as t approaches infinity [ ] . the extent to which a molecule diffuses is directly related to its shape, size, and mass [ ] . in homogeneous isotropic solutions, the root mean square distance (z rms ) traveled by a molecule is given by following equation [ , ] : z rms = ( dt) where d is the diffusion coefficient of the molecule, and t is the diffusion time. making the assumption that the molecules are solid rigid spheres, the value of d can be calculated according to the famous einstein-stokes equation (equation ( )): where k b is the boltzmann's constant ( . × − j/k), t is the absolute temperature, η is the solution viscosity, and r s is the hydrodynamic radius of the molecule [ ] . equation ( ) and equation ( ), however, are not universally applicable; they only apply to molecules that are freely diffusing in isotropic, homogeneous solutions, and importantly that can be accurately described as hard, rigid spheres [ ] . different molecular geometries and additional modes of diffusion (i.e., restricted and anisotropic) require more advanced mathematics and theory [ , ] , but the essential concepts of diffusion remain the same. molecules , , x for peer review of figure . visual representation of how molecules diffuse in solution from a high concentration to a low concentration across an arbitrarily defined point (x = ) after some period of time (t = ∞) [ ] . the red arrow indicates the direction of translational motion as the molecules (green) move from an area of higher concentration (left) to an area of lower concentration (right) [ ] . the earliest pulse sequence used to measure diffusion in nmr spectroscopy is the gradient spin echo sequence (se), developed by stejskal et al. [ ] . the se pulse sequence is shown in figure . the se pulse sequence uses a gradient (g) of the externally applied magnetic field, (pulsed field gradient), the first after the o pulse, and the other after the o refocusing pulse. the first gradient pulse (g ) labels or gradient-encodes the nmr-active nuclei based on their physical position in the sample tube. if the molecules diffuse during the time period they are not in the correct position to experience the second gradient which re-focuses the spins. this is detected via nmr as a signal intensity decrease. after a diffusion time (∆), the second gradient pulse is applied to decode the spatial labeling of nmr-active nuclei, obtaining a well-defined spectra of diffusing molecules in solution [ ] . additional nmr sequences are available for diffusion experiments [ ] , and are detailed in more comprehensive reviews dealing with the subject [ , ] . the signal intensity of the diffusing molecules depends on three factors, as described by equation where i is the observed intensity, i the reference intensity (unattenuated signal intensity), d is, of course, the diffusion coefficient referred to earlier, γ is the gyromagnetic ratio of the observed nucleus, g is the strength of the gradient, δ the length of the gradient, and ∆ the diffusion time [ ] . from equation ( ), it is easy to see that the signal intensity decreases exponentially with time, so it is vital to optimize the values of g, δ, and ∆ for diffusion nmr measurements [ ] . the earliest pulse sequence used to measure diffusion in nmr spectroscopy is the gradient spin echo sequence (se), developed by stejskal et al. [ ] . the se pulse sequence is shown in figure . the se pulse sequence uses a gradient (g) of the externally applied magnetic field, (pulsed field gradient), the first after the • pulse, and the other after the • refocusing pulse. the first gradient pulse (g ) labels or gradient-encodes the nmr-active nuclei based on their physical position in the sample tube. if the molecules diffuse during the time period they are not in the correct position to experience the second gradient which re-focuses the spins. this is detected via nmr as a signal intensity decrease. after a diffusion time (∆), the second gradient pulse is applied to decode the spatial labeling of nmr-active nuclei, obtaining a well-defined spectra of diffusing molecules in solution [ ] . additional nmr sequences are available for diffusion experiments [ ] , and are detailed in more comprehensive reviews dealing with the subject [ , ] . molecules , , x for peer review of figure . spin echo (se) sequence, as discovered by stejskal et al. [ ] . g is the first gradient pulse applied after the first ° pulse, and g is the second gradient pulse applied after the first ° pulse. δ and ∆ are the gradient length, and diffusion time, respectively. in silico (virtual) screening is now a standard technique in drug design and discovery [ ] that has been in use since at least [ ] , though the exact origin of the phrase "in silico" is not clear [ ] . the nearly ubiquitous use of virtual screening is due to its efficiency in searching massive chemical databases in order to generate lead molecules [ ] that inhibit protein-protein interactions [ ] , and its ability to help identity ligand (drug) binding sites on the target of interest [ ] to lend insight to the mechanisms of action for lead compounds [ , ] . virtual screening is often accompanied by in vitro or in vivo techniques for pharmacology drug research [ ] , to increase drug throughput, helping to reduce the time and cost of developing novel drug candidates [ ] . virtual screening has also been used to identify candidates for anti-viral drugs [ ] and anticancer drugs [ ] . several chemical databases are available both for public and academic use [ ] . virtual screening is properly identified as a high-throughput screening (hts) technique [ ] , though using its full capacity as an hts technique is not required for most purposes. virtual screening requires a minimum of two inputs, ( ) a three-dimensional model of the ligand (drug), and ( ) a three-dimensional model of the receptor (protein) [ ] , the latter generated from the atomic studies of proteins via x-ray crystallography or nmr spectroscopy [ ] . virtual screening is not a truly "stand-alone" technique and has often been combined with additional biophysical techniques besides nmr spectroscopy and/or x-ray crystallography [ ] , such as differential scanning fluorimetry [ ] , fluorescence polarization, and surface plasmon resonance [ ] . in this section, we briefly introduce how virtual screening has been combined with nmr spectroscopy, and how they are complementary approaches to each other in drug design. the complete details of how [ ] . g is the first gradient pulse applied after the first • pulse, and g is the second gradient pulse applied after the first • pulse. δ and ∆ are the gradient length, and diffusion time, respectively. the signal intensity of the diffusing molecules depends on three factors, as described by equation ( ) [ ] : where i is the observed intensity, i the reference intensity (unattenuated signal intensity), d is, of course, the diffusion coefficient referred to earlier, γ is the gyromagnetic ratio of the observed nucleus, g is the strength of the gradient, δ the length of the gradient, and ∆ the diffusion time [ ] . from equation ( ), it is easy to see that the signal intensity decreases exponentially with time, so it is vital to optimize the values of g, δ, and ∆ for diffusion nmr measurements [ ] . the drug design approach based on diffusion nmr is basically a screening technique used to differentiate the binding ligands (drug) from non-binding components [ ] . ligands able to bind should have significantly different diffusion coefficients (d) compared to non-binding ligands [ ] , i.e., the diffusion coefficients of binding ligands will be smaller than those of non-binding ligands [ ] . thus, diffusion-based nmr is a way of effectively "filtering" and identifying which ligands are binding [ ] . diffusion-based nmr spectroscopy has advantages in ligand based screening applied to drug discovery. for example, diffusion ordered spectroscopy (dosy) does not require prior separation/purification of the ligand/target solution [ ] . diffusion based nmr allows simultaneous determination of diffusion coefficients in multicomponent systems containing large molecules (i.e., proteins) and possible binding partners (i.e., small drug compounds) [ ] , and no special labeling or contrasting agents are required, though their use is not exclusively inhibited (for an example of the use of labeled compounds in diffusion nmr spectroscopy, see [ ] ). a problem occurs when there is significant chemical shift overlap between the binding molecule signals and the target. this situation makes it hard to distinguish the nmr signals [ ] , and the calculations typically assign an intermediate value to the diffusion rate (i.e., one gets a smear). multidimensional diffusion nmr pulse sequences are available [ ] , which may help resolve spectral overlap in d experiments [ ] . another issue is that molecules in chemical databases may have generally low solubility [ , ] . low solubility decreases the overall signal intensity and therefore makes accurately measuring diffusion experiments far more difficult [ ] . there are many examples demonstrating the successful application of diffusion nmr in examining drugs of pharmaceutical interest [ ] , and ligand-target interactions [ ] . hajduk et al. [ ] exploited the changes in diffusion rates to detect ligands that bind to the fk binding protein and the catalytic domain of stromelysin. nishimura et al. [ ] utilized dosy, in combination with noesy to determine the orientation of two guest molecules, p-ethoxyiodobenzene and -iodo- -methoxynaphthalene, within a host composed of a tetrakis( -hydroxyphenyl)-cavitand and a tetra( -pyridyl)-cavitand. furthermore, matthias et al. [ ] used h molecular diffusion and f spin diffusion to probe the drug loading properties of the rf-peg hydrogel for -fluorouracil (fu) and , -dimethyl- -fluorouracil (dmfu), two anticancer drugs. dosy can be combined with saturation transfer difference (std, discussed earlier in this review) to yield new insights about ligand-target interactions. kramer et al. [ ] combined std with dosy to analyze a mixture composed of wheat germ agglutinin and two derivatives of n-acetyl glucosamine (ligands). using this new technique they were able to obtain high quality spectra of the components in the mixture. tanoli et al. [ ] also combined std and dosy to explore the interactions of smaller molecules with bovine serum albumin. these are just a few examples to show that diffusion nmr spectroscopy has played, and will continue to play, a prominent role in drug design. in silico (virtual) screening is now a standard technique in drug design and discovery [ ] that has been in use since at least [ ] , though the exact origin of the phrase "in silico" is not clear [ ] . the nearly ubiquitous use of virtual screening is due to its efficiency in searching massive chemical databases in order to generate lead molecules [ ] that inhibit protein-protein interactions [ ] , and its ability to help identity ligand (drug) binding sites on the target of interest [ ] to lend insight to the mechanisms of action for lead compounds [ , ] . virtual screening is often accompanied by in vitro or in vivo techniques for pharmacology drug research [ ] , to increase drug throughput, helping to reduce the time and cost of developing novel drug candidates [ ] . virtual screening has also been used to identify candidates for anti-viral drugs [ ] and anticancer drugs [ ] . several chemical databases are available both for public and academic use [ ] . virtual screening is properly identified as a high-throughput screening (hts) technique [ ] , though using its full capacity as an hts technique is not required for most purposes. virtual screening requires a minimum of two inputs, ( ) a three-dimensional model of the ligand (drug), and ( ) a three-dimensional model of the receptor (protein) [ ] , the latter generated from the atomic studies of proteins via x-ray crystallography or nmr spectroscopy [ ] . virtual screening is not a truly "stand-alone" technique and has often been combined with additional biophysical techniques besides nmr spectroscopy and/or x-ray crystallography [ ] , such as differential scanning fluorimetry [ ] , fluorescence polarization, and surface plasmon resonance [ ] . in this section, we briefly introduce how virtual screening has been combined with nmr spectroscopy, and how they are complementary approaches to each other in drug design. the complete details of how virtual screening works, and how it applies to drug design outside of its combination with nmr is well documented in additional reviews [ , , [ ] [ ] [ ] [ ] [ ] . a prime example of the complementarity between nmr screening and virtual docking is found in the work of chen et al. [ ] , in which the authors sought to target the a a adenosine receptor (a a ar) protein, a drug target for the treatment of parkinson's disease [ ] . they used virtual screening and an nmr-based screening method against the same molecules in a fragment library so they could compare the results of both methods. the virtual screen successfully predicted (based on calculated binding affinities) four out of the five orthosteric ligands discovered by nmr that were within the top % of the fragment library, showing that the two separate methods can give similar and reliable results. later on, chen et al. discovered that virtual screening picked up three additional fragments that remained undetected by the nmr-based method, and were, in fact, a a ar ligands; this shows that though neither method is flawless, they are still perfectly complementary approaches for drug design [ , ] . in another scientific work that integrated nmr with virtual screening, di lello et al. [ ] found small molecular inhibitors of the enzyme ubiquitin specific protease (usp ), a key regulator of the tumor suppressor protein, p [ ] . a fragment screen by nmr revealed a series of small molecules that bind in the active site of usp near the catalytic cysteine (amino acid ). a ligand-based virtual screen utilizing the fastrocs program identified~ hit molecules, several of which were further characterized by h- n trosy chemical shift perturbation and line broadening to probe the binding site of the active hits. di lello. also tested the active compounds against eol- cells to verify the hits as identified by virtual screening and further characterized by nmr, showing that the active compounds do indeed inhibit usp activity. through additional study of the active molecules and further optimization of their structures, they eventually discovered a series of ligands that bind in the "palm" region of the catalytic domain of usp , inhibiting its catalytic activity [ ] . this study clearly demonstrates that nmr screening-based techniques can be combined with virtual screening to find viable drugs for targets of interest. additional examples of the successful integration of nmr and virtual screening as applied to protein targets are also found in the literature, further demonstrating the practicality and complementarity of virtual screening and nmr [ , [ ] [ ] [ ] . for example, li et al. [ ] used virtual screening filtered by nmr to identify and characterize non-metal chelating metallo-β-lactamase (mbl) inhibitors, and in particular, verona integron-encoded mbl (vim)- , when previously there were no clinically significant inhibitors of mbl, since mbl enzymes hydrolyse many, if not all, β-lactam antibacterials compounds specifically designed to inhibit their activity [ ] . furthermore, shan et al. [ ] and bertini et al. [ ] both used virtual screening and nmr, in their respective studies. through the combined use of nmr and virtual screening, shan et al. was able to identify, design, and synthesize novel pdz domain inhibitors, which are proteins implicated in tumorigenesis [ ] . bertini et al. was able to combine nmr to study the interaction of ligands with metalloproteinases, using known inhibitors of metalloproteinases as a starting point [ ] . while hsqc noesy nmr data provided structural and spatial constraints for the proposed d models, virtual screening was used to refine the models, and to probe the ligand-protein interaction. in each case (i.e., ligand-protein interaction), bertini et al. was able to obtain a well-defined ligand conformation in the protein binding site, thus offering a viable alternative to other approaches described in the literature [ ] . clearly, combining virtual screening with nmr-based methods is advantageous in studying how ligands (drugs) bind and interact with targets (proteins) of interest. paramagnetic nmr (pnmr) can also play a prominent role in drug discovery [ ] , as pnmr can provide key structural information in situations where crystal structures cannot due to the weak binding of ligands [ ] . pnmr can be used to quantify the binding between ligands and large biomolecules such as proteins, dna, and rna [ ] . pnmr depends on the presence of a group (called the paramagnetic center) with an unpaired electron [ ] , and since many naturally occurring biomolecules and organic compounds lack a paramagnetic center, one such as caged lanthanide (clanp) [ ] , must be introduced artificially [ ] . once the paramagnetic center (often a metal ion) is present, paramagnetic effects can be used to measure the distance and the relative orientation (i.e., angle) between molecules [ ] . this information is crucial when it comes to determining how ligands and substrates bind. thus, pnmr is quite a useful technique for drug discovery when a paramagnetic center is present. the most relevant consequence of pnmr for drug discovery is paramagnetic relaxation enhancement (pre), although there are a number of studies demonstrating the use of pseudocontact shift (pcs) effect in drug discovery research [ ] . paramagnetic relaxation enhancement (pre) is proportional to the inverse sixth power of the distance between the paramagnetic center and the nucleus of interest (i.e., h), although it does not reveal anything about relative orientation [ ] . pre can give quantitative information in the range of - angstroms [ ] . several researchers have taken advantage of this outstanding property to study the structural and dynamic properties of complex biomolecular machineries in their native environment [ ] . for example, iwahara et al. ( ) demonstrated that a protein's binding polarity to dna can be determined by pre, using edta-derivatized deoxythymidine (dt-edta) with a chelated metal ion (such as cu + or mn + ) as a probe. dt-edta with a chelated metal ion is a convenient choice, as it can be inserted into any position of a synthesized oligonucleotide. with data derived from the pre effect, one can easily determine the polarity of the protein (or drug) binding to dna [ ] . several researchers have investigated dna as a drug target [ ] , and the study of iwahara et al. clearly demonstrates, and even indicates, that pre can potentially be used to study the interactions between a drug and dna [ ] , provided that a paramagnetic center such as dt-edta or a metal ion is present. brasuń et al. [ ] also used pre derived distances between a paramagnetic center and a nucleus of interest. they replaced the cys-s-s-cys bridge found in oxytocin and vasopressin with the his-cu + -his motif to investigate if doing so would alter the stability of oxytocin and vasopressin. they determined the distances between the cu + ion and h nuclei (possible because of pre), and used these values to generate three-dimensional models of the his-cu + -his motifs in both oxytocin and vasopressin. in doing so, they indicated that such an approach using pre can help in designing new biologically active compounds [ ] , and hence in drug discovery research, as many drug discovery studies require a reliable models for the successful generations of hit-lead molecules, especially in the case of in silico docking [ ] . this study again proves the usefulness of pre, and therefore, pnmr, in drug discovery research. in two additional studies, huang et al. [ , ] used pre in their individual studies of protein binding and protein dynamics, respectively. in the huang et al. case [ ] , these authors used pre to establish a model of the binding between the g-actin protein, and thymosin β , an actinbinding protein. using pre determined constraints (distances) and h- n hsqc, they were able to establish a well-converging docking structure of the g-actin/thymonsin β complex [ ] . on the other hand huang et al. [ ] did not measure protein binding, but studied the conformational changes and dynamics of select large membrane proteins utilizing f-nmr spectroscopy, and ni + as the paramagnetic center. through a series of extensive experiments, they showed that conformational exchange rates of membrane proteins can be determined from measurements of the metal-enhanced longitudinal relaxation (i.e., pre) of the f nuclei [ ] , thus yielding additional information (i.e., protein conformation dynamics) that could be utilized in drug discovery projects targeting proteins (i.e., understanding how the protein changes shape based on its environment can be used to find potential binding sites for drug candidates). all these examples prove that pnmr is powerful approach in drug discovery research, given that pre can aid in generating trustworthy models of interacting molecules, and that it can help researchers understand better how the molecules interact in the first place. since the late s solid state nmr (ssnmr) has demonstrated its usefulness in complex biomolecular systems such as collagen or lipid bilayers [ ] . however, over the past years ssnmr has gained attention in the field of drug design and is slowly becoming a commonly used technique as its proving to be a powerful tool for structural analysis of membrane proteins and amyloid fibrils [ ] [ ] [ ] . ssnmr is becoming a more attractive alternative for several different reasons. one of them is the fact that it enables the characterization of a chemical compound in a solid-state form such as in a tablet/pill [ ] [ ] [ ] . moreover, ssnmr is not only restricted to analyzing the chemical structure but it can also provide insight into the physical properties of a compound such as polymorphism (different crystalline structures of the same compound), disorder (crystal defects and amorphous solids in the compound) or the presence of cocrystals (multicomponent crystal made of a compound and one or more small organic molecules) [ , ] . ssnmr can also be used to quantify the amount of crystalline against the amount of amorphous material in the sample to establish phase purity (the amount of desired phase separated from other, undesirable phase) [ ] [ ] [ ] . ssnmr differs from liquid state nmr by the presence of anisotropic interactions. in liquids nmr these effects are averaged to zero as a consequence of rapid molecular tumbling. in solid state however, the molecules are not tumbling rapidly and the residual effects of anisotropic (orientation depended) interactions such as anisotropic chemical shift, magnetic dipolar coupling, and quadrupolar coupling could be observed in the form of broad peaks, with could be much wider than the chemical shift range of the nucleus [ , , ] . as a results, there has been a constant effort to improve the sensitivity and resolution of solid state nmr spectra, which increased the potential of ssnmr in future applications [ ] . one of the methods that works for nuclei with spin value of i = / is called magic-angle spinning (mas). it increases the resolution by rapidly rotating the sample around a fixed (or so-called magic) angle of . • [ ] . this method can be combined with decoupling, to remove the dipolar couplings between spins. this is done by applying radiofrequency pulses or cross-polarization (cp) transfer of magnetization from abundant and sensitive nuclei such as h to less sensitive such as c [ , ] . a broader comparison between ssnmr and liquid state nmr is provided in [ ] . as mentioned before, ssnmr can provide information about membranes and membrane proteins. for this reason, ssnmr can be used to detect interactions of ligands with receptors embedded to the membrane which enables the mapping of binding site of a receptor by utilizing cp-mas (cross-polarization magic-angle spinning) nmr and site specific mutagenesis [ ] . ssnmr can provide the conformation of ligands bound to the receptor which can then be used to optimize future drug in terms of better affinity and efficiency [ ] . since ssnmr is also applicable to amyloid research, it can be used for probing polypeptide structures of amyloid and intermolecular contacts between fibrils. the potential is for the design a drug that will inhibit the process of aggregation of proteins and peptides [ ] . lastly, since ssnmr gains insight into physical properties of a chemical compound it can be used for control of the process of formulation and processing of a drug to help assess the purity of a compound [ ] . an example of ssnmr application related to drug design is the work of callari et al., who monitored the effect of drug loading on the properties of micelles [ ] . polymer micelles are widely used as nano-carries for drug delivery, but so far the effects of drug loading on the morphology of a drug carrier had not been thoroughly investigated [ ] . they created a model consisting of a fructose hydrophilic block and a pmaa block (micelle), to which a different amount of platinum complex was anchored. the results from this experiment showed that micelles loaded with a higher amount of platinum complex had reduced cellular uptake, release, and cytotoxicity. the micelles with a lower load (ll) of platinum complex were more effective at targeting cancer cells (of cell lines mda-mb- (breast cancer) and a (lung cancer) than the micelles with a higher load (hl) of the platinum complex. this is evidenced by the lower ic (half maximal inhibitory concentration) values of the ll micelles as compared to the hl micelles. both of those results could be related to the micellar structure and their potential for interaction between the sugar moieties and the cell wall [ ] . another example of practical application of ssnmr is the work of lee and colleagues [ ] in which they investigated the structure of a designed zinc-binding amyloid fibril that catalyzed ester hydrolysis. metals ions such as zinc where found to affect the process of protein aggregation which resulted in arise of amyloid like structures. therefore, understanding the processes of aggregation and the factors related to them is crucial for creation of new drugs for amyloid related diseases [ ] . in the experiment lee et al. used ac-ihvhlqi-conh peptide (referred as hhq) to form fibrils with varying zn + :hhq molar ratios. the results showed that zn + -bound hhq fibrils form parallel-in-register form of packing β-strand in each sheet and his residues are coordinated to zn + via nδ , while half of the his residues are also coordinated to zn + via nε . additionally, zn + binds in a : metal ion/peptide ratio. after further analysis using structural bioinformatics, it was concluded that each zinc ion was coordinated by three histidine nitrogens from two adjacent strands. half of all histidines bridged to zn + ions forming a metal-imidazolate chain [ ] . a "hit" is a molecule identified from a screening technique (hts, fbdd, etc) as having a desirable effect (i.e., decreased cellular growth, high affinity score) on a target [ , ] . however, the question of whether the activity is related to actual binding to the target, or to interference with one of the components of the assay readout mechanism, is uncertain. thus, a validation step is required. hit-validation is therefore the process of confirming, or validating, that the molecule(s) identified previously have on target activity and selectivity [ , ] . one of the highest-impacts of nmr on drug discovery is the use as a hit-validation tool. though the hit-validation or confirmation of drugs is mostly limited to the solution state [ ] , this aspect of nmr truly is a "gold standard" technique in drug discovery. nmr by itself is a powerful tool for drug validation as in the case of sharma et al. ( ) [ ] who sought to identify potential drug-like inhibitors against l-aspartate α-decarboxylase (adc) an enzyme responsible for the decarboxylation of l-aspartate in order to generate β-alanine and carbon dioxide [ ] , in mycobacterium tuberculosis. they began with known inhibitors of adc, and developed a protocol to measure the enzymatic activity of adc. upon addition of adc to a solution of l-aspartate, l-aspartate gradually disappeared because adc was converted to l-aspartate to β-alanine; therefore the peak intensity of l-aspartate decreased, and the peak intensity of β-alanine increased in the presence of adc (no inhibitor drug present). using this newly developed nmr-based protocol allowed direct measurement of adc enzymatic activity, and sharma et al. were able to confirm the enzymatic inhibiting activity of seven previously discovered inhibitors of adc [ ] . this study demonstrated that nmr can be an effective validation tool for known drugs and for new drugs generated by a screening approach. nmr is also able to remove false positives that emerge from biochemical screens [ ] . for example, an aptly named technique called a la assay to detect reactive molecules by nuclear magnetic resonance (alarm nmr) is able to eliminate false positives from hts methods [ ] , and in the presence of a test compound or mixture, measures dithiothreitol (dtt)-dependent c chemical shift changes of the human la antigen [ ] . dahlin et al. provided an updated protocol of alarm nmr to aid researchers in the production of the c-labeled la antigen reporter protein, in testing compounds with the la protein, and in the analysis of obtained nmr spectra. using alarm nmr prioritized hits identified from hts screening [ ] . an example of alarm nmr is found in the work of dahlin et al., where they used this technique to test molecules that were assumed to be inhibitors of histone acetyltransferase (hat) inhibitors, and from their studies, actually discovered that % ( out of ) of the most commonly reported hat inhibitors were actually faulty. they were actually nonselective interference compounds, not necessarily specific to the inhibition of hat [ ] . thus, alarm nmr (and nmr in general) served as a useful validation method, especially for unvalidated hits identified from biochemical screens [ ] or other screening techniques. the last example highlights the need for cross validation, or the combination of two or more techniques to verify identified chemical hits. of course, nmr is not the sole technique used for drug validation. most often, nmr drug validation is coupled with additional methods [ ] such as surface plasmon resonance (spr) [ , ] x-ray crystallography [ ] [ ] [ ] , isothermal calorimetry (itc) [ ] , uv-vis and/or fluorescence spectroscopy [ ] . the work of goudreau et al. is an excellent example of combining nmr with another biophysical technique, in this case x-ray crystallography, for drug validation [ ] . a series of benzodiazepine inhibitors of human immunodeficiency virus (hiv- ) was identified using an in vitro capsid assembly assay, and further characterized by f-nmr. analysis of the chemical shift perturbation and line broadening effect on the f-nmr spectra of the benzodiazepine inhibitors revealed the specificity and reversibility of the binding inhibitors. the same set of f-nmr spectra were used to identify the n-terminal domain of the capsid as the binding site of the benzodiazepine inhibitors. the specific amino acids involved in the binding of the benzodiazepine inhibitors were identified from the chemical shift perturbation of h, n-trosy nmr spectra. later, use of x-ray co-crystallography confirmed binding locations of the benzodiazepine inhibitors and their binding modes, which was useful for further development and optimization of the benzodiazepine inhibitors [ ] . the work of goudreau et al. therefore showed how nmr could be used as a co-validation technique with another biophysical method [ ] . nmr can be also coupled with multiple biophysical techniques to validate a molecule's ability to inhibit protein-protein interactions (ppis) [ ] . an example of the combination of nmr with spr and x-ray crystallography can be found in the work of fry et al., where the authors sought to understand how the nutlin molecule inhibits mdm -p , a protein-protein interaction that has been an important cancer therapy target for several years [ ] [ ] [ ] . fry et al. [ ] gradually deconstructed rg , the first nutlin molecule to enter clinical trials [ ] , into fragments so they could study the inhibitory effect of rg on the mdm -p interaction by spr, nmr, and x-ray crystallography. spr was used to determine the k d values of the rg fragments and confirmed that rg and some of its fragments do bind to mdm , inhibiting the mdm -p interaction. h, n-hsqc nmr chemical shift perturbation was also used to assess and verify binding identified by spr. of the six fragments of rg confirmed by h, n-hsqc nmr as binding to mdm , spr showed binding for five of them; thus, the two separate techniques were in good agreement with each other. the fragments of rg that were confirmed to bind by both spr and h, n-hsqc nmr were further studied with x-ray crystallography, which can tell precisely where and how the molecules bind to the protein. using co-crystallization, fry et al. were able to obtain structures for several of the verified binding fragments in complex with mdm and were able to visualize the binding of the fragments to the mdm protein [ ] . nmr is obviously a powerful drug binding validation tool, but it becomes much more powerful when coupled with additional biophysical techniques, as seen in the work of fry et al. [ ] . dias et al. [ ] took a similar approach as fry et al. [ ] in that they took known inhibitors of a protein-protein interaction, and dissected them into individual fragments to assess a protein's drug-ability. the interaction studied was that between the proteins von hippel-lindau (vhl), and the alpha subunit of hypoxia-inducible factor (hif- α). twelve compounds (known inhibitors and derived fragments) were developed using a crystal structure of hif- α peptide bound to the stable multiprotein complex pvhl-elongin c:elongin b (vcb). each of these compounds was screened using three separate nmr techniques, saturation transfer difference (std), carr-purcell-meiboom-gill (cpmg) relaxation experiments, and waterlogsy (to assess drug binding and to predict drug binding mode. each compound that was unambiguously detected (i.e., the molecule was identified as successfully binding by at least two of the three nmr methods of std, cpmg, and waterlogsy) was subjected to further analysis by itc and x-ray crystallography. itc was used to determine the dissociation constants of binding molecules, and x-ray crystallography was used to confirm the binding mode predicted by the nmr studies. generally speaking, the designed fragments had similar ligands efficacies compared to the parent molecules but had much higher dissociation constants (k d values), meaning that the fragments bound less tightly than the original parent molecule [ ] . with this example, it is possible to see the strength of using nmr as its own hit-validation tool (i.e., three different nmr techniques were used for screening compounds [ ] ), and yet, the follow-up of nmr studies with itc and x-ray crystallography was useful in providing a basis for assessing the drug-ability of a protein-protein interaction [ ] [ ] [ ] [ ] . thus, it is clear to see that nmr is a prominent method of hit-validation in drug discovery research, especially in combination with other biophysical techniques. substantial progress has been made in the nmr field over the past - years, and various methods were established to determine the drug-target complexes. most of them utilize either noe or chemical shift perturbations (csp) although in silico models/programs, using nmr-derivate data also exist. one of the methods called difference of inversion recovery rate with and without target irradiation (direction) is used to map pharmacophores and can be an alternative to std experiments. this method uses the difference between longitudinal relaxation rates of ligand protons with-and with-out irradiation of the protons of the target protein. the direction approach, however cannot be used for slowly exchanging (strong binding) ligands. the practical approach of this method was demonstrated on the experiment when analyzed the interactions between p mapk (p a mitogen-activated protein kinase) and its inhibitor-sb [ , ] . the results from this experiment showed that protons h , h , h , and h of sb , are in close neighborhood with the protons of p mapk when compared with h , h , and methyl protons. it indicates that two aromatic rings (a pyridine ring and fluorophenyl ring) of sb interact tightly with p mapk. the results were later confirmed with proton density map of each ligand's proton, based on the crystal structure of sb -p mapk complex [ ] . moreover, the same authors already created a new and improved protein-ligand docking method by combining the direction obtained nmr data with docking software. [ ] . a second method that can be used to map pharmacophores is called inter-ligand nuclear overhauser effect (iloe). this d nmr experiment detects when two ligands bind simultaneously to adjacent sites on a protein surface although both of the ligands do not have to bind to the same binding pocket (opposite to inpharma, see above) [ , ] . a negative ligand−ligand noe signal will be created when ligands bind in close proximity to each other whereas ligands that do not bind will show no noes, or at most very weak positive ones [ , ] . iloe also enables determination of the ligand orientations with respect to one another [ ] . as in the case of inpharma, iloe can be utilized even in the absence of a d protein structure and used with large proteins. additionally, iloe differs from inpharma in mixing times-for iloe the mixing times are typically in the range of - ms [ ] . application of iloe was first shown on glycolate+nad + in the presence of porcine heart lactatedehydrogenase, and by glucose- -phosphate+nadph in the presence of l. mesenteroides glucose- -phosphatedehydrogenase and from that time it has been widely used [ , , ] . a third method called structural information using overhauser effects and selective labeling (sos-nmr), relies of std experiments performed on ligand complexes with different protein samples that have been fully deuterated excluding a specific type of amino acid. in other words, the data obtained by sos-nmr gives insight into the ligand-binding amino acid composition and when taken into consideration the d structure of targeted protein can be used to establish the structure of protein-ligand complex. this approach has been demonstrated using two complexes-fkbp complexed to -( -pyridyl)-benzimidazole and mura complexed to uridine diphosphaten-acetylglucosamine (udp-glcnac). the results showed that for fkbp and mura, only four and three amino acids (fkbp: ile, val, leu, met; mura: trp, phe, his) were needed to be selectively protonated in perdeuterated samples to establish the ligand-binding site. additionally, on average only amino acids were required for accurate identification of ligand-binding surface. according to authors sos-nmr can greatly improve the early stages of the drug discovery process [ ] . moreover, combining sos-nmr with other methods can even further increase chances for a positive outcome of an experiment [ ] . a completely different approach to this topic was taken by chen et al. [ , ] . instead of using isotope labeling, chen's group decided to use a tert-butyl group contained within ligand- to obtain structural information about the protein-ligand complex [ ] . the tert-butyl group formed an intense singlet in . to . ppm range thanks to rapid methyl rotation and methyl reorientation within that group. when compared with the protein's h-nmr signal, the tert-butyl signal tended to be much narrower and resulted in easy detection without the need for isotopic enrichment even in protein complexes of high molecular mass such as bacillus stearothermophilus dnab hexamer ( kda) [ ] . additionally, the tert-butyl group produces intense noesy cross peaks that can be observed even in the situations where normally cross-peaks of the proteins are barely detectable. this is partially because the signal corresponded to nine protons within tert-butyl group. those aspects enable measurements of pseudo-contact shifts generated by paramagnetic tags attached to the protein. as a result, it allows positioning of the ligand on the protein. an example of this approach, is dengue virus ns b-ns protease from serotype (referred as denpro) in complexed with ligand containing a tert-butyl group. the result of this experiment showed noes between the tert-butyl group of ligand- and residue val from denpro [ ] . solvent accessibility, ligand binding, and mapping of ligand orientation by nmr spectroscopy (salmon) is another method based on the data obtained via nuclear overhauser effect. this method utilizes waterlogsy [ ] to probe for solvent accessibility to the ligand and determine the orientation of the ligand by analyzing signal changes in waterlogsy spectra (positive signal from unbound ligand vs. negative for protein-bound ligands). this method was first used to determine the orientation of prodrug called tretazicar (( -(aziridin- -yl)- , -dinitrobenzamide) known as cb in nqo (quinone oxidoreductase ) binding site. previous attempts had been made to obtain the orientation of tretazicar bounded to nqo , however the results obtained from x-ray crystallography were inconclusive as two orientations of tretazicar could be possible. the information obtained via salmon showed that the side chain of asparagine at position formed a hydrogen bond with -nitrogroup of tretazicar, and that the aziridine moiety of tretazicar pointed toward the solvent [ ] . another variant of waterlogsy method called logsy utilizes the titration slopes as a measure of solvent accessibility. the titration slopes are created by a constant increase of protein concentrations. this method also provides more insight into the process of ligand solvation by checking the influence of protein concentration onto the process. this approach was used on the bromodomain of protein (brd -bd ) by mapping epitopes of two ligands interacting with brd -bd and predicting ligands position. the results showed that the triazolopyridazine moiety of both ligands was implanted into the binding pocket of the brd . additionally, the results from logsy titration showed that methyl-group of ligand , aromatic proton of ligand and aromatic proton of ligand exhibit strong water noe. this information enabled researchers to utilize a chemical replacement strategy (substitute bound water molecules by suitable functional groups) for aromatic proton in a series of ligands containing the triazolopyridazine ring. those protons were replaced with an amino or aminomethyl groups and as a result, the binding affinity of those ligands increased -fold. finally, the results obtained from x-ray crystallography for ligands with such modifications allowed to find the binding mode of the triazolopyridazine ring of ligand (with methyl group pointing internally) and the substituted amino group was found to create hydrogen bond to the side chain of asn of brd -bd [ ] . the most recent approach called nuclear magnetic resonance molecular replacement (nmr ) utilizes spatial data obtained through solution-state nmr in order to locate the binding pocket of a complex structure. for that, it uses a receptor model, e.g., a x-ray structure of a homolog, to conduct an analysis and at the same time excluding the need for protein resonance assignment. to conduct an experiment using such an approach requires a few steps. first, either the protein or ligand used in the complex must be uniformly c and n labeled. then, an experiment to assign the ligand is needed such as d c, h-hmqc or c, h-hmbc. the next step is the evaluation of ligand intra-and ligand-protein intermolecular distances through noe cross peaks obtained from f - n, c-filtered h, h-noesy. lastly, choosing a proper input structure is required which can be either x-ray or nmr structures in apo form, with another bound ligand, or a homolog to the protein of interest. then the nmr program analyzes for all possible partial assignments (such as methyl groups of a protein) and calculates the complex structures for all options [ , ] . this method was already successfully used to resolve complex structures in case of slow and fast exchange ligands [ ] [ ] [ ] [ ] . in silico methods combined with nmr derived information can also be used to determine accurate drug-target complexes. . . . samplex another program that can utilize csp called smoothed automatic mapping of protein from listed extremes (samplex) can help to determine the interaction surface of proteins complexes. samplex takes the chemical shifts of the protein of interests in both the free and bound state and corresponding d structure of a protein in the free state. the programs returns a confidence value for each residue to be in a perturbed or unperturbed state ( . as being in a perturbed state, − . as remaining in their unperturbed state). this approach was tested on five examples, one of which was subtilisin bpn' (serine protease) complexed with its inhibitor-chymotrypsin inhibitor . the results showed that residue , and residues - of chymotrypsin inhibitor- were perturbed and residue was in an ambiguous state. to compare, the x-ray crystallography data showed residues and - to be involved in the interaction. for subtilisin bpn' the program predicted residues , , - , - , , - , - and - to perturbed and residues , and to be in ambiguous state. that information was also confronted with the x-ray crystallography data which shown residues - , - , - , , - to be perturbed [ ] . the interactions between targets (proteins) and ligands (small molecules) can be analyzed independently of the biological systems by using 'cell-based' nmr drug design approaches. three basic approaches [ ] are as follows: ( ) compound-detected in-cell nmr, ( ) target-detected in-cell nmr, and ( ) reporter-detected in-cell nmr. these methods, with the exception of compound detected in-cell nmr, differ according to the isotopically labeled structure (protein, cell structure, etc.), which enables nmr detection. a cartoon representation of each of these methods is given in figure . as we have attempted to emphasize and demonstrate, nmr has a powerful and unique role in drug design. nmr provides detailed structural information about a molecule along with kinetic information over extended time periods, i.e. not just a snapshot [ , ] . moreover, nmr is quantitative and highly reproducible, allowing applications in diverse fields such as relaxometry, combinatorial chemistry, fluxomics, and targeted analysis [ ] . nmr can be combined with other analytical techniques, such as mass spectrometry "in tandem" analysis of the molecules of interest [ ] [ ] [ ] . h d-nmr is used particularly in the analysis of metabolites, while the strength and std nmr is a technique that lies within the compound-detected in-cell nmr method but does not require isotope-labelling of the studied compound. however isotopic labelling of the compound may be used to enhance the quality of the spectra. in the target-detected in-cell nmr only the target of interest is isotopically labeled (i.e., n labeled protein). for instance, target proteins can be isotopically labeled during cell growth in isotopically enriched ( c, n, or both n/ c) media [ ] . the cell type and the labeling method may vary across experiments. different cell types, including bacteria [ ] , oocytes [ ] , yeast cells [ ] , mammalian cells [ ] , hela cells [ ] and even insect cells [ ] have been reported in the literature. the fact that in-cell nmr applies to more than one cell type testifies of the versatility and potential application of this technique. in terms of labeling, n is one of the most commonly used approaches [ ] when the targets of interest are proteins. recently, f labeling has been reported as a useful probe for protein-ligand interactions [ ] . it was shown that f can reveal information about the dynamics of protein-ligand interactions [ ] . methyl groups [ ] have also been used as probes for proteins and complexes in vivo [ ] , proving that labeling specific chemical groups instead of the entire biomolecule (i.e., protein) is feasible. in-cell nmr extends beyond proteins, and has been applied successfully to dna [ , ] and rna molecules [ , ] . telomeric repeats have also been studied using target detected in-cell nmr [ ] . the reporter-detected in-cell nmr technique isotopically labels neither the ligand nor the target, but rather a receptor that indirectly measures the effects of ligand-target binding [ ] . the "reporter" varies according to the experimental context. for instance, dose et al. [ ] used acetylation-and deacetylation-based assays to monitor the activity of histone deacetylase and acetyl-transferase. thongwichian et al. [ ] used peptide-based reporters to identify active kinases and phosphatases in cellular conditions. lastly, doura et al. [ ] designed a f probe that operates in biological conditions in order to study the adherence and dynamics of proteins found in human blood. in many viruses and phages, scaffolding proteins (sps) are required to ensure the correct organization of coat proteins (cps) and other minor capsid proteins into a precursor structure, called a procapsid [ , ] . although sps are critical for viral assembly and therefore potential therapeutic targets their structural properties (with only a few exceptions [ , ] ) are poorly understood. the size limitation of nmr can be used advantageously as a filter to identify disordered segments even in very large supramolecular protein complexes. in this way, nmr can provide a unique perspective on the dynamic and disordered elements of macromolecules not accessible by other techniques. the procapsid encapsulation experiments described by whitehead et al. [ ] were conceptually analogous to in-cell nmr experiments [ ] [ ] [ ] in which signals from small proteins, or flexible segments of proteins, can be observed when they are incorporated inside living cells, as long as the isotope-labeled proteins of interest do not interact strongly with other large cellular components [ ] [ ] [ ] . the so called "in-virus" nmr strategy applied by whitehead et al. [ ] could be more generally used to study the dynamic properties of macromolecules encapsulated into virus particles, including cargo molecules encased in viral capsids for nanotechnology applications. additionally, such studies could assess the level of interaction of cargo molecules with the virus and probe the release properties of cargo nmr [ ] . as we have attempted to emphasize and demonstrate, nmr has a powerful and unique role in drug design. nmr provides detailed structural information about a molecule along with kinetic information over extended time periods, i.e., not just a snapshot [ , ] . moreover, nmr is quantitative and highly reproducible, allowing applications in diverse fields such as relaxometry, combinatorial chemistry, fluxomics, and targeted analysis [ ] . nmr can be combined with other analytical techniques, such as mass spectrometry "in tandem" analysis of the molecules of interest [ ] [ ] [ ] . h d-nmr is used particularly in the analysis of metabolites, while the strength and intensity of the recorded signals is directly proportional to the concentration of the sample [ , , ] . h d-nmr can also be used to follow "real-time" analysis of different molecules [ ] . in the d h-nmr experiment, there are no polarization transfer techniques required (the h atom is already highly sensitive) and covers a spread of interesting nuclei in the molecule(s) being studied [ ] . assuming that the sample can be stored stably for extended periods of time, the non-destructive nature of nmr permits the re-use on the sample for different experiments [ , ] . aiding in the reproducibility of nmr [ ] , sample-recycling offers a significant advantage of nmr [ , ] . while always important in drug design, sensitivity and resolution of nmr are two major factors that need special consideration. since both factors improve with increasing magnetic field strength, we have seen a spike in the demand for ultra-high-field nmr spectrometers. recently, . t (i.e., . ghz for h) magnets have become commercially available, and with recent advances in magnet technology, such as liquid helium recycling and magnetic field shielding [ ] , nmr has begun to offer far better resolution and higher sensitivity while reducing the substantial costs of maintaining the instruments compared to past decades. other steps have been taken to enhance the sensitivity of nmr including: the development of cryoprobes increasing the signal to noise ratio to times, and micro-coil probes that not only increased sensitivity but also reduced the amount of sample required for the measurements [ ] . from another perspective, one can further optimize the process of obtaining spectra by using different methods of measurements. one of these, called sofast, helps to reduce the delay between scans resulting in lowering acquisition time for d experiments such as hmqc utilizing h, n or h, c [ , , ] . the basic principle of this method is to use selective h pulses that will excite only a small portion of the available nuclei pool, while the unperturbed spins provide a magnetization "heat sink" thus improving the spin-lattice relaxation (t ) rate via dipolar interactions [ ] . these methods can be highly efficient when studying drug binding and molecular interactions [ ] . another method, called ultrafast d nmr, enables obtaining a d spectra within a single scan but with the associated cost of reduced sensitivity [ , ] . the principle of this method is to divide the sample into n number of fractions, and apply the appropriate incremental aspect to each fraction, while recording them all simultaneously within the one scan [ ] . this method has been applied to many metabolomic studies [ , , ] as well as in the analysis of natural products [ ] . the difficulty is that the effective concentration of the sample is lowered by the fractionation level. the more slices the sample is split into, the greater the reduction of combined signal that is obtained. lastly, a method termed non-uniform sampling (nus) may provide an advantage by reducing the total time for measurement while maintaining the same resolution of spectra [ ] . nus effectively skips over parts of the total dataset, collecting only around to % of the total. usually sections containing higher concentrations of signal (over noise) are emphasized in the selection scheme, known as non-linear data acquisition. these methods reconstruct the complete data subset by applying various algorithms such as multidimensional decomposition (mdd), which essentially separates the sets of multidimensional data into one dimensional problems that are much easier to solve given the common process of signal overlapping in multidimensional nmr spectra [ ] . other algorithms such as compressed sensing (cs), maximum entropy method (max ent) and iterative soft threshold (ist) each have their own advantages but all focus on decreasing the time needed for collection of spectra [ , ] . the cost is in signal to noise, as no gain is ever absolutely free. the inherent advantages of nmr do not eliminate the disadvantages (table ) , which are namely being limited for some nuclei, and inherent insensitivity for many types of experiments. nmr can provide high quality resolution and sensitivity for some experiments [ , ] but the application can be challenging. when the experiments become multidimensional, there is often a tradeoff between the improved higher resolution and/or the resulting sensitivity and/or the amount of time an experiment takes, i.e., high-quality spectra for multidimensional experiments take much longer than their simple d counterparts [ ] (scheme ). one must always choose between resolution, sensitivity, and the amount time; as you can only ever have two out of the three. advantages disadvantages ability to identify simple chemical compounds. high quality resolution and sensitivity for many d experiments. less time consuming compared to d nmr. compared to d nmr less details can be obtained for more complex molecules. for nuclei other than h and f, relative sensitivity is fairly low-requires extra labeling to obtain better spectra. ability to identify complex molecules and observe different interactions between the nuclei, e.g., correlations between all spins in one spin system using tocsy experiment. requires long times to obtain a proper spectra (up to days). greatly reduces time to obtain d spectra. reduced sensitivity. significantly reduces acquisition time of hmqc. relatively low resolution lowers the time of measurement while keeping the same level of resolution. requires use of reconstruction algorithms since missing data points can lead to artifacts in spectra. multidimensional data are "broken" to one dimensional, which are easier to analyze. ability to resolve overlapping resonances. data must be (approximately) symmetrical (lorentzian shape) to obtain good spectra. good reconstruction of weaker peaks. large computational costs, low performance on noisy data. significant reduction in acquisition time. nominally lorentzian peak shapes may be distorted, and peak intensities may be altered. greatly reduced time to obtain nmr spectra. requires a grid of uniformly sampled data points. often makes stronger binding ligands from weakly binding fragments. less time and resource intensive. can be used only for small fragments of compound of interest. direct observation of target binding to ligand. several types of nmr experiments are possible. inability to distinguish binding modes, difficult to gage the "true" binding site of ligand to protein. only requires a small amount of sample. highly reproducible. allows direct observations of ligand binding. only works for ligands with low binding affinity (fast chemical exchange). inability to distinguish binding modes. in vivo studies are possible, can focus on specific cell parts. special labeling techniques may be required. spectra may be more challenging to interpret. can model protein drug interactions, helps speed up and reduce cost of drug delivery protein models need to be validated through experimental approach. can observe proteins interacting with metal ions, long observation distance ( - angstroms) between paramagnetic left and nearby atoms. paramagnetic left required in the system. enables the characterization of a chemical compound in a solid-state form such as a tablet/pill. provides insight into the physical properties of a compound. significant broadening of the spectral lineshapes due to anisotropic spin interactions. noticeable difference in spectra of binding and non-binding ligands. sets the lower limit of time for which experiments can be performed. unfortunately, in the real world, when working with nmr spectroscopy, we are mainly forced to choose between high or low precision data (scheme ) with fixed available instrument times. scheme can be useful, when choosing nmr technique and/or method/approaches in drug studies. it shows the general representation of different nmr techniques and method/approaches in costtime matrix, bearing in mind that the exact position in the matrix can be influenced by the environmental conditions, pulse sequence and sample preparation (e.g. concentration). regarding, nmr methods/approaches, the exact position depends on the choice of proper nmr technique. for the purposes of scheme , the d techniques were chosen as described in the practical examples. nevertheless, with relatively short times and at low cost, we can acquire numerous data sets and therefore the low precision can be partially compensated for by statistical analysis. fortunately, significant efforts have been undertaken to reduce the amount of time it takes to record multidimensional spectra, especially for d nmr, and still obtain high quality spectra (table ) . novel pulse sequences have been developed to decouple nuclei in pure-shift nmr [ ] . dynamic nuclear polarization (dnp) can induce hyper-polarization in atoms ( c, n) with an inherently low sensitivity [ , ] . parahydrogen-induced polarization (phip) and signal amplification by reversible exchange (sabre) are other polarization techniques used to increase the sensitivity of inherently insensitive nuclei [ ] . unfortunately, in the real world, when working with nmr spectroscopy, we are mainly forced to choose between high or low precision data (scheme ) with fixed available instrument times. scheme can be useful, when choosing nmr technique and/or method/approaches in drug studies. it shows the general representation of different nmr techniques and method/approaches in cost-time matrix, bearing in mind that the exact position in the matrix can be influenced by the environmental conditions, pulse sequence and sample preparation (e.g., concentration). regarding, nmr methods/approaches, the exact position depends on the choice of proper nmr technique. for the purposes of scheme , the d techniques were chosen as described in the practical examples. nevertheless, with relatively short times and at low cost, we can acquire numerous data sets and therefore the low precision can be partially compensated for by statistical analysis. fortunately, significant efforts have been undertaken to reduce the amount of time it takes to record multidimensional spectra, especially for d nmr, and still obtain high quality spectra (table ) . novel pulse sequences have been developed to decouple nuclei in pure-shift nmr [ ] . dynamic nuclear polarization (dnp) can induce hyper-polarization in atoms ( c, n) with an inherently low sensitivity [ , ] . parahydrogen-induced polarization (phip) and signal amplification by reversible exchange (sabre) are other polarization techniques used to increase the sensitivity of inherently insensitive nuclei [ ] . nmr has become a "gold standard" method in drug design due to its speed, simplicity, and reproducibility. the standardized ppm scale allows one to compare all nmr results and gather them in databases for the common use of researchers. although sample labeling was limiting in the beginning, it has now become a strength of nmr that permits the observation of big molecules and/or biomolecular processes "through the door lock". nmr is the only analytical technique that permits qualitative and quantitative analysis without previous sample purification or separation. high precision nmr data still requires long experiment times and has elevated costs; however, these will no doubt be alleviated in the future. estimating the cost of new drug development: is it really $ million? health aff. (millwood) the price of innovation: new estimates of drug development costs animal testing and its alternatives-the most important omics is economics what is the question? nmr in drug design evaluation of ligand-based nmr screening methods to characterize small molecule binding to hiv- glycoprotein- ramped-up nmr: multiplexed nmr-based screening for drug discovery extraction, characterization, and anticoagulant activity of a sulfated polysaccharide from bursatella leachii viscera ultra-low thermal conductivity in na/sb chalcobismuthates: synthesis, crystal structures, optical properties and na nmr spectroscopy layered copper thioaluminate k cu als : synthesis, crystal structure, characterization and solid-state al and k nmr studies identification and characterization of sse , a microtubule depolymerizing agent that overcomes multidrug resistance amide versus amine ratio in the discrimination layer of reverse osmosis membrane by solid state n nmr and dnp nmr current and future perspectives on the structural identification of small molecules in biological systems nmr experiments for the rapid identification of p=o···h-x type hydrogen bonds in nucleic acids separation and structural elucidation of a novel analogue of vardenafil included as an adulterant in a dietary supplement by liquid chromatography-electrospray ionization mass spectrometry, infrared spectroscopy and nuclear magnetic resonance spectroscopy three-dimensional nmr spectroscopy of fluorinated pharmaceutical solids under ultrafast magic angle spinning essential parameters for structural analysis and dereplication by h-nmr spectroscopy uplc-hrms and nmr applied in the evaluation of solid-phase extraction methods as a rational strategy of dereplication of phyllanthus spp. aiming at the discovery of cytotoxic metabolites a light responsive two-component supramolecular hydrogel: a sensitive platform for the fabrication of humidity sensors nuclear magnetic resonance study of nanoscale ionic materials variable-temperature nmr and conformational analysis of oenothein, b nmr at low and ultralow temperatures h-nmr study of the effect of temperature through reversible unfolding on the heme pocket molecular structure and magnetic properties of aplysia limacina cyano-metmyoglobin spectroscopic studies of the intermediates in the conversion of , , , -tetrahydro- , -anthraquinone to , -anthraquinone by reaction with oxygen under basic conditions heterogeneous intraand intermolecular hydroamination catalysts -(phosphonomethoxy)ethyl]adenine (pmea) and of its n , n , and n deaza derivatives with copper(ii) in aqueous solution online reaction monitoring by single-scan d nmr under flow conditions solvent-and anion-dependent li+-o -coupling strength and implications on the thermodynamics and kinetics of li-o batteries nmr approaches in structure-based lead discovery: recent developments and new frontiers for targeting multi-protein complexes screening protein-single stranded rna complexes by nmr spectroscopy for structure determination intramolecularly stapled amphipathic peptides via a boron-sugar interaction structure determination using solution nmr: is it worth the effort? nmr spectroscopy brings invisible protein states into focus fragment-based screening using x-ray crystallography and nmr spectroscopy utilizing nmr and epr spectroscopy to probe the role of copper in prion diseases combining nmr and x-ray crystallography in fragment-based drug discovery: discovery of highly potent and selective bace- inhibitors. in fragment-based drug discovery and x-ray crystallography real-time structure-based drug development-tutorial: triad's nmr-based structural determinations are smart chemistry method development and validation: quantitation of telmisartan bulk drug and its tablet formulation by h-nmr spectroscopy p and n solid-state nmr study for the development of a novel membrane protein drug-screening methodology functional analyses of target proteins for drug development by nmr band-selective excitation nmr spectroscopy and quantitative time-domain analysis using complete reduction to amplitude-frequency table (craft) to determine distribution coefficients during drug development aggregation ability of three phylogenetically distant anammox bacterial species host-dependent nitrogen recycling as a mechanism of symbiont control in aiptasia recommended strategies for spectral processing and post-processing of d h-nmr data of biofluids with a particular focus on urine anti-cancer agents in saudi arabian herbals revealed by automated high-content imaging synthesis and evaluation of modified chalcone based p stabilizing agents chapter three-paclitaxel synthesis and characterization of a homogeneous and silica supported homoleptic cationic tungsten(vi) methyl complex: application in olefin metathesis ft-icr mass spectrometry, and nmr spectroscopy study of heavy fuel oil recommendations and standardization of biomarker quantification using nmr-based metabolomics with particular focus on urinary analysis marine bacterial transparent exopolymer particles (tep) and tep precursors: characterization and ro fouling potential active site arginine controls the stereochemistry of hydride transfer in cyclohexanone monooxygenase synthesis, characterization and conformational study of new α,β-unsaturated acylhydrazones based on calix [ ] arene backbone poly-γ-p-biphenylmethyl-glutamate as enantiodifferentiating alignment medium for nmr spectroscopy with temperature-tunable properties elucidation and partial nmr assignment of monosulfated maitotoxins from the caribbean elucidation of the structure of the -amino- , -dibromochalcone epoxides in solution and solid state synthesis, spectroscopy, and theoretical calculations of some -thiohydantoin derivatives as possible new fungicides identification of polyunsaturated triacylglycerols and cc location isomers in sacha inchi oil by photochemical reaction mass spectrometry combined with nuclear magnetic resonance spectroscopy coordination geometry determination of stannane compounds with phosphinoyldithioformate ligands using multinuclear nmr, sn mössbauer and dft methods clathrate structure determination by combining crystal structure prediction with computational and experimental xe nmr spectroscopy synthesis and biological activity of n-(arylsulfonyl) valine hydrazones and assistance of nmr spectroscopy for definitive d structure sández macho, i. use of h-nmr std, waterlogsy, and langmuir monolayer techniques for characterization of drug-zein protein complexes nmr spectroscopy techniques for screening and identifying ligand binding to protein receptors sequential h-nmr assignment of the complex of aponeocarzinostatin with ethidium bromide and investigation of protein-drug interactions in the chromophore binding site nmr-based metabolomic techniques identify the toxicity of emodin in hepg cells h-nmr-based metabolomics study of the toxicological effects in rats induced by new insights about doxorubicin-induced toxicity to cardiomyoblast-derived h c cells and dexrazoxane cytoprotective effect: contribution of in vitro h-nmr metabonomics nmr-based metabonomic study on the subacute toxicity of aristolochic acid in rats solid state nmr and bioequivalence comparison of the pharmacokinetic parameters of two formulations of clindamycin bace is the major β-secretase for generation of aβ peptides by neurons structural basis for the antifolding activity of a molecular chaperone chapter ten-binding site identification and structure determination of protein-ligand complexes by nmr: a semiautomated approach fragment-based drug design nmr in target driven drug discovery: why not? nmr in integrated biophysical drug discovery for ras: past, present, and future nmr in pharmaceutical discovery and development clinical magnetic resonance spectroscopy principles of pulse nmr spectroscopy spin dynamics: basics of nuclear magnetic resonance nmr spectroscopy explained: simplified theory, applications and examples for organic chemistry and structural biology solid-state nmr paramagnetic relaxation enhancement immersion depth studies in phospholipid bilayers protein dynamics from nmr an introduction to nmr-based approaches for measuring protein dynamics using nmr spectroscopy to investigate the role played by copper in prion diseases monitoring dendrimer conformational transition using f and h o nmr retention of strong intramolecular hydrogen bonds in high polarity solvents in binaphthalene-benzamide derivatives: extensive nmr studies potential hepatoxicity risk of the shell of herpetospermum caudigerum wall in rats based on h-nmr metabonomics metabolomic analysis of anti-hypoxia and anti-anxiety effects of fu fang jin jing oral liquid integrative drug efficacy assessment of danggui and european danggui using nmr-based metabolomics ardá, a. nmr and molecular recognition of n-glycans: remote modifications of the saccharide chain modulate binding features obtaining d atomistic structure of saccharides from raman/roa/nmr spectroscopic techniques the strengths and weaknesses of nmr spectroscopy and mass spectrometry with particular focus on metabolomics research monitoring dna-ligand interactions in living human cells using nmr spectroscopy situ monitoring of bacteria under antimicrobial stress using p solid-state nmr nmr studies of protein structure and dynamics combined hplc, nmr spectroscopy, and ion-trap mass spectrometry with application to the detection and characterization of xenobiotic and endogenous metabolites in human urine bioequivalence assessment of two formulations of ibuprofen lc-nmr-ms in drug discovery asics: an automatic method for identification and quantification of metabolites in complex d h-nmr spectra metabolite identification via the madison metabolomics consortium database a gui r package for proficient automatic profiling of d h-nmr spectra of study datasets open source software for identifying and quantifying metabolites in nmr spectra chapter -experimental aspects of nmr spectroscopy practical aspects of n-dimensional data acquisition and processing water suppression that works. excitation sculpting using arbitrary waveforms and pulsed field gradients distortion-free homonuclear spectra of peptides and nucleic acids in water using excitation sculpting water suppression that works. excitation sculpting using arbitrary wave-forms and pulsed-field gradients gradient-tailored excitation for single-quantum nmr spectroscopy of aqueous solutions solvent signal suppression in nmr experiments on ax systems distortionless enhancement of nmr signals by polarization transfer c-nmr-based metabolic fingerprinting of citrus-type crude drugs chemoselective n tag for sensitive and high-resolution nuclear magnetic resonance profiling of the carboxyl-containing metabolome n-cholamine-a smart isotope tag for combining nmrand ms-based metabolite profiling fast reconstruction of non-uniform sampling multidimensional nmr spectroscopy via a deep neural network n and c-sofast-hmqc editing enhances d-noesy sensitivity in highly deuterated, selectively [ h, c]-labeled proteins chapter -new advances in fast methods of d nmr experiments heterogeneous microtesla sabre enhancement of n nmr signals information from combined h and p nmr studies of cell extracts: differences in metabolism between drug-sensitive and drug-resistant mcf- human breast cancer cells assessment of pharmacological treatment of myocardial infarction by phosphorus- nmr with surface coils application of p nmr spectroscopy and chemical derivatization for metabolite profiling of lipophilic compounds in human serum development of a bioreactor system for cytotoxic evaluation of pharmacological compounds in living cells using nmr spectroscopy the organic set of nmr spectra. in and more essential nmr experiments: a detailed guide pulse pair technique in high resolution nmr a reprint of the historical lecture notes on two-dimensional spectroscopy choosing the best pulse sequences, acquisition parameters, postacquisition processing strategies, and probes for natural product structure elucidation by nmr spectroscopy the phase transfer catalysed synthesis of isoflavone-o-glucosides determination of the relative signs of j( p- p) in complexes of tungsten( ) and molybdenum( ) using two-dimensional [ p- p]-cosy- nuclear magnetic resonance chemical shift correlation two dimentional nmr knowns and unknowns in metabolomics identified by multidimensional nmr and hybrid ms/nmr methods product operators, coherence pathways, and phase cycling. part iii: phase cycling artifacts in two-dimensional nmr chapter -correlations through the chemical bond i: homonuclear shift correlation analytical optimization of active bandwidth and quality factor for tocsy experiments in nmr spectroscopy novel selective tocsy method enables nmr spectral elucidation of metabolomic mixtures elimination of zero-quantum interference in two-dimensional nmr spectra h-nmr and ms based metabolomics study of the intervention effect of curcumin on hyperlipidemia mice induced by high-fat diet pharmacometabonomics analysis reveals serum formate and acetate potentially associated with varying response to gemcitabine-carboplatin chemotherapy in metastatic breast cancer patients based metabolic profiling of cells in response to treatment with a hexacationic ruthenium metallaprism as potential anticancer drug the use of coupled hsqc spectra to aid in stereochemical assignments of molecules with severe proton spectral overlap chapter -nmr spectroscopy methods in metabolic phenotyping. in the handbook of metabolic phenotyping high-resolution nmr techniques in organic chemistry doubly compensated multiplicity-edited hsqc experiments utilizing broadband inversion pulses product operator formalism in nmr acac)(γ-acac)(dms)] treatment revealed by a metabolomic h-nmr study a comprehensive discussion of hsqc and hmqc pulse sequences evaluation of band-selective hsqc and hmbc: methodological validation on the cyclosporin cyclic peptide and application for poly( -hydroxyalkanoate)s stereoregularity determination chapter -nmr molecular recognition studies for the elucidation of protein and nucleic acid structure and function metabominer-semi-automated identification of metabolites from d nmr spectra of complex biofluids an improved n relaxation dispersion experiment for the measurement of millisecond time-scale dynamics in proteins improved hsqc experiments for the observation of exchange broadened signals quantification of metabolites by nmr spectroscopy in the presence of chapter -relaxation and dynamic processes basic principles of nmr a complete introduction to modern nmr spectroscopy nmr in drug discovery high-resolution diffusion and relaxation edited one-and two-dimensional h-nmr spectroscopy of biological fluids measurement of spin relaxation in complex systems -multiple-pulse nmr experiments ultrafast nmr t relaxation measurements: probing molecular properties in real time saturation-inversion-recovery: a method for t measurement chapter -the physical basis of the nuclear magnetic resonance experiment. part ii: pulse and fourier-transform nmr spin-lattice relaxation time in drug discovery and design nmr methods to characterize protein-ligand interactions the interaction of dna with intercalating agents probed by sodium- nmr relaxation rates nmr screening techniques in drug discovery and drug design one-dimensional relaxation-and diffusion-edited nmr methods for screening compounds that bind to macromolecules use of relaxation-edited one-dimensional and two dimensional nuclear magnetic resonance spectroscopy to improve detection of small metabolites in blood plasma the quest for simplicity: remarks on the free-approach models fast evaluation of protein dynamics from deficient n relaxation data effects of diffusion on free precession in nuclear magnetic resonance experiments modified spin-echo method for measuring nuclear relaxation times measuring t and t relaxation rates an exact solution for r , eff in cpmg experiments in the case of two site chemical exchange nmr-based screening in drug discovery extraction of t from nmr linewidths in simple spin systems by use of reference deconvolution quantitative metabolic profiling of nmr spectral signatures of branched chain amino acids in blood serum isoniazid-induced hepatotoxicity and neurotoxicity in rats investigated by h-nmr based metabolomics approach h-nmr-based metabolic profiling of naproxen-induced toxicity in rats h-nmr toxicometabolomics following cisplatin-induced nephrotoxicity in male rats the antitumor effect of formosanin c on hepg cell as revealed by h-nmr based metabolic profiling an nmr-based metabonomic investigation of the subacute effects of melamine in rats metabolomic profiling predicts outcome of rituximab therapy in rheumatoid arthritis response to drug treatment in newly diagnosed epilepsy: a pilot study of h-nmr-and ms-based metabonomic analysis metabolic alteration in obese diabetes rats upon treatment with centella asiatica extract novel , , -thiadiazoles inhibit colorectal cancer via blockade of il- /cox- mediated jak /stat signals as evidenced through data-based mathematical modeling plasma-metabolite-biomarkers for the therapeutic response in depressed patients by the traditional chinese medicine formula xiaoyaosan: a h-nmr-based metabolomics approach metabonomic profiles delineate the effect of traditional chinese medicine sini decoction on myocardial infarction in rats h-nmr spectral identification of medication in cerebrospinal fluid of pediatric meningitis h-nmr-based metabolomics approach to investigating the renal protective effects of genipin in diabetic rats potential metabolomic biomarkers for evaluation of adriamycin efficacy using a urinary h-nmr spectroscopy h-nmr-based serum metabolomics reveals erythromycin-induced liver toxicity in albino wistar rats nmr-based metabonomics study on the effect of gancao in the attenuation of toxicity in rats induced by fuzi nmr-fragment based virtual screening: a brief overview discovering high-affinity ligands for proteins: sar by nmr when fragments link: a bibliometric perspective on the development of fragment-based drug discovery current perspectives in fragment-based lead discovery (fbld) adaptation of high-throughput screening in drug discovery-toxicological screening tests high-throughput screening: the hits and leads of drug discovery-an overview review of applications of high-throughput sequencing in personalized medicine: barriers and facilitators of future progress in research and clinical application review article: high-throughput affinity-based technologies for small-molecule drug discovery design principles for fragment libraries: maximizing the value of learnings from pharma fragment-based drug discovery (fbdd) programs for use in academia how size matters: diversity for fragment library design high-throughput screening: update on practices and success industrial scale high-throughput screening delivers multiple fast acting macrofilaricides virtual high throughput screening using machine learning methods introduction to fragment-based drug discovery. in fragment-based drug discovery and x-ray crystallography the influence of lead discovery strategies on the properties of drug candidates biophysical screening in fragment-based drug design: a brief overview high-throughput screening and fragment-based design: general considerations for lead discovery and optimization nmr screening and hit validation in fragment based drug discovery structural biology in fragment-based drug design fragment-based drug discovery using nmr spectroscopy chapter nine-practical aspects of nmr-based fragment screening fragment-based drug design nmr-based screening: a powerful tool in fragment-based drug discovery nmr in drug discovery: a practical guide to identification and validation of ligands interacting with biological macromolecules applied biophysical methods in fragment-based drug discovery concepts and core principles of fragment-based drug design application of fragment-based drug discovery to versatile targets a three-stage biophysical screening cascade for fragment-based drug discovery protein crystallography and drug discovery: recollections of knowledge exchange between academia and industry twenty years on: the impact of fragments on drug discovery churcher, i. fragment-based drug discovery-from hit discovery to fda approval: lessons learned and future challenge the first drug approved for braf -mutant cancer venetoclax: evidence to date and clinical potential fragment-based drug design: strategic advances and lessons learned krimm, i. fragment linking strategies for structure-based drug design discovery and development of venetoclax, a selective antagonist of bcl- an inhibitor of bcl- family proteins induces regression of solid tumours x-ray and nmr structure of human bcl-x l, an inhibitor of programmed cell death ribociclib for the first-line treatment of advanced hormone receptor-positive breast cancer: a review of subgroup analyses from the monaleesa- trial the csf receptor inhibitor pexidartinib (plx ) reduces tissue macrophage levels without affecting glucose homeostasis in mice structure-guided blockade of csf r kinase in tenosynovial giant-cell tumor characterizing and overriding the structural mechanism of the quizartinib-resistant flt "gatekeeper" f l mutation with plx discovery and chemical development of verubecestat, a bace inhibitor for the treatment of alzheimer's disease discovery of the -imino- , , -thiadiazinane , -dioxide derivative verubecestat (mk- )-a β-site amyloid precursor protein cleaving enzyme inhibitor for the treatment of alzheimer's disease fragment-based drug discovery applied to hsp . discovery of two lead series with high ligand efficiency discovery of ( , -dihydroxy- -isopropylphenyl)-[ -( -methylpiperazin - -ylmethyl)- , -dihydroisoindol- -yl]methanone (at ), a novel inhibitor of the molecular chaperone hsp by fragment based drug design optimization of pyrrolamide topoisomerase ii inhibitors toward identification of an antibacterial clinical candidate (azd ) using fragment-based approaches to identification of n-( -piperidinyl)- -( , -dichlorobenzoylamino)- h-pyrazole- -carboxamide (at ), a novel cyclin dependent kinase inhibitor using fragment-based x-ray crystallography and structure based drug design cyclin-dependent kinase inhibitor at as a potential drug for mycn-dependent neuroblastoma biological characterization of at , a small-molecule inhibitor of cyclin-dependent kinases, in human tumor cell lines development of second-generation cdk inhibitors for the prevention of cisplatin-induced hearing loss practical fragments: fragments in the clinic: edition sar by nmr: putting the pieces together encyclopedia nmr-based approaches for the identification and optimization of inhibitors of protein-protein interactions nmr-based screening of proteins containing c-labeled methyl groups estimating protein−ligand binding affinity using high-throughput screening by nmr exploring the binding of peptidic west nile virus ns b-ns protease inhibitors by nmr blocking the interactions between calcium-bound s a protein and the v domain of rage using tranilast characterization of protein−ligand interactions by high-resolution solid-state nmr spectroscopy using chemical shift perturbation to characterise ligand binding combining automated peak tracking in sar by nmr with structure-based backbone assignment from n-noesy atia-tul-wahab chapter -nuclear overhauser effect accuracy in determining interproton distances using nuclear overhauser effect data from a flexible molecule chapter -more d and d nmr experiments: the nuclear overhauser effect-polarization transfer-spin lock experiments- d nmr use of nmr saturation transfer difference spectroscopy to study ligand binding to membrane proteins waterlogsy as a method for primary nmr screening: practical aspects and range of applicability characterization of ligand binding by saturation transfer difference nmr spectroscopy detecting binding affinity to immobilized receptor proteins in compound libraries by hr-mas std nmr ligand based nmr methods for drug discovery virus-ligand interactions: identification and characterization of ligand binding by nmr spectroscopy method for detecting biologically active compounds from compound libraries evolutionary change of the heme c electronic structure: ferricytochrome c- from pseudomonas aeruginosa and horse heart ferricytochrome c nuclear magnetic resonance studies of the binding of trimethoprim to dihydrofolate reductase application of nmr based binding assays to identify key hydroxy groups for intermolecular recognition determining binding sites in protein-nucleic acid complexes by cross-saturation group epitope mapping by saturation transfer difference nmr to identify segments of a ligand in direct contact with a protein receptor synthetic derivatives of pyrrole and pyrrolidine suitable for the therapy of infections caused by rhinoviruses global prevalence of norovirus in cases of gastroenteritis: a systematic review and meta-analysis economic impact of outbreaks of norovirus infection in hospitals norovirus-host interaction: implications for disease control and prevention x-ray crystallographic structure of the norwalk virus capsid structural insights into antigenic diversity and host specificity. proc. natl. acad. sci structural basis for the recognition of blood group trisaccharides by norovirus epitope mapping of histo blood group antigens bound to norovirus vlps using std nmr experiments reveals fine details of molecular recognition synthesis and nmr studies on the abo histo-blood group antigens: synthesis of type iii and iv structures and nmr characterization of type i-vi antigens nmr experiments reveal the molecular basis of receptor recognition by a calicivirus negative nuclear overhuaser effects as probes of macromolecular structure identification of an e-selectin antagonist in a substance mixture by transfer noe the inpharma method: protein-mediated interligand noes for pharmacophore mapping diffusion nmr spectroscopy in supramolecular and combinatorial chemistry: an old parameter-new insights beyond passive: chaotic transport in stirred fluids . passive transport chapter -momentum, heat and mass transfer in boundary layers diffusion nmr spectroscopy chapter -diffusion nmr spectroscopy the diffusion equation models for diffusion spin diffusion measurements: spin echoes in the presence of a time-dependent field gradient diffusion by nmr the stejskal-tanner equation generalized for any gradient shape-an overview of most pulse sequences measuring free diffusion diffusion ordered nuclear magnetic resonance spectroscopy: principles and applications measuring ligand-protein binding using nmr diffusion experiments chapter -dosy nmr for drug analysis diffusion-weighted nmr spectroscopy allows probing of c labeling of glutamate inside distinct metabolic compartments in the brain spatial encoding and spatial selection methods in high-resolution nmr spectroscopy spatially encoded d and d diffusion-ordered nmr spectroscopy enumeration of billion organic small molecules in the chemical universe database gdb- exploring chemical space for drug discovery using the chemical universe database f dosy diffusion-nmr spectroscopy of fluoropolymers pulsed-field gradient nuclear magnetic resonance measurements (pfg nmr) for diffusion ordered spectroscopy (dosy) mapping guest-encapsulation properties of a self-assembled capsule by dynamic boronic ester bonds properties of small molecular drug loading and diffusion in a fluorinated peg hydrogel studied by h molecular diffusion nmr and f spin diffusion nmr a new nmr method to analyze multi-component enzyme/substrate systems the exploration of interaction studies of smaller size, mostly ignored yet intrinsically inestimable molecules towards bsa; an example of std and dosy nmr. open chem structure-based virtual screening for drug discovery: principles, applications and recent advances from data banks to data bases in silico pharmacology for drug discovery: methods for virtual ligand screening and profiling ultra-high-throughput structure-based virtual screening for small-molecule inhibitors of protein-protein interactions in silico activity profiling reveals the mechanism of action of antimalarials discovered in a high-throughput screen in silico target prediction for elucidating the mode of action of herbicides including prospective validation docking, virtual high throughput screening and in silico fragment-based drug design in silico virtual screening approaches for anti-viral drug discovery virtual screening strategies: recent advances in the identification and design of anti-cancer agents a review of ligand-based virtual screening web tools and screening algorithms in large molecular databases in the age of big data virtual high throughput screening (vhts)-a perspective application of nmr and molecular docking in structure-based drug discovery virtual ligand screening against comparative protein structure models biophysical screening for the discovery of small-molecule ligands the use of virtual screening and differential scanning fluorimetry for the rapid identification of fragments active against mek the holistic integration of virtual screening in drug discovery retrospect and prospect of virtual screening in drug discovery docking and scoring in virtual screening for drug discovery: methods and applications nmr and in silico screening acceleration of the drug discovery process: a combinatorial approach using nmr spectroscopy and virtual screening complementarity between in silico and biophysical screening approaches in fragment-based lead discovery against the a a adenosine receptor adenosine a a receptors in parkinson's disease treatment discovery of small-molecule inhibitors of ubiquitin specific protease (usp ) using integrated nmr and in silico techniques molecular recognition of p and mdm by usp /hausp design and characterization of libraries of molecular fragments for use in nmr screening against protein targets antihypertensive drug valsartan in solution and at the at receptor: conformational analysis, dynamic nmr spectroscopy, in silico docking, and molecular dynamics simulations combining in silico tools and nmr data to validate protein−ligand structural models: application to matrix metalloproteinases nmr-filtered virtual screening leads to non-metal chelating metallo-β-lactamase inhibitors metallo-β-lactamase inhibitors inspired on snapshots from the catalytic mechanism synthesis of potent dishevelled pdz domain inhibitors guided by virtual screening and nmr studies paramagnetic nmr in drug discovery paramagnetic nmr in drug discovery paramagnetic nmr in solution and the solid state clanp-an artificial paramagnetic centre to study proteins by nmr current nmr techniques for structure-based drug discovery requirements on paramagnetic relaxation enhancement data for membrane protein structure determination by nmr paramagnetic nmr as a new tool in structural biology edta-derivatized deoxythymidine as a tool for rapid determination of protein binding polarity to dna by intermolecular paramagnetic relaxation enhancement dna as a target for drug action the structural effects of the cys-s-s-cys bridge exchange by the his-cu(ii)-his motif studied on natural peptides-a promising tool for natural compounds-based design towards reproducible computational drug discovery utilization of paramagnetic relaxation enhancements for structural analysis of actin-binding proteins in complex with actin monitoring dynamics of large membrane proteins by f paramagnetic longitudinal relaxation: domain movement in a glutamate transporter homolog solid-state nmr spectroscopy on complex biomolecules solid-state nmr spectroscopy as a tool for drug design: from membrane-embedded targets to amyloid fibrils new approaches to the characterization of drug candidates by solid-state nmr saikiran solid state nuclear magnetic resonance spectroscopy-a review solid-state nmr spectroscopy in pharmaceutical research and analysis solid-state nuclear magnetic resonance spectroscopy: theory and pharmaceutical applications recent advances in solid-state nuclear magnetic resonance spectroscopy solid-state nmr in drug design and discovery for membrane-embedded targets the effect of drug loading on micelle properties: solid-state nmr as a tool to gain structural insight zinc-binding structure of a catalytic amyloid from solid-state nmr aggregation of biologically important peptides and proteins: inhibition or acceleration depending on protein and metal ion concentrations principles of early drug discovery hit discovery and hit-to-lead approaches chapter twelve-hit-to-lead: hit validation and assessment biophysics: for hts hit validation, chemical lead optimization, and beyond advances in nuclear magnetic resonance for drug discovery validation of drug-like inhibitors against mycobacterium tuberculosis l-aspartate α-decarboxylase using nuclear magnetic resonance expression of bacterial l-aspartate-alpha-decarboxylase in tobacco increases beta-alanine and pantothenate levels and improves thermotolerance methods for identification of false positives in biochemical screens alarm nmr: a rapid and robust experimental method to detect reactive false positives in biochemical screens alarm nmr for hts triage and chemical probe validation assay interference and off-target liabilities of reported histone acetyltransferase inhibitors a combination of f nmr and surface plasmon resonance for site-specific hit selection and validation of fragment molecules that bind to the atp-binding site of a kinase deconstruction of a nutlin: dissecting the binding determinants of a potent protein-protein interaction inhibitor monitoring binding of hiv- capsid assembly inhibitors using f ligand-and n protein-based nmr and x-ray crystallography: early hit validation of a benzodiazepine series is nmr fragment screening fine-tuned to assess druggability of protein-protein interactions de novo design of chiral organotin cancer drug candidates: validation of enantiopreferential binding to molecular target dna and -gmp by uv-visible, fluorescence, h and p nmr inhibiting the p -mdm interaction: an important target for cancer therapy inhibiting mdm -p interaction suppresses tumor growth in patient-derived non-small cell lung cancer xenograft models targeting p -mdm -mdmx loop for cancer therapy. in mutant p and mdm in cancer mdm small-molecule antagonist rg activates p signaling and regresses human tumors in preclinical cancer models small molecules, big targets: drug discovery faces the protein-protein interaction challenge exploring protein-protein interactions as drug targets for anti-cancer therapy with in silico workflows targeting protein-protein interactions for drug discovery protein-protein interaction modulators: advances, successes and remaining challenges sb is a specific inhibitor of a map kinase homologue which is stimulated by cellular stresses and interleukin- novel homologues of csbp/p map kinase: activation, substrate specificity and sensitivity to inhibition by pyridinyl imidazoles an accurate pharmacophore mapping method by nmr spectroscopy protein-ligand docking guided by ligand pharmacophore-mapping experiment by nmr the inter-ligand overhauser effect: a powerful new nmr approach for mapping structural relationships of macromolecular ligands sar by iloes: an nmr-based approach to reverse chemical genetics targeting apoptosis via chemical design: inhibition of bid-induced cell death by small organic molecules epitope mapping and competitive binding of hsa drug site ii ligands by nmr diffusion measurements a saturation transfer nmr-based method for determining the structures of protein−ligand complexes how much nmr data is required to determine a protein-ligand complex structure? o-tert-butyltyrosine, an nmr tag for high-molecular-weight systems and measurements of submicromolar ligand binding affinities sensitive nmr approach for determining the binding mode of tightly binding ligand molecules to protein targets solvent accessibility, ligand binding, and mapping of ligand orientation by nmr spectroscopy direct nmr probing of hydration shells of protein ligand interfaces and its application to drug design protein-ligand structure determination with the nmr molecular replacement tool, nmr nmr-based determination of the d structure of the ligand-protein interaction site without protein resonance assignment the nmr method to determine rapidly the structure of the binding pocket of a protein-ligand complex with high accuracy protein-fragment complex structures derived by nmr molecular replacement using ligand-induced protein chemical shift perturbations to determine protein-ligand structures automatic mapping of perturbed and unperturbed regions of proteins and complexes applications of in-cell nmr in structural biology and drug discovery protein f nmr in escherichia coli d structure determination of a protein in living cells using paramagnetic nmr spectroscopy structure of proteins in eukaryotic compartments direct structural evidence of protein redox regulation obtained by in-cell nmr cell nmr spectroscopy in protein chemistry and drug discovery high-resolution heteronuclear multidimensional nmr of proteins in living insect cells using a baculovirus protein expression system isotope labeling for solution and solid-state nmr spectroscopy of membrane proteins. in isotope labeling in biomolecular nmr f-nmr in target-based drug discovery applications of f-nmr in fragment-based drug discovery methyl groups as probes for proteins and complexes in in-cell nmr experiments nmr-based investigations into target dna search processes of proteins isotope labeling strategies for nmr studies of rna the first successful observation of in-cell nmr signals of dna and rna in living human cells g-quadruplex dna and ligand interaction in living cells using nmr spectroscopy nmr profiling of histone deacetylase and acetyl-transferase activities in real time a multiplexed nmr-reporter approach to measure cellular kinase and phosphatase activities in real-time an adhesive f mri chemical probe allows signal off-to-on-type molecular sensing in a biological environment scaffolding proteins and their role in viral assembly mechanism of scaffolding-assisted viral assembly structure of the coat protein-binding domain of the scaffolding protein from a double-stranded dna virus edited by m. summers structural assembly of the tailed bacteriophage φ nmr mapping of disordered segments from a viral scaffolding protein enclosed in a mda procapsid hydrogen exchange of monomeric α-synuclein shows unfolded structure persists at physiological temperature and is independent of molecular crowding in escherichia coli investigating macromolecules inside cultured and injected cells by in-cell nmr spectroscopy physicochemical properties of cells and their effects on intrinsically disordered proteins (idps) beyond the paradigm: combining mass spectrometry and nuclear magnetic resonance for metabolomics sample collection and preparation of biofluids and extracts for gas chromatography-mass spectrometry gas chromatography-mass spectrometry of biofluids and extracts application of nmr metabolomics to search for human disease biomarkers optimized metabolite extraction from blood serum for h nuclear magnetic resonance spectroscopy molecular thermodynamics using nuclear magnetic resonance (nmr) spectroscopy. inventions a review of nondestructive characterization of composites using nmr chapter -mass spectrometry dehydrodimerization of pterostilbene during electrospray ionization mass spectrometry cryogenic probe c nmr spectroscopy of urine for metabonomic studies sofast-hmqc experiments for recording two-dimensional deteronuclear correlation spectra of proteins within a few seconds sofast-hmqc-an efficient tool for metabolomics ultrafast d nmr: an emerging tool in analytical spectroscopy evaluation of fast d nmr for metabolomics real-time separation of natural products by ultrafast d nmr coupled to on-line hplc compressed sensing and the reconstruction of ultrafast d nmr data: principles and biomolecular applications application of ex situ dynamic nuclear polarization in studying small molecules dynamic nuclear polarization for sensitivity enhancement in modern solid-state nmr determinants for optimal enhancement in ex situ dnp experiments hyperpolarized nmr spectroscopy: d-dnp, phip, and sabre techniques this article is an open access article distributed under the terms and conditions of the creative commons attribution (cc by) license the authors would like to thank king abdullah university of science and technology (kaust) for financial support. the authors declare no conflict of interest. key: cord- -wgad eoh authors: francesconi, valeria; cichero, elena; schenone, silvia; naesens, lieve; tonelli, michele title: synthesis and biological evaluation of novel (thio)semicarbazone-based benzimidazoles as antiviral agents against human respiratory viruses date: - - journal: molecules doi: . /molecules sha: doc_id: cord_uid: wgad eoh respiratory rna viruses are responsible for recurrent acute respiratory illnesses that still represent a major medical need. previously we developed a large variety of benzimidazole derivatives able to inhibit these viruses. herein, two series of (thio)semicarbazone- and hydrazone-based benzimidazoles have been explored, by derivatizing -acetyl benzimidazoles previously reported by us, thereby evaluating the influence of the modification on the antiviral activity. compounds , , and , bearing the -(thio)semicarbazone and -hydrazone functionalities in combination with the -benzyl ring on the benzimidazole core structure, acted as dual inhibitors of influenza a virus and human coronavirus. for respiratory syncytial virus (rsv), activity is limited to the -thiosemicarbazone ( ) and -hydrazone ( ) compounds carrying the -[(benzotriazol- / -yl)methyl]benzimidazole scaffold. these molecules proved to be the most effective antiviral agents, able to reach the potency profile of the licensed drug ribavirin. the molecular docking analysis explained the sar of these compounds around their binding mode to the target rsv f protein, revealing the key contacts for further assessment. the herein-investigated benzimidazole-based derivatives may represent valuable hit compounds, deserving subsequent structural improvements towards more efficient antiviral agents for the treatment of pathologies caused by these human respiratory viruses. the benzimidazole scaffold is a structural isostere of naturally occurring nucleobases. many approved drugs bear a benzimidazole ring as their main unit or important substructure [ , ] . benzimidazole-based agents are also considered for antimicrobial purposes, such as antituberculosis, antiprotozoan or antiviral activities [ , ] . maribavir, a benzimidazole riboside with strong activity against cytomegalovirus (hcmv), is undergoing phase iii evaluation (clinicaltrials.gov nct and nct ) ( figure ). different substitutions on the benzimidazole nucleus of maribavir were reported to entail activity against flaviviruses, hiv, hepatitis b virus, hepatitis c virus (hcv) or respiratory syncytial virus (rsv) [ ] . besides, a few non-nucleoside benzimidazole derivatives interfering with rsv f protein-mediated fusion, namely, jnj- (r- ), bms- and tmc , have reached a far stage of (pre)clinical development, but have been halted after a negative outcome [ , ] (figure ). two fda-approved inhibitors of the hcv ns a protein, pibrentasvir and after a negative outcome [ , ] (figure ). two fda-approved inhibitors of the hcv ns a protein, pibrentasvir and velpatasvir, contain a benzimidazole nucleus (figure ). the benzimidazole derivative b , under clinical evaluation for the treatment of neurodegenerative diseases, was shown to delay hcv infection in a humanized mouse model [ ] (figure ). the literature further contains examples of other benzimidazole derivatives with activity against unrelated viruses, such as influenza virus or poliovirus. another chemical entity with relevance for antimicrobial drug development is the (thio)semicarbazone moiety. compounds containing this structure may interfere with processes that are essential for microbe survival, such as deoxyribonucleotide synthesis, bacterial cell wall synthesis or maintenance of thiol content [ , ] . interest in their antiviral properties dates back to the discovery of methysazone (marboran ® ), which was used to treat smallpox prior to its global eradication [ ] . more recent studies have reported activity against other poxviruses, herpes simplex virus or influenza virus [ ] . transformation of some -acetyl- -phenylbenzimidazoles into the corresponding (thio)semicarbazone analogues yielded new agents with activity against hcv and the another chemical entity with relevance for antimicrobial drug development is the (thio)semicarbazone moiety. compounds containing this structure may interfere with processes that are essential for microbe survival, such as deoxyribonucleotide synthesis, bacterial cell wall synthesis or maintenance of thiol content [ , ] . interest in their antiviral properties dates back to the discovery of methysazone (marboran ® ), which was used to treat smallpox prior to its global eradication [ ] . more recent studies have reported activity against other poxviruses, herpes simplex virus or influenza virus [ ] . transformation of some -acetyl- -phenylbenzimidazoles into the corresponding (thio)semicarbazone analogues yielded new agents with activity against hcv and the related bovine viral diarrhea virus (bvdv) [ ] . inhibition of the bvdv rna polymerase was reported for , -dimethoxy- -indanone-derived thiosemicarbazones [ ] . during the few last years, we synthesized a large variety of benzimidazole-based derivatives and we explored their antitumor [ ] , analgesic [ ] or antiviral potential. we focused on two series, i.e., -benzylbenzimidazoles (series ) [ ] and -[(benzotriazol- / -yl)methyl)benzimidazoles (series ) [ , ] -substituted with a basic chain. among them, we obtained analogues with promising activity against diverse rna viruses ( figure ). molecules , , x for peer review of related bovine viral diarrhea virus (bvdv) [ ] . inhibition of the bvdv rna polymerase was reported for , -dimethoxy- -indanone-derived thiosemicarbazones [ ] . during the few last years, we synthesized a large variety of benzimidazole-based derivatives and we explored their antitumor [ ] , analgesic [ ] or antiviral potential. we focused on two series, i.e., -benzylbenzimidazoles (series ) [ ] and -[(benzotriazol- / -yl)methyl)benzimidazoles (series ) [ , ] -substituted with a basic chain. among them, we obtained analogues with promising activity against diverse rna viruses ( figure ). in the light of our previous findings, we deemed it interesting to explore new additional substitution patterns on the benzimidazole main core, with the aim of gaining a better understanding of the potential of this nucleus towards the development of novel series of antiviral agents. since the -acetyl substituted benzimidazoles were generally endowed with low efficacy, we explored alternative substitutions at position of the benzimidazole ring, by synthesizing (thio)semicarbazone and hydrazone derivatives ( figure ). these new compounds were evaluated for their activities against a broad panel of viruses and cytotoxicity in several mammalian cell lines. the main results are presented in tables and , from which we omitted the compounds having no antiviral nor cytotoxic activity (at µ m, the highest concentration tested). these investigations were accompanied by docking studies of the title compounds in complex with the rsv fusion f protein, which allowed the identification of the most relevant features involved in the protein-inhibitor recognition. in the light of our previous findings, we deemed it interesting to explore new additional substitution patterns on the benzimidazole main core, with the aim of gaining a better understanding of the potential of this nucleus towards the development of novel series of antiviral agents. since the -acetyl substituted benzimidazoles were generally endowed with low efficacy, we explored alternative substitutions at position of the benzimidazole ring, by synthesizing (thio)semicarbazone and hydrazone derivatives ( figure ). these new compounds were evaluated for their activities against a broad panel of viruses and cytotoxicity in several mammalian cell lines. the main results are presented in tables and , from which we omitted the compounds having no antiviral nor cytotoxic activity (at µm, the highest concentration tested). these investigations were accompanied by docking studies of the title compounds in complex with the rsv fusion f protein, which allowed the identification of the most relevant features involved in the protein-inhibitor recognition. the starting -acetyl- -benzylbenzimidazoles (series ) have been synthesized according to the literature [ ] , by the reaction of the proper -acetyl- , -phenylenediamine with the hydrochloride of the iminoester, previously obtained from the corresponding nitrile under pinner conditions. the condensation at • c of a mixture of the proper , -phenylenediamine with the (benzotriazol- / -yl)acetic acid has given the -acetyl- -[(benzotriazol- / -yl)methyl]benzimidazoles [ ] . our target compounds - have been obtained following the synthetic routes presented in schemes and . thiosemicarbazones - , , , and , and semicarbazones - and have been prepared by refluxing an ethanolic/aqueous solution of thiosemicarbazide or semicarbazide hydrochloride in the presence of glacial acetic acid or sodium acetate, respectively. the reaction at reflux of the proper -acetyl benzimidazole with a slight excess of hydrazine hydrate for h has afforded the target hydrazone derivatives - (scheme ). the structures of the novel compounds have been confirmed using h and c nmr, and elemental analysis. the purity of compounds (checked by elemental analysis) has been in all cases > %. thiosemicarbazones are known to display thione-thiol tautomerism, since they contain the hydrazidic proton (-c(=s)nh-n=) that can shift onto the sulfur atom, leading to a thiol form stabilized by conjugation (-c(-sh)=n-n=). indeed, on the h nmr spectra our compounds do not exhibit the signal at . ppm, attributable to -sh proton, suggesting that the thione form is the only tautomer. the schiff base of our thiosemicarbazone moiety acquires e isomerism since the hydrazinic proton (-c(=s)nh-n=) falls in the - ppm range [ ] . in fact this signal appears at ca . ppm, whilst the thioamide protons (-c(=s)nh ) exhibit two different chemical shifts at ca . ppm and . ppm. the same consideration can be made for semicarbazones with the exception of the signal of amide group (conh ) which appears as a broad singlet (ca . ppm) with the two protons being indistinguishable. the different behaviors of the two series may be explained by the restricted free rotation brought about by the formation of the carbon-nitrogen double bond character relative to thione-thiol tautomerism [ ] . in addition, it is noteworthy that the sulfur atom of the thione tautomer has a greater atomic radius than oxygen atom of the corresponding semicarbazone, thereby making the thioamide protons magnetically different for steric hindrance. the reaction at reflux of the proper -acetyl benzimidazole with a slight excess of hydrazine hydrate for h has afforded the target hydrazone derivatives - (scheme ). the structures of the novel compounds have been confirmed using h and c nmr, and elemental analysis. the purity of compounds (checked by elemental analysis) has been in all cases > %. thiosemicarbazones are known to display thione-thiol tautomerism, since they contain the hydrazidic proton (-c(=s)nh-n=) that can shift onto the sulfur atom, leading to a thiol form stabilized by conjugation (-c(-sh)=n-n=). indeed, on the h nmr spectra our compounds do not exhibit the signal at . ppm, attributable to -sh proton, suggesting that the thione form is the only tautomer. the schiff base of our thiosemicarbazone moiety acquires e isomerism since the hydrazinic proton (-c(=s)nh-n=) falls in the - ppm range [ ] . in fact this signal appears at ca . ppm, whilst the thioamide protons (-c(=s)nh ) exhibit two different chemical shifts at ca . ppm and . ppm. the same consideration can be made for semicarbazones with the exception of the signal of amide group (conh ) which appears as a broad singlet (ca . ppm) with the two protons being indistinguishable. the different behaviors of the two series may be explained by the restricted free rotation brought about by the formation of the carbon-nitrogen double bond character relative to thione-thiol tautomerism [ ] . in addition, it is noteworthy that the sulfur atom of the thione tautomer has a greater atomic radius than oxygen atom of the corresponding semicarbazone, thereby making the thioamide protons magnetically different for steric hindrance. as explained above, the new compounds were synthesized from -acetyl substituted -benzylbenzimidazoles and -[(benzotriazol- / -yl)methyl]benzimidazoles (series and ; figure ). we previously published that series has high potential against rsv, with the best performing derivatives having nanomolar activity [ , ] . these compounds carried at position of the benzimidazole ring different basic chains, such as the most efficacious quinolizidinylalkyl [(octahydro- h-quinolizin- -yl)alkyl] chains or the dialkylaminoalkyl ones [ , ] . the new compounds ( - ) were evaluated against a broad panel of rna and dna viruses in suitable mammalian cell culture assays [ ] [ ] [ ] [ ] . the antiviral activity data obtained by microscopic inspection of the viral cpe (data not shown) were in agreement with those obtained by the colorimetric mts cell viability test (table ) (table ) . the following careful sar analysis could be made. for rsv, activity is restricted to the -(thio)semicarbazone ( ) and hydrazone ( ) compounds carrying the -[(benzotriazol- / -yl)methyl]benzimidazole scaffold, in line with the previously synthesized analogues (see above), which show comparable potency in the low micromolar range. regarding influenza a and coronaviruses, the activity is promoted by (thio)semicarbazone and hydrazone functionalities, especially when combined with the benzyl ring ( , , , and ) compared to the bulkier (benzotriazol- / -yl)methyl skeleton ( ) . the nature of the substituent in the para position of the benzyl ring (h, cl, och ) does not seem to have significant impact on the antiviral activity, since the unsubstituted derivatives ( , ; r = h) had comparable potency of those decorated with electron-withdrawing ( ; r = cl) or electron-donor groups ( ; r = och ). finally, most compounds were devoid of cytotoxicity at µm, the highest concentration tested. two compounds, and , produced cytotoxicity in two of the four cell lines. the other molecules were either not toxic or exhibited a cc value of about µm in one of the four cell lines. interestingly, influenza a and human coronavirus shared sensitivity to the same inhibitors, , , and , whose definition of the mechanism of action is beyond the scope of this exploratory work. as is well-known from literature, the antiviral activity against rsv is limited to several benzimidazole derivatives ( figure ) [ , ] , and also to the more recent analogue jnj- ( figure ) [ ] , which were demonstrated to impair the viral replication machinery by blocking the f protein-induced membrane fusion. from , jnj- entered phase studies in adults and infants for therapy of rsv infections (clinicaltrials.gov identifier nct , nct , nct ). due to the substantial structural similarity between the newly synthesized compounds and the above anti-rsv (pre)clinical candidates, molecular modeling studies were performed in order to reveal the most important features underlying the f protein/ligand interactions. interestingly, the presence of the alkyl-morpholine chain linked to the dihydrobenzimidazole core of the compound tmc allowed us to explore another rsv f protein binding pocket, which is able to anchor the inhibitor at the exposed surface of the protein (see figure ). during the last few years, a number of crystallographic structures of the prefusion rsv glycoprotein became available focusing on several benzimidazole-based or bioisosteres inhibitors as co-crystallized ligands [ ] [ ] [ ] . a number of them highlighted a small number of contacts responsible for the inhibitor positioning at the exposed surface area of the protein, occupying one hydrophobic region of the biological target especially, including the f residue. as shown in figure , the inhibitors bms- and jnj- move the , -dihydroimidazo [ , -c] pyridin- -one ring into the proximity of the aforementioned f and f amino acids, detecting the first one π-π stacking with these residues. the substitution of the alkoxy chain of bms- with the jnj- sulfone moiety makes the compound more hydrophobic than the prototype, flatting the jnj- indole core towards f , f and f . interestingly, the presence of the alkyl-morpholine chain linked to the dihydrobenzimidazole core of the compound tmc allowed us to explore another rsv f protein binding pocket, which is able to anchor the inhibitor at the exposed surface of the protein (see figure ). interestingly, the presence of the alkyl-morpholine chain linked to the dihydrobenzimidazole core of the compound tmc allowed us to explore another rsv f protein binding pocket, which is able to anchor the inhibitor at the exposed surface of the protein (see figure ). indeed, the x-ray crystallographic data of this ligand in complex with the f protein reveal polar contacts between the tmc protonated nitrogen atom of the morpholine ring and a polar area of the protein, including d and e . on the other hand, the pyridine group and the terminal phenyl ring were properly oriented toward t , detecting one h-bond with the biological target, and around the hydrophobic pocket previously discussed for bms- and jnj- characterized by f and f . on this basis, it is thought that interactions with f are mandatory to exert rsv f protein inhibition; the most promising compounds are better stabilized at the surface of the protein by additional h-bonds with the near polar pocket. herein we discuss this computational work with the aim of exploring an adequate rsv f protein inhibition activity of the newly synthesized antiviral compounds, by means of molecular docking simulations taking into account the x-ray crystallographic structure of the rsv f protein in complex with jnj- (pdb code = kww; resolution = . Å) [ ] . this crystallographic data have been chosen based on the structural similarity shown by the reference compound jnj- with the in-house, benzimidazole-based hydrazones and thiosemicarbazones. currently, jnj- is the more promising clinical candidate, whose potential therapeutic is undergoing evaluation in patients at high risk to developing acute rsv lower respiratory tract infections (clinicaltrials.gov identifier nct , nct , nct ). the main issues to be addressed were to clarify the role played by the hydrazone moiety and by the thiosemicarbazone at the position five of benzimidazole main core, especially when accompanied by a benzyl or a benzotriazole- -yl or benzotriazole- -yl ring linked at position of the inhibitor scaffold. this was done through docking studies of all the newly synthesized compounds. according to our calculations (table ) , only compounds and properly bind the exposed surface of the rsv f protein-they are anchored in the hydrophobic pocket by π-π stacking and cation-π interactions with f , f and f . as shown in figure , both the inhibitors moved the benzimidazole core in proximity of the aforementioned aromatic residues (π-π interactions) while the hydrazone group of and the protonated amine chain of were projected near f , featuring cation-π stacking and h-bonds with the oxygen atom of the f carbonyl group. while maintaining the hydrazone moiety in tandem with the substitution of the benzotriazolyl group with a decorated phenyl one led to the inactive compound , unable to be stabilized at the surface of the protein, the replacement of the hydrazone group with the thiosemicarbazone one led while maintaining the hydrazone moiety in tandem with the substitution of the benzotriazolyl group with a decorated phenyl one led to the inactive compound , unable to be stabilized at the surface of the protein, the replacement of the hydrazone group with the thiosemicarbazone one led to the analogue . interestingly, this featured h-bonds between the thiosemicarbazone function and e , while the benzotriazolyl group and the amine chain moved far from the corresponding heteroaromatic group and hydrazone moiety of the analogue (see figure s ). on the other hand, even if this kind of positioning was in agreement with that previously discussed for the effective analogue , it was unable to detect the same contacts displayed by probably because of the different docking mode driven by the benzotriazol- -yl skeleton in place of the benzotriazole- -yl one of . accordingly, compound exhibited a comparable docking mode with that of the analogue , lacking the key contacts with f at the thiosemicarbazone moiety or amine chain group ( figure s ) . finally, the introduction of the smaller phenyl ring instead of the benzotriazolyl core at position two of the benzimidazole main ring (compound ) moved the amine chain towards the protein's deep crevice delimited by t while the phenyl moiety detected π-π stacking with f , missing any h-bonds with this residue. conversely, the thiosemicarbazone group was h-bonded to e (see figure s ). accordingly, this compound was poorly effective if compared with the analogue . in this work we also evaluated, by computational prediction, a number of descriptors related to absorption, distribution, metabolism and excretion properties (adme). this represents a useful in silico strategy for accelerating the discovery of drug-like compounds [ ] . for the most promising antiviral compounds, and , we calculated the number of h-bonding acceptor and donor groups and rotable bonds, the logarithmic ratio of the octanol-water partitioning coefficient (clogp), the human intestinal absorption (hia), the volume of distribution (vd), the binding to plasmatic proteins (%ppb) and albumin (logka hsa) and the oral bioavailability percentage (%f). as shown in table , all the compounds were characterized by a favorable profile in terms of lipophilicity-that being an logp below (lipinski rules)-and also displayed the ability to be fully adsorbed at the human intestinal membrane. with respect to the reference compound, and featured higher oral bioavailability and lower binding to plasmatic proteins. table . absorption, distribution, metabolism and excretion (adme) descriptors related to absorption and distribution properties. docking studies allowed us to rationalize the sar observed in these anti-rsv benzimidazoles and to recognize the compounds and as the best suited f protein inhibitors. the preliminary information concerning their pharmacokinetic properties pointed to a favorable drug-like profile, enabling these anti-rsv agents to undergo a further optimization process. chemicals, solvents and reagents were used as obtained from commercial sources (alfa aesar and sigma-aldrich) without further purification. column chromatography (cc): silica gel (sio ) (merck). mps: büchi apparatus, uncorrected. h nmr and c nmr spectra were recorded on varian gemini- spectrometer using dmso-d as a solvent. the chemical shifts (δ) in ppm were measured relative to tetramethylsilane (tms). j in hz. elemental analyses were performed on flash chns (thermo scientific) instrument in the microanalysis laboratory of the department of pharmacy, university of genoa. benz = benzimidazole ring; bzt = benzotriazole ring; arom = phenyl ring. to a solution of the proper -acetyl benzimidazole ( . mmol) in ethanol ( ml), a solution of thiosemicarbazide ( . mmol) in water ( . ml) and glacial acetic acid ( . ml) was added. the mixture was refluxed for h under stirring. the reaction mixture was then evaporated under vacuum, yielding an oily residue that was treated with warm water to get rid of the remaining thiosemicarbazide. the crude was purified by cc (sio , ch cl + % dea), affording the final product as a white solid. to a solution of the proper -acetyl benzimidazole ( . mmol) in ethanol ( ml), a solution of semicarbazide hydrochloride ( . mmol) previously dissolved in ml of a n solution of sodium acetate was added. the mixture was refluxed for h under stirring. after evaporation of the solvent, the oily residue was treated with warm water to get rid of the remaining semicarbazide. the crude was purified by cc (sio , ch cl + % dea), obtaining the title compound as a white solid. a solution of nh nh · h o ( . mmol) in ml of water was refluxed for h with a solution of the proper -acetyl benzimidazole ( . mmol) in . ml of ethanol with stirring. at room temperature, ml of water were added and the solution was kept at - • c overnight. the expected product was directly separated from the solution as an amorphous solid that was filtered and crystallized as a white solid from anhydrous et o. cell proliferation assay from promega) was added, and after h of incubation at • c, the od nm values were measured in a plate reader. the compounds' ec ( % antivirally effective concentration) values were calculated by interpolation using semi-log dose response curves. for the mts data, the percentage of protection against virus was defined as: ((od cpd )virus − (od contr )virus))/((od contr )mock − (od contr )virus) × , where (od cpd )virus is the od for a given concentration of the compound in virus-infected cells; (od contr )virus is the od for the untreated virus control; and (od contr )mock is the od for the untreated mock-infected control. the values for cc ( % cytotoxic concentration) were also calculated by interpolation using semi-log dose response curves. the percentage of cytotoxicity was defined as: ( − (od cpd )mock/((od contr )mock) × , where (od cpd )mock is the od for a given concentration of the compound in mock-infected wells. all the compounds were built, parameterized (gasteiger-huckel method) and energy minimized within moe using mmff forcefield [ ] . all ligands were used in their protonated state. docking calculations within the x-ray structure of rsv f protein (pdb code = kww) were done using the leadit . . software suite (www.biosolveit.com) including the flexx scoring algorithm, which is based on binding free energy calculations by means of gibbs-helmholtz equation [ ] [ ] [ ] . the software detects the binding site defining a radius of Å far from the co-crystallized ligand, in order to set up a spherical search space for the docking approach. the standard settings for the docking strategy were followed, choosing the so-called hybrid approach (enthalpy and entropy criteria); the related scoring function evaluation is described in the literature [ ] . the derived docking poses were prioritized by the score values of the lowest energy pose of the compounds docked to the protein structure. all ligands were refined and rescored by assessment with the algorithm hyde, included in the leadit . . software. the hyde module considers dehydration enthalpy and hydrogen bonding [ , ] . finally, the reliability of the selected docking poses was assessed using a short~ ps run of molecular dynamics (md) at constant temperature, followed by an all-atom energy minimization (lowmodemd implemented in moe software). this represents a conformational search method that uses implicit vibrational analysis to focus a md trajectory along the low-mode vibrations [ ] [ ] [ ] . this has the effect of searching for minima along the valleys and troughs on the potential energy surface, thereby performing an exhaustive conformational analysis of the ligand-receptor binding site complex, as we previously discussed about other case studies [ ] [ ] [ ] . adme properties have been predicted by means of advanced chemistry development (acd) percepta platform (www.acdlabs.com) named acd/labs percepta software (version . ). all of the calculated parameters were derived and evaluated by percepta on the basis of training libraries, implemented in the software, which include a consistent number of molecules, whose pharmacokinetic and toxicity profiles are known. in summary, this study reports the synthesis of a series of (thio)semicarbazone-and hydrazone-containing benzimidazoles for the development of novel antiviral agents which have shown the ability to inhibit the replication of three human respiratory viruses. acute viral respiratory illnesses are usually the result of infections with a heterogeneous group of respiratory viruses, including some of the most notable rna viruses, such as influenza virus, coronavirus and rsv. the relative infections continue to cause frequent morbidity, and sometimes cause severe outcomes, including about . million deaths worldwide each year, particularly among children under five years, the elderly and immunocompromised individuals [ , ] . this scenario enlightens the serious and important need for identifying new scaffolds that are useful in discovering innovative, highly potent and safe antiviral agents [ ] . interestingly, our antiviral data suggest compounds , , and work as dual virus inhibitors of influenza and coronavirus strains. in these series the (thio)semicarbazone and hydrazone moieties have proven to mediate the observed antiviral activity of -benzylbenzimidazoles and -[(benzotriazol- / -yl)methyl]benzimidazole scaffolds, since the respective chemical precursor -acetylbenzimidazoles were found to be less effective or ineffective antiviral agents. it is worth noting that, although the efficacy against human coronavirus ( e) is moderate, these compounds are the first benzimidazole derivatives to be found as active against this virus. their chemical optimization might acquire greater importance in light of the current outbreak of the novel coronavirus ( -ncov); who is calling for the urgent setting up of a complex network of strategies, which also look for accelerating the development of diagnostics, vaccines and therapeutics to contain the pandemic proportion of -ncov infections [ ] . moreover, compounds and proved to be the most potent and safe antivirals among these series, being able to inhibit rsv replication with the same degree of potency of ribavirin, w is the only drug available to treat rsv infections, but its limited efficacy and low margin of safety restrict its use to children at high risk [ ] . docking studies also performed on the best performing rsv f protein inhibitors reported in the literature supported the sar observed in these series of compounds, and enlightened the efficient binding modes of and at the exposed surface of the rsv f protein, establishing π-π stacking and cation-π interactions with the hydrophobic pocket formed by f , f and f residues. therefore, the above adequately substituted (thio)semicarbazone-and hydrazone-based benzimidazoles, inhibiting the replication of the aforementioned viruses, may be considered as promising new hits, worthy of further structural optimization for an improved antiviral profile, as a result of the chemical variation of the benzimidazole core: (a) by exploring different side chains in position and/or (b) replacing the benzyl or (benzotriazolyl)methyl moieties with different aromatic or heteroaromatic rings. in particular, with the aim of obtaining more effective and drug-like anti-rsv agents, the design process will be driven by the previously built comfa and comsia models, filtering tools predicting the safety trend of any new analogue prior to synthesis [ ] . meanwhile, as some compounds are able to target both influenza virus and coronavirus, their underlying possible common mechanisms of viral inhibition are worth investigation. yield: %; m (s, h, ch -ar), . - . (m, h, ch ch -n(ch ch ) ), . (s, h, och ), . - . (m superimposed to dmso, h, h ch ch -n(ch ch ) and h ch ch -n(ch ch ) ), . (s, h yield: %; m.p. . - . • c. h nmr ( mhz, dmso-d ): . (s, h, nh), . (s, h, h( )benz.), . (d, j = . hz ch ch -n(ch ch ) ), . (s superimposed, h )benz.), . (pseudo s, h, h( , , , )arom.), . (broad s, h, nh ), . (s, h, ch -ar), . (pseudo s, h, ch ch ch -n(ch ch ) ), . - . (m superimposed, h, h ch ch ch -n(ch ch ) and h ch ch -n(ch ch ) ), . (s superimposed, h, ch c=n-), . (pseudo s, h, ch ch ch -n(ch ch ) ), . (pseudo s, h, n(ch ch ) ). c-nmr ( mhz yield: %; m.p. - • c. h nmr ( mhz, dmso-d ): (broad s, h, nh ), . (s, h, ch -ar), . - . (m superimposed, h, ch ch ch -n(ch ch ) ), . (s, h, och ), . - . (m superimposed, h, h ch ch ch -n(ch ch ) and h n(ch ch ) ), . (s, h, ch c=n-) ( c). anal. calcd. for c h n o : % c . , h . , n . (m, h, ch ch -n(ch ) ), . (s, h, ch c=n-), . (s, h, n(ch ) ). c-nmr ( mhz, dmso-d ): . , . , . , . ( c), . , . , . , . ( c), . , . , . ( c), . , . , . , . , . ( c), . , . . anal. calcd. for c h n o: % c . , h . , n . ; found % c . , h . , n a/ned/ / (a/h n ) and b/ned/ / (all from r. fouchier, rotterdam, the netherlands) were tested in madin-darby canine kidney (mdck) cells, a kind gift from m. matrosovich (marburg, germany) at the same time, serial compound dilutions were added. the plates were incubated (at • c for influenza and coronavirus, and at • c for the other viruses) for to days, until full-blown cytopathic effect (cpe) was visible. at that time, microscopy was performed to score the viral cpe and compound cytotoxicity (assessed in mock-infected plates). next, the mts cell viability reagent pharmacological significance of heterocyclic h-benzimidazole scaffolds: a review functionalized benzimidazole scaffolds: privileged heterocycle for drug design in therapeutic medicine therapeutic evolution of benzimidazole derivatives in the last quinquennial period comprehensive review in current developments of benzimidazole-based medicinal chemistry benzimidazole nucleosides: antiviral and antitumour activities, methods of synthesis drug candidates and model systems in respiratory syncytial virus antiviral drug discovery overview of current therapeutics and novel candidates against influenza, respiratory syncytial virus, and middle east respiratory syndrome coronavirus infections identification of a new benzimidazole derivative as an antiviral against hepatitis c virus thio and semicarbazone derivatives against tropical infective diseases: chagas disease, human african trypanosomiasis (hat), leishmaniasis, and malaria new quinolone-based thiosemicarbazones showing activity against plasmodium falciparum and mycobacterium tuberculosis to-day's drugs: methisazone investigation of the salicylaldehyde thiosemicarbazone scaffold for inhibition of influenza virus pa endonuclease -acetyl- -arylbenzimidazoles as antiviral agents m. synthesis, antiviral evaluation and molecular docking studies of n -aryl substituted/unsubstituted thiosemicarbazones derived from -indanones as potent anti-bovine viral diarrhea virus agents primary anti-proliferative activity evaluation of -(quinolizidin- '-yl)methyl-and -(ω-tert-amino)alkyl-substituted -phenyl-, -benzyl-and -[(benzotriazol- / -yl)methyl]benzimidazoles on human cancer cell lines exploring the effectiveness of novel benzimidazoles as cb ligands: synthesis, biological evaluation, molecular docking studies and admet prediction antiviral activity of benzimidazole derivatives. ii. antiviral activity of -phenylbenzimidazole derivatives antiviral activity of benzimidazole derivatives. i. antiviral activity of -substituted- -[(benzotriazol- / -yl)methyl]benzimidazoles benzimidazole-based derivatives as privileged scaffold developed for the treatment of the rsv infection: a computational study exploring the potency and cytotoxicity profiles dialchilamminoalchilbenzimidazoli d'interesse farmacologico. iii dialchilamminoalchilbenzimidazoli d'interesse farmacologico. iv organoplatinum(ii) complexes with -acetylthiophene thiosemicarbazone: synthesis, characterization, crystal structures, and in vitro antitumor activity synthesis, nmr structural characterization and molecular modeling of substituted thiosemicarbazones and semicarbazones using dft calculations to prove the syn/anti isomer formation novel inhibitors of influenza virus fusion: structure-activity relationship and interaction with the viral hemagglutinin host dihydrofolate reductase (dhfr)-directed cycloguanil analogues endowed with activity against influenza virus and respiratory syncytial virus synthesis, biological evaluation and molecular modeling of novel azaspiro dihydrotriazines as influenza virus inhibitors targeting the host factor dihydrofolate reductase (dhfr) synthesis and anti-coronavirus activity of a series of -thia- -azaspiro orally active fusion inhibitor of respiratory syncytial virus respiratory syncytial virus-the discovery and optimization of orally bioavailable fusion inhibitors. drugs fut advances in respiratory virus therapeutics -a meeting report from the th isirv antiviral group conference molecular mechanism of respiratory syncytial virus fusion inhibitors therapeutic efficacy of a respiratory syncytial virus fusion inhibitor admet in silico modelling: towards prediction paradise? gasteiger-huckel method. chemical computing group inc. montreal. h a r canada the computer program ludi: a new method for the de novo design of enzyme inhibitors the development of a simple empirical scoring function to estimate the binding constant for a protein-ligand complex of known three-dimensional structure a fast flexible docking method using an incremental construction algorithm docking assay of small molecule antivirals to p of hcv towards an integrated description of hydrogen bonding and dehydration: decreasing false positives in virtual screening with the hyde scoring function substantial improvements in large-scale redocking and screening using the novel hyde scoring function a molecular mechanism of p-loop pliability of rho-kinase investigated by molecular dynamic simulation hessian-free low-mode conformational search for large-scale protein loop optimization: application to c-jun n-terminal kinase jnk lowmodemd-implicit low-mode velocity filtering applied to conformational search of macrocycles and protein loops in silico evaluation of human small heat shock protein hsp : homology modeling, mutation analyses and docking studies novel biguanide-based derivatives scouted as taar agonists: synthesis, biological evaluation, adme prediction and molecular docking studies tricyclic pyrazoles. part . synthesis, biological evaluation and modelling of tricyclic pyrazole carboxamides as potential cb receptor ligands with antagonist/inverse agonist properties battle against respiratory viruses (brave) initiative. available online research needs for the battle against respiratory viruses (brave) novel coronavirus ( -ncov) situation reports: situation report- chemotherapy of respiratory syncytial virus infections: the final breakthrough this article is an open access article distributed under the terms and conditions of the creative commons attribution funding: this research received no external funding. the authors declare no conflict of interest. key: cord- -coby m e authors: marton, soledad; reyes-darias, josé a.; sánchez-luque, francisco j.; romero-lópez, cristina; berzal-herranz, alfredo title: in vitro and ex vivo selection procedures for identifying potentially therapeutic dna and rna molecules date: - - journal: molecules doi: . /molecules sha: doc_id: cord_uid: coby m e it was only relatively recently discovered that nucleic acids participate in a variety of biological functions, besides the storage and transmission of genetic information. quite apart from the nucleotide sequence, it is now clear that the structure of a nucleic acid plays an essential role in its functionality, enabling catalysis and specific binding reactions. in vitro selection and evolution strategies have been extremely useful in the analysis of functional rna and dna molecules, helping to expand our knowledge of their functional repertoire and to identify and optimize dna and rna molecules with potential therapeutic and diagnostic applications. the great progress made in this field has prompted the development of ex vivo methods for selecting functional nucleic acids in the cellular environment. this review summarizes the most important and most recent applications of in vitro and ex vivo selection strategies aimed at exploring the therapeutic potential of nucleic acids. nucleic acids, particularly rna, are extremely versatile molecules. apart from their role as carriers of genetic information they can also express a phenotype, e.g., they may show a catalytic activity, have a specific binding function, or have the capacity to recruit specific molecules. the appearance of sequence variation in a nucleic acid can, in some cases, provide an advantage to an organism: a key feature in natural evolution. over the last years, advances in molecular biology and biotechnology have seen the development of methods that allow the effect of such naturally arising variation to be mimicked in the laboratory. it was in the s when spiegelman's group first observed evolution in vitro [ ] . these authors reported that changes in the rna genome of the qβ bacteriophage during replication led to the formation of rna molecules that were more efficiently copied by the viral replicase. these genomes lacked unnecessary sequences and were synthesized at a greater rate. however, these finding fell into oblivion until , when the great potential of in vitro selection and evolution techniques was reported by three independent groups [ ] [ ] [ ] [ ] . since then, numerous authors have used these technologies to study the chemical and catalytic properties of nucleic acids (for further information see [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] . they have also helped uphold the rna world hypothesis, and it seems likely that they may soon have applications in biomedicine. indeed, new selection methods -known as ex vivo selection procedures -are now being used to identify molecules that target viruses, subcellular fractions and even whole cells. these techniques overcome some of the limits imposed by in vitro technology and provide new environments and conditions in which to explore the properties and functions of nucleic acids. this review highlights the most recent advances in in vitro and ex vivo selection procedures for nucleic acids, and discusses their potential application in biomedicine. in vitro selection strategies have been used to select nucleic acids with a large variety of properties. although each strategy differs according to the feature or phenotype sought, all in vitro selection methods follow the same three-step pattern ( figure ). genetic variability is introduced into the system (generally by chemical synthesis) to yield nucleic acid populations, the heterogeneity of which is determined by fixing the number of nucleotides to be mutagenized and the mutation rate per nucleotide. in most cases, completely random synthesis involving a fixed number of nucleotides yields a starting population of variant molecules that, a priori, differ only in the sequence and the structure of the variable region. constant sequences, or primer binding sites (pbs), flanking the variable region are incorporated during the design of the starting population to facilitate the amplification of desired molecules, although this limits the structural diversity of the rna populations to specific conformations [ ] . different approaches have been tried to minimize the effect of pbss, e.g., the addition of customized primers or adapters by ligation before amplification, and their removal prior to the selection step [ ] [ ] [ ] . the selection strategy needs to be specifically designed according to the phenotype sought. the initial pool of variants usually contains very few active molecules corresponding to the desired phenotype, and their enrichment can only be made possible by properly designing the selection step. the selection of inactive molecules may also be important since the analysis of these molecules can provide very valuable information on the sequence and structural requirements of active molecules [ ] . in vitro selection strategies have been widely used for the selection of nucleic acids capable of catalyzing specific chemical reactions, i.e., dnazymes and ribozymes [ ] . these strategies have also been successfully used to identify dna and rna molecules with affinity for a specific ligand (i.e., aptamers) [ ] , being known as selex, which stands for systematic evolution of ligands by exponential enrichment [ ] . sequence variants are separated into active, those that satisfy selection criteria, and inactive molecules. active molecules are selectively amplified by pcr resulting in the production of a new pool of molecules that can be the input of a new selection cycle. an additional reverse transcription and in vitro transcription steps are required, before and after pcr, respectively, when the selection is performed on rna molecules. alternatively, active and inactive resulting pools can be cloned and analyzed. variants that show the required phenotype need to be replicated to ensure their passage into the next generation and therefore their persistence in the population. specific primer binding sites are used to amplify the selected molecules. when necessary a rna polymerase promoter is incorporated at the ' end of the pbs during the amplification step. in addition to these general steps, the amplified molecules may require additional manipulations prior to their introduction into the next round of selection when the selected molecule is ssdna, both dna strands must be separated and the positive one isolated, e.g., by incorporating a biotinylated residue into the unwanted strand [ , ] via asymmetric pcr [ ] . as a result of the described selection cycle, a population enriched in the sought-after molecules (but not composed of them entirely) is produced; a new selection cycle can then be undertaken. by iteratively executing the process of selection and amplification the complexity of the original population is reduced and enriched with candidates of interest. during this process, the stringency of selection can be increased to achieve the isolation of the molecules with the desired phenotype. the great advances made in in vitro selection procedures over recent decades have helped us improve our knowledge of the plasticity of nucleic acids and their potential applications as therapeutic tools. indeed, such have been the advances that selection strategies within living cells are now contemplated. these new methodologies are named according to the specific procedure employed in each method, e.g., ex vivo, in vivo, in cell selection, or cell-selex etc. these are here all referred to as ex vivo selection strategies. ex vivo selection methodologies follow the general pattern described above for in vitro techniques. these systems have mainly been used in the isolation of dna and rna molecules that interfere with the activity of a target molecule. when the expression of an oligonucleotide leads to a desired cell phenotype, it allows for the selection of these cells and the subsequent isolation of the oligonucleotide itself. following these principles a strategy was used to identify a transcriptional activator regulated by tmr (tetramethylrosamine) [ ] . for this purpose, a chimera was constructed by tethering a tmr aptamer to the transcriptional activator for ms protein (also known as n - ) [ ] . variability was introduced by randomizing seven nucleotides within the linker region of the two rna domains. selection was performed in yeast cells containing a construct coding for the his and lacz genes under the control of the lexa operator, and expressing a lexa-ms coat protein fusion. this is a hybrid protein that binds to the operator and to the n - ms rna hairpin present in the rna construct. only cells expressing an active transcriptional activator were capable of growth in the absence of histidine, and were the only ones capable of expressing β-galactosidase. the stringency of the selection process was increased by the presence of varying amounts of a competitive inhibitor of the his p activity. analysis of the selected yeast clones revealed specific rna sequences that responded to tmr enhancing the transcription activator effect [ ] . the selection of an artificial ribosome switched on or off via the external addition of a small molecule to the growth medium deserves special mention. the escherichia coli s ribosomal rna was fused to an aptazyme, a ribozyme capable of binding thiamine pyrophosphate (ttp) through an aptamer domain. the binding of ttp activates the ribozyme, reducing gene expression by cleaving the s ribosomal rna and thus working as a riboswitch. this allows for the identification and selection of specific e. coli colonies according to the phenotype expressed [ ] . the ttp riboswitch was also included in the `utr region of a reporter gene, nt upstream of the shine-dalgarno sequence. this linker between these elements was randomized and e. coli colonies selected depending on the expression of the reporter gene in response to ttp [ ] . ex vivo selection methods in cells are limited by the number of sequences that can be studied, this number being determined by the number of available cells. another problem is the possibility of there being more than one sequence variant per cell, which could lead to many false positive. ellington's group developed a very interesting strategy that allowed the direct selection of active molecules instead of selecting cell clones [ ] . the cleavage products of the autocatalytic ribozymes synthesized in the cell nucleus were extracted via hybridization to a biotinylated oligonucleotide, allowing the direct identification of active molecules. the selection of nucleic acids against whole cell targets has been successfully used to select nucleic acids that target cell surface receptors. this technology also known as cell-selex could have a role to play in the treatment of cancer. for most types of cancer cell there is a shortage of highly specific surface markers that can be used with diagnostic and therapeutic intent. aptamers generated from whole living cells are the optimal molecular probe for characterizing target cells at the molecular level. when bound to the membrane receptors of cell lines they provide an effective means of identifying disease markers. the pharmacokinetic and pharmacodynamic properties of a nucleic acid, and its resistance to nucleases, all condition its effectiveness as a therapeutic molecule. after selection, the most effective molecules can be modified to improve their nuclease resistance as well as their affinity for their targets, their cellular uptake and selectivity. a great diversity of post-selection modifications has been described. only those of interest for therapeutic purpose are discussed here. these include modifications of oligonucleotide size and sequence, and mainly certain chemical modifications (for a review see [ , ] . advances in chemical synthesis have allowed the production of conjugates that combine an oligonucleotide sequence with compounds such as fluorophores, peptides, carbohydrates and lipids ( figure ). these modified oligonucleotides show advantageous properties with respect to the native form. the ligands are usually linked to the ' or ' termini, which are the most accessible regions for chemical conjugation reactions; in addition, any disruption of the nucleic acid's folding and functional properties are minimized [ ] . conjugations at the ' position of ribose or involving the internucleotidic phosphodiester bonds are also possible. fluorophore conjugation is already used in clinical diagnosis, e.g., in fluorescence in situ hybridization (fish) and molecular beacons. fish can detect specific genes in cells, while molecular beacons act like switches, emitting fluorescent light when bound to their target sequence. cell uptake has been improved by the conjugation of oligonucleotides to peptides capable of translocating them across the cell membrane by a non-receptormediated endocytotic mechanism. some of the most frequently used peptides are residues - of the third helix of the antennapedia homeodomain (penetratin), the highly arginine/lysine rich region of the hiv- tat protein, the hydrophobic signal peptide, the nuclear localization sequence (nls), and transportan [ ] . besides improving cellular uptake, peptide-oligonucleotide conjugates show increased stability to nucleases degradation and enhanced binding [ ] . carbohydrate-oligonucleotide conjugates (cocs) have similar applications. in addition they confer cell or tissue specificity by their binding to sugar receptors (i.e., lectins) present at the cell surface capable of recognizing and internalizing (by endocytosis) glycoproteins bearing specific carbohydrates moieties [ ] . lipophilic oligonucleotide conjugates (locs), such as cholesterol, reduce the hydrophobic character of the oligonucleotide, and some bind to blood lipoprotein carriers. lipophilic oligonucleotides are also used for designing supramolecular assemblages such as micelles, vesicles and liposome networks [ ] . the ability of certain rna polymerases to incorporate modified nucleotides to the growing chain, such as the '-modified ribonucleosides, makes it possible to use the selection procedure with populations of chemically modified oligonucleotides [ , ] . chemical modifications of the ' hydroxyl group of rna, such as ' fluoro, amino, methoxy and amido modifications, are noteworthy for their potential therapeutic applications since they increase rna stability, conferring greater resistance to nucleases. another important modification for therapeutic purposes is the use of locked nucleotides (lnas) in the nucleotidic chain. lnas contain a methylene link between the '-o and '-c of the ribose ring which locks the sugar moiety in the ' endo conformation [ ] . this generates the most stable hybrids ever characterized, with a Δt m of + to + per lna residue upon binding to dna and rna respectively, thus conferring nuclease resistance [ , ] . the introduction of lna modifications into in vitro selection techniques has so far been restricted to post selection incorporation (for a review see [ ] ). nevertheless, it has recently been shown that locked nucleotides can be incorporated enzymatically into both dna and rna [ ] [ ] [ ] . spiegelmers (spiegel = mirror in german), unnatural but biostable l-forms of d-aptamers, were developed to allow oligonucleotides to escape nuclease attack. naturally occurring proteins are lchiral, therefore natural nucleases digest d-oligonucleotides while l-nucleosides escape this fate. to obtain spiegelmers, natural oligonucleotides are used during the selection cycle against unnatural dproteins that mirror the natural structure of the l-target [ , ] . the selection process yields l-form aptamer sequences that by virtue of the law of symmetry recognize their natural target. spiegelmers have been selected against peptide hormones such as gonadotropin-releasing hormone (gnrh) [ ] , and ghrelin, an endogenous ligand for growth hormone secretagogue receptor a [ ] . both have a neutralizing effect in vivo against these hormones after systemic administration. in vitro selection strategies have been extensively and successfully used to characterize known ribozymes and dnazymes, and to isolate new catalytic nucleic acids with unsuspected activities. in fact, the first observation of a dna molecule catalyzing a chemical reaction (dnazyme) was made when using an in vitro selection strategy [ ] . although ribozymes and dnazymes have been extensively assayed as potential therapeutic agents, and different clinical trials have already tested their efficiency against various diseases [ ] [ ] [ ] [ ] , very few reports have described the direct application of in vitro selection strategies in the development of potentially therapeutic catalytic nucleic acids. ellington's group recently described a procedure aimed at identifying cleaving ribozymes active within the cell milieu, but this has not yet been used with therapeutic intent [ ] . most of the work referred to herein describes the use of in vitro and ex vivo selection strategies for the identification of aptamers of therapeutic potential. assays with catalytic nucleic acids engineered by so-called 'rational design' are beyond the scope of this review. the idea that aptamers can modulate the activity of target proteins emerged from basic studies of viruses. in the s, research on hiv and adenovirus led to the discovery that these viruses contain several structural rna domains that bind to viral or cellular proteins with high affinity and specificity. not surprisingly, functional analyses of these viral rna ligands demonstrated that the viruses had evolved these aptamers either to modulate the activity of proteins essential for their replication [ ] or to inhibit the activity of proteins involved in cellular antiviral responses [ ] . the first study performed to determine whether an rna aptamer could be used to inhibit the activity of a pathogenic protein was published in . this work reported that the tar aptamer, evolved by hiv to recruit viral and cellular proteins to viral transcripts, could be turned against the virus to inhibit its replication [ ] . the in vitro aptamer selection strategies developed during the s prompted the idea of sullenger's group that therapeutic aptamers might be possible. several such aptamers have now completed various stages of preclinical development, and a number of others are currently being tested clinically (table ) . indeed, one aptamer is already on the market as a therapeutic drug several modifications of the general selection process scheme have been described in order to achieve different goals. for example, the toggle-selex strategy is used for the selection of potentially therapeutic nucleic acids [ ] . toggle-selex was designed for the isolation of aptamers with a broad range of specificities for closely related targets, such as the homologous proteins of different species. these aptamers were obtained by performing the selection procedure for related targets (i.e., homologous proteins) in alternating cycles. such alternation ensures that the rna or dna variants resulting from selection will bind to both proteins, most likely to domains conserved between them. sullenger's group described an in vitro selection strategy in which rna aptamers that bind both human and porcine thrombin were selected by "toggling" the protein target between the human and porcine forms in successive rounds of selection [ ] . this yielded a family of aptamers, all of which bound both thrombin types with high affinity. toggle- , a characteristic member, inhibited two of thrombin's most important functions: plasma clot formation and platelet activation [ ] . this strategy could facilitate the isolation of ligands with properties required for gene therapy or other therapeutic or diagnostic applications. to date, the only aptamer approved by the fda [ ] , known as pegaptanib or macugen, was approved in december for the treatment of wet type age-related macular degeneration (amd). this aptamer binds to vascular endothelial growth factor, vegf [ , ] , the main isoform of a family of growth factors involved in promoting blood vessel development and maintenance via tyrosine kinase receptor signaling. vegf is also involved in several pathological processes such as amd, diabetic retinopathy and cancer [ ] . a phase ii clinical trial to evaluate the use of this aptamer in the fight against diabetic retinopathy is currently underway [ ] . the selection procedure involved a '-fluoro-pyrimidine ( '-fy)-modified rna pool. additional modifications were made after selection by adding '-o-methyl ( '-mr) to all purine residues of the aptamer except two, by adding a ' cap, and by adding polyethylene glycol (peg; kd) to the ' end ( figure ). the macugen aptamer binds to the heparin-binding domain of vegf [ , ] and efficiently inhibits the growth of blood vessels [ , ] . this agent is of particular interest with respect to preventing tumor angiogenesis. different aptamers are currently being tested for the treatment of other degenerative diseases. niu's group attempted to interfere with the function of the glur ampa receptor associated with cerebral ischemia and amylotrophic lateral sclerosis [ ] . a selected aptamer known as an acts as a glutamate antagonist preventing glutamate-induced activation of the cationic channel. interestingly, the aptamer adopts two mutually exclusive non-interchangeable isoforms that are both necessary for proper inhibition to occur [ ] . recently, new rna aptamers have been isolated that bind to the nogo- neural receptor as antagonists of myelin-derived ligands. nogo- signaling blocks neurite outgrowth, but the binding of the aptamers allow axon growth in rat ganglion cells in vitro [ ] . this aptamer is of special interest in the search for agents that aid neural repair, e.g., after spinal cord trauma. aptamers can also be targeted against disease-causing proteins such the scrapie isoform of the prion protein (prp sc ). a '-fy rna aptamer known as saf- [ ] has been shown to prevent its aggregation in cell free systems, while a '-amino- '-deoxypyrimidine-modified aptamer known as dp slows down its aggregation in neuroblastoma cells [ ] . both aptamers target the same conserved region involved in prion interactions. neutrophil elastase (hne) is involved in the pathogenesis of inflammatory diseases such as acute respiratory distress syndrome (ards), septic shock, emphysema and arthritis, as well as ischemiareperfusion injuries [ ] . a covalent inhibitor of hne, a diphenyl phosphate derivative of valine, has been coupled to an rna library to enhance its binding to hne [ ] . after in vitro selection, an rna aptamer conjugated with dna:valp (rna . :dna:valp) was isolated that binds hne with high affinity. the bound molecule, unlike the aptamer rna . or dna:valp alone, also inhibits lung inflammation in an ex vivo rat model of ards [ ] . more efficient inhibitors of hne were obtained from a valyl phosphonate:dna pool [ ] . after selection, aptamer inhibitor ed inhibited hne formation two orders of magnitude greater than rna. . :dna:valp [ ] . a truncated dna aptamer version named nx , composed of two annealed dna oligonucleotides, was tested in a rat model of lung inflammation and was found to inhibit neutrophil infiltration by % at a dose of nmol [ ] . immunoglobulin e (ige) plays an important role in protecting mammals from parasites [ ] . however, its overproduction due to exposure to environmental antigens can result in allergies, atopic dermatitis and allergic asthma [ ] . dna selection was performed against human ige to produce aptamers that bind it with high affinity [ ] . these aptamers inhibited the binding of ige to its receptor fcεri, and also prevented ige-mediated cellular degranulation in the serum of patients with allergy to grass pollen. in patients exposed to grass pollen extract, the ic s for the dna aptamers were - µm, but when triggered by anti-ige antibodies they reached - nm. these dna aptamers represent a novel class of ige inhibitors that may prove useful in the fight against allergic diseases. cancer has been one of the diseases most targeted by aptamers. alteration of the signaling pathways results in the escape of tumor cells from the control of cell division and apoptosis. the formation of metastases is also promoted. all of these processes have been the target of aptamers. the tyrosine kinase receptors (tkr) has been targeted by two aptamers: ret (rearranged during transfection) and her (human epidermal growth factor receptor). the ret aptamer, d , a '-fy aptamer, was isolated by ex vivo selection against the extracellular mutant ret c y receptor [ ] . briefly, an rna population was incubated against a parental pc cell line and variants expressing different ret mutants as a negative selection step. the unbound fraction of rna molecules was recovered and incubated against pc cells expressing the recombinant ret c y receptor. the selected d aptamer inhibits neurite outgrowth and reverts the neoplastic phenotype of nih/men a cells in vitro [ ] and in three dimensional collagen gel matrix cultures [ ] . the her- aptamer, a '-fy aptamer, known as a , was isolated against the her- monomeric extracellular domain; this prevents her- signaling via her- heterodimerization in cell culture [ ] . cell adhesion misregulation involved in metastasis has also been prevented by aptamers. the plasminogen activator inhibitor- (pai- ) is overexpressed in breast cancer cells and binds to vitronectin, leading to the loss of adhesion. a '-fy aptamer known as sm- , specific for plasminogen activator inhibitor- (pai- ), restores cell adhesion to vitronectin-coated plates in vitro [ , ] . another type of therapeutic signaling modulation is the targeting of nuclear factor κb (nf-κb) inside cells. maher's group isolated two rna aptamers, α-p and r , by two independent in vitro selection procedures. these bind nf-κb p and p isoforms in vitro respectively [ , ] . these aptamers underwent further ex vivo selection using the yeast three-hybrid system [ , ] . the inhibition of nf-κb might be of therapeutic interest in different types of cancer, hiv- infection and inflammatory diseases. in fact, gene therapy with different anti-nf-κb aptamers administered via an adenoviral vector suppresses doxorubicin resistance in vivo in a lung tumor xenograft mouse model [ , ] . the inhibition of nucleophosmin oligomerization by an aptamer promotes higher p levels and, consistently, sensitizes cells to dna-damaging-agent-induced apoptosis in cell culture [ ] . the stimulation of the immune system can also be used in anti-cancer therapy. the activation of cd + t cells within a tumor would promote its cytotoxic involution. in this respect, the modulation of cytotoxic t-cell antigen- (ctla- ), - bb and ox receptors by aptamers has been explored. multimeric aptamer forms frequently improve aptamer signaling properties and have proven especially importance in this area. it has been shown that an antagonist rna aptamer against ctla- , a negative regulator of t-cell activation, inhibits its function [ ] , while an agonistic aptamer against - bb, a major co-stimulatory receptor, leads to the activation of t cells [ ] . immunity against the tumor is induced in vivo in both cases. a specific aptamer for the dimeric murine ox combined with a dendritic cell-based tumor vaccine promotes tumor immunity in a xenograft melanoma model in mice [ ] . aptamers have also been shown able to promote tumor cell death. when expressed in cells, an aptamer selected against nucleophosmin was shown to prevent the latter's oligomerization. higher p levels were therefore promoted that led to apoptosis. in addition, the sensitivity of cells to dnadamaging agents in cell culture was increased [ ] . research into anti-angiogenesis aptamers has provided some very interesting results. sullenger's group reported the selection of specific rna aptamers against the ang and ang genes [ , ] . aptamer ang - binds to ang and inhibits its signaling pathway, leading to the reduced survival of huvec cells in vitro [ ] . similarly, intraocularly administered aptamer - . binds to ang and inhibits angiogenesis in rat corneal micropockets [ ] . a ' deoxythymidine cap protects this aptamer from rnases. the pegylated version of this molecule was shown to inhibit tumor angiogenesis and growth in an in vivo murine metastatic colon cancer model following systemic administration [ ] . while the large majority of aptamers have been isolated by selex [ ] , the anti-proliferative dna aptamer as- was developed based on observations that guanosine-rich oligonucleotides have antiproliferative effects in tumor cells [ ] . molecular studies have shown that this aptamer binds to the cell surface protein nucleolin and inhibits the activity of nf-kb, a ubiquitous transcription factor, through intracellular complex formation [ ] . clinical studies of as- have focused on patients with renal, pancreatic and other solid tumors. the aptamer was administered to patients as a continuous infusion for or days. selectins are a family of cell adhesion molecules expressed by leukocytes, endothelial cells and platelets [ ] . they are involved in a number of inflammatory diseases as well as tissue injury and infection. dna selection against the l-selectin/igg fusion protein (ls-rg) was performed to find aptamers that could be tested in vivo [ ] . aptamers ld , ld and ld all bound with a kd of . nm at ºc. truncated versions of these aptamers inhibited sl-rg binding to its ligand sialyl lewis x (slex) with an ic of nm. aptamer ld t blocked l-selectin-mediated adhesion of human lymphocytes and neutrophils and inhibited human cell trafficking to peripheral and mesenteric lymph nodes in scid mice. platelet-derived growth factor (pdgf) is a ubiquitous mitogen and chemotactic growth factor in the form of three disulphide-linked dimers made of two homologous chains, a and b. it is involved in wound healing and is linked to the progression of numerous diseases, including atherosclerosis and glomerulonephritis [ , ] . a hallmark of malignant transformation is the loss of dependence on exogenous mitogenic stimulation; many tumor cell lines are thought to produce and secrete pdgf for this reason [ ] . dna selection against recombinant human pdgf-ab yielded dna specific aptamers of the pdgf b-chain that bound with subnanomolar affinity [ ] . the consensus secondary structure motif for most of the high-affinity ligands is a three-way helix junction with a threenucleotide loop at the branch point. the pdgf aptamers inhibited the mitogenic effects of pdgf-bb in cells that expressed pdgf β receptors [ , ] . peg-modified aptamers in a rat model of mesangioproliferative glomerulonephritis led to a % reduction in mitoses by day , and % by day . there was also a % reduction of proliferating mesangial cells by day and a markedly reduced glomerular expression of the endogenous pdgf b-chain. aptamer-treated animals also showed a reduced influx of monocytes/macrophages and the overproduction of glomerular extracellular matrix on day [ ] . further studies revealed the inhibition of other disease mechanisms by the above aptamer in experimental glomerulonephritis [ , ] . the expression of pdgf β receptors in tumors is associated with increased interstitial fluid pressure (ifp) in the dermis. this reduces the gradient between capillaries and the interstitium and impedes the exchange of solutes, such as anticancer agents, over the capillary membrane [ ] . increasing this gradient may facilitate the transport of anticancer drugs to tumors [ ] . to reduce the ifp, the pdgf-b aptamers [ ] were tested in a rat tumor model. the treated animals had an ifp of . mm hg compared to . mm hg in scrambled-rna-treated animals [ ] . another dna aptamer known as e - that targets the pdgf-b subtype is currently undergoing phase i clinical trials as an antivascular endothelial growth factor compound [ ] . blood fluidity and blood vessel resistance are involved in numerous vascular diseases such as coronary and thrombotic syndromes and myocardial infarction. certainly, these factors must be carefully controlled during coronary surgery. the proliferation of cardiac and vascular cells is key in the development of vessel resistance in diseases such as cardiac intimal hyperplasia, cardiac hypertrophy and atherosclerosis, as well as in the development of malignancies [ , ] . a recent study has reported the development of an rna aptamer able to specifically recognize members of the e f transcription factors involved in cell proliferation. the binding of the '-fy aptamer - mainly to e f , reduces intimal hyperplasia and the pathological proliferation and migration of vascular smooth muscle cells (vsmcs) after bypass surgery in a mouse model [ ] . this aptamer avoids the side effects derived from cross reactivity with other members of the e f family. in a different approach, selex has been performed with the e f protein to find in vitro selected rna aptamers that bind to and inhibit e f activity. clone e rna was found to bind to e f and blocked the latter's attachment to its dna binding site [ ] . by impeding e f activity, the e f rna aptamer inhibited s-phase induction by % compared to controls. thus, both natural and in vitro selected aptamers appear able to limit cell proliferation. the coagulation signaling cascade offers several targets for the modulation of blood fluidity. the conversion of pro-thrombin to thrombin is delayed by a long half-life ( h) '-fy aptamer targeting the catalyst factor viia in a dose dependent manner [ ] . nevertheless, the rapid restoration of cascade integrity is needed to prevent the harmful effects of coagulation deficiency. with this aim, an rna-based aptamer-antidote system has been developed [ , ] . the reg- rna aptamer targets factor xia. an antisense rna molecule was designed to specifically bind the '-half of the aptamer (the antidote). binding of the antidote abolishes the aptamer's binding to its target, thereby reversing the anticoagulant effect ( figure ). neither antidote nor aptamer have been seen to cause any adverse effect in phase i clinical trials (ia and ib) when given to healthy people and patients with stable cardiovascular disease receiving antiplatelet therapy [ , ] . the results of another phase i clinical trial (ic) indicate that the anticoagulation effect can be modulated by varying the dose of antidote rna [ ] . in a recent phase iia trial, percutaneous coronary injection of the aptamer increased the activated clotting time (act) in patients immediately after its administration, reaching values close to those obtained with heparin. act values were restored min after the administration of the antidote [ ] . thrombin is a natural target for anticoagulation therapy and numerous aptamers have been generated with different capacities to inhibit its activity in vitro [ , ] . the archemix corp., in collaboration with arca biopharma inc. (formerly nuvelo inc.), has tested nu- , a dna aptamer against thrombin. in phase i clinical trials, nu- was administered intravenously as a continuous infusion to healthy volunteers. the results showed an increase in activated clotting time with a return to baseline when administration ceased (see archemix corp. website). a phase ii clinical study is currently underway with the goal of using nu- in coronary artery bypass graft surgery and percutaneous coronary intervention. figure . mechanism of the apatamer-antidote pair for anticoagulant therapy.the intrinsic pathway of the blood coagulation cascade involves the activation of factor x. anticoagulation system reg consists of rb (drug), an injectable rna aptamer that specifically binds to activated factor ix (ixa) and prevents the proteolytic cleavage of factor x; and rb (antidote), a rna antisense oligonucleotide that neutralizes the anticoagulating effect of the aptamer rb . in the presence of the antidote, the aptamer is released from factor ixa and clotting parameters return to normal. together with activated factor viii (viiia), factor ixa catalyzes the cleavage of factor x (pro-enzyme) to yield activated factor x (xa), which is required for the blood clotting cascade. a dna/rna aptamer conjugated to peg, known as arc- , generated against vwf (von willebrand factor), a central factor in the adhesion of platelets to the endothelial surface at vascular injury sites [ ] , has been examined in a phase i trial in healthy volunteers. the aptamer increased platelet function in a whole-blood assay sensitive to vwf-mediated platelet inhibition. moreover, a slow intravenous bolus followed by h of continuous infusion inhibited more than % of vwf function, which returned to baseline over - h after administration was suspended. nf-κb is involved in inflammation responses and modulates the synthesis of chemokines, interferons, major histocompatibility complex (mhc) proteins, growth factors, and the cell adhesion molecules that play a role in ischemia-reperfusion injuries seen in most myocardial infarctions [ ] . a natural double stranded dna aptamer was found that binds to nf-κb with high affinity. in a rat cardiac ischemia-reperfusion model, this aptamer significantly reduced the expected injury [ ] . in a rat cardioplegic arrest model, animals transfected with the nf-κb dna aptamer showed improved recovery of left ventricular function as well as coronary flow three days post-transfection compared to scrambled-dna controls ( % vs. %) [ ] . the aptamer-treated group also showed a lower percentage of neutrophil adhesion to endothelial cells ( % vs. %) and a lower level of interleukin il- ( vs. ng/mg). the same aptamer was also studied in a murine model of nephritis, in which it abolished glomerular inflammation and the expression of inflammatory markers il- α, il- β, il- , icam- (intracellular adhesion molecule ), and vcam- (vascular cell adhesion molecule ) [ ] . a rat global brain ischemia model showed inhibition of tnf-α, il- β and icam- expression in nf-κb aptamer-treated animals after h of ischemia. moreover, days after ischemia, neuronal damage was significantly attenuated in the nf-κb-aptamer-treated group compared to controls [ ] . the proteins of different pathogens have also attracted the attention of researchers as targets for inhibitory nucleic acids. a recent study reported the use of a '-fy aptamer against the extracellular domain of the erythrocyte membrane protein (pfemp ) of the parasitic protozoan plasmodium falciparum, achieving the efficient inhibition of erythrocyte rosseting in blood cultures [ ] . rna viruses, especially hiv- and hcv, have been the main targets for therapeutic nucleic acids with catalytic activity. rossi's group designed a very interesting ex vivo selection procedure to identify anti-hiv hammerhead ribozymes. a pool of hammerhead catalytic domains containing randomized binding arm sequences was assayed against an hiv- chimera containing the thymidine kinase gene. after cell transfection of the ribozyme population expressed under the control of the u snorna promoter, gancyclovir-resistant cells were selected. such antibiotic resistance suggests the presence of an active anti-hiv ribozyme [ ] . banerjea's group designed a strategy to identify accessible cleavage sites within the hiv- gag rna and to pick out dnazymes effective against this target [ ] . two dnazyme variant populations were synthesized. the specificity of the first was limited to all possible augs in the target rna, whereas the second population was designed to cleave any potential target site. these options were made possible by either fixing the nucleotides immediately preceding the catalytic motif to ca (for aug cleavage) or totally randomizing the seven bases on either side of the catalytic motif. dnazymes selected from both populations showed target-specific cleavage activities in the presence of mg + , and significantly interfered with hiv- -specific gene expression. this strategy could be used for the selection of effective target sites in any target rna [ ] . viral proteins are favorite targets for the development of therapeutic aptamers. rna aptamers have been selected against different viral enzymes and proteins involved in host-cell interactions, such as hiv- reverse transcriptase (rt), hiv- glycoprotein , hcv rna-dependent rna polymerase (rdrp), sars coronavirus ntpase/helicase, and the hemagglutinin protein of human influenza virus b, all of which show efficient viral inhibition [ ] [ ] [ ] [ ] [ ] [ ] . human influenza b virus hemagglutinin protein has been inhibited in vitro by the rna pool resulting from rounds of in vitro selection [ ] . further studies are required for the identification of independent aptamers. recently, a dna aptamer (termed del) has been described that adopts a novel type of dimeric quadruplex folding and shows anti-hiv integrase activity in the nanomolar range in vitro by blocking several catalytic amino acid residues essential for integrase function. several other g-rich dna aptamers have been identified as remarkable hiv -in inhibitors with ic values in the nanomolar range. t is one such aptamer, and has been well studied in recent years, [ , ] . astier-gi's group described the characterization of two dna aptamers ( v and v) that specifically bind to hepatitis c virus (hcv) rna polymerase (ns b), inhibiting its activity in vitro [ ] . aptamer v competed with the rna template for binding to the enzyme and blocked both the initiation and elongation phases of rna synthesis, whereas aptamer v exclusively inhibited initiation events. the authors also determined that, in addition to an in vitro inhibitory effect on rna synthesis, aptamer v interfered with the multiplication of hcv jfh in huh cells. the efficient cellular entry of these short dnas, and the inhibitory effect observed in human cells infected with hcv, indicate that aptamers are useful tools for the study of hcv rna synthesis; their therapeutic against hcv infection is also attractive [ ] . an alternative and innovative potential therapeutic approach that has attracted much hope is the targeting of the structural domains of viral genomes which are frequently concentrated within untranslated terminal regions (utrs). the hiv- trans-activation response (tar) element is a polyfunctional rna domain mainly involved in the activation of transcription by its binding to the viral protein tat. aptamer r binds the tar element in vitro by a loop-loop interaction [ ] . the intracellular expression of the aptamer by the nucleolar u rna promoter inhibits hiv- infection by more than %. this strategy takes advantage of hiv- rna nucleolar-trafficking to efficiently colocalize the aptamer and target [ ] . the same r aptamer has been improved by the inclusion of extra binding rna domains targeting additional '-utr sites [ ] . our own results show that presynthesized aptamers, or aptamers produced intracellularly via u rna promoter-driven expression, both with multiple binding sites targeting the whole hiv- '-utr, efficiently inhibit hiv- replication in cell culture (sánchez-luque et al., unpublished). there have been different attempts to isolate rna aptamers against different domains of the hcv internal ribosome entry site (ires) located within the '-utr [ ] [ ] [ ] . aptamer - binds domain iii-iv of hcv ires inhibiting its activity in cell-free systems [ ] . this inhibition was further improved by conjugation of - with - in a chimeric molecule that targets domain ii [ ] . rna aptamer ap isolated against the hcv (-) strand ires domain i partially inhibits hcv rna replicase-mediated (+) strand synthesis, probably by interfering with (-) strand binding [ ] . a step forward was the design of an innovative selection approach to identify chimeric ribozyme/aptamer rna molecules against the entire hcv-ires. each selection cycle includes two consecutive selection steps for binding and cleavage of the viral rna [ ] . after six selection rounds, seven families of inhibitor rnas were identified simultaneously targeting two sites within the hcv-ires, one for each inhibitory domain. these chimeric rna inhibitors promoted ires inhibition by up to % in cell extracts, identifying new targets for the development of anti-hcv agents. further characterization revealed up to % inhibition of viral translation and replication in a human liver cell line [ , ] . the ability of selex to generate aptamers against any kind of target provides the possibility of their being used as therapeutic antidotes or palliatives. ricin is a toxic heterodimeric lectin from the castor bean plant ricinus communis. it disrupts protein synthesis by inactivation of the ribosomes. an aptamer isolated against the a chain partially restores protein synthesis levels in cell free translation systems and cell cultures [ ] . a different approach has been explored for cocaine and anticonvulsant mk- alleviation. both compounds preferentially bind the open isoform of nicotine acetylcholine receptors (nachrs) found at neuromuscular junctions, autonomic ganglia and in the central nervous system. rna aptamers developed against nachrs with equal binding affinity for both open and closed sodium-potasium channel isoforms partially restore isoform equilibrium, alleviating drug channel inhibition in cells [ ] [ ] [ ] . aptamers can distinguish between different isoforms of the same protein or different members of the same family. aptamer-sirna/toxin conjugates have been developed to deliver therapeutic agents within a specific target cell. ex vivo selection procedures performed against a specific cell type have yielded aptamers that are very efficiently taken up by those cells; they could therefore be used as a specific delivery tool. the tta aptamer of tenascin c, selected against the human glioma u cell line [ , ] , was efficiently taken up by human tumor cells in a xenograft glioblastoma and breast tumor model [ ] . aptamers targeting the prostate-specific membrane antigen (psma) are those most used as delivery tools. coffey's group reported two '-fy rna aptamers, a and a , that bind specifically recombinant psma [ ] . since psma is continually recycled from the cell surface, these aptamers are appropriate vehicles for delivering therapeutic compounds via the endosomal pathway. small interfering rnas against pro-survival factors over-expressed in prostate cancer cells, such as plk- and bcl- and eukaryotic elongation factor , have been delivered by the a aptamer [ ] [ ]. the aptamer-driven uptake of plk- bcl- sirnas leads to the death of psma-positive cells and tumor regression following intra-tumoral injection in mice xenograft models [ ] . the improvement of the delivery vehicles were achieved in several ways and tumor regression through the almost complete loss of cell viability was observed when the eukaryotic elongation factor sirna was delivered by a dimeric aptamer [ ] . the delivery of pharmacological compounds by aptamers has also been studied by different approaches. doxorubicin (dox) is a chemotherapeutic intercalating agent used against cancer. dox intercalates between the aromatic rings of the gc pairs of the a aptamer, enhancing its cytotoxicity against psma-expressing cells and reducing the same in non-expressing cells [ ] . the same results have been reported when using the a -genolin conjugate, a toxin that blocks protein synthesis [ ] . side toxicity of the chemotherapeutic compound can be further prevented by encapsulating it within nanoparticles or nanoconjugates [ ] [ ] [ ] . aptamer b , specific for hiv- gp [ ] , has also been used to deliver sirna against infected cells. hiv- gp is found on the surface of infected cells and promotes cell fusion. consequently, besides neutralizing infective particles, the b aptamer can be used for anti-hiv- sirna delivery to infected cells. the first attempt involved a chimera of the b aptamer and sirna targeting the overlapping rev tat region [ ] . the chimera was internalized in an aptamer-dependent manner, with inhibition dependent on the interference machinery. the aptamer-sirna linkage was further improved for the easy combination of sirnas to the aptamer and better sirna processing by the cellular machinery. this improved chimera resulted in the specific inhibition of hiv- replication and infectivity in pbmc and cultured cem t cells [ ] . finally, aptamers can also be used to colocalize rna inhibitors with their specific target molecules at the subcellular level. aptamers can target rna domains, e.g., in viral rna genomes; they can therefore improve trans-cleaving ribozymes by anchoring them to their target. chimeric molecules composed of a hammerhead ribozyme and an aptamer both targeting the hcv ires have efficiently inhibited ires activity in human hepatocyte cell cultures [ , ] . the procedures used to identify dna and rna molecules of interest in large populations of variant nucleic acid molecules have contributed significantly to the development of nucleic acid-based therapeutic drugs. aptamers show high specificity for their targets and have low toxicity and immunogenicity profiles. since the s, the design and isolation of specific aptamers using selection and evolution techniques has been optimized and even automated. this has lead to great advances in our knowledge of aptamers as therapeutic agents and has expanded our bank of inhibitory nucleic acids and their possible targets, which now include cytokines, cell receptors, viruses and even whole cells. an aptamer drug is now on the market, and several mid and late clinical trials in progress that appears to confirm the great potential of these tools. with the improvement and optimization of selection strategies, and the ongoing discoveries made in this field, success in the development of nucleic acidbased therapeutics protocols might be predicted. an extracellular darwinian experiment with a selfduplicating nucleic acid molecule in vitro genetic analysis of the tetrahymena selfsplicing intron in vitro selection of rna molecules that bind specific ligands selection in vitro of an rna enzyme that specifically cleaves singlestranded dna systematic evolution of ligands by exponential enrichment: rna ligands to bacteriophage t dna polymerase in vitro selection and evolution of rna: applications for catalytic rna, molecular recognition, and drug discovery in vitro selection of hairpin ribozymes in vitro selection of catalytic polynucleotides in vitro selection of functional nucleic acids in vitro selection of functional nucleic acid sequences in vitro selection procedures for identifying dna and rna aptamers targeted to nucleic acids and proteins berzal-herranz, a. rna selection and evolution in vitro: powerful techniques for the analysis and identification of new molecular tools aptamers: a new class of oligonucleotides in the drug discovery pipeline? in vitro rna random pools are not structurally diverse: a computational analysis in vitro selection using a dual rna library that allows primerless selection short bioactive spiegelmers to migraineassociated calcitonin gene-related peptide rapidly identified by a novel approach: tailored-selex minimal primer and primer-free selex protocols for selection of aptamers from random dna libraries in vitro selection of active hairpin ribozymes by sequential rna-catalyzed cleavage and ligation reactions ribozyme catalysis of metabolism in the rna world selection-by-function: efficient enrichment of cathepsin e inhibitors from a dna library an allosteric synthetic dna engineering a ligand-dependent rna transcriptional activator in vivo evolution of an rna-based transcriptional activator aptazyme-mediated regulation of s ribosomal rna reengineering a natural riboswitch by dual genetic selection direct selection for ribozyme cleavage activity in cells chemical modification of sirnas for in vivo use therapeutic applications of dna and rna aptamers recent developments in oligonucleotide conjugation cell penetrating peptides: overview and applications to the delivery of oligonucleotides sequence-specific activity of antisense oligonucleotides conjugated to poly (l-lysine) carriers the cluster glycoside effect nucleotide and oligonucleotide based amphiphiles: a successful marriage of nucleic acids with lipids prion-proteinspecific aptamer reduces prpsc formation a nuclease-resistant rna aptamer specifically inhibits angiopoietin- -mediated tie activation and function the first analogues of lna (locked nucleic acids): phosphorothioate-lna and '-thio-lna structural studies of lna:rna duplexes by nmr: conformations and implications for rnase h activity the conformations of locked nucleic acids (lna) locked nucleic acids: promising nucleic acid analogs for therapeutic applications in vitro incorporation of lna nucleotides polymerase chain reaction and transcription using locked nucleic acid nucleotide triphosphates efficient enzymatic synthesis of lna-modified dna duplexes using kod dna polymerase mirror-image rna that binds dadenosine mirror-design of loligonucleotide ligands binding to l-arginine in vivo properties of an anti-gnrh spiegelmer: an example of an oligonucleotide-based therapeutic substance class inhibition of ghrelin action in vitro and in vivo by an rna-spiegelmer a general purpose rna-cleaving dna enzyme ribozyme structures and mechanisms gaining target access for deoxyribozymes dnazymes as potential therapeutic molecules inhibition of hiv- replication by rna-based strategies regulatory pathways governing hiv- replication identification and characterization of a hela nuclear protein that specifically binds to the trans-activation-response (tar) element of human immunodeficiency virus overexpression of tar sequences renders cells resistant to human immunodeficiency virus replication generation of species cross-reactive aptamers using "toggle pegaptanib, a targeted anti-vegf aptamer for ocular vascular disease antivascular endothelial growth factor therapy for neovascular agerelated macular degeneration inhibition of von willebrand factor-mediated platelet activation and thrombosis by the anti-von willebrand factor a -domain aptamer arc first-in-human evaluation of anti von willebrand factor therapeutic aptamer arc in healthy volunteers first-in-human experience of an antidote-controlled anticoagulant using rna aptamer technology: a phase a pharmacodynamic evaluation of a drug-antidote pair for the controlled regulation of factor ixa activity a randomized, repeat-dose, pharmacodynamic and safety study of an antidote-controlled factor ixa inhibitor phase b randomized study of antidote-controlled modulation of factor ixa activity in patients with stable coronary artery disease discovery and development of the g-rich oligonucleotide as as a novel treatment for cancer the nucleolin targeting aptamer as destabilizes bcl- messenger rna in human breast cancer cells discovery and development of anticancer aptamers inhibition of platelet-derived growth factor b signaling enhances the efficacy of antivascular endothelial growth factor therapy in multiple models of ocular neovascularization sequential loss of tumor vessel pericytes and endothelial cells after inhibition of platelet-derived growth factor b by selective aptamer ax derivation of rna aptamer inhibitors of human complement c selection of single-stranded dna molecules that bind and inhibit human thrombin in vivo anticoagulant properties of a novel nucleotide-based thrombin inhibitor and demonstration of regional anticoagulation in extracorporeal circuits pilot study of the efficacy of a thrombin inhibitor for use during cardiopulmonary bypass pegaptanib sodium for ocular vascular disease '-fluoropyrimidine rna-based aptamers to the -amino acid form of vascular endothelial growth factor (vegf ). inhibition of receptor binding and vegf-induced vascular permeability through interactions requiring the exon -encoded domain the biology of vegf and its receptors a phase ii randomized doublemasked trial of pegaptanib, an anti-vascular endothelial growth factor aptamer, for diabetic macular edema inhibition of vegf blocks tgf-beta production through a pi k/akt signalling pathway rna aptamers selected against the glur glutamate receptor channel one rna aptamer sequence, two structures: a collaborating pair that inhibits ampa receptors aptamer antagonists of myelin-derived inhibitors promote axon growth characterization of '-fluoro-rna aptamers that bind preferentially to disease-associated conformations of prion protein and inhibit conversion the role of neutrophil elastase in chronic inflammation in vitro selection of rna-based irreversible inhibitors of human neutrophil elastase highly potent irreversible inhibitors of neutrophil elastase generated by selection from a randomized dna-valine phosphonate library protective effects of an aptamer inhibitor of neutrophil elastase in lung inflammatory injury high-affinity ige receptor on eosinophils is involved in defence against parasites the human ige network high-affinity oligonucleotide ligands to human ige inhibit binding to fc epsilon receptor i neutralizing aptamers from whole-cell selex inhibit the ret receptor tyrosine kinase distribution and bioactivity of the ret-specific d aptamer in three-dimensional collagen gel cultures inhibition of heregulin signaling by an aptamer that preferentially binds to the oligomeric form of human epidermal growth factor receptor- antimetastatic potential of pai- -specific rna aptamers rna aptamers as conformational probes and regulatory agents for plasminogen activator inhibitor- selection and characterization of an rna decoy for transcription factor nf-kappa b selection and characterization of anti-nf-kappab p rna aptamers yeast genetic selections to optimize rna decoys for transcription factor nf-kappa b characterization of anti-nf-kappab rna aptamer-binding specificity in vitro and in the yeast three-hybrid system h rna polymerase iii promoter-driven expression of an rna aptamer leads to high-level inhibition of intracellular protein activity rna aptamertargeted inhibition of nf-kappa b suppresses non-small cell lung cancer resistance to doxorubicin rna aptamers interfering with nucleophosmin oligomerization induce apoptosis of cancer cells multivalent rna aptamers that inhibit ctla- and enhance tumor immunity multivalent - bb binding aptamers costimulate cd + t cells and inhibit tumor growth in mice assembling ox aptamers on a molecular scaffold to create a receptor-activating aptamer inhibition of rat corneal angiogenesis by a nuclease-resistant rna aptamer specific for angiopoietin- inhibition of in vivo tumor angiogenesis and growth via systemic delivery of an angiopoietin -specific rna aptamer aptamers: an emerging class of therapeutics antiproliferative activity of grich oligonucleotides correlates with protein binding agro inhibits activation of nuclear factor-kappab (nf-kappab) by forming a complex with nf-kappab essential modulator (nemo) and nucleolin novel approach to specific growth factor inhibition in vivo: antagonism of platelet-derived growth factor in glomerulonephritis by aptamers tenascin-c aptamers are generated using tumor cells and purified protein a subpopulation of smooth muscle cells in injured rat arteries expresses platelet-derived growth factor-b chain mrna platelet-derived growth factor (pdgf) and pdgf receptor are induced in mesangial proliferative nephritis in the rat structural and functional studies on platelet-derived growth factor inhibitory dna ligands to platelet-derived growth factor b-chain in vivo transfection of cis element "decoy" against nuclear factor-kappab binding site prevents myocardial infarction specific antagonism of pdgf prevents renal scarring in experimental glomerulonephritis the effects of platelet-derived growth factor antagonism in experimental glomerulonephritis are independent of the transforming growth factor-beta system transport of molecules in the tumor interstitium: a review delivery of molecular medicine to solid tumors inhibition of platelet-derived growth factor receptors reduces interstitial hypertension and increases transcapillary transport in tumors emerging pharmacologic therapies for wet age-related macular degeneration braking the cycle e f: a link between the rb tumor suppressor protein and viral oncoproteins distinct roles of e f proteins in vascular smooth muscle cell proliferation and intimal hyperplasia inhibition of cell proliferation by an rna ligand that selectively blocks e f function blocking the initiation of coagulation by rna aptamers to factor viia rna aptamers as reversible antagonists of coagulation factor ixa antidote-mediated control of an anticoagulant aptamer in vivo nucleic acid aptamers as antithrombotic agents: opportunities in extracellular therapeutics von willebrand factor and the endothelium in vascular disease rel/nf-kappa b/i kappa b family: intimate tales of association and dissociation a novel strategy for myocardial protection using in vivo transfection of cis element 'decoy' against nfkappab binding site: evidence for a role of nfkappab in ischemia-reperfusion injury transcription factor decoy for nfkappab inhibits cytokine and adhesion molecule expressions in synovial cells derived from rheumatoid arthritis nuclear factor-kappa b decoy attenuates neuronal damage after global brain ischemia: a future strategy for brain protection during circulatory arrest in vitro selection of rna aptamers against a conserved region of the plasmodium falciparum erythrocyte membrane protein use of a u snorna-containing ribozyme library to identify ribozyme targets in hiv- in vitro-selected rna cleaving dna enzymes from a combinatorial library are potent inhibitors of hiv- gene expression rna pseudoknots that inhibit human immunodeficiency virus type reverse transcriptase bent pseudoknots and novel rna inhibitors of type human immunodeficiency virus (hiv- ) reverse transcriptase neutralization of infectivity of diverse r clinical isolates of human immunodeficiency virus type by gp -binding 'f-rna aptamers in vitro selection of rna aptamers that bind the rna-dependent rna polymerase of hepatitis c virus: a possible role of gc-rich rna motifs in ns b binding isolation of inhibitory rna aptamers against severe acute respiratory syndrome (sars) coronavirus ntpase/helicase an efficient rna aptamer against human influenza b virus hemagglutinin mechanism of inhibition of hiv- integrase by g-tetrad-forming oligonucleotides in vitro structure-activity of tetrad-forming oligonucleotides as a potent anti-hiv therapeutic drug astier-gin, t. inhibition of hepatitis c virus (hcv) rna polymerase by dna aptamers: mechanism of inhibition of in vitro rna synthesis and effect on hcv-infected cells in vitro selection identifies key determinants for loop-loop interactions: rna aptamers selective for the tar rna element of hiv- endogenous expression of an anti-tar aptamer reduces hiv- replication bimodal loop-loop interactions increase the affinity of rna aptamers for hiv- rna structures apical loop-internal loop interactions: a new rna-rna recognition motif identified through in vitro selection against rna hairpins of the hepatitis c virus mrna rna aptamers targeted to domain ii of hepatitis c virus ires that bind to its apical loop region a hepatitis c virus (hcv) internal ribosome entry site (ires) domain iii-iv-targeted aptamer inhibits translation by binding to an apical loop of domain iiid increased inhibitory ability of conjugated rna aptamers against the hcv ires isolation and characterization of rna aptamers specific for the hcv minus-ires domain i berzal-herranz, a. interfering with hepatitis c virus ires activity using rna molecules identified by a novel in vitro selection method berzal-herranz, a. inhibition of hepatitis c virus internal ribosome entry site-mediated translation by an rna targeting the conserved iiif domain berzal-herranz, a. inhibition of hepatitis c virus replication and internal ribosome entry site-dependent translation by an rna molecule protective effects of anti-ricin a-chain rna aptamer against ricin toxicity in vitro selection of rna molecules that displace cocaine from the membranebound nicotinic acetylcholine receptor mechanism-based discovery of ligands that counteract inhibition of the nicotinic acetylcholine receptor by cocaine and mk- minimal rna aptamer sequences that can inhibit or alleviate noncompetitive inhibition of the muscle-type nicotinic acetylcholine receptor a tenascin-c aptamer identified by tumor cell selex: systematic evolution of ligands by exponential enrichment tumor targeting by an aptamer identification and characterization of nucleasestabilized rna molecules that bind human prostate cancer cells via the prostate-specific membrane antigen cell type-specific delivery of sirnas with aptamer-sirna chimeras cell-specific induction of apoptosis by rationally designed bivalent aptamer-sirna transcripts silencing eukaryotic elongation factor . curr. cancer drug targets an aptamer-doxorubicin physical conjugate as a novel targeted drug-delivery platform aptamer:toxin conjugates that specifically target prostate tumor cells targeted nanoparticle-aptamer bioconjugates for cancer chemotherapy in vivo formulation of functionalized plga-peg nanoparticles for in vivo targeted drug delivery targeted delivery of cisplatin to prostate cancer cells by aptamer functionalized pt(iv) prodrug-plga-peg nanoparticles novel dual inhibitory function aptamer-sirna delivery system for hiv- therapy selection, characterization and application of new rna hiv gp aptamers for facile delivery of dicer substrate sirnas into hiv infected cells the work of berzal-herranz's group is funded by grant bfu - from the spanish ministry of science and innovation, grant cts- from the junta de andalucía, and by feder funds from the e.u. key: cord- - trqex authors: dana, dibyendu; pathak, sanjai k. title: a review of small molecule inhibitors and functional probes of human cathepsin l date: - - journal: molecules doi: . /molecules sha: doc_id: cord_uid: trqex human cathepsin l belongs to the cathepsin family of proteolytic enzymes with primarily an endopeptidase activity. although its primary functions were originally thought to be only of a housekeeping enzyme that degraded intracellular and endocytosed proteins in lysosome, numerous recent studies suggest that it plays many critical and specific roles in diverse cellular settings. not surprisingly, the dysregulated function of cathepsin l has manifested itself in several human diseases, making it an attractive target for drug development. unfortunately, several redundant and isoform-specific functions have recently emerged, adding complexities to the drug discovery process. to address this, a series of chemical biology tools have been developed that helped define cathepsin l biology with exquisite precision in specific cellular contexts. this review elaborates on the recently developed small molecule inhibitors and probes of human cathepsin l, outlining their mechanisms of action, and describing their potential utilities in dissecting unknown function. lysosomes play critical roles in human biology receiving, trafficking, processing, and degrading biological molecules from seminal cellular processes, such as endocytosis, phagocytosis, autophagy and secretion. discovered by the ground-breaking work of de duve, these single-membrane enclosed cytosolic organelle maintain an acidic (~ . ph environment, and house close to sixty proteolytic enzymes [ , ] . among these are the eleven members of the cysteine cathepsin enzymes with a versatile expression and functional profile: cathepsin l (l ), b, c, f, h, k, o, v (l ), x, s, and w [ ] . these enzymes closely mimic the ca clan of the papain structure and catalytic cycle and mediate numerous crucial cellular events. for example, they participate in processes involving cell death, protein degradation, post-translational modifications of proteins, extracellular matrix (ecm) remodeling, autophagy, and immune signaling. given that their functions are aberrantly dysregulated in several human diseases, many are considered prime targets for therapeutic development [ ] . several elegant reviews have recently emerged describing the importance of cysteine cathepsins in both normal physiology and human diseases [ ] [ ] [ ] [ ] [ ] [ ] [ ] . the focus of this review is specifically on human cathepsin l, a ubiquitously expressed endopeptidase whose involvement in several human diseases has emerged in recent years. these include liver fibrosis, type i and ii diabetes, cardiac and bone, immune and kidney disorders [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] in addition, its role in a wide variety of highly invasive forms of cancer is the propeptides act as an important regulatory on/off switch as well as a folding catalyst in cathepsin activation. not surprisingly, the nature of propeptides among cysteine cathepsins is highly divergent by both chain lengths and primary sequences. it is thought that this uniqueness is functionally relevant given its ubiquitous presence in most tissues and allows for the selective suppression of enzyme activity (hence unintended autoactivation) during the transport to the the propeptides act as an important regulatory on/off switch as well as a folding catalyst in cathepsin activation. not surprisingly, the nature of propeptides among cysteine cathepsins is highly divergent by both chain lengths and primary sequences. it is thought that this uniqueness is functionally relevant given its ubiquitous presence in most tissues and allows for the selective suppression of enzyme activity (hence unintended autoactivation) during the transport to the endolysosomal compartment. molecules , , of in cathepsin l, two inhibitory propeptides, one containing amino acid (thr -glu ) and the other containing amino acid (glu -asp ) exist. a crystal structure of human procathepsin l revealed that the amino acid inhibitory propeptide chain spans in the opposite directions of substrate binding and forms several high-affinity non-covalent interactions with the surrounding residues in active site [ , ] . interestingly, this opposite direction binding of inhibitory propeptide segment is evolutionarily conserved in other members of cysteine cathepsins, including in cathepsin b. molecules , , of endolysosomal compartment. in cathepsin l, two inhibitory propeptides, one containing amino acid (thr -glu ) and the other containing amino acid (glu -asp ) exist. a crystal structure of human procathepsin l revealed that the amino acid inhibitory propeptide chain spans in the opposite directions of substrate binding and forms several high-affinity non-covalent interactions with the surrounding residues in active site [ , ] . interestingly, this opposite direction binding of inhibitory propeptide segment is evolutionarily conserved in other members of cysteine cathepsins, including in cathepsin b. biogenesis of human cathepsin l. after the full length cathepsin l mrna is transcribed, it is translated in ribosomes. following this, the full-length peptide enters the ribosomes-bound endoplasmic reticulum lumen where signal peptide is removed. pro-cathepsin l then enters the golgi network where it undergoes n-linked glycosylation at asn , followed by mannose phosphorylation and formation of appropriate disulfide linkages. in the last step, modified procathepsin l is shuttled to lysosome by endolysosomal pathways, generating the double chain form of active and mature human cathepsin l. the dominant pathway of regulation of activated and mature cathepsin l is by endogenous protein inhibitors, cystatins, that like propeptide compete with the physiological substrates for binding to the enzyme active site (table ) [ , ] . interestingly, protein inhibitory agents of cathepsin l have also been reported in other organisms. for example, kotsyfakis m. et al. reported the existence of two cathepsin l inhibitory proteins in the carrier of the main vector of lyme disease-carrying parasite, ixodus scapularis. named so because of its abilities to specifically inhibit cathepsin l (ic = . nm; ki = pm) activity, sialostatin l abrogates the protective proteolytic activity of host cells at the infestation sites, thereby promoting the tick's survival [ ] . in addition, it also possess a potent anti-inflammatory and immunosuppressive activity by inhibiting cytotoxic killer t cells [ ] . after the full length cathepsin l mrna is transcribed, it is translated in ribosomes. following this, the full-length peptide enters the ribosomes-bound endoplasmic reticulum lumen where signal peptide is removed. pro-cathepsin l then enters the golgi network where it undergoes n-linked glycosylation at asn , followed by mannose phosphorylation and formation of appropriate disulfide linkages. in the last step, modified procathepsin l is shuttled to lysosome by endolysosomal pathways, generating the double chain form of active and mature human cathepsin l. the dominant pathway of regulation of activated and mature cathepsin l is by endogenous protein inhibitors, cystatins, that like propeptide compete with the physiological substrates for binding to the enzyme active site (table ) [ , ] . interestingly, protein inhibitory agents of cathepsin l have also been reported in other organisms. for example, kotsyfakis m. et al. reported the existence of two cathepsin l inhibitory proteins in the carrier of the main vector of lyme disease-carrying parasite, ixodus scapularis. named so because of its abilities to specifically inhibit cathepsin l (ic = . nm; k i = pm) activity, sialostatin l abrogates the protective proteolytic activity of host cells at the infestation sites, thereby promoting the tick's survival [ ] . in addition, it also possess a potent anti-inflammatory and immunosuppressive activity by inhibiting cytotoxic killer t cells [ ] . table . reported physiological inhibitory ligands of human cathepsin l with their inhibition constants. over the years, several classes of physiological and synthetic inhibitors have been discovered targeting cathepsin l. herein, the focus is on non-physiological inhibitors that could broadly be classified as reversible and irreversible inhibitors. reversible and irreversible inhibitor could be distinguished by means of the mechanistic approaches they utilize for enzyme inactivation. reversible inhibitors 'generally' engage with the target protein using non-covalent interaction; however, in certain cases, they form a quasi-covalent bond which eventually disengages from the active site, say, upon dilution. irreversible inhibitors, on the other hand, permanently modify the protein of interest via the formation of a stable covalent bond. while both reversible and irreversible inhibitors have their pros and cons, it is generally believed that reversible inhibitors are preferred candidates in drug discovery and irreversible inhibitors not so due to their adverse immune responses [ ] . there are, however, several examples of successful drugs being used in clinics that work by an irreversible inhibitory mechanism [ ] . irreversible inhibitors are also widely utilized in functional biology (e.g., in the development of activity-based probes). the majority of synthetic cathepsin l inhibitors have recognizable peptide sequences, often derived from its physiological substrates, that bind to the active site of the protein and often contain strategically placed electrophilic warheads that trap the nucleophilic cys residue (figure ) for activity. knowledge of enzyme structure and its associated substrate specificity thus has played an important role in designing selective inhibitors of cathepsins [ , ] . in a seminal study, choe et al. studied substrate specificity using a highly diversified positional scanning synthetic combinatorial library comprised of , fluorogenic tetrapeptides; this allowed to differentiate individual enzymes binding propensity based on their distinct amino acid preferences [ ] . by capitalizing on this strategy, they successfully developed a selective substrate and substrate-based inhibitor of cathepsin k. the other important contribution to our understanding of subsite binding preferences of cathepsin l enzyme stems from several timely crystal structure studies that helped reveal key difference in the structural landscape of enzyme subsites [ ] [ ] [ ] . in an important study, shenoy et al. solved the crystal structure of ligand bound cathepsin l and documented the structural compositions of ligand binding sites ( figure ) [ ] . their analysis of z-phe-tyr (o-tert-butyl)-c(o)c(h)o bound cathepsin l revealed that (a) s subsite is relatively wide and unrestricted and composed of asp , ser , and cys , (b) s subsite is guided by leu , trp , ala and gly where trp associates with trp and phe and forms an aromatic cluster that accommodates the tert-butyl group (c) side chains of leu and met help form the s subsite that engages in non-polar interactions with the phenyl side chain, and finally, (d) the carboxybenzyl group finds interaction with the gly residue of s subsite ( figure ). however, it turns out that different ligands may find slightly altered binding interactions with the enzyme subsites, depending on their structural features. for example, shenoy et al. showed that although z-phe-tyr (o-tert-butyl)-dmk binds to the same subsites of cathepsin l as observed for the z-phe-tyr (o-tert-butyl)-c(o)c(h)o ligand and finds some alternative interactions with residues from the active site pocket. this finding has been corroborated by other studies as well that suggest that structural features of ligands can influence the subsite composition of the cathepsin l enzyme [ , , , ] . such information has been duly capitalized to develop different classes of inhibitors that are discussed next. in the following section, we discuss the types of cathepsin l-targeting chemotypes that have been utilized for development of small molecule inhibitors. available universal cathepsin inhibitor of this class, showed only a marginal selectivity towards cathepsin l compared to cathepsin b and a moderate -fold selectivity over cathepsin h, as reported by barrett et al. [ ] . although they developed several synthetic analogs of e- with improved potency, they lacked desirable selectivity toward cathepsin l. they proposed the probable binding orientation of e- that follows a non-substrate-like orientation, i.e., it occupies only non-prime sites ( figure ) at the enzyme pocket. phe arg cbz amc sh s s ' s ' s s the key subsite-forming amino acid residues with their corresponding numbers are shown in blue whereas the catalytic diad forming residues are depicted in red. (the authors used pdb: of file to construct the figure [ ] ). in another study, gour-salin et al. reported a number of epoxysuccinyl amino acid benzyl esters in which they systematically varied the amino acid attached to the epoxide ring. this was intended to investigate its effect in determining selectivity toward cathepsin l or s [ ] . their results surprisingly indicate that the specificity of these analogs did not follow the trend, generally observed for substrate, possibly due to e- like binding orientation at the enzyme pocket. among all cathepsin l compared to cathepsin b and a moderate -fold selectivity over cathepsin h, as reported by barrett et al. [ ] . although they developed several synthetic analogs of e- with improved potency, they lacked desirable selectivity toward cathepsin l. they proposed the probable binding orientation of e- that follows a non-substrate-like orientation, i.e., it occupies only non-prime sites ( figure ) at the enzyme pocket. phe arg cbz amc sh s s ' s ' s s the key subsite-forming amino acid residues with their corresponding numbers are shown in blue whereas the catalytic diad forming residues are depicted in red. (the authors used pdb: of file to construct the figure [ ] ). in another study, gour-salin et al. reported a number of epoxysuccinyl amino acid benzyl esters in which they systematically varied the amino acid attached to the epoxide ring. this was intended to investigate its effect in determining selectivity toward cathepsin l or s [ ] . their results surprisingly indicate that the specificity of these analogs did not follow the trend, generally observed for substrate, possibly due to e- like binding orientation at the enzyme pocket. among all the key subsite-forming amino acid residues with their corresponding numbers are shown in blue whereas the catalytic diad forming residues are depicted in red. (the authors used pdb: of file to construct the figure [ ] ). epoxysuccinate inhibitors have historically played a crucial role in deciphering the cysteine protease biology. this class of compounds contains an epoxide ring as an electrophilic warhead that traps the active site catalytic cysteine residue of the protein. e- (l-trans-epoxysuccinyl-leucylamido( -guanidino)butane) (entry ; table ), perhaps the most studied commercially available universal cathepsin inhibitor of this class, showed only a marginal selectivity towards cathepsin l compared to cathepsin b and a moderate -fold selectivity over cathepsin h, as reported by barrett et al. [ ] . although they developed several synthetic analogs of e- with improved potency, they lacked desirable selectivity toward cathepsin l. they proposed the probable binding orientation of e- that follows a non-substrate-like orientation, i.e., it occupies only non-prime sites ( figure ) at the enzyme pocket. in another study, gour-salin et al. reported a number of epoxysuccinyl amino acid benzyl esters in which they systematically varied the amino acid attached to the epoxide ring. this was intended to investigate its effect in determining selectivity toward cathepsin l or s [ ] . their results surprisingly indicate that the specificity of these analogs did not follow the trend, generally observed for substrate, possibly due to e- like binding orientation at the enzyme pocket. among all synthesized compounds, katunuma: clik) with the help of computational modeling based on the stereo-structure [ ] . three of the developed clik inhibitors were hydrolytically stable and showed highly selective inhibition for hepatic cathepsin l in vivo. further, they elucidated the inhibition mechanism of this class of compounds based on the crystal structure of papain-clik (entry , table ) complex [ ] . this crystal structure revealed that clik , unlike e- , binds to both prime and non-prime sites of the active site pocket. notably, the specificity toward cathepsin l was attributed to the existence of phenylalanine residue at the s site, and a hydrophobic interaction mediated by n-terminal pyridine ring ( figure ). the other classes of alkylating agents that initially played an important role in deciphering the mechanistic aspects of cathepsin inhibition were peptidyldiazomethane (entry , table ) and peptidylchloromethane (entry , table ). crawford et al. reported highly potent inhibitors from these two chemotypes that spans the active site and shows significant selectivity improvement over other cysteine proteases [ ] . however, these classes of inhibitors suffer from stability issues and have found limited utility in in vivo assays. peptidylhydroxylamines were first introduced as mechanism-based inhibitors of serine and cysteine proteinases [ ] [ ] [ ] [ ] . bromme et al. adapted this scaffold and prepared a library of n-peptidyl-o-acyl hydroxylamines which exhibited rapid and selective inactivation of several lysosomal cysteine proteinases [ ] . this class of inhibitors occupied the active site of the enzyme and irreversibly inactivated cysteine cathepsins as the free enzyme activity was not recovered when the enzyme-inhibitor complex was exposed to exhaustive ultrafiltration (up to an enzyme/free inhibitor ratio of < : . ) or chromatography on sephadex g- . among developed inhibitors, z-phe-phe-nho-ma (entry , table ) inhibited cathepsin l with most potency and showed significant selectivity, -fold and -fold, over cathepsin s and cathepsin b, respectively. interestingly the stability and efficacy of this class of inhibitors were determined by the nature of substitutions on hydroxylamine oxygen as their electron-withdrawing tendencies showed a positive correlation with inactivation kinetics. although this class of inhibitors exhibited desirable traits with respect to both potency and selectivity, they suffered majorly from aqueous stability issue; half-lives (t / ) only in the range of min in aqueous solution. in another study, bromme et al. developed a series of n-peptidyl-o-acyl hydroxamates with lysine in p position with improved inhibitory profile for cysteine proteases over their serine counterparts [ ] . the maximum inhibition was observed by z-phe-lys-nho-nbz (entry , table ), with -fold selectivity over cathepsin s, and > -fold selectivity over cathepsin b. the authors postulated that the active site cys residue attacks the carbonyl of the hydroxamate and forms a tetrahedral intermediate, whereas the nitrogen of the hydroxamate, which primarily remains deprotonated (pka < ), presumably engages in an electrostatic interaction with the active site his residue ( figure ). inhibitors further provided the option of varying the leaving group that targets s ′ site; more hydrophobic substituents were preferred at this position as comparison of ki/ki values of the inactivation exhibited the following trend for aa: gly < ala < val < leu < phe < -no -ph. the nitrophenyl analog (entry , table ) which incur both hydrophobicity and electron-withdrawing property exerted the maximum potency and selectivity for cathepsin l over other tested cysteine proteases. hydrophobic substituents were preferred at this position as comparison of ki/ki values of the inactivation exhibited the following trend for aa: gly < ala < val < leu < phe < -no -ph. the nitrophenyl analog (entry , table ) which incur both hydrophobicity and electron-withdrawing property exerted the maximum potency and selectivity for cathepsin l over other tested cysteine proteases. hydrophobic substituents were preferred at this position as comparison of ki/ki values of the inactivation exhibited the following trend for aa: gly < ala < val < leu < phe < -no -ph. the nitrophenyl analog (entry , table ) which incur both hydrophobicity and electron-withdrawing property exerted the maximum potency and selectivity for cathepsin l over other tested cysteine proteases. in a follow-up study, bromme et al. synthesized and tested a series of new inhibitors, with the general formula of z-phe-gly-nho-co-aa (aa: amino acid), against papain class of enzymes [ ] . this class of inhibitors covalently modified active-site cys residue via sulfenamidation, like in their n-peptidyl-o-acyl hydroxamate counterparts, as shown by the mass spectrometric analysis. these inhibitors further provided the option of varying the leaving group that targets s ′ site; more hydrophobic substituents were preferred at this position as comparison of ki/ki values of the inactivation exhibited the following trend for aa: gly < ala < val < leu < phe < -no -ph. the nitrophenyl analog (entry , table ) which incur both hydrophobicity and electron-withdrawing property exerted the maximum potency and selectivity for cathepsin l over other tested cysteine proteases. in a follow-up study, bromme et al. synthesized and tested a series of new inhibitors, with the general formula of z-phe-gly-nho-co-aa (aa: amino acid), against papain class of enzymes [ ] . this class of inhibitors covalently modified active-site cys residue via sulfenamidation, like in their n-peptidyl-o-acyl hydroxamate counterparts, as shown by the mass spectrometric analysis. these inhibitors further provided the option of varying the leaving group that targets s site; more hydrophobic substituents were preferred at this position as comparison of k i /k i values of the inactivation exhibited the following trend for aa: gly < ala < val < leu < phe < -no -ph. the nitrophenyl analog (entry , table ) which incur both hydrophobicity and electron-withdrawing property exerted the maximum potency and selectivity for cathepsin l over other tested cysteine proteases. another class of peptidyl inhibitors that spans the active site and utilizes the catalytic machinery of cathepsin l for effective attenuation of enzyme activity is peptidyl acyloxymethanes. krantz et al. developed a library of inhibitory compounds with a general sequence, z-phe-x-ch oco-r; here they systematically varied the amino acid residue at p (denoted as x) and p (denoted as r) positions [ ] . among the synthesized compounds, z-phe-cys(sbn)-ch oco- , -(cf ) -ph (entry , table ) exhibited almost a diffusion-controlled inactivation kinetics toward cathepsin l, however it showed only marginal selectivity over cathepsin b and cathepsin s. by analyzing the structure-activity relationships of the library, the author elucidated the importance of s site in cathepsin l that could potentially be used to harness selectivity among other cysteine cathepsins. in a separate study, torkar et al. designed a library of peptides for cathepsin l that spanned the active site and attenuated the enzyme activity via a non-covalent interaction [ ] . they initially evaluated the compounds' activities against cathepsin l and cathepsin b, and compared their hits against cathepsin k and s. the authors discovered five most selective non-covalent, peptidyl inhibitors of cathepsin l, and transformed them into irreversible inhibitors by strategically appending electrophilic warhead ( figure )-acyloxymethyl ketone (aomk) groups (entry , table ). however, the attachment of the aomk group drastically impacted the selectivity profiles of these inhibitors, suggesting the importance of the adjuvant effect of prime site targeting in determining the efficacy and selectivity of this class of compounds. this class of inhibitors found wide-spread utilities in detecting protease activity and were utilized to develop activity-based probe; this will be discussed in the later sections. a very interesting class of inhibitors, the concept of which was derived from e- , is peptidyl aziridines. martichonok et al. developed a series of aziridine derivatives of e- and tested them against papain and cathepsin l and b [ ] . contrarily to e- , in which (l)-diastereomer is more potent than (d)-isomer, aziridine analogs exhibited the opposite trend while still inactivating the enzymes. more importantly, the efficacy of this class of inhibitors was strongly ph-dependent and showed maximal inhibitory potency at ph ; this is attributed to the protonated form of aziridine ring that is more susceptible to nucleophilic attack by catalytic cys residue. among the developed library, ho-(d)-az-leu-nh-iam (entry , table ) analog exhibited maximal inhibitory potency towards cathepsin l with moderate selectivity over cathepsin b; the corresponding l isomer, ho-(l)-az-leu-nh-iam (entry ) was almost -fold less activity while maintaining the similar trend in general. since only protonated form of aziridine ring undergoes nucleophilic attack, presumably it does not involve water molecule-mediated ring-opening like its epoxide counterpart e- . although promising, a full potential of this class of compounds could presumably be achieved only below ph when aziridine ring nitrogen gets completely protonated. this class of inhibitors thus lacks practical utility as the catalytic cysteine of cathepsin l starts to lose its activity below ph and the majority of the cell-based assays are performed mostly at a ph higher than . . in a notable study, schirmeister et al. developed three different classes of inhibitors with aziridine- , -dicarboxylic acid (azi), an electrophilic warhead, installed at different positions of the peptide chain ( figure ) [ ] . they performed a thorough sar analysis of this type of inhibitors, all off which exhibited time-dependent irreversible inactivation of cathepsin l with no-recovery of enzyme activity, even after extensive dialysis of the enzyme-inhibitor complex. among type-i inhibitors, n-acylated aziridines with aziridine as c-terminal amino acid, a mixture of diastereomeric peptides with procathepsin b sequence leu-gly-gly (entry , table ), exhibited enhanced inhibition toward cathepsin l. this was attributed to an overall unique folding of cathepsin l with a shallowness of the s pocket due to the presence of an additional met residue. type ii class of inhibitors ( figure ) resemble classic aziridine scaffold, n-unsubstituted aziridines with aziridine as n-terminal amino acid, analogously to e- where nitrogen of aziridine remained unsubstituted. among the type ii inhibitors tested, eto-(r,r)-azi-leu-obzl (entry , table ) inactivated cathepsin l with higher second-order rate constant than eto-(s,s)-azi-leu-obzl, although the latter showed better selectivity over cathepsin b. a very interesting class of inhibitors, the concept of which was derived from e- , is peptidyl aziridines. martichonok et al. developed a series of aziridine derivatives of e- and tested them against papain and cathepsin l and b [ ] . contrarily to e- , in which (l)-diastereomer is more potent than (d)-isomer, aziridine analogs exhibited the opposite trend while still inactivating the enzymes. more importantly, the efficacy of this class of inhibitors was strongly ph-dependent and showed maximal inhibitory potency at ph ; this is attributed to the protonated form of aziridine ring that is more susceptible to nucleophilic attack by catalytic cys residue. among the developed library, ho-(d)-az-leu-nh-iam (entry , table ) analog exhibited maximal inhibitory potency towards cathepsin l with moderate selectivity over cathepsin b; the corresponding l isomer, ho-(l)-az-leu-nh-iam (entry ) was almost -fold less activity while maintaining the similar trend in general. since only protonated form of aziridine ring undergoes nucleophilic attack, presumably it does not involve water molecule-mediated ring-opening like its epoxide counterpart e- . although promising, a full potential of this class of compounds could presumably be achieved only below ph when aziridine ring nitrogen gets completely protonated. this class of inhibitors thus lacks practical utility as the catalytic cysteine of cathepsin l starts to lose its activity below ph and the majority of the cell-based assays are performed mostly at a ph higher than . . in a notable study, schirmeister et al. developed three different classes of inhibitors with aziridine- , -dicarboxylic acid (azi), an electrophilic warhead, installed at different positions of the peptide chain ( figure ) [ ] . they performed a thorough sar analysis of this type of inhibitors, all off which exhibited time-dependent irreversible inactivation of cathepsin l with no-recovery of enzyme activity, even after extensive dialysis of the enzyme-inhibitor complex. among type-i inhibitors, n-acylated aziridines with aziridine as c-terminal amino acid, a mixture of diastereomeric peptides with procathepsin b sequence leu-gly-gly (entry , table ), exhibited enhanced inhibition toward cathepsin l. this was attributed to an overall unique folding of cathepsin l with a shallowness of the s pocket due to the presence of an additional met residue. type ii class of inhibitors ( figure ) resemble classic aziridine scaffold, n-unsubstituted aziridines with aziridine as n-terminal amino acid, analogously to e- where nitrogen of aziridine remained unsubstituted. among the type ii inhibitors tested, eto-(r,r)-azi-leu-obzl (entry , table ) inactivated cathepsin l with higher second-order rate constant than eto-(s,s)-azi-leu-obzl, although the latter showed better selectivity over cathepsin b. the type iii inhibitor class (figure ) is comprised of n-acylated bispeptidyl derivatives of aziridine, where aziridine ring rests in the middle of the peptide. boc-phe-(r,r)-(eto)-azi-leu-pro-obzl (entry , table ) of this series was -fold more potent than the (s,s) analog; however both exhibited only a marginal selectivity over cathepsin b with diminished eudysmic ratio. the authors then superimposed and analyzed the structures of certain epoxide and aziridines and postulated that these inhibitors can assume different orientations in the active site while still binding within the enzyme pockets. this scaffold was further explored by vicik et al. who extended the previous work and developed a series of compounds in which boc-(s)-leu-(s)-azy-(s,s)-azi(obn) (figure ), type i analog, spanned from s to s pocket and inactivated cathepsin l with more than -fold selectivity over cathepsin b [ ] . this motif was also used for affinity labeling of cathepsin l, which will be discussed in the later sections. these classes of compounds, especially n-unsubstituted aziridinyl peptides and in special cases n-acylated ones, exhibited a high selectivity and potency and provided the premise for the further development of chemical biology tools much needed for functional studies. table ) of this series was -fold more potent than the (s,s) analog; however both exhibited only a marginal selectivity over cathepsin b with diminished eudysmic ratio. the authors then superimposed and analyzed the structures of certain epoxide and aziridines and postulated that these inhibitors can assume different orientations in the active site while still binding within the enzyme pockets. this scaffold was further explored by vicik et al. who extended the previous work and developed a series of compounds in which boc-(s)-leu-(s)-azy-(s,s)-azi(obn) ( figure ), type i analog, spanned from s to s ′ pocket and inactivated cathepsin l with more than -fold selectivity over cathepsin b [ ] . this motif was also used for affinity labeling of cathepsin l, which will be discussed in the later sections. these classes of compounds, especially n-unsubstituted aziridinyl peptides and in special cases n-acylated ones, exhibited a high selectivity and potency and provided the premise for the further development of chemical biology tools much needed for functional studies. another promising scaffold that acts as a michael acceptor and hijacks the catalytic residue of cysteine cathepsins is peptidyl aryl vinylsulfones. this scaffold was first introduced by palmer et al. as an irreversible inhibitor of cysteine cathepsins [ ] . subsequently, they extended their study by performing an sar analysis of this class of inhibitors which showed pan-cathepsin inhibition with table ) of this series was -fold more potent than the (s,s) analog; however both exhibited only a marginal selectivity over cathepsin b with diminished eudysmic ratio. the authors then superimposed and analyzed the structures of certain epoxide and aziridines and postulated that these inhibitors can assume different orientations in the active site while still binding within the enzyme pockets. this scaffold was further explored by vicik et al. who extended the previous work and developed a series of compounds in which boc-(s)-leu-(s)-azy-(s,s)-azi(obn) ( figure ), type i analog, spanned from s to s ′ pocket and inactivated cathepsin l with more than -fold selectivity over cathepsin b [ ] . this motif was also used for affinity labeling of cathepsin l, which will be discussed in the later sections. these classes of compounds, especially n-unsubstituted aziridinyl peptides and in special cases n-acylated ones, exhibited a high selectivity and potency and provided the premise for the further development of chemical biology tools much needed for functional studies. another promising scaffold that acts as a michael acceptor and hijacks the catalytic residue of cysteine cathepsins is peptidyl aryl vinylsulfones. this scaffold was first introduced by palmer et al. as an irreversible inhibitor of cysteine cathepsins [ ] . subsequently, they extended their study by performing an sar analysis of this class of inhibitors which showed pan-cathepsin inhibition with occasional selectivity towards cathepsin s for certain scaffolds [ ] . however, in a separate study by mendieta et al., the authors developed a structurally novel library of twenty peptidyl -aryl vinylsulfones ( figure ) in which they introduced extensive diversity at the r position. subsequently, they also varied the r position while keeping either morpholine or n-methyl piperazine group intact [ ] . docking studies with most active and selective inhibitor (entry , another promising scaffold that acts as a michael acceptor and hijacks the catalytic residue of cysteine cathepsins is peptidyl aryl vinylsulfones. this scaffold was first introduced by palmer et al. as an irreversible inhibitor of cysteine cathepsins [ ] . subsequently, they extended their study by performing an sar analysis of this class of inhibitors which showed pan-cathepsin inhibition with occasional selectivity towards cathepsin s for certain scaffolds [ ] . however, in a separate study by mendieta et al., the authors developed a structurally novel library of twenty peptidyl -aryl vinylsulfones ( figure ) in which they introduced extensive diversity at the r position. subsequently, they also varied the r position while keeping either morpholine or n-methyl piperazine group intact [ ] . docking studies with most active and selective inhibitor (entry , table ) of this class revealed that the inhibitor extends from s to s sites of cathepsin l and the β-vinylsulfone moiety resides in a close proximity of cys- residue thereby favoring the formation of michael adduct. the authors postulated that considering the efficacy of peptidyl aryl vinyl sulfones, strong anti-cancer candidates could be harnessed by cultivating this scaffold. table ) of this class revealed that the inhibitor extends from s to s ′ sites of cathepsin l and the βvinylsulfone moiety resides in a close proximity of cys- residue thereby favoring the formation of michael adduct. the authors postulated that considering the efficacy of peptidyl aryl vinyl sulfones, strong anti-cancer candidates could be harnessed by cultivating this scaffold. one other very potent class of inhibitors that also includes a michael acceptor is peptidyl aryl vinylsulfonate esters, a superior michael acceptor than vinyl sulfone. they served as potent inhibitors of cruzain-a parasitic cysteine protease from t. cruzi that is homologous to cathepsin l [ , ] . this scaffold was explored by dana et al., who determined the superiority of aryl vinylsulfonate ester over aryl vinylsulfone and aryl vinylsulfonamide counterparts towards cathepsin l inhibition [ ] . thus, they synthesized and screened the efficacy of a library of aryl vinylsulfonate ester compounds against cathepsin l; -bromo phenyl vinylsulfonate was found to be the champion ligand presumably due to favorable interactions between the -bromo phenyl moiety with the prime site residues of cathepsin l. they further designed a hybrid inhibitor, kd- , (entry , table ) by strategically appending the -bromophenyl vinylsulfonate moiety as electrophilic warhead to a modestly potent reversible cathepsin l inhibitor [ ] ; this design was based on the hypothesis that the developed compound will target both the prime site and the non-prime site residues for interaction. kd- indeed exhibited almost a diffusion-controlled inactivation kinetics while maintaining an excellent selectivity profile toward cathepsin l ( figure ). furthermore, kd- was cell-permeable and inhibited the intracellular activity of cathepsin l in human mda-mb- breast cancer cell lines. kd- also enhanced the integrity of cell-cell junctions by effectively attenuating the migratory potential of the cells, as demonstrated by the scratch assay. the authors anticipated that this class of inhibitors may find extensive usage in deciphering context-specific cathepsin l biology. one other very potent class of inhibitors that also includes a michael acceptor is peptidyl aryl vinylsulfonate esters, a superior michael acceptor than vinyl sulfone. they served as potent inhibitors of cruzain-a parasitic cysteine protease from t. cruzi that is homologous to cathepsin l [ , ] . this scaffold was explored by dana et al., who determined the superiority of aryl vinylsulfonate ester over aryl vinylsulfone and aryl vinylsulfonamide counterparts towards cathepsin l inhibition [ ] . thus, they synthesized and screened the efficacy of a library of aryl vinylsulfonate ester compounds against cathepsin l; -bromo phenyl vinylsulfonate was found to be the champion ligand presumably due to favorable interactions between the -bromo phenyl moiety with the prime site residues of cathepsin l. they further designed a hybrid inhibitor, kd- , (entry , table ) by strategically appending the -bromophenyl vinylsulfonate moiety as electrophilic warhead to a modestly potent reversible cathepsin l inhibitor [ ] ; this design was based on the hypothesis that the developed compound will target both the prime site and the non-prime site residues for interaction. kd- indeed exhibited almost a diffusion-controlled inactivation kinetics while maintaining an excellent selectivity profile toward cathepsin l ( figure ). furthermore, kd- was cell-permeable and inhibited the intracellular activity of cathepsin l in human mda-mb- breast cancer cell lines. kd- also enhanced the integrity of cell-cell junctions by effectively attenuating the migratory potential of the cells, as demonstrated by the scratch assay. the authors anticipated that this class of inhibitors may find extensive usage in deciphering context-specific cathepsin l biology. molecules , , of table ) of this class revealed that the inhibitor extends from s to s ′ sites of cathepsin l and the βvinylsulfone moiety resides in a close proximity of cys- residue thereby favoring the formation of michael adduct. the authors postulated that considering the efficacy of peptidyl aryl vinyl sulfones, strong anti-cancer candidates could be harnessed by cultivating this scaffold. one other very potent class of inhibitors that also includes a michael acceptor is peptidyl aryl vinylsulfonate esters, a superior michael acceptor than vinyl sulfone. they served as potent inhibitors of cruzain-a parasitic cysteine protease from t. cruzi that is homologous to cathepsin l [ , ] . this scaffold was explored by dana et al., who determined the superiority of aryl vinylsulfonate ester over aryl vinylsulfone and aryl vinylsulfonamide counterparts towards cathepsin l inhibition [ ] . thus, they synthesized and screened the efficacy of a library of aryl vinylsulfonate ester compounds against cathepsin l; -bromo phenyl vinylsulfonate was found to be the champion ligand presumably due to favorable interactions between the -bromo phenyl moiety with the prime site residues of cathepsin l. they further designed a hybrid inhibitor, kd- , (entry , table ) by strategically appending the -bromophenyl vinylsulfonate moiety as electrophilic warhead to a modestly potent reversible cathepsin l inhibitor [ ] ; this design was based on the hypothesis that the developed compound will target both the prime site and the non-prime site residues for interaction. kd- indeed exhibited almost a diffusion-controlled inactivation kinetics while maintaining an excellent selectivity profile toward cathepsin l ( figure ). furthermore, kd- was cell-permeable and inhibited the intracellular activity of cathepsin l in human mda-mb- breast cancer cell lines. kd- also enhanced the integrity of cell-cell junctions by effectively attenuating the migratory potential of the cells, as demonstrated by the scratch assay. the authors anticipated that this class of inhibitors may find extensive usage in deciphering context-specific cathepsin l biology. recently, another interesting discovery that provided a wealth of information on cathepsin l-inhibitor interactions came from marine cyanobacterial extracts. miller et al. first reported gallinamide a as potent irreversible inhibitor of cathepsin l with an ic value of nm and a -to -fold greater selectivity over cathepsin v and b, respectively [ ] . they further performed molecular docking and molecular dynamics simulations and learned that the peptidyl backbone of the inhibitor spans the active site whereas the side chains engage in favorable interactions with different active site pockets, placing the michael acceptor enamide in close proximity to the catalytic cys residue. in a follow-up study, boudreau et al. performed molecular docking studies to predict the potential modifications of a gallinamide a scaffold that would harness favorable enzyme-inhibitor interactions and enable the development of compounds with improved inhibitory efficacy [ ] . they synthesized a panel of compounds by retaining gallinamide a and only varying the amino acids at p , p , and p positions. (figure ). this led to the discovery of the most potent analog of this series (entry , table ) with sub-nanomolar ic value ( pm) and fast time dependent inactivation kinetics, suggesting an improved binding and reactivity of the inhibitor with the enzyme active site. the authors found that this class of compounds effectively inactivated cruzain, a homologous cysteine protease from t. cruzi, using cell-based assay. gallinamide a and its analogs thus provide a remarkable inhibitory scaffold that could potentially be harnessed to build selective enzyme inhibitors for a variety of therapeutic applications. recently, another interesting discovery that provided a wealth of information on cathepsin linhibitor interactions came from marine cyanobacterial extracts. miller et al. first reported gallinamide a as potent irreversible inhibitor of cathepsin l with an ic value of nm and a -to -fold greater selectivity over cathepsin v and b, respectively [ ] . they further performed molecular docking and molecular dynamics simulations and learned that the peptidyl backbone of the inhibitor spans the active site whereas the side chains engage in favorable interactions with different active site pockets, placing the michael acceptor enamide in close proximity to the catalytic cys residue. in a follow-up study, boudreau et al. performed molecular docking studies to predict the potential modifications of a gallinamide a scaffold that would harness favorable enzymeinhibitor interactions and enable the development of compounds with improved inhibitory efficacy [ ] . they synthesized a panel of compounds by retaining gallinamide a and only varying the amino acids at p , p ′, and p ′ positions. (figure ). this led to the discovery of the most potent analog of this series (entry , table ) with sub-nanomolar ic value ( pm) and fast time dependent inactivation kinetics, suggesting an improved binding and reactivity of the inhibitor with the enzyme active site. the authors found that this class of compounds effectively inactivated cruzain, a homologous cysteine protease from t. cruzi, using cell-based assay. gallinamide a and its analogs thus provide a remarkable inhibitory scaffold that could potentially be harnessed to build selective enzyme inhibitors for a variety of therapeutic applications. figure . gallinamide was reconstructed with altered amino acid sequence at p , p ′, and p ′ positions. the acrylic group, the michael acceptor, is shown in red. one classical inhibitor that has been used over a long period of time to dissect cysteine proteinase activity is leupeptin, a microbial product. leupeptin is a peptidyl aldehyde that occupies the active site cleft of cysteine cathepsins and forms a thiohemiacetal intermediate by trapping catalytic cysteine residue; this complex hydrolyzes over time, thus showing the covalent and reversible nature of the inhibitor (figure a ). leupeptin unfortunately suffers from non-specific inhibition of both serine and cysteine proteinases, thus making it unfavorable for clinical usage and chemical biology applications. this issue was, however, addressed by woo et al. who designed and synthesized six peptidyl aldehyde analogs that were more potent than leupeptin (ic = . nm) and exhibited improved selectivity towards cathepsin l over cathepsin b and calpain ii [ ] . the most potent cathepsin l inhibitor of this series was z-phe-phe-h (ic = . nm) (figure b ) that showed more than -fold selectivity over cathepsin b. interestingly; their data demonstrated the importance of aromatic amino acids, such as phenylalanine and tyrosine, at the p position in determining the potency and selectivity towards cathepsin l; o-alkylation of tyrosine group diminishes the inhibitory efficiency as in z-phe-tyr(bu)-h (ic : . nm). in a follow-up publication, they further tested the efficacy of z-phe-tyr-h (ic : . nm, -fold selective over cathepsin b) (figure b ) in vitro and in vivo [ ] . this compound effectively inhibited parathyroid hormone-stimulated osteoclastic bone resorption in pit formation assays, and suppressed bone weight loss of ovariectomized mouse in a dose-dependent manner when administered intraperitoneally. figure . gallinamide was reconstructed with altered amino acid sequence at p , p , and p positions. the acrylic group, the michael acceptor, is shown in red. one classical inhibitor that has been used over a long period of time to dissect cysteine proteinase activity is leupeptin, a microbial product. leupeptin is a peptidyl aldehyde that occupies the active site cleft of cysteine cathepsins and forms a thiohemiacetal intermediate by trapping catalytic cysteine residue; this complex hydrolyzes over time, thus showing the covalent and reversible nature of the inhibitor (figure a ). leupeptin unfortunately suffers from non-specific inhibition of both serine and cysteine proteinases, thus making it unfavorable for clinical usage and chemical biology applications. this issue was, however, addressed by woo et al. who designed and synthesized six peptidyl aldehyde analogs that were more potent than leupeptin (ic = . nm) and exhibited improved selectivity towards cathepsin l over cathepsin b and calpain ii [ ] . the most potent cathepsin l inhibitor of this series was z-phe-phe-h (ic = . nm) (figure b ) that showed more than -fold selectivity over cathepsin b. interestingly; their data demonstrated the importance of aromatic amino acids, such as phenylalanine and tyrosine, at the p position in determining the potency and selectivity towards cathepsin l; o-alkylation of tyrosine group diminishes the inhibitory efficiency as in z-phe-tyr(bu)-h (ic : . nm). in a follow-up publication, they further tested the efficacy of z-phe-tyr-h (ic : . nm, -fold selective over cathepsin b) (figure b ) in vitro and in vivo [ ] . this compound effectively inhibited parathyroid hormone-stimulated osteoclastic bone resorption in pit formation assays, and suppressed bone weight loss of ovariectomized mouse in a dose-dependent manner when administered intraperitoneally. in another interesting study, yasuma et al. developed a library of compounds by varying the amino acid substituents at p , p , p position of the inhibitor and carried out a thorough sar study [ ] . their study revealed that the configuration of the stereogenic center (s-configuration is favored over r-configuration) at the p position, and not the steric factor, was key to the inhibitory efficacy. apparently, the substituent at p position does not interact with s position residues; rather, proper stereogenicity allows the placement of the inhibitor in vicinity of the catalytic cysteine residue for interaction. s subsite of cathepsin l, on the other hand, preferred a hydrophobic and moderate-size group; α-branched alkyl chains but not the bulkier groups like phenylalanine was favorable. further, the s subsite showed a preference for hydrophobic and bulky moieties such as -and naphthalenylsulfonyl substituents. among synthesized compound, n-( -naphthalenylsulfonyl-lisoleucyl-l-tryptophanal (ic = . nm, -fold selective over cathepsin b; figure ) attenuated the release of ca + and hydroxyproline from bone in an in vitro bone culture system and further restricted bone loss in ovariectomized mice dosed orally. a further modification of this scaffold was reported by lynas et al. [ ] here, authors designed and developed di-and tri-peptidyl α-keto-β-aldehydes, based on substrate and inhibitor specificity profiles of cathepsin l. the compound z-phe-tyr(obut)-cocho (entry , table ) turned out as highly potent and selective inhibitor of cathepsin l with ki value of . nm. this molecule was further adapted by shenoy et al. to assess the structural basis for cathepsin l inhibition [ ] . in their study, the authors crystallized the glyoxal inhibitor with cathepsin l; the β-aldehyde forms a tetrahedral thiohemiacetal and α-keto oxygen atom is stabilized by the oxyanion hole. the tyr(obut) group was found to occupy s site while phenyl and carboxybenzyl groups occupied s and s sites, respectively. this class of inhibitors has successfully been deployed in the functional biology of cathepsin l. azepanone-based compounds were first reported as orally bioavailable and extremely potent inhibitors of cathepsin k, as shown by the pharmacokinetic studies in the rat [ ] . marquis et al. subsequently adopted the template and extended their work to acquire a selective inhibitor of cathepsin l with similar potency [ ] . this class of inhibitors are armored with keto functional group in another interesting study, yasuma et al. developed a library of compounds by varying the amino acid substituents at p , p , p position of the inhibitor and carried out a thorough sar study [ ] . their study revealed that the configuration of the stereogenic center (s-configuration is favored over r-configuration) at the p position, and not the steric factor, was key to the inhibitory efficacy. apparently, the substituent at p position does not interact with s position residues; rather, proper stereogenicity allows the placement of the inhibitor in vicinity of the catalytic cysteine residue for interaction. s subsite of cathepsin l, on the other hand, preferred a hydrophobic and moderate-size group; α-branched alkyl chains but not the bulkier groups like phenylalanine was favorable. further, the s subsite showed a preference for hydrophobic and bulky moieties such as -and -naphthalenylsulfonyl substituents. among synthesized compound, n-( -naphthalenylsulfonyl-l-isoleucyl-l-tryptophanal (ic = . nm, -fold selective over cathepsin b; figure ) attenuated the release of ca + and hydroxyproline from bone in an in vitro bone culture system and further restricted bone loss in ovariectomized mice dosed orally. in another interesting study, yasuma et al. developed a library of compounds by varying the amino acid substituents at p , p , p position of the inhibitor and carried out a thorough sar study [ ] . their study revealed that the configuration of the stereogenic center (s-configuration is favored over r-configuration) at the p position, and not the steric factor, was key to the inhibitory efficacy. apparently, the substituent at p position does not interact with s position residues; rather, proper stereogenicity allows the placement of the inhibitor in vicinity of the catalytic cysteine residue for interaction. s subsite of cathepsin l, on the other hand, preferred a hydrophobic and moderate-size group; α-branched alkyl chains but not the bulkier groups like phenylalanine was favorable. further, the s subsite showed a preference for hydrophobic and bulky moieties such as -and naphthalenylsulfonyl substituents. among synthesized compound, n-( -naphthalenylsulfonyl-lisoleucyl-l-tryptophanal (ic = . nm, -fold selective over cathepsin b; figure ) attenuated the release of ca + and hydroxyproline from bone in an in vitro bone culture system and further restricted bone loss in ovariectomized mice dosed orally. a further modification of this scaffold was reported by lynas et al. [ ] here, authors designed and developed di-and tri-peptidyl α-keto-β-aldehydes, based on substrate and inhibitor specificity profiles of cathepsin l. the compound z-phe-tyr(obut)-cocho (entry , table ) turned out as highly potent and selective inhibitor of cathepsin l with ki value of . nm. this molecule was further adapted by shenoy et al. to assess the structural basis for cathepsin l inhibition [ ] . in their study, the authors crystallized the glyoxal inhibitor with cathepsin l; the β-aldehyde forms a tetrahedral thiohemiacetal and α-keto oxygen atom is stabilized by the oxyanion hole. the tyr(obut) group was found to occupy s site while phenyl and carboxybenzyl groups occupied s and s sites, respectively. this class of inhibitors has successfully been deployed in the functional biology of cathepsin l. azepanone-based compounds were first reported as orally bioavailable and extremely potent inhibitors of cathepsin k, as shown by the pharmacokinetic studies in the rat [ ] . marquis et al. subsequently adopted the template and extended their work to acquire a selective inhibitor of cathepsin l with similar potency [ ] . this class of inhibitors are armored with keto functional group figure . n-( -naphthalenylsulfonyl-l-isoleucyl-l-tryptophanal orients itself into the active site of cathepsin l and finds favorable interactions within the s , s , and s pockets of the enzyme. a further modification of this scaffold was reported by lynas et al. [ ] here, authors designed and developed di-and tri-peptidyl α-keto-β-aldehydes, based on substrate and inhibitor specificity profiles of cathepsin l. the compound z-phe-tyr(obut)-cocho (entry , table ) turned out as highly potent and selective inhibitor of cathepsin l with k i value of . nm. this molecule was further adapted by shenoy et al. to assess the structural basis for cathepsin l inhibition [ ] . in their study, the authors crystallized the glyoxal inhibitor with cathepsin l; the β-aldehyde forms a tetrahedral thiohemiacetal and α-keto oxygen atom is stabilized by the oxyanion hole. the tyr(obut) group was found to occupy s site while phenyl and carboxybenzyl groups occupied s and s sites, respectively. this class of inhibitors has successfully been deployed in the functional biology of cathepsin l. azepanone-based compounds were first reported as orally bioavailable and extremely potent inhibitors of cathepsin k, as shown by the pharmacokinetic studies in the rat [ ] . marquis et al. subsequently adopted the template and extended their work to acquire a selective inhibitor of cathepsin l with similar potency [ ] . this class of inhibitors are armored with keto functional group that act as an electrophilic warhead and traps cysteine proteases by forming a transient covalent bond with the active-site cys residue, rendering inactivated enzyme. the authors initiated their work by scrupulously studying the cathepsin k-inhibitor complex that revealed the influence of p and p substituents of the inhibitor in determining the efficacy and selectivity profile of the compound. based on these observations, they designed and synthesized a series of compound and secured a highly potent cathepsin l inhibitor (k i,app : . nm; entry , table ) that exerted remarkable selectivity over cathepsin k and fairly modest selectivity over both cathepsin b and s. interestingly, sar showed that replacement of p leucine and p benzofuran of cathepsin k inhibitor with bulkier hydrophobic aromatic groups yielded an improved potency and the selectivity towards cathepsin l ( figure ). molecular docking studies further supported this observation as cathepsin k was found to have a shallower s pocket than cathepsin l, thus incorporation of bulkier napthyl group at p position favored cathepsin l inhibition but not cathepsin k. on the other hand, inclusion of another napthyl group at p position promoted a steric clash rather than furthering the desired hydrophobic interactions within the s pocket of cathepsin k, thus incurring a better selectivity profile towards cathepsin l over cathepsin k. this template has proven to be an important tool to study cysteine cathepsins as it has been further extended to achieve potent cathepsin s-selective inhibitor with cellular activity [ ] . molecules , , of that act as an electrophilic warhead and traps cysteine proteases by forming a transient covalent bond with the active-site cys residue, rendering inactivated enzyme. the authors initiated their work by scrupulously studying the cathepsin k-inhibitor complex that revealed the influence of p and p substituents of the inhibitor in determining the efficacy and selectivity profile of the compound. based on these observations, they designed and synthesized a series of compound and secured a highly potent cathepsin l inhibitor (ki,app: . nm; entry , table ) that exerted remarkable selectivity over cathepsin k and fairly modest selectivity over both cathepsin b and s. interestingly, sar showed that replacement of p leucine and p benzofuran of cathepsin k inhibitor with bulkier hydrophobic aromatic groups yielded an improved potency and the selectivity towards cathepsin l ( figure ). molecular docking studies further supported this observation as cathepsin k was found to have a shallower s pocket than cathepsin l, thus incorporation of bulkier napthyl group at p position favored cathepsin l inhibition but not cathepsin k. on the other hand, inclusion of another napthyl group at p position promoted a steric clash rather than furthering the desired hydrophobic interactions within the s pocket of cathepsin k, thus incurring a better selectivity profile towards cathepsin l over cathepsin k. this template has proven to be an important tool to study cysteine cathepsins as it has been further extended to achieve potent cathepsin s-selective inhibitor with cellular activity [ ] . nitrile group containing inhibitors have been widely recognized as covalent and reversible inhibitors of a certain class of enzymes that depend on cysteine-mediated nucleophilic attack for catalysis; the nitrile residue traps the sulfur and forms a thioimidate bond ( figure ) that hydrolyzes over the time rendering free enzyme. odanacatib is one of the prime examples of this class of compounds that has been evaluated as a clinical agent, although with limited success [ , ] . because of nitrile's tunable target engagement nature, this scaffold has been adapted to target other relevant enzymes, including cathepsin. hardegger et al. utilized nitrile warhead and examined the effect of halogen bonding in protein-ligand interactions [ ] . they developed a series of compounds and performed a thorough sar analysis in which the nitrile electrophile faced towards s site and trapped the catalytic cysteine. in the developed analogs, the substituents that occupied the s site were systematically varied by strategically altering the substituents at the para-position of the phenyl group ( figure ). the authors observed an improvement in inhibition profile with the placement of halogen at the para-position of phenyl ring which followed a trend cl < br < i (entry , table ), with the f substituent being an outlier. further analysis of the enzyme-inhibitor co-crystal structures revealed that halogen at the para position of the phenyl ring suitably interacted with gly at the s site; fluorine analog pointed away to avoid the electronic repulsion from the oxygen lone pairs of gly . the authors have also performed computational analysis which taken together with the crystal nitrile group containing inhibitors have been widely recognized as covalent and reversible inhibitors of a certain class of enzymes that depend on cysteine-mediated nucleophilic attack for catalysis; the nitrile residue traps the sulfur and forms a thioimidate bond ( figure ) that hydrolyzes over the time rendering free enzyme. odanacatib is one of the prime examples of this class of compounds that has been evaluated as a clinical agent, although with limited success [ , ] . because of nitrile's tunable target engagement nature, this scaffold has been adapted to target other relevant enzymes, including cathepsin. hardegger et al. utilized nitrile warhead and examined the effect of halogen bonding in protein-ligand interactions [ ] . they developed a series of compounds and performed a thorough sar analysis in which the nitrile electrophile faced towards s site and trapped the catalytic cysteine. in the developed analogs, the substituents that occupied the s site were systematically varied by strategically altering the substituents at the para-position of the phenyl group ( figure ). the authors observed an improvement in inhibition profile with the placement of halogen at the para-position of phenyl ring which followed a trend cl < br < i (entry , table ) , with the f substituent being an outlier. further analysis of the enzyme-inhibitor co-crystal structures revealed that halogen at the para position of the phenyl ring suitably interacted with gly at the s site; fluorine analog pointed away to avoid the electronic repulsion from the oxygen lone pairs of gly . the authors have also performed computational analysis which taken together with the crystal data suggests o· x-c angle and the distance between the interacting atoms primarily influenced the protein-ligand interaction. this work provides an important roadmap for developing improved chemical biology tools where a halogen-protein interaction has successfully been utilized [ ] . molecules , , of data suggests o· x-c angle and the distance between the interacting atoms primarily influenced the protein-ligand interaction. this work provides an important roadmap for developing improved chemical biology tools where a halogen-protein interaction has successfully been utilized [ ] . to examine what effect amide···heteroarene π-stacking interactions may have on chalcogen bonding in the s pocket of cathepsin l, giroud et al. utilized triazine-nitrile scaffold [ ] . the authors synthesized a diverse set of triazine-nitrile compounds with a diversified heteroarenes targeting s pocket; the s and s substituents were kept constant. among the developed compound library, -benzothienyl analog (entry , table ) exhibited maximum inhibitory potential; benzofuranyl, -benzothiazolyl, and -imidazopyridinyl, which are of similar geometry, also followed a similar inhibitory pattern ( figure ). molecular modelling based on co-crystal structures showed favorable chalcogen interaction to the backbone carbonyl of asn (d(s···o = casn ) = . Å and the angle α(oasn ···s-c) = °) at the s pocket; this was further supported by a conformational strain analysis, as chalcogen-enzyme interactions compensated for higher torsional strain in the s-containing ligands when compared to the benzofuranyl and imidazopyridinyl ligands. their study demonstrated the importance of both intermolecular interactions and conformational strain in assessing the effect of heterobicyclic ligands at the s pocket that could be potentially be utilized to develop cathepsin l selective inhibitors. data suggests o· x-c angle and the distance between the interacting atoms primarily influenced the protein-ligand interaction. this work provides an important roadmap for developing improved chemical biology tools where a halogen-protein interaction has successfully been utilized [ ] . to examine what effect amide···heteroarene π-stacking interactions may have on chalcogen bonding in the s pocket of cathepsin l, giroud et al. utilized triazine-nitrile scaffold [ ] . the authors synthesized a diverse set of triazine-nitrile compounds with a diversified heteroarenes targeting s pocket; the s and s substituents were kept constant. among the developed compound library, -benzothienyl analog (entry , table ) exhibited maximum inhibitory potential; benzofuranyl, -benzothiazolyl, and -imidazopyridinyl, which are of similar geometry, also followed a similar inhibitory pattern ( figure ). molecular modelling based on co-crystal structures showed favorable chalcogen interaction to the backbone carbonyl of asn (d(s···o = casn ) = . Å and the angle α(oasn ···s-c) = °) at the s pocket; this was further supported by a conformational strain analysis, as chalcogen-enzyme interactions compensated for higher torsional strain in the s-containing ligands when compared to the benzofuranyl and imidazopyridinyl ligands. their study demonstrated the importance of both intermolecular interactions and conformational strain in assessing the effect of heterobicyclic ligands at the s pocket that could be potentially be utilized to develop cathepsin l selective inhibitors. to examine what effect amide···heteroarene π-stacking interactions may have on chalcogen bonding in the s pocket of cathepsin l, giroud et al. utilized triazine-nitrile scaffold [ ] . the authors synthesized a diverse set of triazine-nitrile compounds with a diversified heteroarenes targeting s pocket; the s and s substituents were kept constant. among the developed compound library, -benzothienyl analog (entry , table ) exhibited maximum inhibitory potential; -benzofuranyl, -benzothiazolyl, and -imidazopyridinyl, which are of similar geometry, also followed a similar inhibitory pattern ( figure ). molecular modelling based on co-crystal structures showed favorable chalcogen interaction to the backbone carbonyl of asn (d(s···o = casn ) = . Å and the angle α(oasn ···s-c) = • ) at the s pocket; this was further supported by a conformational strain analysis, as chalcogen-enzyme interactions compensated for higher torsional strain in the s-containing ligands when compared to the benzofuranyl and imidazopyridinyl ligands. their study demonstrated the importance of both intermolecular interactions and conformational strain in assessing the effect of heterobicyclic ligands at the s pocket that could be potentially be utilized to develop cathepsin l selective inhibitors. in a subsequent study, kuhn et al. systematically compared the effectiveness of four different approaches: (a) selection by a medicinal chemist (b) manual modeling (c) docking followed by manual filtering, and (d) free energy calculations (fep). this systematic protocol enabled them to prioritize building blocks for effective targeting of cathepsin l enzyme [ ] . the authors developed a series of analogs by varying only s substituents and keeping s and s fixed ( figure ). after analyzing the affinity by enzyme kinetics, they found that the fep method was superior over other well-established methodologies; this method not only predicted the most relevant ligands but also identified the topological requirements of the substituents for a more effective engagement in the s pocket. among the developed compounds, cyclopentylmethyl substituent in the s pocket (entry , table ) incurred the most favorable interaction as it optimally filled the front part of the pocket. this strategy certainly provided an edge over other conventional methodologies in predicting the optimal ligands for the s pocket targeting. these findings could benefit the ongoing effort of achieving a suitable therapeutic candidate for cathepsin l enzyme. benzofuranyl, -benzothiazolyl, and -imidazopyridinyl, which are of similar geometry, also followed a similar inhibitory pattern (figure ). molecular modelling based on co-crystal structures showed favorable chalcogen interaction to the backbone carbonyl of asn (d(s···o = casn ) = . Å and the angle α(oasn ···s-c) = °) at the s pocket; this was further supported by a conformational strain analysis, as chalcogen-enzyme interactions compensated for higher torsional strain in the s-containing ligands when compared to the benzofuranyl and imidazopyridinyl ligands. their study demonstrated the importance of both intermolecular interactions and conformational strain in assessing the effect of heterobicyclic ligands at the s pocket that could be potentially be utilized to develop cathepsin l selective inhibitors. in a subsequent study, kuhn et al. systematically compared the effectiveness of four different approaches: (a) selection by a medicinal chemist (b) manual modeling (c) docking followed by manual filtering, and (d) free energy calculations (fep). this systematic protocol enabled them to prioritize building blocks for effective targeting of cathepsin l enzyme [ ] . the authors developed a series of analogs by varying only s substituents and keeping s and s fixed (figure ). after analyzing the affinity by enzyme kinetics, they found that the fep method was superior over other well-established methodologies; this method not only predicted the most relevant ligands but also identified the topological requirements of the substituents for a more effective engagement in the s pocket. among the developed compounds, cyclopentylmethyl substituent in the s pocket (entry , table ) incurred the most favorable interaction as it optimally filled the front part of the pocket. this strategy certainly provided an edge over other conventional methodologies in predicting the optimal ligands for the s pocket targeting. these findings could benefit the ongoing effort of achieving a suitable therapeutic candidate for cathepsin l enzyme. the thiosemicarbazone moiety was first recognized as a relevant covalent and reversible warhead of cathepsin l homologous enzyme cruzain, a protease from t. cruzi. the mechanism of inactivaction involves the formation of a transient covalent bond with the catalytic cys residue ( figure ) [ ] . in an interesting study, kishore kumar et al. first utilized this idea and synthesized a small library of compounds in which the most active class of inhibitors were comprised of one metabromo substituted aryl ring along with another one with optimally substituted functionalities [ ] . the inhibitor places itself in the active site cleft of cathepsin l where meta-bromo substituted aryl ring occupies the s site and thiosemicarbazone motif lies near the active site cysteine. however, when the motif was extended to capture s ′ site interaction by placing the aryl/alkyl group at the terminal nitrogen of thiosemicarbazone, the inhibitory potency was completely diminished. overall, this class of inhibitors showed a good selectivity over cathepsin b and exhibited low cytotoxicity when tested on human cancer cell lines. in follow up studies, kishore et al. and parker et al. further expanded the scope of thiosemicarbazone scaffold and developed diversely the thiosemicarbazone moiety was first recognized as a relevant covalent and reversible warhead of cathepsin l homologous enzyme cruzain, a protease from t. cruzi. the mechanism of inactivaction involves the formation of a transient covalent bond with the catalytic cys residue (figure ) [ ] . in an interesting study, kishore kumar et al. first utilized this idea and synthesized a small library of compounds in which the most active class of inhibitors were comprised of one meta-bromo substituted aryl ring along with another one with optimally substituted functionalities [ ] . the inhibitor places itself in the active site cleft of cathepsin l where meta-bromo substituted aryl ring occupies the s site and thiosemicarbazone motif lies near the active site cysteine. molecules , , of in a subsequent study, kuhn et al. systematically compared the effectiveness of four different approaches: (a) selection by a medicinal chemist (b) manual modeling (c) docking followed by manual filtering, and (d) free energy calculations (fep). this systematic protocol enabled them to prioritize building blocks for effective targeting of cathepsin l enzyme [ ] . the authors developed a series of analogs by varying only s substituents and keeping s and s fixed (figure ). after analyzing the affinity by enzyme kinetics, they found that the fep method was superior over other well-established methodologies; this method not only predicted the most relevant ligands but also identified the topological requirements of the substituents for a more effective engagement in the s pocket. among the developed compounds, cyclopentylmethyl substituent in the s pocket (entry , table ) incurred the most favorable interaction as it optimally filled the front part of the pocket. this strategy certainly provided an edge over other conventional methodologies in predicting the optimal ligands for the s pocket targeting. these findings could benefit the ongoing effort of achieving a suitable therapeutic candidate for cathepsin l enzyme. the thiosemicarbazone moiety was first recognized as a relevant covalent and reversible warhead of cathepsin l homologous enzyme cruzain, a protease from t. cruzi. the mechanism of inactivaction involves the formation of a transient covalent bond with the catalytic cys residue ( figure ) [ ] . in an interesting study, kishore kumar et al. first utilized this idea and synthesized a small library of compounds in which the most active class of inhibitors were comprised of one metabromo substituted aryl ring along with another one with optimally substituted functionalities [ ] . the inhibitor places itself in the active site cleft of cathepsin l where meta-bromo substituted aryl ring occupies the s site and thiosemicarbazone motif lies near the active site cysteine. however, when the motif was extended to capture s site interaction by placing the aryl/alkyl group at the terminal nitrogen of thiosemicarbazone, the inhibitory potency was completely diminished. overall, this class of inhibitors showed a good selectivity over cathepsin b and exhibited low cytotoxicity when tested on human cancer cell lines. in follow up studies, kishore et al. and parker et al. further expanded the scope of thiosemicarbazone scaffold and developed diversely functionalized analogs that exhibited an enhanced inhibitory potency and promising cellular activities while still retaining the selectivity over cathepsin b [ , ] . in their latest study, parker et al. strategically transformed an active inhibitor with limited aqueous solubility into a water-soluble prodrug (entry , table ), by phosphorylation of phenolic hydroxy group; this group was readily hydrolyzable by alkaline phosphatases, rendering the active pharmacophore [ ] . the phosphate prodrug exhibited a remarkable -fold increase in solubility over the parent drug and did not disintegrate in aqueous solution, even after prolonged exposure at the physiological temperature. furthermore, this compound did not show any significant cytotoxicity on normal primary huvecs cells in comparison to other fda-approved cytotoxic drugs, doxorubicin and paclitaxel. this prodrug thus far has shown promise to be a desirable clinical candidate and the authors have proposed to evaluate its in vivo efficacy in a preclinical setup. as noted earlier, cathepsin l, like other cysteine cathepsins, contains an inhibitory propeptide domain that spans the active site of the enzyme in the inverse direction to the regular substrate binding mode. chowdhury et al., in their seminal study, exploited this concept by examining the effect of a series of synthesized tripeptidyl compounds that mimicked cathepsin l inhibitory propeptide [ ] . importantly, the developed tripeptidyl motifs also exhibited nanomolar potency; however, a moderate truncation of the full-length propeptide drastically lost all activities [ ] . notably, while the full-length propeptide showed only -fold selectivity over cathepsin k, the most potent analog of this series (entry , table ) demonstrated a far-improved selectivity ( -fold). the authors further investigated the binding mode of this class of inhibitors by means of co-crystal structure and molecular modeling. this revealed that (a) arginine residue of the inhibitor occupied the s pocket, (b) phenyl alanine residue found favorable hydrophobic interactions within the s pocket, (c) -phenylethyl group pointed toward s pocket, (d) the methionine residue showed optimal interaction within s pocket, and (e) the biphenyl acetyl group extended to the s pocket for favorable interactions [ , ] . this class of inhibitors has shown resistance to enzyme-dependent hydrolysis and demonstrates the reversible mode of enzyme inactivation. overall, this inhibitor class provides a wealth of information on inhibitor binding to cathepsin l and provides a general template for the development of therapeutic candidates for other relevant enzymes as well. in an effort to discover small molecule inhibitors of cathepsin l, myers et al. performed high throughput screening (hts) of the nih molecular libraries small molecule repository (mlsmr); they identified , -disubstituted oxadiazoles (figure a ) as potent hit compounds [ ] . surprisingly, upon synthesis and purification of the putative inhibitory lead compounds, a complete loss of activity was observed. the authors then investigated the compounds' integrity from nih mlsmr library by lc-ms; this showed the presence of additional impurities. to trace back the active impurity, the authors hypothesized the presence of impurities resulting from an acid-catalyzed ring-opening reaction of thiocarbazate. the resulting azapeptides was likely the active pharmacophore that inhibited enzyme via acylation of active site cys; this was validated by synthesis of azapeptides and performing the enzyme assay [ ] . the (s)-stereoisomer of newly synthesized compound (entry , table ; figure b ) indeed attenuated the activity of cathepsin l with an ic value of nm. in follow up studies, the authors further developed a series of compounds with structural diversity and performed a computational analysis to recognize the basis of potent enzyme inhibition [ , ] one of the thiocarbazate analogs developed this way (figure c) showed improved potency over the parent compound. molecular modeling studies performed with parent compound (entry , table ) in complex with papain indicated that indole motif preferably bound to s subsite, -nhboc group engaged in favorable hydrophobic interactions within the s subsite and -ethylphenyl anilide extended to s pocket. to further probe the importance of thiocarbazate moiety, the authors synthesized compound containing oxocarbazate (entry , table ) and azapeptide (entry , table ) motifs. the oxocarbazate showed a fairly improved ic value ( nm) towards cathepsin l, whereas the azapeptide was at best only a modest inhibitor (ic = µm). consistently, the binding mode of oxocarbazate exerted similarity to that of thiocarbazate when investigated by molecular modeling studies [ ] . ) motifs. the oxocarbazate showed a fairly improved ic value ( nm) towards cathepsin l, whereas the azapeptide was at best only a modest inhibitor (ic = µm). consistently, the binding mode of oxocarbazate exerted similarity to that of thiocarbazate when investigated by molecular modeling studies [ ] . in a separate study, shah et al. carried out a thorough enzymatic analysis of the champion thiocarbazate compound (entry , table ) that showed a time-dependent improvement in the inhibition profile; the ic value went down to nm when preincubated with cathepsin l for h [ ] . lc-ms and kinetic analysis of enzyme-inhibitor complex (inhibition rate constants: kon = , m − s − and koff = . × − s − , and binding constant: ki = . nm) demonstrated a slow-binding kinetics and reversibility of inhibition. the selectivity over other members of the enzyme family was modest. interestingly, the compound inhibited propagation of malaria parasite plasmodium falciparum [ic = . µm], and leishmania major [ic = . µm], and did not exhibit any significant toxicity against human aortic endothelial cells and zebrafish. although thiocarbazate motif showed promise as an inhibitory scaffold, the lack of reasonable stability (it decomposes even in dmso) and only modest inhibitory activity in cell-based assays probably ceased any further development of the scaffold [ ] . the authors also extended their studies to evaluate the potential of oxocarbazate inhibitor that showed an improved ic value of . nm upon h preincubation with the enzyme. like as in the case of thiocarbazate, they performed an enzyme kinetic analysis of the enzyme-inhibitor complex and obtained the following parameters: inhibition rate constants: kon = , m − s − and koff = . × − s − , and binding constant: ki = . nm [ ] . the inhibitor blocked sars-cov (ic = ± nm) and ebola virus (ic = ± nm) entry into the human embryonic kidney (hek) t cells, a process that utilizes cathepsin l-mediated proteolysis for host cell infection. the oxocarbazate, when treated with hek t lysate in the presence of dcg- , an activity-based cysteine cathepsin probe, showed reduced cathepsin l labeling when assessed by a western-blot analysis; this further in a separate study, shah et al. carried out a thorough enzymatic analysis of the champion thiocarbazate compound (entry , table ) that showed a time-dependent improvement in the inhibition profile; the ic value went down to nm when preincubated with cathepsin l for h [ ] . lc-ms and kinetic analysis of enzyme-inhibitor complex (inhibition rate constants: k on = , m − s − and k off = . × − s − , and binding constant: k i = . nm) demonstrated a slow-binding kinetics and reversibility of inhibition. the selectivity over other members of the enzyme family was modest. interestingly, the compound inhibited propagation of malaria parasite plasmodium falciparum [ic = . µm], and leishmania major [ic = . µm], and did not exhibit any significant toxicity against human aortic endothelial cells and zebrafish. although thiocarbazate motif showed promise as an inhibitory scaffold, the lack of reasonable stability (it decomposes even in dmso) and only modest inhibitory activity in cell-based assays probably ceased any further development of the scaffold [ ] . the authors also extended their studies to evaluate the potential of oxocarbazate inhibitor that showed an improved ic value of . nm upon h preincubation with the enzyme. like as in the case of thiocarbazate, they performed an enzyme kinetic analysis of the enzyme-inhibitor complex and obtained the following parameters: inhibition rate constants: k on = , m − s − and k off = . × − s − , and binding constant: k i = . nm [ ] . the inhibitor blocked sars-cov (ic = ± nm) and ebola virus (ic = ± nm) entry into the human embryonic kidney (hek) t cells, a process that utilizes cathepsin l-mediated proteolysis for host cell infection. the oxocarbazate, when treated with hek t lysate in the presence of dcg- , an activity-based cysteine cathepsin probe, showed reduced cathepsin l labeling when assessed by a western-blot analysis; this further corroborated the results obtained from the virus pseudotype infection assay. overall, oxocarbazate inhibitor not only provided a promising template for further exploitation but also rendered a new direction for intervening sars and ebola virus infections. ubiquitous expression of human cathepsin l in most human tissues possesses a significant challenge in targeting this enzyme for therapeutic development. this problem is further exacerbated with recent findings that alternative spliced isoforms could exist at distinct cellular locations (e.g., nucleus, cytosol, and ecm space) [ , , ] . while several unique functional roles of cathepsin l are known, it has also been reported that some of its function can also be accomplished by other members of the cathepsin family (i.e., functional redundancy); for example, both cathepsin l and b can mediate a mutually compensatory role in the inflammatory response signaling pathways [ ] . in this regard, accurate function of cathepsin l must be first determined in different cell types individually before considering significant investment in drug development. since the function of an enzyme, such as cathepsin l, depends primarily on its activity profile and given that the activity profiles of differentially processed cathepsin l isoforms may be very different, probes capable of reporting accurate activity status in different cell types (and cellular location) are anticipated to advance our understanding if cathepsin l biology. over the years, the concept of activity-based probes (abps) has emerged as a valuable chemical biology tool for monitoring the enzyme activity (not just the expression profile alone) in cells at the proteome levels [ ] [ ] [ ] [ ] . in most cases, existing covalent inhibitors containing a recognition motif are adopted and transformed to abps by conjugating optimal detection modalities; these include fluorescent probes, affinity labels, radiotracer, and many others ( figure ) . indeed, the use of abps has rather established cathepsins as key diagnostic marker for various disease conditions, and have even enabled optical surgical navigation, leading to an improved surgical precision [ , ] . in the following sections, we discussed cathepsin l-selective molecular probes that have been developed and utilized for monitoring its activity. a thorough overview of cathepsin probe development for imaging purpose could be found elsewhere [ , [ ] [ ] [ ] . ubiquitous expression of human cathepsin l in most human tissues possesses a significant challenge in targeting this enzyme for therapeutic development. this problem is further exacerbated with recent findings that alternative spliced isoforms could exist at distinct cellular locations (e.g., nucleus, cytosol, and ecm space) [ , , ] . while several unique functional roles of cathepsin l are known, it has also been reported that some of its function can also be accomplished by other members of the cathepsin family (i.e., functional redundancy); for example, both cathepsin l and b can mediate a mutually compensatory role in the inflammatory response signaling pathways [ ] . in this regard, accurate function of cathepsin l must be first determined in different cell types individually before considering significant investment in drug development. since the function of an enzyme, such as cathepsin l, depends primarily on its activity profile and given that the activity profiles of differentially processed cathepsin l isoforms may be very different, probes capable of reporting accurate activity status in different cell types (and cellular location) are anticipated to advance our understanding if cathepsin l biology. over the years, the concept of activity-based probes (abps) has emerged as a valuable chemical biology tool for monitoring the enzyme activity (not just the expression profile alone) in cells at the proteome levels [ ] [ ] [ ] [ ] . in most cases, existing covalent inhibitors containing a recognition motif are adopted and transformed to abps by conjugating optimal detection modalities; these include fluorescent probes, affinity labels, radiotracer, and many others ( figure ) . indeed, the use of abps has rather established cathepsins as key diagnostic marker for various disease conditions, and have even enabled optical surgical navigation, leading to an improved surgical precision [ , ] . in the following sections, we discussed cathepsin l-selective molecular probes that have been developed and utilized for monitoring its activity. a thorough overview of cathepsin probe development for imaging purpose could be found elsewhere [ , [ ] [ ] [ ] . radio-labeled inhibitors have long been used as a primary mode for detecting active cysteine proteases both in vitro and in vivo. docherty et al. first used a chloromethyl inhibitor containing radioactive iodine, i-tyr-ala-lys-argch cl, and detected cathepsin b in crude granule fraction of islet cells [ ] . in their follow up study, they also presumably identified cathepsin l in insulin secretory granule using the same radio-isotopically labeled inhibitor [ ] . the mechanism of detection involves covalent modification of the target protein that shows up as a distinct band upon performing autoradiography. this chloromethyl containing inhibitor turned out to be non-selective due to its reactivity towards trypsin, a serine protease. this was followed by the discovery of a radioactive-peptidyldiazomethane compound (entry p , table ), a selective cysteine proteinase inhibitor [ ] . mason et al. adopted the peptidyldiazomethane scaffold, which potently inhibited both cathepsin l and b. this scaffold showed improved inhibition profile upon iodination, as demonstrated by crawford et al. [ ] this inhibitory agent was then transformed to a radio-labeled probe via incorporation of i [ ] . the developed probe was utilized to detect both cathepsin l and b in kirsten-virus-transformed knih t cells. the incubation of cellular extracts with p followed by gel electrophoresis showed the presence of two protein bands at and kda, showing two active forms of cathepsin l. active cathepsin b was also detected at around - kda. interestingly, pulse-chase experiments with [ s]methionine-labeled proteins only detected two separate bands at kda and kda, which correspond to the intracellular inactive precursors of cathepsin l and b respectively. this indicated that the inactive precursor proteins did not react with p , demonstrating its unique ability to quantify only active protein. further, p was utilized to probe active cathepsin l and b in different human tissues as well as in lysosomes and whole cells [ , ] . in a follow-up study, xing et al. developed fmoc-[i ]tyr-ala-chn (entry p , table ) that selectively detected cathepsin l and b over cathepsin s, exhibiting a faster rate of inactivation towards cathepsin l [ ] . the developed compound successfully probed the amount of active cathepsin l and b in different cell-lines; two unknown proteins also got labeled in certain cases. overall, these probes enabled the detection of active cathepsin enzymes with their cellular location, thereby advancing the knowledge of cathepsin l biology. radio-labeled inhibitors have long been used as a primary mode for detecting active cysteine proteases both in vitro and in vivo. docherty et al. first used a chloromethyl inhibitor containing radioactive iodine, i-tyr-ala-lys-argch cl, and detected cathepsin b in crude granule fraction of islet cells [ ] . in their follow up study, they also presumably identified cathepsin l in insulin secretory granule using the same radio-isotopically labeled inhibitor [ ] . the mechanism of detection involves covalent modification of the target protein that shows up as a distinct band upon performing autoradiography. this chloromethyl containing inhibitor turned out to be non-selective due to its reactivity towards trypsin, a serine protease. this was followed by the discovery of a radioactive-peptidyldiazomethane compound (entry p , table ), a selective cysteine proteinase inhibitor [ ] . mason et al. adopted the peptidyldiazomethane scaffold, which potently inhibited both cathepsin l and b. this scaffold showed improved inhibition profile upon iodination, as demonstrated by crawford et al. [ ] this inhibitory agent was then transformed to a radio-labeled probe via incorporation of i [ ] . the developed probe was utilized to detect both cathepsin l and b in kirsten-virus-transformed knih t cells. the incubation of cellular extracts with p followed by gel electrophoresis showed the presence of two protein bands at and kda, showing two active forms of cathepsin l. active cathepsin b was also detected at around - kda. interestingly, pulse-chase experiments with [ s]methionine-labeled proteins only detected two separate bands at kda and kda, which correspond to the intracellular inactive precursors of cathepsin l and b respectively. this indicated that the inactive precursor proteins did not react with p , demonstrating its unique ability to quantify only active protein. further, p was utilized to probe active cathepsin l and b in different human tissues as well as in lysosomes and whole cells [ , ] . in a follow-up study, tyr-ala-chn (entry p , table ) that selectively detected cathepsin l and b over cathepsin s, exhibiting a faster rate of inactivation towards cathepsin l [ ] . the developed compound successfully probed the amount of active cathepsin l and b in different cell-lines; two unknown proteins also got labeled in certain cases. overall, these probes enabled the detection of active cathepsin enzymes with their cellular location, thereby advancing the knowledge of cathepsin l biology. radio-labeled inhibitors have long been used as a primary mode for detecting active cysteine proteases both in vitro and in vivo. docherty et al. first used a chloromethyl inhibitor containing radioactive iodine, i-tyr-ala-lys-argch cl, and detected cathepsin b in crude granule fraction of islet cells [ ] . in their follow up study, they also presumably identified cathepsin l in insulin secretory granule using the same radio-isotopically labeled inhibitor [ ] . the mechanism of detection involves covalent modification of the target protein that shows up as a distinct band upon performing autoradiography. this chloromethyl containing inhibitor turned out to be non-selective due to its reactivity towards trypsin, a serine protease. this was followed by the discovery of a radioactive-peptidyldiazomethane compound (entry p , table ), a selective cysteine proteinase inhibitor [ ] . mason et al. adopted the peptidyldiazomethane scaffold, which potently inhibited both cathepsin l and b. this scaffold showed improved inhibition profile upon iodination, as demonstrated by crawford et al. [ ] this inhibitory agent was then transformed to a radio-labeled probe via incorporation of i [ ] . the developed probe was utilized to detect both cathepsin l and b in kirsten-virus-transformed knih t cells. the incubation of cellular extracts with p followed by gel electrophoresis showed the presence of two protein bands at and kda, showing two active forms of cathepsin l. active cathepsin b was also detected at around - kda. interestingly, pulse-chase experiments with [ s]methionine-labeled proteins only detected two separate bands at kda and kda, which correspond to the intracellular inactive precursors of cathepsin l and b respectively. this indicated that the inactive precursor proteins did not react with p , demonstrating its unique ability to quantify only active protein. further, p was utilized to probe active cathepsin l and b in different human tissues as well as in lysosomes and whole cells [ , ] . in a follow-up study, tyr-ala-chn (entry p , table ) that selectively detected cathepsin l and b over cathepsin s, exhibiting a faster rate of inactivation towards cathepsin l [ ] . the developed compound successfully probed the amount of active cathepsin l and b in different cell-lines; two unknown proteins also got labeled in certain cases. overall, these probes enabled the detection of active cathepsin enzymes with their cellular location, thereby advancing the knowledge of cathepsin l biology. radio-labeled inhibitors have long been used as a primary mode for detecting active cysteine proteases both in vitro and in vivo. docherty et al. first used a chloromethyl inhibitor containing radioactive iodine, i-tyr-ala-lys-argch cl, and detected cathepsin b in crude granule fraction of islet cells [ ] . in their follow up study, they also presumably identified cathepsin l in insulin secretory granule using the same radio-isotopically labeled inhibitor [ ] . the mechanism of detection involves covalent modification of the target protein that shows up as a distinct band upon performing autoradiography. this chloromethyl containing inhibitor turned out to be non-selective due to its reactivity towards trypsin, a serine protease. this was followed by the discovery of a radioactive-peptidyldiazomethane compound (entry p , table ), a selective cysteine proteinase inhibitor [ ] . mason et al. adopted the peptidyldiazomethane scaffold, which potently inhibited both cathepsin l and b. this scaffold showed improved inhibition profile upon iodination, as demonstrated by crawford et al. [ ] this inhibitory agent was then transformed to a radio-labeled probe via incorporation of i [ ] . the developed probe was utilized to detect both cathepsin l and b in kirsten-virus-transformed knih t cells. the incubation of cellular extracts with p followed by gel electrophoresis showed the presence of two protein bands at and kda, showing two active forms of cathepsin l. active cathepsin b was also detected at around - kda. interestingly, pulse-chase experiments with [ s]methionine-labeled proteins only detected two separate bands at kda and kda, which correspond to the intracellular inactive precursors of cathepsin l and b respectively. this indicated that the inactive precursor proteins did not react with p , demonstrating its unique ability to quantify only active protein. further, p was utilized to probe active cathepsin l and b in different human tissues as well as in lysosomes and whole cells [ , ] . in a follow-up study, tyr-ala-chn (entry p , table ) that selectively detected cathepsin l and b over cathepsin s, exhibiting a faster rate of inactivation towards cathepsin l [ ] . the developed compound successfully probed the amount of active cathepsin l and b in different cell-lines; two unknown proteins also got labeled in certain cases. overall, these probes enabled the detection of active cathepsin enzymes with their cellular location, thereby advancing the knowledge of cathepsin l biology. the detecting agents-radionuclide (p , p ), biotin (p ), fluorophore (p , p , p ), clickable acetylene (p ), and lanthanide containing dota (p )-is red-color coded and the photoactivatable benzoyl group (p ) and quencher (p ) is coded in blue. # : no inhibition. gelhaus et al. first developed biotinylated aziridine- , -dicarboxylate and demonstrated its antiplasmodial activity using cell-based studies [ ] . since, the biotinylated compound inhibited plasmodial protease falcipain and cathepsin l, authors suggested that this scaffold could be utilized for the development of cell-permeable, non-radioactive reagents that selectively labels enzymes involved in parasite pathogenicity. later on, vicik et al. adopted this motif (entry p , table ) and developed an affinity label to probe cathepsin l activity [ ] . the aziridine analog irreversibly inactivates the enzyme via covalent modification, as discussed previously. the conjugated biotin moiety is utilized for affinity pull down and target identification. when (s,s) isomer of the biotinylated probe was incubated with cathepsin l and subjected to gel electrophoresis, electrotransferred to a membrane, and exposed to streptavidin-alkaline phosphatase conjugate, a strong labeling of the enzyme-inhibitor complex was observed. however, when the enzyme was treated with e- , an active site-directed competitive and irreversible cathepsin inhibitor, prior to incubation with p , the labeling was diminished, clearly demonstrating that p competes for the active site of cathepsin l. in line with this observation, a desthiobiotinylated analog also exhibited the same trend but with reduced labeling due to its weaker binding affinity to streptavidin. although p has a the detecting agents-radionuclide (p , p ), biotin (p ), fluorophore (p , p , p ), clickable acetylene (p ), and lanthanide containing dota (p )-is red-color coded and the photoactivatable benzoyl group (p ) and quencher (p ) is coded in blue. # : no inhibition. gelhaus et al. first developed biotinylated aziridine- , -dicarboxylate and demonstrated its antiplasmodial activity using cell-based studies [ ] . since, the biotinylated compound inhibited plasmodial protease falcipain and cathepsin l, authors suggested that this scaffold could be utilized for the development of cell-permeable, non-radioactive reagents that selectively labels enzymes involved in parasite pathogenicity. later on, vicik et al. adopted this motif (entry p , table ) and developed an affinity label to probe cathepsin l activity [ ] . the aziridine analog irreversibly inactivates the enzyme via covalent modification, as discussed previously. the conjugated biotin moiety is utilized for affinity pull down and target identification. when (s,s) isomer of the biotinylated probe was incubated with cathepsin l and subjected to gel electrophoresis, electrotransferred to a membrane, and exposed to streptavidin-alkaline phosphatase conjugate, a strong labeling of the enzyme-inhibitor complex was observed. however, when the enzyme was treated with e- , an active site-directed competitive and irreversible cathepsin inhibitor, prior to incubation with p , the labeling was diminished, clearly demonstrating that p competes for the active site of cathepsin l. in line with this observation, a desthiobiotinylated analog also exhibited the same trend but with reduced labeling due to its weaker binding affinity to streptavidin. although p has a the detecting agents-radionuclide (p , p ), biotin (p ), fluorophore (p , p , p ), clickable acetylene (p ), and lanthanide containing dota (p )-is red-color coded and the photoactivatable benzoyl group (p ) and quencher (p ) is coded in blue. # : no inhibition. gelhaus et al. first developed biotinylated aziridine- , -dicarboxylate and demonstrated its antiplasmodial activity using cell-based studies [ ] . since, the biotinylated compound inhibited plasmodial protease falcipain and cathepsin l, authors suggested that this scaffold could be utilized for the development of cell-permeable, non-radioactive reagents that selectively labels enzymes involved in parasite pathogenicity. later on, vicik et al. adopted this motif (entry p , table ) and developed an affinity label to probe cathepsin l activity [ ] . the aziridine analog irreversibly inactivates the enzyme via covalent modification, as discussed previously. the conjugated biotin moiety is utilized for affinity pull down and target identification. when (s,s) isomer of the biotinylated probe was incubated with cathepsin l and subjected to gel electrophoresis, electrotransferred to a membrane, and exposed to streptavidin-alkaline phosphatase conjugate, a strong labeling of the enzyme-inhibitor complex was observed. however, when the enzyme was treated with e- , an active site-directed competitive and irreversible cathepsin inhibitor, prior to incubation with p , the labeling was diminished, clearly demonstrating that p competes for the active site of cathepsin l. in line with this observation, a desthiobiotinylated analog also exhibited the same trend but with reduced labeling due to its weaker binding affinity to streptavidin. although p has a fluorescent cathepsin l specific (some degree of labelling was seen for cat v and b) the detecting agents-radionuclide (p , p ), biotin (p ), fluorophore (p , p , p ), clickable acetylene (p ), and lanthanide containing dota (p )-is red-color coded and the photoactivatable benzoyl group (p ) and quencher (p ) is coded in blue. # : no inhibition. gelhaus et al. first developed biotinylated aziridine- , -dicarboxylate and demonstrated its antiplasmodial activity using cell-based studies [ ] . since, the biotinylated compound inhibited plasmodial protease falcipain and cathepsin l, authors suggested that this scaffold could be utilized for the development of cell-permeable, non-radioactive reagents that selectively labels enzymes involved in parasite pathogenicity. later on, vicik et al. adopted this motif (entry p , table ) and developed an affinity label to probe cathepsin l activity [ ] . the aziridine analog irreversibly inactivates the enzyme via covalent modification, as discussed previously. the conjugated biotin moiety is utilized for affinity pull down and target identification. when (s,s) isomer of the biotinylated probe was incubated with cathepsin l and subjected to gel electrophoresis, electrotransferred to a membrane, and exposed to streptavidin-alkaline phosphatase conjugate, a strong labeling of the enzyme-inhibitor complex was observed. however, when the enzyme was treated with e- , an active site-directed competitive and irreversible cathepsin inhibitor, prior to incubation with p , the labeling was diminished, clearly demonstrating that p competes for the active site of cathepsin l. in line with this observation, a desthiobiotinylated analog also exhibited the same trend but with reduced labeling due to its weaker binding affinity to streptavidin. although p has a the detecting agents-radionuclide (p , p ), biotin (p ), fluorophore (p , p , p ), clickable acetylene (p ), and lanthanide containing dota (p )-is red-color coded and the photoactivatable benzoyl group (p ) and quencher (p ) is coded in blue. # : no inhibition. gelhaus et al. first developed biotinylated aziridine- , -dicarboxylate and demonstrated its antiplasmodial activity using cell-based studies [ ] . since, the biotinylated compound inhibited plasmodial protease falcipain and cathepsin l, authors suggested that this scaffold could be utilized for the development of cell-permeable, non-radioactive reagents that selectively labels enzymes involved in parasite pathogenicity. later on, vicik et al. adopted this motif (entry p , table ) and developed an affinity label to probe cathepsin l activity [ ] . the aziridine analog irreversibly inactivates the enzyme via covalent modification, as discussed previously. the conjugated biotin moiety is utilized for affinity pull down and target identification. when (s,s) isomer of the biotinylated probe was incubated with cathepsin l and subjected to gel electrophoresis, electrotransferred to a membrane, and exposed to streptavidin-alkaline phosphatase conjugate, a strong labeling of the enzyme-inhibitor complex was observed. however, when the enzyme was treated with e- , an active site-directed competitive and irreversible cathepsin inhibitor, prior to incubation with p , the labeling was diminished, clearly demonstrating that p competes for the active site of cathepsin l. in line with this observation, a desthiobiotinylated analog also exhibited the same trend but with reduced labeling due to its weaker binding affinity to streptavidin. although p has a the detecting agents-radionuclide (p , p ), biotin (p ), fluorophore (p , p , p ), clickable acetylene (p ), and lanthanide containing dota (p )-is red-color coded and the photoactivatable benzoyl group (p ) and quencher (p ) is coded in blue. # : no inhibition. gelhaus et al. first developed biotinylated aziridine- , -dicarboxylate and demonstrated its anti-plasmodial activity using cell-based studies [ ] . since, the biotinylated compound inhibited plasmodial protease falcipain and cathepsin l, authors suggested that this scaffold could be utilized for the development of cell-permeable, non-radioactive reagents that selectively labels enzymes involved in parasite pathogenicity. later on, vicik et al. adopted this motif (entry p , table ) and developed an affinity label to probe cathepsin l activity [ ] . the aziridine analog irreversibly inactivates the enzyme via covalent modification, as discussed previously. the conjugated biotin moiety is utilized for affinity pull down and target identification. when (s,s) isomer of the biotinylated probe was incubated with cathepsin l and subjected to gel electrophoresis, electro-transferred to a membrane, and exposed to streptavidin-alkaline phosphatase conjugate, a strong labeling of the enzyme-inhibitor complex was observed. however, when the enzyme was treated with e- , an active site-directed competitive and irreversible cathepsin inhibitor, prior to incubation with p , the labeling was diminished, clearly demonstrating that p competes for the active site of cathepsin l. in line with this observation, a desthiobiotinylated analog also exhibited the same trend but with reduced labeling due to its weaker binding affinity to streptavidin. although p has a modest binding affinity (k i = . µm) to cathepsin l, it exerted a -fold selectivity over cathepsin b. certainly, the affinity labeling technique not only served as an abp for cathepsin l but also provides a premise for developing aziridine-based chemical tools for functional proteomics. although covalent modifiers of proteins have been vastly exploited as chemical tools for target identification and functional proteomics, photoaffinity probes offer unique mode of action. they bind proteins non-covalently (affinity based solely on non-covalent interactions) first and form a non-selective covalent bond with the closest amino acid residue only upon irradiation. torkar et al. took advantage of this technique and developed the first photoaffinity-based probe (entry p , table ) to detect active cathepsin l selectively over other members of the family [ ] . the photoaffinity-based probe was designed based on existing peptidyl acetyloxymethyl ketone (aomk), a known covalent modifier of cysteine proteases. during the design, the aomk group at the c-terminus was replaced by a short di(ethylene glycol) moiety that increased the aqueous solubility and altered the character of the inhibitor from irreversible to a reversible one. the probe was comprised of a lysine residue that was strategically placed to append fluorescent cyanine- (cy ) group for detection. a photoactivatable benzoylphenylalanine amino acid was placed to accommodate the s pocket of cathepsin l. the developed probe detected recombinant cathepsin l upon incubation and subsequent irradiation for min at nm as demonstrated by sds-page. the protein band became invisible when enzyme was incubated with known cathepsin l inhibitors, e- and gb -nh , respectively, prior to the probe treatment [ , ] . the probe also showed preferential selectivity towards cathepsin l when compared to other two fluorescent-based probes. the probe p exhibited a remarkable selectivity for cathepsin l over all other cathepsins in light-mediated labeling experiments with recombinant proteins. the relative labeling percentages (cathepsin b and k: %; cathepsin s: %) were insignificant, except for cathepsin v ( %), the closest homolog of cathepsin l, relative to cathepsin l. interestingly, u -mg glioma cell extracts did not present any cathepsin l for detection with the p probe. however, when the same cell extracts were treated with recombinant cathepsin l added externally and subjected to a labeling experiment, the probe selectively detected the desired protein in complex proteome. the mechanism of protein labeling by p was attributed to putative bond formation between benzophenone and non-conserved met at the s site of cathepsin l. although the developed probe lacks cell-penetrability, the authors envisioned that the technique might have diagnostic and prognostic value where cathepsin l overexpression is high, such as in malignant tissues. although several fluorescent-based molecular probes have been reported for cysteine cathepsins, the superiority of two-photon-based over one-photon based imaging technique inspired na et al. to develop probes with a better cellular imaging profile [ ] . notably, the two-photon fluorescence imaging technique provides increased tissue penetration depth with reduced photobleaching, and a lower tissue autofluorescence. the authors first fabricated a microarray with different peptidyl aldehydes and screened against gfp-labeled cathepsin l enzyme; this led to the identification of two inhibitory hits that potently inactivated enzyme with ic values of . and . nm. the lead inhibitors were further structurally modified to include (a) a two-photon dye, dl- (a , -bis( -hydroxystyryl)pyrimidine derivative), at p position, and (b) disperse red dye, a fluorescence quencher, in the place of aldehyde moiety. the resulting imaging probes (entry p in table is one of such examples) showed a time-dependent increase in fluorescence signals when treated with hepg cell lysates, a mammalian liver cancer cell-line known to overexpresses cysteine cathepsins. enhanced fluorescence signal is due to the release of the quencher upon successful proteolytic cleavage. furthermore, to assess the suitability of p as imaging agents for live cells, hepg cells were incubated with the probe and subjected to a live-cell imaging analysis. there were strong fluorescence signals from endolysomal compartments that disappeared completely when cells were pretreated with e- , validating the target specificity of the probe. as the probe was developed using cathepsin l as a model enzyme, it likely will lack specificity and perhaps interact with other members of the family. still, however, this motif could potentially be utilized to develop selective abps targeting respective cathepsins and assess their activities in a tissue environment, as proposed by the authors. activity-based probes with a single photon fluorescent tag have been successfully utilized for the functional analysis of target proteins both in vitro and in vivo. poreba et al., in their pursuit of developing cathepsin l selective imaging agent, took advantage of this technology [ ] . they realized the importance of developing selective cathepsin l substrate that will bind to the active site of the enzyme over other homologous proteins. to do so, they employed hybrid combinatorial substrate library (hycosul) technology that provides information on optimal chemical space inside the enzyme active sites by strategically scanning diverse peptide library containing both natural and unnatural amino acids. this led to the acquisition of a panel of compounds with desired properties. the potent hits discovered by hycosul technology were further optimized to gain selectivity over other cathepsins while identifying the most efficient substrate based on michaelis menten parameters. unfortunately, when the chosen peptide substrate transformed to an activity-based probe by appending an acyloxymethylketone warhead and a biotin tag, it showed cross-reactivity with cathepsin b. despite biotinylated probe's somewhat compromised selectivity, the authors still assessed its activity-based labeling profile in hek t cells; as expected, cathepsin l labeling was primarily observed with minor amounts of cathepsin b. further optimization by swapping the biotin and arg with cyanine- and cys(bzl) (entry p , table ) groups was carried out next. the newly developed fluorescent abp, p , showed an enhanced selectivity (in comparison to pan-cathepsin probes) against a panel of other recombinant cathepsins (cathepsin v, b, s, and k), as well as cellular extracts derived from hek t and mda-mb- cells. the developed probe served as an effective imaging agent for cellular cathepsin l activity in human mda-mb- breast cancer cells when incubated for h. the selectivity of the probe started to recede with longer incubation time. the importance of optimizing the probe concentration and time-course of the reaction was evident from these experiments. interestingly, p only detected active cathepsin l and not procathepsin l; this certainly signifies the effectiveness of the developed probes as an activity-based probe. the authors further examined the colocalization of the probe with both cathepsin l and b in mda-mb- cells by treating the cells with respective cathepsin antibodies and performing a quantitative pixel analysis from a set of fluorescence microscopy images. this supported the previous observation as the highest weighted colocalization coefficient was obtained for cathepsin l and not for cathepsin b. the developed compound certainly harbors the key traits of an effective activity-based probe, as the authors duly envisioned its significance in deciphering cathepsin l biology in the coming years. as noted above, fluorescence-based imaging probes have been successfully developed to gain access to unknown functionalities of cathepsin enzymes. however, the bulkiness and often multiple charges associated with the fluorophore and/or quencher structures on these probes likely render them poorly cell-permeable and reduce their target affinity. to address this issue, dana et al. adopted a previously reported peptidyl vinylsulfonate inhibitor kd- -a highly potent, selective, covalent and irreversible inhibitor of cathepsin l discussed in section . -and tactically appended a small alkynyl group at the para-position of the cbz group [ ] . this led to the development of a clickable and tagless activity-based probe (catabp) of cathepsin l that retained the key desirable traits of kd- ; i.e., cell permeability, charge profile, molecular weight, potency and selectivity profile [ ] . this strategy eliminated the requirements of including bulky and charged fluorophore/quencher moiety to the probe. one of the inherent advantages of this approach is that the labeling can be performed in live cellular environment with high efficiency. after cell lysis, the labeled cathepsin l can be quantified by performing click chemistry protocol with a fluorescent azide containing dye, resolving the protein using gel-electrophoresis, and directly scanning the gel for fluorescence signal. as anticipated, the developed kdp- probe (entry p , table ), exhibited rapid inactivation kinetics, retained selectivity for cathepsin l, and labeled recombinant active cathepsin l in an activity-dependent manner; the heat-denatured and e- treated cathepsin l showed no labeling when subjected to the identical labeling protocol. a mass-spectrometric analysis of the enzyme-probe complex concluded that the probe was active-site directed, and covalently modified the catalytic cys residue for inactivation. since the kdp- probe was developed with the intention of capturing active cathepsin l in vivo and in human live cell culture, the probe was tested for its cytotoxicity in mda-mb- cells; no cytotoxicity was observed in these cells, even at a concentration as high as µm. incubation of mda-mb- cells overexpressing cathepsin l with kdp- attenuated the intracellular cathepsin l activity in a dose-dependent manner; this was demonstrated by live-cell imaging and wound healing assay. finally, kdp- also interfered with the hatching process of post-fertilized zebrafish embryos, further validating probe's in vivo activity; notably, cathepsin l serves as the key regulator of the hatching process [ ] [ ] [ ] . in conclusion, kdp- demonstrated many desirable attributes of a good probe and is anticipated to find extensive applications in probing cathepsin l function in cells from diverse origins. although fluorophores-containing molecules have been appreciated as useful imaging probes, commonly used fluorophores often suffer from spectral overlapping that limits the number of targets that can be analyzed concomitantly [ ] . to address this issue, poreba et al. recently developed protease-selective lanthanide-labeled probes compatible with mass cytometry which allows subsequent analysis by both mass and imaging mass cytometry (imc) [ ] . these metal-tagged, time of flight activity-based probes (tof) allowed them to determine cellular activities and location of three lysosomal proteases. thus, using cathepsin l, b, and legumain as the model systems, they elegantly crafted an activity-based probe by incorporating (a) a protease-selective peptide sequence for specific enzyme recognition, (b) the acyloxymethylketone as electrophilic warhead to trap the target enzyme, and (c) the dodecanetetraacetic acid (dota) for metal chelation that is tethered to the peptide sequence via a linker (entry p , table ). they further incorporated three different lanthanides, tb, lu, and gd (a mixture of naturally occurring six isotopes), to validate their approach and to further evaluate the influence of isotopes on enzyme binding specificity. the newly developed probes exerted promising selectivity toward both recombinant proteases and proteases from cancer cell lines, hct- and mda-mb- .the authors were able to simultaneous detect the activities of proteases in hct- cells. moreover, each of the protease-specific probes exerted a similar labelling efficiency in hct- cells, regardless of their metal counterparts, which further reinforces the compatibility of this class of probes as cytometry-labelling agents. they extended their investigation to thp- cells, a non-adherent monocyte-like cell line, that expresses both cathepsin b and l but contains very low levels of legumain enzyme. probe treated thp- showed a clear labeling of both cathepsin b and l with no detectable activity of legumain. this finding was consistent with their transcriptional data. finally, the developed probes not only allowed to detect the activome of cathepsin l, b, and legumain in peripheral blood mononuclear cells (pbmc) but also enabled to categorize nk cells in two distinct populations based on protease activome levels. this strategy, unlike many existing technologies, thus allowed the simultaneous detection of target proteases, thereby providing a more holistic understanding of the activome. this certainly merits the future development of tof-based probes for multiplexed enzyme activity detection. the inhibition of cathepsin l has continue to emerge at the forefront of drug development for several human diseases. yet, no inhibitory agents targeting cathepsin l have advanced to clinical trials. while inhibitors of cathepsin b and s are currently being evaluated in clinical trials, recent failure of odanacatib, a cathepsin k inhibitor for osteoporosis, in late stage clinical trial has made pharmaceutical industries wary of targeting cathepsins. the key challenge remains gaining inhibitor selectivity with respect to the other members of cathepsins and directing them to the targeted cell types for selective functional perturbation. with its ubiquitous expression profile, the function of cathepsin l in individual cell types must be precisely defined first; this is especially important since several isoforms of cathepsin l have been reported in the distinct cellular locations, and their activity and functional profile in individual cell types still remain poorly documented. while recently developed cathepsin inhibitors and probes have significantly advanced our understanding of cathepsin l function in both normal and disease cells, more efforts are needed for the development of isoform-selective reagents for further advancement; perhaps new allosteric modules on cathepsin l enzyme can be explored and exploited for precision targeting. fortunately, several structural coordinates for cathepsin l enzyme forms, some with a diverse set of complexing ligands and some without, are now available (table ). these could aid in the development of isoform-selective inhibitory probes that will enable researchers to assess the context-specific needs of targeting cathepsin l in different cellular states. in recent years, disease-associated protease activatable prodrugs have gained much recognition and garnered breakthrough therapies in the area of antibody-drug conjugation (adc) [ ] . the majority of the adcs are constructed by tethering a cytotoxic drug with an antibody via a protease-sensitive module for targeted delivery. cathepsin b-specific module, for example, has been successfully implemented for this role, which led to the development of fda approved therapies; for example, brentuximab vedotin (adcetris ® ) for cd -positive relapsed or refractory hodgkin s lymphoma. in addition, protease cleavable prodrug strategy has also inspired the development of cathepsin b selective probe and even enabled real-time monitoring of drug release [ , ] . interestingly, ueki et al. slightly maneuvered this approach to acquire a prodrug which gets serially activated by histone deacetylase (hdac) and cathepsin l, and subsequently delivers the cytotoxic payload, puromycin, to cancer cells [ ] . this strategy has thus enabled selective targeting of cancer cells, specifically with high hdac and cathepsin l activities. taken together, these developments surely lay the foundation for the development of future cathepsin l-based therapies. much excitement remains as new cathepsin l functions continue to emerge from specific cell types in the coming years. axl x-ray . [ ] axm x-ray . [ ] f x-ray . [ ] i h x-ray . [ ] mae x-ray . [ ] maj x-ray . [ ] mqy x-ray . [ ] ezp x-ray . [ ] ezx x-ray . [ ] f x-ray . [ ] funding: s.k.p. gratefully acknowledges the financial support from the national science foundation (nsf); grant no. , , for this work. acknowledgments: authors also wish to thank senthil perumal for providing critical feedback during the preparation of this manuscript. the authors declare no conflict of interest. the lysosome turns fifty the proteome of lysosomes comprehensive search for cysteine cathepsins in the human genome specialized roles for cysteine cathepsins in health and disease cysteine cathepsins: from structure, function and regulation to new frontiers cysteine cathepsin proteases: regulators of cancer progression and therapeutic response cysteine cathepsins in extracellular matrix remodeling: extracellular matrix degradation and beyond. matrix boil the future of cysteine cathepsins in disease management cysteine proteases as therapeutic targets: does selectivity matter? a systematic review of calpain and cathepsin inhibitors cysteine cathepsins: their role in tumor progression and recent trends in the development of imaging probes cysteine cathepsins in tumor-associated immune cells cathepsin l: critical role in ii degradation and cd t cell selection in the thymus cathepsin l and b as potential markers for liver fibrosis: insights from patients and experimental models cathepsin l is essential for onset of autoimmune diabetes in nod mice impaired cathepsin l gene expression in skeletal muscle is associated with type diabetes cathepsin l deficiency reduces diet-induced atherosclerosis in low-density lipoprotein receptor-knockout mice cathepsin-l contributes to cardiac repair and remodelling post-infarction the cathepsin-l inhibitor caa protects against myocardial ischaemia-reperfusion injury cathepsin l-deficient mice exhibit abnormal skin and bone development and show increased resistance to osteoporosis following ovariectomy distinct roles of cathepsin k and cathepsin l in osteoclastic bone resorption synthesis of peptide aldehyde derivatives as selective inhibitors of human cathepsin l and their inhibitory effect on bone resorption cathepsin l is crucial for a th -type immune response during leishmania major infection proteolytic processing of dynamin by cytoplasmic cathepsin l is a mechanism for proteinuric kidney disease podocyte migration during nephrotic syndrome requires a coordinated interplay between cathepsin l and alpha integrin cathepsin l targeting in cancer treatment cathepsin l, target in cancer treatment? cysteine cathepsins: multifunctional enzymes in cancer redundancy between cysteine cathepsins in murine experimental autoimmune encephalomyelitis loss of cathepsin b and l leads to lysosomal dysfunction, npc-like cholesterol sequestration and accumulation of the key alzheimer's proteins cathepsin inhibition-induced lysosomal dysfunction enhances pancreatic beta-cell apoptosis in high glucose uniprot: a worldwide hub of protein knowledge lysosomal enzyme phosphorylation. recognition of a protein-dependent determinant allows specific phosphorylation of oligosaccharides present on lysosomal enzymes lysosomal acid phosphatase is transported to lysosomes via the cell surface an active -kda cathepsin l is secreted directly from ht fibrosarcoma cells and not via lysosomal exocytosis ph-induced conformational transitions of the propeptide of human cathepsin l. a role for a molten globule state in zymogen activation evidence that aspartic proteinase is involved in the proteolytic processing event of procathepsin l in lysosomes splice variants of human cathepsin l mrna show different expression rates characterization of human cathepsin l promoter and identification of binding sites for nf-y, sp and sp that are essential for its activity complete and limited proteolysis in cell cycle progression increased expression and activity of nuclear cathepsin l in cancer cells suggests a novel mechanism of cell transformation a cathepsin l isoform that is devoid of a signal peptide localizes to the nucleus in s phase and processes the cdp/cux transcription factor structure of human procathepsin l reveals the molecular basis of inhibition by the prosegment crystallization and preliminary x-ray diffraction studies of human procathepsin l antiinflammatory and immunosuppressive activity of sialostatin l, a salivary cystatin from the tick ixodes scapularis the tick salivary protein sialostatin l inhibits the th -derived production of the asthma-promoting cytokine il- and is effective in the prevention of experimental asthma potency and selectivity of the cathepsin l propeptide as an inhibitor of cysteine proteases cystatin-like cysteine proteinase inhibitors from human liver tight-binding inhibition of cathepsin s by cystatins the place of human gamma-trace (cystatin c) amongst the cysteine proteinase inhibitors structural and functional characterization of two allelic variants of human cystatin d sharing a characteristic inhibition spectrum against mammalian cysteine proteinases major histocompatibility complex class ii-associated p invariant chain fragment is a strong inhibitor of lysosomal cathepsin l human low-mr kininogen contains three copies of a cystatin sequence that are divergent in structure and in inhibitory activity for cysteine proteinases cystatin f is a glycosylated human low molecular weight cysteine proteinase inhibitor antimicrobial peptide ll- is both a substrate of cathepsins s and k and a selective inhibitor of cathepsin l a cathepsin l-specific inhibitor preferentially inhibits degradation of autophagosomal lc and gabarap in hela and huh- cells podocyte biology and pathogenesis of kidney disease dynamin rings: not just for fission the actin cytoskeleton of kidney podocytes is a direct target of the antiproteinuric effect of cyclosporine a the multifaceted role of the lysosomal protease cathepsins in kidney disease acceleration of polycystic kidney disease progression in cpk mice carrying a deletion in the homeodomain protein cux regulation of cell proliferation and differentiation in the kidney dilated cardiomyopathy in mice deficient for the lysosomal cysteine peptidase cathepsin l lysosomal, cytoskeletal, and metabolic alterations in cardiomyopathy of cathepsin l knockout mice cell type-specific functions of the lysosomal protease cathepsin l in the heart clinical significance of cathepsin l and cathepsin b in dilated cardiomyopathy lysosomal cysteine peptidase cathepsin l protects against cardiac hypertrophy through blocking akt/gsk beta signaling in vitro activation of pro-cathepsin b by three serine proteinases: leucocyte elastase, cathepsin g, and the urokinase-type plasminogen activator secreted cathepsin l generates endostatin from collagen xviii osteoclastic bone degradation and the role of different cysteine proteinases and matrix metalloproteinases: differences between calvaria and long bone homing of progenitor cells to ischemic tissues enhancing the outcome of cell therapy for cardiac repair: progress from bench to bedside and back homing and engraftment of progenitor cells: a prerequisite for cell therapy high glucose reduces cathepsin l activity and impairs invasion of circulating progenitor cells cathepsin l inactivates human trypsinogen, whereas cathepsin l-deletion reduces the severity of pancreatitis in mice parkinson's disease involves autophagy and abnormal distribution of cathepsin l autophagic neuron death biogenesis and proteolytic processing of lysosomal dnase ii acquisition of t regulatory function in cathepsin l-inhibited t cells by eye-derived ctla- alpha during inflammatory conditions cathepsin l in secretory vesicles functions as a prohormone-processing enzyme for production of the enkephalin peptide neurotransmitter decreased arthritis severity in cathepsin l-deficient mice is attributed to an impaired t helper cell compartment roles for cathepsins s, l, and b in insulitis and diabetes in the nod mouse plasma levels of cathepsins l, k, and v and risks of abdominal aortic aneurysms: a randomized population-based study cathepsin l in tumor angiogenesis and its therapeutic intervention by the small molecule inhibitor kgp cysteine cathepsins and the cutting edge of cancer invasion distinct roles for cysteine cathepsin genes in multistage tumorigenesis multiple roles for cysteine cathepsins in cancer transformation-dependent secretion of a low molecular weight protein by murine fibroblasts strategies for discovering and derisking covalent, irreversible enzyme inhibitors the resurgence of covalent drugs lysosomal cysteine proteases (cathepsins): promising drug targets substrate profiling of cysteine proteases using a combinatorial peptide library identifies functionally unique specificities the crystal structure of human cathepsin l complexed with e- design of noncovalent inhibitors of human cathepsin l. from the -residue proregion to optimized tripeptides structural basis for reversible and irreversible inhibition of human cathepsin l by their respective dipeptidyl glyoxal and diazomethylketone inhibitors halogen bonding at the active sites of human cathepsin l and mek kinase: efficient interactions in different environments repurposing a library of human cathepsin l ligands: identification of macrocyclic lactams as potent rhodesain and trypanosoma brucei inhibitors l-trans-epoxysuccinyl-leucylamido( -guanidino)butane (e- ) and its analogues as inhibitors of cysteine proteinases including cathepsins b, h and l e- analogs as inhibitors of cathepsin l and cathepsin s: importance of the s -p interactions for potency and selectivity structure based development of novel specific inhibitors for cathepsin l and cathepsin s in vitro and in vivo inhibition mechanism of cathepsin l-specific inhibitors based on the crystal structure of papain-clik complex the design of peptidyldiazomethane inhibitors to distinguish between the cysteine proteinases calpain ii, cathepsin l and cathepsin b diacylhydroxylamines as enzyme-activated inhibitors for serine proteases. die pharm dipeptidylpeptidase iv-inactivation with n-peptidyl-o-aroyl hydroxylamines potent and selective inactivation of proteinases with n-peptidyl-o-acylhydroxylamines reactions between dipeptidyl peptidase iv and diacyl hydroxylamines: mechanistic investigations potent and selective inactivation of cysteine proteinases with n-peptidyl-o-acyl hydroxylamines novel n-peptidyl-o-acyl hydroxamates: selective inhibitors of cysteine proteinases n-peptidyl-o-carbamoyl amino acid hydroxamates: irreversible inhibitors for the study of the s specificity of cysteine proteinases -guanidino)butane] analogues as inhibitors of cysteine proteinases: investigation of s subsite interactions peptidyl (acyloxy)methanes as quiescent affinity labels for cysteine proteinases identification of new peptide amides as selective cathepsin l inhibitors: the first step towards selective irreversible inhibitors? ) as inhibitors of cysteine proteases new peptidic cysteine protease inhibitors derived from the electrophilic α-amino acid aziridine- , -dicarboxylic acid novel peptidyl aryl vinyl sulfones as highly potent and selective inhibitors of cathepsins l and b development of a highly potent, selective, and cell-active inhibitor of cysteine cathepsin l-a hybrid design approach design of gallinamide a analogs as potent inhibitors of the cysteine proteases human cathepsin l and trypanosoma cruzi cruzain development of peptidyl α-keto-β-aldehydes as new inhibitors of cathepsin l-comparisons of potency and selectivity profiles with cathepsin b azepanone-based inhibitors of human cathepsin l systematic investigation of halogen bonding in protein-ligand interactions fluorine scan of inhibitors of the cysteine protease human cathepsin l: dipolar and quadrupolar effects in the pi-stacking of fluorinated phenyl rings on peptide amide bonds prospective evaluation of free energy calculations for the prioritization of cathepsin l inhibitors synthesis and biological evaluation of a water-soluble phosphate prodrug salt and structural analogues of kgp , a lead inhibitor of cathepsin l synthesis and biochemical evaluation of benzoylbenzophenone thiosemicarbazone analogues as potent and selective inhibitors of cathepsin l functionalized benzophenone, thiophene, pyridine, and fluorene thiosemicarbazone derivatives as inhibitors of cathepsin l design, synthesis, and biological evaluation of potent thiosemicarbazone based cathepsin l inhibitors a combined crystallographic and molecular dynamics study of cathepsin l retrobinding inhibitors identification and synthesis of a unique thiocarbazate cathepsin l inhibitor design, synthesis, and evaluation of inhibitors of cathepsin l: exploiting a unique thiocarbazate chemotype kinetic characterization and molecular docking of a novel, potent, and selective slow-binding inhibitor of human cathepsin l a small-molecule oxocarbazate inhibitor of human cathepsin l blocks severe acute respiratory syndrome and ebola pseudotype virus infection into human embryonic kidney t cells rational design of aziridine-containing cysteine protease inhibitors with improved potency: studies on inhibition mechanism vinyl sulfones as mechanism-based cysteine protease inhibitors peptidyl vinyl sulphones: a new class of potent and selective cysteine protease inhibitors: s p specificity of human cathepsin o in comparison with cathepsins s and l relative rates of michael reactions of '-(phenethyl)thiol with vinyl sulfones, vinyl sulfonate esters, and vinyl sulfonamides relevant to vinyl sulfonyl cysteine protease inhibitors vinyl sulfonate esters and vinyl sulfonamides: potent, irreversible inhibitors of cysteine proteases the marine cyanobacterial metabolite gallinamide a is a potent and selective inhibitor of human cathepsin l peptidyl aldehyde derivatives as potent and selective inhibitors of cathepsin l suppressive effect of n-(benzyloxycarbonyl)-l-phenylalanyl-l-tyrosinal on bone resorption in vitro and in vivo azepanone-based inhibitors of human and rat cathepsin k azepanone-based inhibitors of human cathepsin s: optimization of selectivity via the p substituent the discovery of odanacatib (mk- ), a selective inhibitor of cathepsin k clinical and translational pharmacology of the cathepsin k inhibitor odanacatib studied for osteoporosis inhibition of the cysteine protease human cathepsin l by triazine nitriles: amide···heteroarene π-stacking interactions and chalcogen bonding in the s pocket novel, nonpeptidic cyanamides as potent and reversible inhibitors of human cathepsins k and l discovery of trypanocidal thiosemicarbazone inhibitors of rhodesain and tbcatb cysteine protease inhibition by azapeptide esters molecular docking of cathepsin l inhibitors in the binding site of papain cathepsin l expression is up-regulated by hypoxia in human melanoma cells: role of its -untranslated region multiple cathepsins promote pro-il- beta synthesis and nlrp -mediated il- beta activation activity-based protein profiling: from enzyme chemistry to proteomic chemistry finding enzymes that are actively involved in cancer activity-based probes for protein tyrosine phosphatases global analysis of protein tyrosine phosphatase activity with ultra-sensitive fluorescent probes optimization of a protease activated probe for optical surgical navigation preclinical evaluation of cathepsin-based fluorescent imaging system for cytoreductive surgery activity-based probes that target diverse cysteine protease families functional imaging of proteases: recent advances in the design and application of substrate-based and activity-based probes activity-based profiling of proteases identification of a , molecular weight islet cell protease as cathepsin b cathepsin b-related proteases in the insulin secretory granule the identification of active forms of cysteine proteinases in kirsten-virus-transformed mouse fibroblasts by use of a specific radiolabeled inhibitor the use of benzyloxycarbonyl i]iodotyrosylalanyldiazomethane as a probe for active cysteine proteinases in human tissues inhibition of cysteine proteinases in lysosomes and whole cells quantification of cathepsins b and l in cells synthesis and antiplasmodial activity of a cysteine protease-inhibiting biotinylated aziridine- , -dicarboxylate aziridide-based inhibitors of cathepsin l: synthesis, inhibition activity, and docking studies a novel photoaffinity-based probe for selective detection of cathepsin l active form. chembiochem eur microarray-guided discovery of two-photon ( p) small molecule probes for live-cell imaging of cysteinyl cathepsin activities selective imaging of cathepsin l in breast cancer by fluorescent activity-based probes cell penetrable, clickable and tagless activity-based probe of human cathepsin l the activome: multiplexed probing of activity of proteolytic enzymes using mass cytometry-compatible activity-based probes (tof-probes) noninvasive optical imaging of cysteine protease activity using fluorescently quenched activity-based probes dynamic imaging of protease activity with fluorescently quenched activity-based probes expression of a zebrafish cathepsin l gene in anterior mesendoderm and hatching gland in situ hybridization screen in zebrafish for the selection of genes encoding secreted proteins zebrafish klf is essential for anterior mesendoderm/pre-polster differentiation and hatching toolbox of fluorescent probes for parallel imaging reveals uneven location of serine proteases in neutrophils prodrug-inspired probes selective to cathepsin b over other cysteine cathepsins real-time monitoring of drug release selective cancer targeting with prodrugs activated by histone deacetylases and a tumour-associated protease crystal structure of the parasite protease inhibitor chagasin in complex with a host target cysteine protease crystal structure and silica condensing activities of silicatein alpha-cathepsin l chimeras exploring inhibitor binding at the s subsites of cathepsin l structural basis for the recognition and cleavage of histone h by cathepsin l unreduced cathepsin l in complex with stefin a optimization of triazine nitriles as rhodesain inhibitors: structure-activity relationships, bioisosteric imidazopyridine nitriles, and x-ray crystal structure analysis with human cathepsin l. chemmedchem r)- -( -chloro- -methoxy-benzenesulfonyl)- -[ -( -chloro-pyridin- -yl)-azetidine- -carbonyl]-pyrrolidine- -carboxylic acid ( -cyano-cyclopropyl)-amide caught in the act: the crystal structure of cleaved cathepsin l bound to the active site of cathepsin l key: cord- -a fqmaly authors: vázquez, javier; lópez, manel; gibert, enric; herrero, enric; luque, f. javier title: merging ligand-based and structure-based methods in drug discovery: an overview of combined virtual screening approaches date: - - journal: molecules doi: . /molecules sha: doc_id: cord_uid: a fqmaly virtual screening (vs) is an outstanding cornerstone in the drug discovery pipeline. a variety of computational approaches, which are generally classified as ligand-based (lb) and structure-based (sb) techniques, exploit key structural and physicochemical properties of ligands and targets to enable the screening of virtual libraries in the search of active compounds. though lb and sb methods have found widespread application in the discovery of novel drug-like candidates, their complementary natures have stimulated continued efforts toward the development of hybrid strategies that combine lb and sb techniques, integrating them in a holistic computational framework that exploits the available information of both ligand and target to enhance the success of drug discovery projects. in this review, we analyze the main strategies and concepts that have emerged in the last years for defining hybrid lb + sb computational schemes in vs studies. particularly, attention is focused on the combination of molecular similarity and docking, illustrating them with selected applications taken from the literature. predicting with chemical accuracy the biological activity that a small drug-like compound can attain against its target is a major challenge in drug discovery. in the late stages of lead optimization, this task can be accomplished by resorting to enhanced sampling techniques, such as free energy calculations [ ] [ ] [ ] , which can estimate the binding affinity between a ligand and its macromolecular target. remarkably, the effort spent in developing robust algorithms in conjunction with efficient configurational sampling methods permit to estimate the binding affinity with a chemical accuracy close to the kcal/mol limit [ ] [ ] [ ] [ ] , though at the expense of a significant computational cost that prevents their application in large datasets. nevertheless, attempts have been made to alleviate this limitation through the development of automated workflows for the in silico prediction of binding affinities [ , ] , which will facilitate the usage of these sophisticated techniques to nonexpert researchers in computational chemistry. several strategies have been proposed to combine lbvs and sbvs in order to reinforce the mutual complementarity of these approaches and palliate their individual weaknesses [ ] [ ] [ ] . the major shortcoming in lbvs is the bias toward the reference template, which may result in overfitting to the input structures. when a pharmacophore is used to guide the screening of the compounds, the chemical features of the ligands present in the training set may affect the optimal choice of the pharmacophoric restraints. moreover, the available activity data may turn out to be inadequate for selecting a structural and functional pool of compounds, often limited by the absence of data relative to poorly active or inactive compounds, which may be valuable to calibrate the merits of the pharmacophore model in distinguishing between actives and inactives. on the other hand, accounting for protein flexibility is a major drawback for docking methods. the binding site of a protein is flexible and can adopt diverse conformational states, generally at the level of side chain residues but often also involving structural changes in loops and the remodeling of secondary structural elements induced upon ligand binding [ ] [ ] [ ] [ ] . furthermore, the outcome of docking studies may be largely affected by the identification of water molecules that mediate the interactions of the ligand in the binding pocket, making it necessary to explore the potential role of bridging waters or networks of ordered waters in docking calculations [ ] [ ] [ ] [ ] [ ] . on the other hand, providing an accurate score and even estimating the binding affinity at a reasonable cost compatible with the screening of large chemical libraries is still challenging for docking methods [ ] [ ] [ ] [ ] . finally, the outcome of lbvs and sbvs also appear to exhibit a strong target dependency [ , ] . for the sake of brevity, a detailed discussion of these weaknesses is omitted here, and the reader is addressed to previous studies in the literature [ ] [ ] [ ] [ ] . in this context, searching for computational strategies that can mitigate the limitations of lb and sb methods has been actively pursued in the last years. one alternative is to focus the screening effort on targeted chemical libraries that would facilitate the task of hit identification [ ] [ ] [ ] [ ] [ ] . this can be achieved via automated algorithms of molecular generation or de novo design, often assisted by artificial intelligence techniques, which aim to create sets of compounds endowed with properties similar to the structural and chemical features found in real cases, including a bias toward specific ranges of physicochemical properties or toward compounds active against a given target. alternatively, a balanced combination of lb and sb methods may be devised to exploit synergistically the merits of these vs techniques, while counterbalancing their limitations, in order to increase the success rate in the screening of large chemical libraries. several strategies have been proposed to combine lbvs and sbvs in order to reinforce the mutual complementarity of these approaches and palliate their individual weaknesses [ ] [ ] [ ] . the major shortcoming in lbvs is the bias toward the reference template, which may result in overfitting to the input structures. when a pharmacophore is used to guide the screening of the compounds, the chemical features of the ligands present in the training set may affect the optimal choice of the pharmacophoric restraints. moreover, the available activity data may turn out to be inadequate for selecting a structural and functional pool of compounds, often limited by the absence of data relative to poorly active or inactive compounds, which may be valuable to calibrate the merits of the pharmacophore model in distinguishing between actives and inactives. on the other hand, accounting for protein flexibility is a major drawback for docking methods. the binding site of a protein is flexible and can adopt diverse conformational states, generally at the level of side chain residues but often also involving structural changes in loops and the remodeling of secondary structural elements induced upon ligand binding [ ] [ ] [ ] [ ] . furthermore, the outcome of docking studies may be largely affected by the identification of water molecules that mediate the interactions of the ligand in the binding pocket, making it necessary to explore the potential role of bridging waters or networks of ordered waters in docking calculations [ ] [ ] [ ] [ ] [ ] . on the other hand, providing an accurate score and even estimating the binding affinity at a reasonable cost compatible with the screening of large chemical libraries is still challenging for docking methods [ ] [ ] [ ] [ ] . finally, the outcome of lbvs and sbvs also appear to exhibit a strong target dependency [ , ] . for the sake of brevity, a detailed discussion of these weaknesses is omitted here, and the reader is addressed to previous studies in the literature [ ] [ ] [ ] [ ] . in this context, searching for computational strategies that can mitigate the limitations of lb and sb methods has been actively pursued in the last years. one alternative is to focus the screening effort on targeted chemical libraries that would facilitate the task of hit identification [ ] [ ] [ ] [ ] [ ] . this can be achieved via automated algorithms of molecular generation or de novo design, often assisted by artificial intelligence techniques, which aim to create sets of compounds endowed with properties similar to the structural and chemical features found in real cases, including a bias toward specific ranges of physicochemical properties or toward compounds active against a given target. alternatively, a balanced combination of lb and sb methods may be devised to exploit synergistically the merits of these vs techniques, while counterbalancing their limitations, in order to increase the success rate in the screening of large chemical libraries. here, our attention is focused on the methodologies and computational approaches undertaken to enrich the outcome of vs by combining lb and sb techniques. in particular, we review the main strategies that have been proposed combining molecular similarity and docking. the strengths and weaknesses of the combined approaches are illustrated by selecting representative studies reported in the literature, primarily dealing with the efforts reported in the last five years. overall, the aim of this review is to provide useful guidelines for the application of combined lb and sb methods in drug discovery. different schemes can be adopted to combine lb and sb methods. the classification proposed by drwal and griffith will be adopted in this review [ ] . accordingly, the discussion of the combined lb and sb strategies can be completed following three main categories: sequential, parallel, and hybrid, which are summarized in figure . here, our attention is focused on the methodologies and computational approaches undertaken to enrich the outcome of vs by combining lb and sb techniques. in particular, we review the main strategies that have been proposed combining molecular similarity and docking. the strengths and weaknesses of the combined approaches are illustrated by selecting representative studies reported in the literature, primarily dealing with the efforts reported in the last five years. overall, the aim of this review is to provide useful guidelines for the application of combined lb and sb methods in drug discovery. different schemes can be adopted to combine lb and sb methods. the classification proposed by drwal and griffith will be adopted in this review [ ] . accordingly, the discussion of the combined lb and sb strategies can be completed following three main categories: sequential, parallel, and hybrid, which are summarized in figure . (i) sequential approaches divide the vs pipeline in consecutive steps with the aim to perform a progressive filtering in the library of chemical compounds toward the most promising candidates, which will be selected for biological testing at the end of this multi-step process. generally, prefiltering is performed at the beginning of the vs process using lb techniques due to their reduced computational cost, whereas the most computationally demanding sb methods are exploited in the final stages of the selection process. thus, this strategy attempts to optimize the tradeoff between the computational expensiveness and the complexity of the formalism that underlies the filtering technique along the vs process. however, they do not exploit all the available information at once and maintain the limitations of the individual methods. (ii) in the parallel approach, both lb and sb methods are run independently, and the best candidates identified from each separate method are selected for biological testing. swann et al. reported a prospective application of this approach in [ ] , and subsequent studies have examined distinct functional forms for combining the ranks obtained from lb and sb methods (see below). in particular, the compounds obtained in the final rank order lead to meaningful increases in both performance and robustness over the single-modality approaches, but the results also demonstrate the sensitivity of the performance to the target structural details (i.e., the nature of the template ligand in measurements of molecular similarity and the reference protein pocket in docking studies) [ , ] . (iii) finally, the hybrid strategies comprise approaches that represent a true combination of lb and sb techniques into a standalone method. two main combinations have been followed to (i) sequential approaches divide the vs pipeline in consecutive steps with the aim to perform a progressive filtering in the library of chemical compounds toward the most promising candidates, which will be selected for biological testing at the end of this multi-step process. generally, prefiltering is performed at the beginning of the vs process using lb techniques due to their reduced computational cost, whereas the most computationally demanding sb methods are exploited in the final stages of the selection process. thus, this strategy attempts to optimize the tradeoff between the computational expensiveness and the complexity of the formalism that underlies the filtering technique along the vs process. however, they do not exploit all the available information at once and maintain the limitations of the individual methods. (ii) in the parallel approach, both lb and sb methods are run independently, and the best candidates identified from each separate method are selected for biological testing. swann et al. reported a prospective application of this approach in [ ] , and subsequent studies have examined distinct functional forms for combining the ranks obtained from lb and sb methods (see below). in particular, the compounds obtained in the final rank order lead to meaningful increases in both performance and robustness over the single-modality approaches, but the results also demonstrate the sensitivity of the performance to the target structural details (i.e., the nature of the template ligand in measurements of molecular similarity and the reference protein pocket in docking studies) [ , ] . (iii) finally, the hybrid strategies comprise approaches that represent a true combination of lb and sb techniques into a standalone method. two main combinations have been followed to achieve this goal: (i) interaction-based methods and (ii) similarity-docking methods. the former translates the observed protein-ligand interactions into pharmacophoric features and quantitative structure-activity relationship (qsar) models [ , , ] , which have been used for several applications, such as vs, the profiling of ligands, the analysis of pseudo-receptors, and de novo designs [ ] [ ] [ ] [ ] . on the other hand, the combination of molecular similarity and docking techniques has been examined in the last years as an alternative procedure to assess the reliability of predicted poses of ligands by measuring the overlay against suitable templates [ ] [ ] [ ] [ ] [ ] [ ] . high-throughput vs may be computationally demanding when large sets of compounds have to be evaluated. in this scenario, decomposing the vs pipeline into a multi-step process can be valuable to reduce progressively the number of compounds and enrich the chemical library toward the most promising scaffolds before screening with more expensive methods. lb techniques are generally used in the prefiltering step, as illustrated in different works that have exploited d fingerprints [ , ] , d molecular similarity [ ] [ ] [ ] and pharmacophore models [ ] [ ] [ ] . to enhance the drug-likeness of the compounds, knowledge-based in silico admet or pan-assay interference compounds (pains; [ ] ) filters can also be applied. for cases with a reduced number of compounds, the results obtained from the sbvs can be further refined, resorting to the structural stability observed in md simulations [ ] [ ] [ ] [ ] . as an example that illustrates the sequential application of lb and sb techniques, khan et al. [ ] performed multi-step lbvs and sbvs to identify g protein-coupled estrogen receptor- (gper- ) modulators ( figure a) . lbvs was performed based on a gper- selective agonist ( -(( ar, s, bs)- -( -bromobenzo[d] [ , ] dioxol- -yl)- a, , , b-tetrahydro- h-cyclopenta(c)quinolin- yl)ethan- -one) as a query model for screening of the emolecules library (about . million compounds used in [ ] ) using rapid overlay of chemical structures (rocs; [ ] ) and electrostatic potential screening (eon; [ ] ). then, after generation of a gper- homology model, fred [ , ] was used to screen the top-scored hits from lbvs. next, the top-ranked hits retrieved by molecular docking were clustered based on the similarity between their scaffolds. finally, the prospective validation in sk-br- and mcf- cell lines resulted in two compounds with an ec (i.e., effective drug concentration that gives half-maximal response) antiproliferative activity in the micromolar range. alternative lb and sb sequential protocols have also been adopted, as in the study by dawood et al. [ ] , where the sbvs was followed by a lbvs ( figure b ). an in-house database of phytochemicals used in traditional egyptian medicine was screened to search for inhibitors of the human aromatase enzyme. the initial size of the library allowed the direct use of molecular docking using glide [ ] [ ] [ ] . subsequently, a lb pharmacophore was used to filter the ranked compounds with phase [ , ] . in vitro testing revealed that the methylene chloride extract of artemisia annua showed the most significant aromatase inhibitory activity with an ic of . µg/ml, thus opening a path for the use of secondary metabolites in the search for new therapeutic leads. potential screening (eon; [ ] ). then, after generation of a gper- homology model, fred [ , ] was used to screen the top-scored hits from lbvs. next, the top-ranked hits retrieved by molecular docking were clustered based on the similarity between their scaffolds. finally, the prospective validation in sk-br- and mcf- cell lines resulted in two compounds with an ec (i.e., effective drug concentration that gives half-maximal response) antiproliferative activity in the micromolar range. the application of different lb and sb methods generates distinct sets of ranked compounds for the same target. given that there is no single method that consistently ranks a database of compounds in the best decreasing order, a combination of the ranks obtained from multiple lb and sb searches into a single ranking could lead to a better overall enrichment and a wider diversity of hit structures [ ] . in this context, lbvs approaches have been combined under the framework of data fusion [ , ] , targeting the search for new entities, drug repurposing, polypharmacology, and safety profile analysis [ ] [ ] [ ] [ ] . with regard to sbvs, distinct methods have been examined to yield a "consensus scoring" [ ] [ ] [ ] [ ] , relying on three main strategies: (i) the same docked poses have been evaluated with different scoring functions to build the final ranking, (ii) the results obtained for an ensemble of different protein structures of the same target have been combined to obtain a final score, and (iii) multiple docking methods have been used against a single-protein structure [ , , ] . efforts have also addressed the development of parallel protocols for combining lb and sb methods [ , , ] . table summarizes different fusion strategies that have been adopted to combine the results of lb and sb techniques in benchmarking studies. the parallel selection seems to perform better than other rank fusion metrics, although the quality of the structural information and the specific physicochemical features of the target system may influence the overall performance. for instance, tan et al. [ ] evaluated the performance of a parallel protocol that combined docking and similarity search calculations using d fingerprints on nine target enzymes. the results were combined through rank fusion, where the ranks from docking and similarity searching were added to generate the final ranking, or the parallel selection method, where compounds are alternately selected according to the ranks obtained separately for lb and sb screenings. these combinations yielded an overall improvement in compound recall in % of the calculations. furthermore, parallel selection was found to be more effective than rank fusion. table . description of selected fusion algorithms implemented in parallel ligand-based (lb) and structure-based (sb) strategies. vs: virtual screening. adds together the ranks from the different vs methods rank lists. standard statistical measures, weighted or not, are used (i.e., sum, average, and median or max. value) to combine rank positions. [ , , , ] pareto ranking ranks a compound based on how many other compounds are better in all screening methods. ties could be broken using the sum rank, as example. [ ] parallel selection compounds are alternatively selected among the top-ranked compounds obtained from each screening method until the desired number of compounds is reached. [ , ] swann et al. examined the combination of lbvs ( d graph-based extended connectivity fingerprint (ecfp ) [ ] and rocs) and sbvs (chemical gaussian overlay, cgo [ ] ) within a probabilistic framework that returns a quantitative likelihood (or probability) of observing bioactivity for the selected compounds [ ] . the analysis of the results obtained for a set of targets showed that the retrieval rates for the cumulative probability (obtained from the fusion of the individual lb and sb values) are equal to or better than the highest retrieval rate achieved with any single method. similar trends were observed for an additional external validation set of six targets, and, importantly, the method was successful in the identification of novel hit compounds in a prospective study performed against four targets not included in the training and validation sets. a number of studies dealing with the application of the parallel strategy in the search of novel hits have been reported in the last years [ ] [ ] [ ] [ ] . an illustrative example is the work by vucicevic et al. [ ] , who reported the identification of compounds with anticancer potential effects through a parallel lb and sb screening protocol ( figure a ). starting from a large virtual library with more than × compounds, those molecules that showed good ranking in both approaches were selected for biological testing. the most active compound exhibited a cytotoxic profile similar to the positive control and enhanced the apoptotic response to doxorubicin, thus representing an adjuvant chemotherapeutic strategy for doxorubicin-insensitive cancers. [ ] and rocs) and sbvs (chemical gaussian overlay, cgo [ ] ) within a probabilistic framework that returns a quantitative likelihood (or probability) of observing bioactivity for the selected compounds [ ] . the analysis of the results obtained for a set of targets showed that the retrieval rates for the cumulative probability (obtained from the fusion of the individual lb and sb values) are equal to or better than the highest retrieval rate achieved with any single method. similar trends were observed for an additional external validation set of six targets, and, importantly, the method was successful in the identification of novel hit compounds in a prospective study performed against four targets not included in the training and validation sets. a number of studies dealing with the application of the parallel strategy in the search of novel hits have been reported in the last years [ ] [ ] [ ] [ ] . an illustrative example is the work by vucicevic et al. [ ] , who reported the identification of compounds with anticancer potential effects through a parallel lb and sb screening protocol ( figure a ). starting from a large virtual library with more than × compounds, those molecules that showed good ranking in both approaches were selected for biological testing. the most active compound exhibited a cytotoxic profile similar to the positive control and enhanced the apoptotic response to doxorubicin, thus representing an adjuvant chemotherapeutic strategy for doxorubicin-insensitive cancers. finally, a more recent example is the work by costa et al. [ ] , where they performed a parallel vs application followed by md simulations in the search of a novel compound able to inhibit human immunodeficiency virus type (hiv- ) reverse transcriptase (rt) rna-dependent dna polymerase activity ( figure b ). more than , natural compounds commercially available in the zinc database were screened. as a result, hit molecules were chosen and tested in biochemical assays. however, instead of merging the output rankings, compounds shared in both lb and sb vss were selected. among them, three compounds were identified as novel nonnucleoside rt inhibitors in the low micromolar range. as a final remark, let us note that, compared to sequential methods, parallel lb and sb screenings imply a larger computational cost, as several vs techniques have to be simultaneously finally, a more recent example is the work by costa et al. [ ] , where they performed a parallel vs application followed by md simulations in the search of a novel compound able to inhibit human immunodeficiency virus type (hiv- ) reverse transcriptase (rt) rna-dependent dna polymerase activity ( figure b ). more than , natural compounds commercially available in the zinc database were screened. as a result, hit molecules were chosen and tested in biochemical assays. however, instead of merging the output rankings, compounds shared in both lb and sb vss were selected. among them, three compounds were identified as novel non-nucleoside rt inhibitors in the low micromolar range. as a final remark, let us note that, compared to sequential methods, parallel lb and sb screenings imply a larger computational cost, as several vs techniques have to be simultaneously run in order to derive their respective rankings, which will subsequently be used to generate the final selection. as noted above, hybrid lb and sb strategies can be grouped into two major categories, which are denoted as (i) interaction-based approaches and (ii) similarity-docking methods. these methods rely on the identification of patterns of protein-ligand interactions, which are subsequently used in the screening of compounds through the usage of pseudo-receptor and pseudoquery methods. since sb information is not effectively incorporated in pseudo-receptor models, we limit ourselves to giving a brief description for the sake of completeness but omit a detailed discussion, which can be found elsewhere [ ] . pseudo-receptor methods rely on the mapping of the potential interactions that may be formed by a set of reference ligands suitably aligned in their bioactive conformation to mimic their overlaid arrangement in the binding pocket [ ] [ ] [ ] . this process leads to a rough definition of the overall shape and key anchoring points of the binding pocket, which can be exploited for the screening of chemical libraries. the performance of these models is strongly affected by the chemical space of the ligand dataset and the overlay of the ligands. the model can only account for those features present in the starting set of ligands, and the superposition of ligands is sensitive to minor modifications in the chemical scaffold, especially for highly flexible ligands. in contrast with the preceding approaches, pseudoquery methods exploit the experimental structures of protein-ligand complexes in order to extract a profile of the interaction pattern established by the ligands bound to the protein target. this pattern is generally translated into fingerprints that encode ligand-target interactions or, alternatively, into pharmacophoric features and then used in similarity searches to find ligands that match the interaction pattern [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] (see table for a brief description of several formalisms). in addition, the search of novel hits can be performed, imposing constraints related to the shape and volume of the binding site. table . selection of pseudoquery methods categorized by the underlying protein-interaction model. interaction fingerprint-based sift [ ] fingerprint encoding seven predefined types of target-ligand interactions. plip [ ] a web service for the detection and visualization of seven protein-ligand interaction types considering a d space. flip [ ] for each residue, different interactions are represented in bits. padif [ ] fingerprints with the inclusion of information relative to the strength of interactions and unfavorable ones. ligandscout [ ] pharmacophores derived from six types of nonbonded protein-ligand interactions and volume constraints. flap [ ] four-point pharmacophore fingerprints with a shape component. ichem [ ] converts the protein-ligand interaction pattern in fingerprints and graphs. tifp [ ] encodes a string of unique triplets (two interacting atoms and an interaction pseudo-atom). the pioneering methods included key elements of the protein-ligand complex, such as the formation of hydrogen bonds, hydrophobic or aromatic interactions, or contacts with acidic and basic groups, often supplemented by isocontours of the binding site. as an example, salentin et al. [ ] resorted to plip to perform a pharmacophoric search of over more than , complexes using protein-ligand interaction profiles, leading to the disclosure of the fda-approved malaria drug amodiaquine as the top-ranking hit, which was subsequently validated as a potential anticancer agent showing inhibitory activity on the target protein hsp . this demonstrates the potential of pseudoquery methods for drug repurposing. recent methods have evolved to include solvation and entropy effects. for instance, tran-nguyen et al. [ ] included in their pseudoquery pharmacophoric tool the desolvation component of the protein-ligand interaction energy using a poisson−boltzmann treatment. furthermore, the analysis was decomposed in three consecutive steps: (i) the detection of druggable cavities at the surface of the protein target and the identification of pharmacophoric features, (ii) the generation of cavity-based pharmacophore queries in the d space, and (iii) molecular alignment exploiting the cavity-based feature. the proposed pharmacophoric model was benchmarked using dud-e [ ] . unique chemotypes were retrieved from high-throughput vs, being as efficient as state-of-the-art docking [ ] and shape-matching [ ] methods in both pose prediction and ranking power. as noted above, pseudoquery methods have also exploited interaction fingerprint patterns (ifp) containing information about the contacts of the ligand with the protein, thus condensing the d structural binding information into a d binary string, leading to a drastic reduction in the cost of vs. this is exemplified by the structural interaction fingerprint (sift; [ ] ) method, where crystallographic ligands are divided into two groups of fragments: (i) atoms involved in protein-ligand interactions (interaction fragments, ifs) and (ii) fragments generated by the random deletion of ligand atoms used as a control. then, for each ligand and the corresponding fragments, maccs (molecular access system) structural keys [ ] were calculated and used as a fingerprint for similarity searching. the results of their validation work suggested that ifs used as templates can increase the similarity search performance of conventional structural fingerprints. sift is included in arpeggio [ ] , a web server for the analysis of protein interactions with small-molecule ligands, proteins, and dna. the concept of if has been adopted in alternatives models to outperform conventional scoring functions in predicting the correct poses for drug-like compounds [ ] , similarity-based screening [ , [ ] [ ] [ ] [ ] [ ] , binding/unbinding kinetics [ ] , and drug resistance [ ] . an important challenge in vs is to create accurate scoring and ranking functions to identify hit compounds active against specific targets. in this context, similarity measurements between compounds can be used to assist molecular docking to score sampled poses [ ] [ ] [ ] [ ] [ ] [ ] [ ] and to discriminate between active and inactive molecules [ , ] . exploiting the synergy between molecular similarity and docking has received increasing interest in the last years, leading to hybridized tools, such as homdock [ ] and hybrid [ ] . a direct application of merging molecular similarity and docking is related to improving the prediction of the ligand pose in the binding pocket. at this point, exploiting the experimental information on the binding mode of active compounds has been shown to enhance the performance of predicting the pose of drug-like compounds [ ] [ ] [ ] . for instance, the participants of the drug design data resource (d r) grand challenge were challenged in predicting the binding poses of cathepsin s ligands. kumar and zhang [ ] tested the performance of three methods (popss [ ] , cdvs [ ] , and popss-lite) based on the concept of ligand d shape similarity. popss evaluates the shape similarity with existing crystallographic compounds bound to the target protein for predicting the poses of query ligands with unknown binding modes. the ligand with the highest shape similarity score was selected and placed into the binding pocket. after ligand placement, side-chain residues of the binding pocket were repacked based on the query ligand conformation, followed by monte carlo energy minimization of the protein-ligand complex. finally, ligand-bound structures were scored using the rosetta energy function [ ] . for cdvs and popss-lite, d shape similarity calculations were also used to identify the ligand pose. however, once the suitable ligand-receptor pair was identified, cdvs performed a standard docking using glide, and popss-lite refined the pose with an energy minimization. popss-lite exhibited an excellent performance in this challenge, leading to the lowest mean root mean square deviation (rmsd) values between the native and predicted poses. moreover, cdvs and popss were located among the best methods tested for both metrics. shape similarity between the ligand conformation and the crystallographic ligand is the most common scheme adopted for guiding the pose prediction. however, other similarity measurements and methodological refinements have been explored. as an example jacquemard et al. defined a benchmark constituted by high-quality structures representing proteins and compared the performance of three rescoring schemes applying the similarity of ifp, graph matching of interaction patterns (grim [ ] ), and rocs [ ] (figure ). grim and rocs were more efficient than ifp rescoring based on d fingerprints, even when the comparison involved structurally dissimilar molecules. in addition, the speed of calculation for all the methods was improved, facilitating the processing of a large number of poses. the search for methodological innovations is also exemplified by kumar and zhang [ ] , as they modified popss to account for water-mediated protein-ligand interactions using a continuum benchmark constituted by high-quality structures representing proteins and compared the performance of three rescoring schemes applying the similarity of ifp, graph matching of interaction patterns (grim [ ] ), and rocs [ ] (figure ). grim and rocs were more efficient than ifp rescoring based on d fingerprints, even when the comparison involved structurally dissimilar molecules. in addition, the speed of calculation for all the methods was improved, facilitating the processing of a large number of poses. the search for methodological innovations is also exemplified by kumar and zhang [ ] , as they modified popss to account for water-mediated protein-ligand interactions using a continuum poisson-boltzmann (pb) solvation model, leading to the popss-pb model. popss-pb demonstrated an excellent performance in d r gc , with mean and median rmsds of . (ranked th out of ) and . (ranked th out of ) Å, improving the performance obtained for popss and popss-lite. finally, varela-rial et al. [ ] also evaluated in the d r grand challenge an algorithm named skeledock to define the binding mode based on the structure of a protein−ligand complex. finally, varela-rial et al. [ ] also evaluated in the d r grand challenge an algorithm named skeledock to define the binding mode based on the structure of a protein−ligand complex. the algorithm defines graphs for the query and the template molecules, and then, these graphs are compared to extract a common subgraph, which describes a continuous set of atoms whose element (node) and bonds (edges) are equivalent in the two molecules ( figure ). thus, a mapping that links atoms in query and template compounds can be identified, facilitating the conformational adjustment of the atoms in the query ligand onto those in the template molecule, whereas atoms in the query molecule with no equivalent counterpart in the template are positioned by using a tethered template docking protocol. the algorithm was ranked th out of according to the mean rmsd ( . Å) and th according to the median rmsd ( . Å). (node) and bonds (edges) are equivalent in the two molecules ( figure ). thus, a mapping that links atoms in query and template compounds can be identified, facilitating the conformational adjustment of the atoms in the query ligand onto those in the template molecule, whereas atoms in the query molecule with no equivalent counterpart in the template are positioned by using a tethered template docking protocol. the algorithm was ranked th out of according to the mean rmsd ( . Å) and th according to the median rmsd ( . Å). in addition, to assist the prediction of the ligand pose in the binding cavity, similarity measurements can also be used as a weighting factor in the reranking of the docked compounds. in fact, considering that lb d-shape matching algorithms often produced better enrichments than docking, assessing the overlay of docked poses relative to known crystallographic ligands could be valuable to retrieve active compounds in screening studies. to this end, the scoring function in docking calculations could be supplemented with d molecular similarity measurements to build the final ranking in the vs process, taking into account that positive candidates accommodated in the active site are expected to share similar structural and physicochemical features that resemble those of known actives in cocrystal structures and improve the ranking of the screened ligands. the first implementations of this lb and sb scheme in docking programs were performed by marialke et al. [ ] and mcgann [ ] in the development of homdock and hybrid, respectively. homdock is a combination of optimization methods with graph-based molecular alignment (gma; [ ] ) that superposes a query molecule on a rigid template. gma places candidate ligands over the template and optimizes their placement in the field of the protein. then, the ligands are ranked according to their interaction with the protein and/or their structural similarity with the ligand. on in addition, to assist the prediction of the ligand pose in the binding cavity, similarity measurements can also be used as a weighting factor in the reranking of the docked compounds. in fact, considering that lb d-shape matching algorithms often produced better enrichments than docking, assessing the overlay of docked poses relative to known crystallographic ligands could be valuable to retrieve active compounds in screening studies. to this end, the scoring function in docking calculations could be supplemented with d molecular similarity measurements to build the final ranking in the vs process, taking into account that positive candidates accommodated in the active site are expected to share similar structural and physicochemical features that resemble those of known actives in cocrystal structures and improve the ranking of the screened ligands. the first implementations of this lb and sb scheme in docking programs were performed by marialke et al. [ ] and mcgann [ ] in the development of homdock and hybrid, respectively. homdock is a combination of optimization methods with graph-based molecular alignment (gma; [ ] ) that superposes a query molecule on a rigid template. gma places candidate ligands over the template and optimizes their placement in the field of the protein. then, the ligands are ranked according to their interaction with the protein and/or their structural similarity with the ligand. on the other hand, hybrid uses an exhaustive search algorithm, treating ligand and protein structures as rigid bodies. both the protein and ligand flexibility are addressed through multiple conformers. subsequently, the cgo ligand-based scoring function is applied. cgo scores based on how well the docked molecule matches the shape and d arrangement of the chemical features of the crystallographic ligand bound to the active site. in another vein, anighoro and bajorath published a series of comparative studies where the best poses of commercial docking software are directly scored using d similarity methods, such as the whole-ligand d shape similarity and protein-ligand ifp similarity [ , , ] . the protocol was validated by performing retrospective vs calculations for different targets, including dihydrofolate reductase, glucocorticoid receptor, hiv- protease, vascular endothelial growth factor receptor- , adenosine a a receptor, and β adrenergic receptor. as noted in figure , the hybridized approach yielded better performance in retrieving active compounds against the targets included in the validation set. thus, the results showed that ranking by whole-ligand d similarity calculations outperformed the force field-based ranking tested for both global performance and early enrichments. it was also shown that a ligand was less suitable as a reference for d similarity calculations if it contained large solvent-exposed groups not directly interacting with the target. finally, they highlighted the importance that reference ligands should be engaged in interactions within the binding site as much as possible. as rigid bodies. both the protein and ligand flexibility are addressed through multiple conformers. subsequently, the cgo ligand-based scoring function is applied. cgo scores based on how well the docked molecule matches the shape and d arrangement of the chemical features of the crystallographic ligand bound to the active site. in another vein, anighoro and bajorath published a series of comparative studies where the best poses of commercial docking software are directly scored using d similarity methods, such as the whole-ligand d shape similarity and protein-ligand ifp similarity [ , , ] . the protocol was validated by performing retrospective vs calculations for different targets, including dihydrofolate reductase, glucocorticoid receptor, hiv- protease, vascular endothelial growth factor receptor- , adenosine a a receptor, and β adrenergic receptor. as noted in figure , the hybridized approach yielded better performance in retrieving active compounds against the targets included in the validation set. thus, the results showed that ranking by whole-ligand d similarity calculations outperformed the force field-based ranking tested for both global performance and early enrichments. it was also shown that a ligand was less suitable as a reference for d similarity calculations if it contained large solvent-exposed groups not directly interacting with the target. finally, they highlighted the importance that reference ligands should be engaged in interactions within the binding site as much as possible. figure . roc plots obtained for four validation targets (dhfr: dihydrofolate reductase, gr: glucocorticoid receptor, hiv pr: hiv- protease, and vegfr : vascular endothelial growth factor receptor- ) using shape-based similarity (rocs; green), docking (autodock [ ] and moe [ ] ; cyan), and the hybridized method (yellow). reprinted with permission from the american chemical society [ ] . under the same framework, our group has recently presented a d similarity scheme to enrich the docking performance based on the usage of lipophilic descriptors [ ] determined from quantum mechanical-based continuum solvation models [ ] . the d similarity was determined by comparing the d distribution of atomic lipophilicity computed by pharmscreen [ , ] (figure ) , and the similarity measurements were exploited in conjunction with the poses obtained by using three docking programs: glide, rdock [ ] , and gold [ ] . two hybrid algorithms were examined over sets: (i) rescoring ranking (rr), where the final ranking was determined according to the score obtained from the d lipophilic similarity of the best pose generated by the docking method and the co-crystallized ligand, and (ii) consensus ranking (cr), where the final score of the compounds was obtained by merging the rankings provided directly from the docking method and from the rr. the results obtained support the synergy of the hybrid lb and sb approaches, as the cr consistently showed better performance than using only either the lb or sb methods. in addition, the results suggest that cr may overcome the existence of multiple binding modes differing from the experimental pose of the co-crystallized ligand. receptor- ) using shape-based similarity (rocs; green), docking (autodock [ ] and moe [ ] ; cyan), and the hybridized method (yellow). reprinted with permission from the american chemical society [ ] . under the same framework, our group has recently presented a d similarity scheme to enrich the docking performance based on the usage of lipophilic descriptors [ ] determined from quantum mechanical-based continuum solvation models [ ] . the d similarity was determined by comparing the d distribution of atomic lipophilicity computed by pharmscreen [ , ] (figure ) , and the similarity measurements were exploited in conjunction with the poses obtained by using three docking programs: glide, rdock [ ] , and gold [ ] . two hybrid algorithms were examined over sets: (i) rescoring ranking (rr), where the final ranking was determined according to the score obtained from the d lipophilic similarity of the best pose generated by the docking method and the co-crystallized ligand, and (ii) consensus ranking (cr), where the final score of the compounds was obtained by merging the rankings provided directly from the docking method and from the rr. the results obtained support the synergy of the hybrid lb and sb approaches, as the cr consistently showed better performance than using only either the lb or sb methods. in addition, the results suggest that cr may overcome the existence of multiple binding modes differing from the experimental pose of the co-crystallized ligand. while selecting different lb and sb strategies may provide alternative approaches to enrich the results of vs, the identification of a novel lead compound may be conditioned by the structural diversity of the chemical space encoded in compound libraries. therefore, the choice of a representative dataset well-suited to the specific structural, physicochemical, and biological features of the macromolecular target may be crucial for the successful outcome of a vs campaign, especially keeping in mind that the available chemical libraries comprise only a small portion of the synthesizable chemical universe of compounds. to facilitate this task, there has been continued progress in the availability of experimental data in the public domain during the last two decades [ ] , which is exemplified in the consolidation of curated databases of bioactive molecules with drug-like properties, as exemplified with chembl [ ] , pubchem [ ] , and drugbank [ , ] , where the user may find a comprehensive compilation of diverse information, such as the chemical structure of the compounds, physicochemical properties, biological assays data, related targets, pharmacokinetics and pharmacodynamics properties, metabolic and signaling pathways, and patents. currently, selection of the compounds can be performed through a variety of chemical databases, which can be categorized into three main groups: public, commercial (provided by vendors), and proprietary (table ; see references and for a detailed discussion). table . examples of public and commercial databases (data taken from [ , ] in this context, rather than focusing the computational effort on the massive screening of larger databases, one might consider the possibility to enhance the success of lb and sb strategies in the search of novel hit compounds by resorting to the screening of targeted chemical libraries. an example is the work by miyao et al., who reported an algorithm for the exhaustive generation of chemical structures based on inverse quantitative structure-property (qspr)/activity (qsar) relationships to build datasets of compounds endowed with a suitable range of desired properties [ ] . the synthetic feasibility of the compounds may also be accounted from the availability of information about known chemical reactions [ ] [ ] [ ] . more recently, the de novo design algorithm for exploring chemical space (daecs) exploits the combination of a two-dimensional distribution of the chemical properties with the projection of the biological activity for a set of training compounds in order to generate structures in a specific target area of the chemical space [ , ] . a library of novel designed structures is constructed through an iterative process that involves the selection of seed structures characterized with selected chemical features and the generation of novel compounds by means of introducing slight structural changes from the seed dataset. the design of target chemical libraries is actually an undertaking of increasing interest, as illustrated by a number of recent studies that have reported the implementation of artificial intelligence-based algorithms [ ] [ ] [ ] [ ] [ ] [ ] [ ] . one of them is the development of release (reinforcement learning for structural evolution), which integrates a generative deep neural network with a predictive one into a joint framework for the design of novel compounds satisfying certain chemical requirements, as illustrated with the biased selection of compounds fulfilling a specific range of physical properties (i.e., melting temperature and lipophilicity) or inhibitory activity against the desired target protein (janus protein kinase ) [ ] . another example is the transfer-learning-based generation algorithm proposed by amabilino et al. [ ] , where recurrent neural networks are used as smiles (simplified molecular-input line-entry system) generators and trained on a smaller set of molecules with the biological activity of interest for the design of focused libraries. on the other hand, the reinvent code proposed by olivecrona et al. [ ] also relies on a recurrent neural network model that operates on a smiles representation of molecules for the automated creation of molecules with predicted biological activity. very recently, this algorithm was adapted to enable the pair-based multi-objective optimization of several molecular features based on pareto dominance [ ] and applied to the de novo design of datasets of inhibitors targeting neuraminidase, acetylcholinesterase, and the main protease of the severe acute respiratory syndrome coronavirus . another issue that deserves a brief discussion concerns the discovery of ligands able to modulate protein-protein interactions (ppis) in the early stages of drug discovery. given the estimated , ppis that comprise the human interactome, the stabilization and inhibition of ppis may represent a valuable strategy to alter the oligomerization equilibria of supramolecular protein complexes, thus altering their physiological functions in the cell [ ] [ ] [ ] , which may thus be exploited in the search of novel therapeutic approaches [ , ] . nevertheless, the success of these studies may be affected by the availability of chemical libraries with ligands suitable to interact with druggable pockets at the interface of protein-protein complexes. in this context, it is worth noting the efforts made toward the design of specific databases enriched in protein-protein modulators, such as ppi-hitprofiler, which was developed to provide for any drug-like compound collection a focused chemical library enriched in putative ppi inhibitors [ ] , p i hunter , which is a learning machine tool for filtering potential ppi modulators [ ] , and fr-ppichem, which reflects the collective effort of a french consortium to provide a unique chemical library for ppi inhibition [ ] . a comparative analysis of different ppi-focused libraries was reported in the study by zhang et al. [ ] , where they noticed that ppi inhibitors tend to be larger and more hydrophobic than standard drugs and that ppi-focused libraries, although designed using different strategies, tend to share common chemical subspaces. efforts have also been conducted for the application of combined strategies in the identification of ppi modulators. as an example, singh et al. [ ] proposed a vs protocol using a hybrid sb and lb method, highlighting the benefits of d topological descriptors to assess the post-docking output. as a validation set, ppi targets with known active and inactive compounds were considered. in a first step, the compounds were docked using surflex, and the docked poses were post-processed to calculate the shape similarity and the structural interaction fingerprint similarity to the co-crystallized ppi inhibitor. notably, the hybrid protocol showed an improved performance for numerous targets, supporting the application of these combined techniques for prospective studies of ppi modulators. in a different context, it is also worth mentioning here the systemic chemogenomics/qsar procedure introduced by cruz-monteagudo et al. [ ] , which aimed to generate a disease-relevant pool of ligands by combining phenotypic data with lb and sb information in a sequential process that ended up with a phenotypic vs performed with qsar models (figure ). the first step is the selection of a representative set of ligands targeting a disease with a measurable therapeutic phenotype, such as compounds successfully evaluated in clinical trials. then, information about the ligand-target interaction, key genes, or protein targets involved in the molecular interactions and reaction networks are compiled to build a disease-relevant chemogenomics space, which should encompass potential targets directly or indirectly (i.e., cascading effects) implicated in the physiological response. gene ontology is subsequently used to encode the systemic effect of each ligand in fingerprints containing both chemical descriptors and biological information. the codified compounds are split into two classes: ligands that significantly interact with at least one target (phenotype-positive class) and compounds with no significant interaction with any of the targets associated with the desired phenotype (phenotype-negative class). finally, a qsar-based vs methodology is performed. this protocol was utilized in a retrospective study aimed at prioritizing ligands acting as neuroprotective agents in parkinson's disease, and a significant fraction of the drug candidates used as starting points could be recovered at early fractions of the screened data. compounds are split into two classes: ligands that significantly interact with at least one target (phenotype-positive class) and compounds with no significant interaction with any of the targets associated with the desired phenotype (phenotype-negative class). finally, a qsar-based vs methodology is performed. this protocol was utilized in a retrospective study aimed at prioritizing ligands acting as neuroprotective agents in parkinson's disease, and a significant fraction of the drug candidates used as starting points could be recovered at early fractions of the screened data. as a final remark, it is worth emphasizing the relevance of curating the activity data in the analysis of the compounds and the preparation of targeted chemical libraries, minimizing the bias introduced by spurious hits, which can arise from a number of unexpected factors, such as covalent modification of specific protein residues or changes in the redox state of a range of ligands. in particular, a large number of cases have been attributed to self-aggregation of the ligand in an aqueous solution [ ] [ ] [ ] . the tendency of small organic molecules to spontaneously form colloidal self-aggregates can lead to undesired artifacts in the screening of drug-like compounds, resulting in the identification of false positives [ , ] . the colloidal aggregates formed by these types of compounds exhibit common trends, such as the lack of robust structure-activity relationships, as well as the identification of time-dependent noncompetitive-like inhibition [ , ] , likely reflecting the nonspecific inhibitory mechanisms related to adsorption of the target protein onto the aggregates or the induction of conformational alterations that affect the protein's activity [ ] . in these cases, a critical parameter to be considered is the critical aggregation concentration of the compound, which turns out to be in the micromolar range for a significant number of aggregating drug-like compounds [ , ] . overall, this discussion suffices to emphasize the need to perform a detailed curation of the biological data and of the conditions used in experimental assays, as this information may have an unexpected influence on the efficacy of the bioactive compounds. as a final remark, it is worth emphasizing the relevance of curating the activity data in the analysis of the compounds and the preparation of targeted chemical libraries, minimizing the bias introduced by spurious hits, which can arise from a number of unexpected factors, such as covalent modification of specific protein residues or changes in the redox state of a range of ligands. in particular, a large number of cases have been attributed to self-aggregation of the ligand in an aqueous solution [ ] [ ] [ ] . the tendency of small organic molecules to spontaneously form colloidal self-aggregates can lead to undesired artifacts in the screening of drug-like compounds, resulting in the identification of false positives [ , ] . the colloidal aggregates formed by these types of compounds exhibit common trends, such as the lack of robust structure-activity relationships, as well as the identification of time-dependent noncompetitive-like inhibition [ , ] , likely reflecting the nonspecific inhibitory mechanisms related to adsorption of the target protein onto the aggregates or the induction of conformational alterations that affect the protein's activity [ ] . in these cases, a critical parameter to be considered is the critical aggregation concentration of the compound, which turns out to be in the micromolar range for a significant number of aggregating drug-like compounds [ , ] . overall, this discussion suffices to emphasize the need to perform a detailed curation of the biological data and of the conditions used in experimental assays, as this information may have an unexpected influence on the efficacy of the bioactive compounds. in the past decades, vs has been a powerful alternative to high-throughput screening assays due to the reduced expensiveness, the continued progress in computer resources, and the refinement in lb and sb techniques, often leading to hit rate enrichments that outperform the results obtained with experimental screenings. being the most widely used strategy for disclosing novel hit compounds in the early stages of drug discovery, the success of vs campaigns is also severely affected by the intrinsic shortcomings of both lb and sb methods, which makes it necessary to search for novel computational strategies that exploit the merits of the individual techniques synergistically. in the last years, we have witnessed a flourishment of different combined lb and sb approaches, ranging from the hierarchical application of techniques in multi-step filtering process to novel methods that integrate lb and sb techniques into a standalone framework. the progress is encouraging, but it can be anticipated that the adoption of these integrated strategies will depend on two main factors. first, an extensive benchmarking of the distinct combination strategies, including a diverse sets of targets covering distinctive structural and physicochemical features, the calibration of different descriptors for similarity measurements, and docking algorithms in retrospective studies should be necessary, eventually complemented with the prospective application to drug discovery projects. these studies should be valuable to judge not only the improvement obtained with the usage of integrated methods relative to either pure lb or sb techniques but, also, to identify the optimal combination strategy in light of the druggability characteristics of the target protein. second, the ability to implement the combined lb and sb strategies in automated modeling platforms should provide user-friendly access to the screening of targeted-oriented chemical libraries, guidelines for an appropriate design of the combination strategy, and graphical display facilities to analyze the results. the implementation in modern software will be necessary to facilitate the adoption of the combined strategies by the drug discovery community. the authors declare no conflict of interest. advancing drug discovery through enhanced free energy calculations free energy methods in drug design: prospects of "alchemical perturbation" in medicinal chemistry accuracy assessment and automation of free energy calculations for drug design exploring the effectiveness of binding free energy calculations relative binding free energy calculations in drug discovery: recent advances and practical considerations accurate estimation of the standard binding free energy of netropsin with dna bffe: a user-friendly graphical interface facilitating absolute binding free-energy calculations qligfep: an automated workflow for small molecule free energy calculations in q dynamic docking: a paradigm shift in computational drug discovery dynamic undocking and the quasi-bound state as tools for drug discovery high-throughput docking using quantum mechanical scoring. front. chem. , , single-molecule pulling simulations can discern active from inactive enzyme inhibitors the light and dark sides of virtual screening: what is there to know? improved method of structure-based virtual screening via interaction-energy-based learning cheminformatics analysis of organic substituents: identification of the most common substituents, calculation of substituent properties, and automatic identification of drug-like bioisosteric groups virtual screening of chemical libraries virtual screening: an endless staircase? virtual screening: an in silico tool for interlacing the chemical universe with the proteome a review of protein-small molecule docking methods an overview of scoring functions used for protein-ligand interactions in molecular docking key topics in molecular docking for drug design software for molecular docking: a review structural diversity of small molecule libraries quasi-orthogonal basis sets of molecular graph descriptors as a chemical diversity measure analysis and comparison of d fingerprints: insights into database screening performance using eight fingerprint methods flap: grid molecular interaction fields in virtual screening. validation using the dud data set mimic: a molecular-field matching program. exploiting applicability of molecular similarity approaches virtual screening using molecular fields. application to the dud data set development and validation of molecular overlays derived from three-dimensional hydrophobic similarity with pharmscreen comparison of shape-matching and docking as virtual screening tools rapid shape-based ligand alignment and virtual screening method based on atom/feature-pair similarities and volume overlap scoring efficient generation, storage, and manipulation of fully flexible pharmacophore multiplets and their use in -d similarity searching combining ligand-and structure-based methods in drug design projects combined virtual screening strategies hydroxybenzothiazoles as new nonsteroidal inhibitors of ® -hydroxysteroid dehydrogenase type ( ® -hsd ) discovery of novel potential selective hdac inhibitors by combine ligand-based, structure-based virtual screening and in-vitro biological evaluation integrating structure-based and ligand-based approaches for computational drug design combination of ligand-and structure-based methods in virtual screening combined strategies in structure-based virtual screening protein flexibility and ligand recognition: challenges for molecular modeling protein flexibility in docking and surface mapping bridging molecular docking to molecular dynamics in exploring ligand-protein recognition process: an overview beware of docking! waterdock . : water placement prediction for holo-structures with a pymol plugin prediction of ordered water molecules in protein binding sites from molecular dynamics simulations: the impact of ligand binding on hydration networks intriguing role of water in protein-ligand binding studies by neutro crystallography on trypsin complexes water in protein hydration and ligand recognition the current impact of water thermodynamics for small-molecule drug discovery molecular docking and structure-based drug design strategies classification of current scoring functions empirical scoring functions for structure-based virtual screening: applications, critical aspects, and challenges exponential consensus ranking improves the outcome in docking and receptor ensemble docking docking compared to d-pharmacophores: the scoring function challenge molecular similarity analysis in virtual screening: foundations, limitations and novel approaches challenges, applications, and recent advances of protein-ligand docking in structure-based drug design comparative assessment of scoring functions on an updated benchmark: . evaluation methods and general results understanding the challenges of protein flexibility in drug design comprehensive evaluation of ten docking programs on a diverse set of protein-ligand complexes: the prediction accuracy of sampling power and scoring power chemical-space-based de novo design method to generate drug-like molecules deep reinforcement learning for de novo drug design approaching target selectivity by de novo drug design de novo generation of hit-like molecules from gene expression signatures using artificial intelligence ligbuilder v : a multi-target de novo drug design approach. front a unified, probabilistic framework for structure-and ligand-based virtual screening structure-and ligand-based virtual screening on dud-e + : performance dependence on approximations to the binding pocket function-specific virtual screening for gpcr ligands using a combined scoring method similarity searching using fingerprints of molecular fragments involved in protein-ligand interactions utilizing target-ligand interaction information in fingerprint searching for ligands of related targets protein-ligand-based pharmacophores: generation and utility assessment in computational ligand profiling identification of potential aryl hydrocarbon receptor ligands by virtual screening of industrial chemicals pseudoreceptor models in drug design: bridging ligand-and receptor-based virtual screening ligand generation in the absence of receptor information combined structure-and ligand-based virtual screening to evaluate caulerpin analogs with potential inhibitory activity against monoamine oxidase b three-dimensional similarity in molecular docking: prioritizing ligand poses on the basis of experimental binding modes a hybrid virtual screening protocol based on binding mode similarity binding mode information improves fragment docking local interaction density (lid), a fast and efficient tool to prioritize docking poses assessing the performance of mixed strategies to combine lipophilic molecular similarity and docking in virtual screening a combination of d similarity search, pharmacophore, and molecular docking techniques for the identification of vascular endothelial growth factor receptor- inhibitors virtual screening-driven discovery of dual -ht / -ht a receptor ligands with pro-cognitive properties discovery of novel aminopiperidinyl amide cxcr modulators through virtual screening and rational drug design sequential ligand-and structure-based virtual screening approach for the identification of potential g protein-coupled estrogen receptor- (gper- ) modulators. rsc adv discovery of cyanopyridine scaffold as novel indoleamine- , -dioxygenase (ido ) inhibitors through virtual screening and preliminary hit optimisation virtual screening for potential allosteric inhibitors of cyclin-dependent kinase from traditional chinese medicine identification of novel cdk inhibitors by a multistage virtual screening method based on svm, pharmacophore and docking model ligand-based and e-pharmacophore modeling, d-qsar and hierarchical virtual screening to identify dual inhibitors of spleen tyrosine kinase (syk) and janus kinase (jak ) new substructure filters for removal of pan assay interference compounds (pains) from screening libraries and for their exclusion in bioassays exploring the stability of ligand binding modes to proteins by molecular dynamics simulations exploring the stability of ligand binding modes to proteins by molecular dynamics simulations: a cross-docking study molecular dynamics simulations and kinetic measurements to estimate and predict protein-ligand residence times structural stability predicts the binding mode of protein-ligand complexes openeye scientic software. eon. . . . fred pose prediction and virtual screening accuracy hybrid docking performance on standardized datasets integrated in silico-in vitro strategy for screening of some traditional egyptian plants for human artomatase inhibitors glide: a new approach for rapid, accurate docking and scoring. . method and assessment of docking accuracy glide: a new approach for rapid, accurate docking and scoring. . enrichment factors in database screening extra precision glide: docking and scoring incorporating a model of hydrophobic enclosure for protein-ligand complexes phase: a new engine for pharmacophore perception, d qsar model development, and d database screening: . methodology and preliminary results phase: a novel approach to pharmacophore modeling and d database searching virtual screening data fusion using both structure-and ligand-based methods enhancing the effectiveness of ligand-based virtual screening using data fusion combination of similarity rankings using data fusion fragment-based discovery of a chemical probe for the pwwp domain of nsd multi-aspect candidates for repositioning: data fusion methods using heterogeneous information sources a review of computational drug repositioning approaches in silico target fishing: addressing a "big data" problem by ligand-based similarity rankings with data fusion comparison of data fusion methods as consensus scores for ensemble docking using a consensus docking approach to predict adverse drug reactions in combination drug therapies for gulf war illness consensus scoring: a method for obtaining improved hit rates from docking databases of three-dimensional structures into proteins consensus scoring for protein-ligand interactions constructing and validating high-performance miec-svm models in virtual screening for kinases: a better way for actives discovery discovery of novel rock inhibitors via integrated virtual screening strategy and bioassays integrating structure-and ligand-based virtual screening: comparison of individual, parallel, and fused molecular docking and similarity search calculations on multiple targets extended-connectivity fingerpirints potential broad spectrum inhibitors of the coronavirus clpro: a virtual screening and structure-based drug design study a combined ligand-and structure-based approach for the identification of rilmenidine-derived compounds which synergize the antitumor effects of doxorubicin identification of novel acetylcholinesterase inhibitors designed by pharmacophore-based virtual screening, molecular docking and bioassay novel natural non-nucleoside inhibitors of hiv- reverse transcriptase identified by shapeand structure-based virtual screening techniques pseudo-receptor modeling: a new concept for the three-dimensional construction of receptor binding sites morpheus: a conformation-activity relationships and receptor modeling package improving the quality of d-qsar by using flexible-ligand receptor models ligandscout: -d pharmacophores derived from protein-bound ligands and their use as virtual screening filters a common reference framework for analyzing/comparing proteins and ligands. fingerprints for ligands and proteins (flap): theory and application protein-ligand interaction prediction: an improved chemogenomics approach novel method for generating structure-based pharmacophores using energetic analysis combining machine learning and pharmacophore-based interaction fingerprint for in silico screening pharmacophore search of the zinc database all in one: cavity detection, druggability estimate, cavity-based pharmacophore perception, and virtual screening structural interaction fingerprint (sift): a novel method for analyzing three-dimensional protein-ligand binding interactions plip: fully automated protein-ligand interaction profiler flip: an assisting software in structure based drug design using fingerprint of protein-ligand interaction profiles a novel interaction fingerprint derived from per atom score contributions: exhaustive evaluation of interaction fingerprint performance in docking based virtual screening ichem: a versatile toolkit for detecting, comparing, and predicting protein-ligand interactions encoding protein-ligand interaction patterns in fingerprints and graphs from malaria to cancer: computational drug repositioning of amodiaquine using plip interaction patterns directory of useful decoys, enhanced (dud-e): better ligands and decoys for better benchmarking surflex-dock . : robust performance from ligand energetic modeling, ring flexibility, and knowledge-based search arpeggio: a web server for calculating and visualising interatomic interactions in protein structures optimizing fragment and scaffold docking by use of molecular interaction fingerprints apif: a new interaction fingerprint based on atom pairs and its application to virtual screening interacting with gpcrs: using interaction fingerprints for virtual screening protein-ligand interaction fingerprints for accurate prediction of dissociation rates of p mapk type ii inhibitors revealing acquired resistance mechanisms of kinase-targeted drugs using an on-the-fly, function-site interaction fingerprint approach application of shape similarity in pose selection and virtual screening in csardock exercise integration of ligand and structure based approaches for csar- a pose prediction approach based on ligand d shape similarity improving ligand d shape similarity-based pose prediction with a continuum solvent model prospective evaluation of shape similarity based pose prediction method in d r grand challenge optimization of high throughput virtual screening by combining shape-matching and docking methods posit: flexible shape-guided docking for pose prediction binding mode similarity measures for ranking of docking poses: a case study on the adenosine a areceptor compound ranking based on fuzzy three-dimensional similarity improves the performance of docking into homology models of g-protein-coupled receptors similarity based docking d r grand challenge : evaluation of protein-ligand pose and affinity predictions d r grand challenge : blind prediction of protein-ligand poses, affinity rankings, and relative binding free energies d r grand challenge : blind prediction of protein-ligand poses and affinity rankings shape similarity guided pose prediction: lessons from d r grand challenge a cross docking pipeline for improving pose prediction and virtual screening performance the rosetta all-atom energy function for macromolecular modeling and design skeledock: a web application for scaffold docking in playmolecule graph-based molecular alignment (gma) autodock and autodocktools : automated docking with selective receptor flexibility molecular operating environment . ; chemical computing group ulc: montreal, qc, canada lipophilicity in drug design: an overview of lipophilicity descriptors in d-qsar available online: www.pharmacelera.com (accessed on rdock: a dast, versatile and open source porgram for docking ligands to proteins and nucleic acids development and validation of a genetic algorithm for flexible docking cheminformatics in drug discovery, an industrial perspective cibrí an-uhalte, e.; et al. the chembl database in pubchem: a public information system for analyzing bioactivities of small molecules drugbank: a comprehensive resource for in silico drug discovery and exploration the next level in chemical space navigation: going far beyond enumerable compound libraries virtual compound libraries in computer-assisted drug discovery exhaustive structure generation for inverse-qspr/qsar dogs: reaction-driven de novo design of bioactive compounds customizable generation of synthetically accessible, local chemical subspaces the synthesizability of molecules proposed by generative models development of a new de novo design algorithm for exploring chemical space creating the new from the old: combinatorial libraries generation with machine-learning-based compound structure optimization generative network complex for the automated generation of drug-like molecules guidelines for recurrent neural network transfer learning-based molecular generation of focused libraries molecular de-novo design through deep reinforcement learning de novo drug design of targeted chemical libraries based on artificial intelligence and pair-based multiobjective optimization ppi inhibitor and stabilizer development in human diseases site-directed fragment-based screening for the discovery of protein-protein interaction stabilizers modulators of - - protein-protein interactions modulation of protein-protein interactions for the development of novel therapeutics modulating protein-protein interaction networks in protein homeostasis designing focused chemical libraries enriched in protein-protein interaction inhibitors using machine-learning methods p ichem: focused chemical libraries dedicated to orthosteric modulation of protein-protein interactions fr-ppichem: an academic compound library dedicated to protein-protein interactions focused chemical libraries-design and enrichment: an example of protein-protein interaction chemical space fast rescoring protocols to improve the performance of structure-based virtual screening performed on protein-protein interfaces systemic qsar and phenotypic virtual screening: chasing butterflies in drug discovery a high-throughput screen for aggregation-based inhibition in a large compound library a detergent-based assay for the detection of promiscous inhibitors internal structure and preferential protein binding of colloidal aggregates colloidal aggregation affects the efficacy of anticancer drugs in cell culture revealing drug self-associations into nano-entities case studies of minimizing nonspecific inhibitors in hts campaigns that use assay-ready plates how do small molecule aggregates inhibit enzyme activity? a molecular dynamics study stoichiometry and physical chemistry of promiscuous aggregate-based inhibitors key: cord- -ldkuwcc authors: he, hui-qiong; ye, richard d. title: the formyl peptide receptors: diversity of ligands and mechanism for recognition date: - - journal: molecules doi: . /molecules sha: doc_id: cord_uid: ldkuwcc the formyl peptide receptors (fprs) are g protein-coupled receptors that transduce chemotactic signals in phagocytes and mediate host-defense as well as inflammatory responses including cell adhesion, directed migration, granule release and superoxide production. in recent years, the cellular distribution and biological functions of fprs have expanded to include additional roles in homeostasis of organ functions and modulation of inflammation. in a prototype, fprs recognize peptides containing n-formylated methionine such as those produced in bacteria and mitochondria, thereby serving as pattern recognition receptors. the repertoire of fpr ligands, however, has expanded rapidly to include not only n-formyl peptides from microbes but also non-formyl peptides of microbial and host origins, synthetic small molecules and an eicosanoid. how these chemically diverse ligands are recognized by the three human fprs (fpr , fpr and fpr ) and their murine equivalents is largely unclear. in the absence of crystal structures for the fprs, site-directed mutagenesis, computer-aided ligand docking and structural simulation have led to the identification of amino acids within fpr and fpr that interact with several formyl peptides. this review article summarizes the progress made in the understanding of fpr ligand diversity as well as ligand recognition mechanisms used by these receptors. the formyl peptide receptors (fprs) are a group of g protein-coupled chemoattractant receptors that play important roles in host defense and inflammation [ , ] . they are so named because their cognate agonists are peptides bearing formylated methionine (fmet), such as those derived from bacterial and mitochondrial proteins [ , ] . the present review focuses on the fpr family members in humans and mice and their interaction with a wide variety of ligands. the reader is referred to recently published reviews on fpr signaling [ ] , synthetic non-peptide ligands for fprs [ ] and characterization of fpr knockout mice [ ] . the reader is also referred to a general overview of fprs and their nomenclature [ ] . three genes coding for human g protein-coupled formyl peptide receptors have been cloned, including fpr , fpr and fpr [ ] [ ] [ ] [ ] [ ] [ ] . among them, fpr and fpr share an overall high sequence identity ( figure ) and certain overlapping functions. [ ] in three fpr isoforms are shown in color; (b) schematic diagram for the location of the positive selection sites in the formyl peptide receptors, based on the sequence of human fpr . the amino acids at these positions in each of the three receptors are marked on the right. fpr and fpr are expressed in both monocytes and neutrophils, while fpr is found in monocytes but not neutrophils. besides myeloid cells, fpr is expressed in astrocytes, microglial cells, hepatocytes and immature dendritic cells. fpr shows an even wider distribution pattern than fpr and is expressed in a variety of non-myeloid cells including astrocytoma cells, epithelial cells, hepatocytes, microvascular endothelial cells, neuroblastoma cells, in addition to phagocytic leukocytes. in recent studies, fpr has been associated with anti-bacterial inflammation and metastasis of malignant glioma cells, while fpr is implicated in the pathogenesis of chronic inflammatory diseases such as systemic amyloidosis, alzheimer's disease, atherosclerosis, systemic lupus erythematosus and ovarian cancer metastasis et al [ ] [ ] [ ] [ ] [ ] . more examples and details of fpr involvement in multiple diseases are provided in recent reviews [ , ] . since the human fpr gene (fpr ) was first cloned, orthologs have been identified in other mammalian species, including rabbits, guinea pigs, horses, rats and mice (reviewed in [ ] ). the genes coding for fpr members comprise a large family that implicates a complex evolutionary path with evidence of positive selection [ ] . despite overall sequence homology, these genes vary considerably in their numbers and sequences between humans and other species. for example, the mouse fpr gene family has eight known members (mfpr , mfpr , mfpr-rs , mfpr-rs , mfpr-rs , mfpr-rs , mfpr-rs and mfpr-rs ) clustered on mouse chromosome a . . among these genes, mfpr-rs (ψmfpr-rs ) is found as a pseudogene that does not code for a functional receptor. although several mouse fpr genes are present in neutrophils, work on mfprs has been focused mostly on the gene products of mfpr and mfpr , which are widely expressed in mouse phagocytic leukocytes and show high similarity to their human counterparts. targeted deletion of mfpr or mfpr renders mice more susceptible to bacterial infection without changing their viability and fertility [ , ] . under unstimulated conditions, mice lacking mfpr or mfpr behave normally. however, in models of human diseases, both mfpr −/− and mfpr −/− mice respond differently from their wildtype littermates, [ ] in three fpr isoforms are shown in color; (b) schematic diagram for the location of the positive selection sites in the formyl peptide receptors, based on the sequence of human fpr . the amino acids at these positions in each of the three receptors are marked on the right. fpr and fpr are expressed in both monocytes and neutrophils, while fpr is found in monocytes but not neutrophils. besides myeloid cells, fpr is expressed in astrocytes, microglial cells, hepatocytes and immature dendritic cells. fpr shows an even wider distribution pattern than fpr and is expressed in a variety of non-myeloid cells including astrocytoma cells, epithelial cells, hepatocytes, microvascular endothelial cells, neuroblastoma cells, in addition to phagocytic leukocytes. in recent studies, fpr has been associated with anti-bacterial inflammation and metastasis of malignant glioma cells, while fpr is implicated in the pathogenesis of chronic inflammatory diseases such as systemic amyloidosis, alzheimer's disease, atherosclerosis, systemic lupus erythematosus and ovarian cancer metastasis et al. [ ] [ ] [ ] [ ] [ ] . more examples and details of fpr involvement in multiple diseases are provided in recent reviews [ , ] . since the human fpr gene (fpr ) was first cloned, orthologs have been identified in other mammalian species, including rabbits, guinea pigs, horses, rats and mice (reviewed in [ ] ). the genes coding for fpr members comprise a large family that implicates a complex evolutionary path with evidence of positive selection [ ] . despite overall sequence homology, these genes vary considerably in their numbers and sequences between humans and other species. for example, the mouse fpr gene family has eight known members (mfpr , mfpr , mfpr-rs , mfpr-rs , mfpr-rs , mfpr-rs , mfpr-rs and mfpr-rs ) clustered on mouse chromosome a . . among these genes, mfpr-rs (ψmfpr-rs ) is found as a pseudogene that does not code for a functional receptor. although several mouse fpr genes are present in neutrophils, work on mfprs has been focused mostly on the gene products of mfpr and mfpr , which are widely expressed in mouse phagocytic leukocytes and show high similarity to their human counterparts. targeted deletion of mfpr or mfpr renders mice more susceptible to bacterial infection without changing their viability and fertility [ , ] . under unstimulated conditions, mice lacking mfpr or mfpr behave normally. however, in models of human diseases, both mfpr −/− and mfpr −/− mice respond differently from their wildtype littermates, indicating regulatory roles of these receptors in host defense and inflammation. targeted gene disruption of mfpr also suggested a homeostatic role for the mfpr in the production of glucocorticoids and anxiety-like behavior [ ] . as the suggested ortholog of human fpr , mfpr was identified as a low-affinity receptor for the classical formyl tri-peptide fmet-leu-phe (fmlf) [ ] , but this receptor responds well to several n-formyl peptides derived from other bacteria (e.g., fmifl, fmivil, fmivtlf) and mitochondria (e.g., fmmyalf) [ , ] . in general, mfpr is not a high-affinity receptor for either fmlf or other native formyl peptides tested so far [ ] . however, it responds to endogenous peptide agonists for fpr , including the amyloidogenic proteins saa (serum amyloid a) [ ] and aβ [ ] . likewise, this receptor can be inhibited by fpr -specific antagonists when expressed in neutrophil or by stably transfected cells. besides this, mouse mfpr has also been reported as a receptor for f l [ ] that is a potent agonist for human fpr [ ] . published reports indicate that other members of the murine mfpr family are not coding for stereotypic formyl peptide receptors. in recent studies, mfpr-rs (ψmfpr-rs ) has been characterized as a constitutively expressed gene associated with mouse longevity [ ] . the gene products of five other mfprs, including mfpr-rs , mfpr-rs , mfpr-rs , mfpr-rs , and mfpr-rs , are recently described as mouse vomeronasal sensory receptors [ , ] . phylogenetic analysis suggested that they belong to a chemosensory gpcr family, which is characterized with an olfactory function and has the ability to identify pathogenic states. among them, the property of mfpr-rs is still uncertain as it is also expressed in neutrophils. it was believed that mfpr-rs might have some functional overlap with human fpr because a variant of mfpr-rs was reported to encode a mouse receptor for lipoxin a (lxa ) [ ] , an eicosanoid ligand for fpr . however, functional and pharmacology assays conducted in stably transfected cells suggested that mfpr-rs responds poorly to most agonists that activate human and murine fpr receptors [ ] . these observations add to studies using knockout animal models and together indicate that murine fprs (especially mfpr and mfpr ) share numerous structural and pharmacological properties with human fprs, but the evolutionary correlation between them is by no means linear. the fpr family is well known for the structural diversity of their ligands, including a variety of ligands with different chemical properties and origins ranging from natural peptides to synthetic non-peptide compounds [ , ] (table ) . fprs are therefore classified as a group of pattern recognition receptors (prrs) that recognize pathogen-associated molecular patterns (pamps) and damage-associated molecular pattern (damps) [ ] [ ] [ ] . among all the ligands for the fprs, n-formylated peptides, particularly fmlf (an e. coli-derived chemotactic peptide), are most often studied. fmlf is among the first characterized and also the shortest formyl peptides with full agonistic activities [ ] [ ] [ ] . these peptides are initiated with n-formyl methionine and are generally cleavage products of bacterial and mitochondrial proteins. n-formylated peptides constitute the most commonly studied class of fpr agonists that trigger a variety of biological activities in myeloid cells, such as chemokinesis, chemotaxis, calcium flux, cytokine production and superoxide anion generation. through binding with the high affinity receptor fpr , fmlf and other n-formylated peptides serve as potent chemoattractants, which also include activated complements (c a, c a) and chemokines, in recruiting and guiding leukocytes to the site of bacterial infection and to damaged tissues. a wealth of literatures have described the proinflammatory properties of n-formyl peptides. as early as years ago, right after the discovery of n-formyl peptides, studies have shown that fmlf was involved in the pathogenesis of multiple inflammatory diseases such as colitis [ ] [ ] [ ] , pouchitis, ulcerative colitis and crohn's disease [ , ] and juvenile peridotitis [ ] . studies also reported that inhalation or injection of fmlf can cause rapid neutropenia and bronchial inflammation in human and other mammals [ ] [ ] [ ] [ ] . [ ] f. n-phenylureas ag- [ ] fpr > fpr [ , ] e. pyridazin- ( h)-one derivative [ ] molecules , , of [ ] [ ] human fpr is a low affinity receptor for fmlf and many potent formyl peptide agonists for fpr . however, fpr displays relatively high affinity for n-formylated peptides of specific composition and longer length. for instance, fpr responds better to peptides carrying positive charges at the c-terminus (e.g., fmlfk, fmlfik) than peptides with negative charges (e.g., fmlfe and fmlf) [ ] . studies also showed that fpr responds well to formyl peptides of microbial origin other than e. coli, such as fmifl (s. aureus) and fmivil (l. monocytogenes) in calcium mobilization and camp release assays [ ] . in general, most bacteria-derived formyl peptides are more potent at fpr than fpr . a prominent exception is the recently described cytolytic peptides psm (phenol-soluble modulins) that are α-helical peptides composed of - amino acids [ ] . although they are predominantly secreted from community-associated methicillin resistant staphylococcus aureus (ca-mrsa) in formylated form, psmα peptides are more selective for fpr than for fpr [ ] [ ] [ ] . another exception is mitocryptide- , a neutrophil-activating formyl peptide produced from mitochondrial cytochromes. mitocryptide- can directly bind to fpr and activate it with an ec of . nm in calcium flux assay, whereas it manifests no interaction with fpr [ ] . in addition, another group of formyl peptides derived from mitochondria (fmmyalf, fmyfiniltl, and fmlkliv) were found equally potent on fpr and fpr with ec values ranging from nm to nm [ ] . despite the different potency and selectivity of these formyl peptides, the n-formyl group is important for optimal interaction with fpr and fpr . it is suggested that fpr is a highly effective receptor recognizing formyl peptides and mediating phagocyte functions, while fpr has an overall lower affinity but it can discriminate between formyl peptides with different sizes and features. in contrast, no bacterial or mitochondrial formyl peptide has been described as agonist for fpr by far. noting that the prototypic fpr agonist fmlf is not a potent activator for murine fprs, including mfpr and mfpr , when compared to human fpr . in agreement with the research that murine neutrophils are more susceptible to bacteria other than e. coli [ ] , mfpr was found more responsive to formyl peptides derived from listeria monocytogenes (fmivtlf), staphylococcus aureus (fmifl), and mitochondria (fmmyalf) [ , ] . except for formyl peptides, a number of different peptides/proteins, including microbial and host-derived non-formyl peptides/proteins and peptides from synthetic libraries, have been identified to be agonists for fprs. it appears that the absence of n-formyl group and lack of overall sequence similarity with formyl peptides mentioned above do not impair their agonistic activity at fprs. in general, the ligand diversity is more prominent for fpr than fpr . the non-formylated peptides/proteins derived from microbe are represented by virus envelope proteins (gp and gp ) of the human immunodeficiency virus type (hiv- ), which contains at least five fpr-recognizing peptide sequences composed of - amino acids. most of these sequences were found more potent at fpr than at fpr and fpr [ ] [ ] [ ] [ ] [ ] , except only one is selective for fpr and mfpr [ , , ] . a cecropin-like peptide (hp - ) from helicobacter pylori was also identified as an agonist for fprs [ , ] . the studies demonstrated that in h. pylori infection, hp - was able to induce chemotaxis of monocytes and basophils, and evoke superoxide release via signaling through fpr and fpr . recently, two antimicrobial peptides (amps) isolated from scolopendra subspinipes mutilans were reported to elicit neutrophil chemotactic migration through fpr [ ] . the host-derived non-formyl peptide agonists for fprs are generally peptides and proteins associated with human diseases and inflammation. this group of agonists activates fprs independently of the n-formyl group and they also show a preference for fpr . a prominent example of them is serum amyloid a (saa), an acute-phase protein that is implicated in chronic inflammation and amyloidosis. as the first identified endogenous peptide agonist for fpr [ ] , documented studies have shown that it exerts pro-inflammatory activities through fpr in phagocytes, epithelial cells and t lymphocytes, including stimulating production of inflammatory mediators and enhancing the expression of cytokine receptors [ ] [ ] [ ] [ ] [ ] . recently, questions have been raised regarding the proinflammatory cytokine-like properties of native saa, since most studies of saa and fpr are performed using a commercially available recombinant protein that contains amino acid substitutions of saa in saa [ , , ] . however, research conducted with saa isoforms purified from an e. coli expression system suggested that major saa isoforms have cytokine-inducing activity and minor substitutions of amino acids affected their potency at fpr [ ] . in vivo studies have shown th -induction activity of saa in gut epithelium, although whether these activities are attributed to fpr or other saa receptors remain unclear [ , ] . another amyloidogenic disease-associated fpr agonist is the prion protein fragment prp , which was reported to interact with fpr in glial cells and induce calcium mobilization, chemotaxis as well as production of pro-inflammatory cytokines [ ] . in addition to saa and prp , other two amyloidogenic disease-associated peptides were found to be agonists for fpr : the -amino acid form of aβ amyloid peptide (aβ ) and humanin. although both peptides use fpr to induce migration and activation of monocytic phagocytes in the brain, aβ and humanin display divergent roles in the development of alzheimer's disease: aβ is a major cause of fibrillary formation and deposition in brain of ad patients [ , ] , whereas humanin is neuroprotective on the contrary [ ] . studies suggested that humanin reduces aggregation and fibrillary formation by inhibiting aβ /fpr interaction in mononuclear phagocytes [ ] . fpr and mfpr are also functional receptors for humanin in humans and mice, respectively [ , ] . annexin a , a glucocorticoid-regulated protein, and its n-terminal peptides (ac - and ac - ) are fpr agonists that have dual roles in inflammation. at high concentrations, the annexin a peptides fully activate fpr like the conventional agonists that induce pro-inflammatory responses. in contrast, at low concentrations they only show partial activity at fpr [ ] , leading to neutrophil desensitization and inhibiting neutrophil migration induced by other chemoattractants. all three fpr members are implicated in the various functions of annexin a peptides. some researchers thought that these peptides use fpr for anti-inflammatory actions [ ] , while others suggested the presence of receptors other than fpr and fpr as being responsible for the resolving effects [ ] . the exact mechanism for the pro-/anti-inflammatory activity of annexin a peptides is not entirely clear. the antimicrobial peptide ll- and its murine homolog cramp (cathelicidin-related anti-microbial peptide) were identified as fpr agonists [ , ] . ll- is a cleavage product of the neutrophil granule protein cathelicidin found in leukocytes and epithelial cells. as a pro-inflammatory mediator, ll- activates fpr and evokes superoxide generation in human fibroblasts [ ] . it also induces directional migration of human monocytes, neutrophils, and t lymphocytes [ ] . however, ll- also inhibits saa signaling in human neutrophils, including il- production and chemotaxis through fpr [ ] . ll- seems to be a pleiotropic peptide and its interaction with fpr is implicated in wound healing, cell proliferation [ ] , angiogenesis [ ] and anti-or pro-tumorigenesis [ ] [ ] [ ] . in a recent study, the proinflammatory circuits of ll- and leukotriene b was reported. in human neutrophils, ll- /fpr promotes leukotriene b production, which in turn elicits further ll- release through blt (leukotriene b receptor ) [ ] . upar is the cell surface receptor for urokinase-type plasminogen activator (upa) that is a serine protease involved in regulation of fibrinolysis, cell adhesion, migration and tissue repair. upar can be cleaved by different proteases, including upa, generating soluble upar fragments that can be recognized by different fpr members. for instance, upar d d - , a cleaved peptide corresponding to residues from to of upar, binds to and activates fpr in monocytes, inducing cell migration [ ] . moreover, upar - ( srsry ) interacts with fpr [ ] , while upar [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] activates both fpr and fpr in basophils [ ] . in a more recent study, a new host-derived chemotaxis agonist fam d, for the fpr and fpr was identified [ ] . fam d, a cytokine-like protein, is constitutively expressed in the gastrointestinal tract with significant induction in dextran sulfate sodium (dss)-induced colitis [ ] . fam d was found to strongly attract the chemotaxis of human peripheral blood neutrophils and monocytes. through functional screen of a group of chemoattractant receptors, fpr and fpr were determined to be the receptors for fam d. in hek cells transfected to express fpr or fpr , fam d induced significant cell chemotaxis and calcium mobilization that was desensitized by other fpr agonists (fmlf and wkymvm), and vice versa. the activation of erk / and p mapk signaling caused by fam d in neutrophils was blocked by an antagonist of fpr (wrw ). this study suggested that fam d/fpr interaction might play a role in intestinal homeostasis and gastrointestinal inflammation. the fpr receptors also have other endogenous peptide ligands, such as cathepsin g for fpr , chemokine ccl (amino acids and its n-terminal fragment shaagtide for fpr , a vasoactive intestinal neuropeptide vip (vasoactive intestinal polypeptide) for fpr , and a heme-binding protein fragment f l for fpr (table ). in addition, exogenous allergens including house dust mite and birch pollen extracts were recently shown as being agonistic for fpr and fpr . the detailed properties of these fpr agonists are discussed in previous reviews [ , , ] . among all the synthetic peptide agonists for fprs, a hexapeptide, trp-lys-tyr-met-val-d-met-nh (wkymvm) isolated from a random peptide library [ ] , was identified to be a strong activator for fpr , fpr and fpr [ , ] . wkymvm conjugated with fitc in the second residue lys was shown slightly more efficacious in binding to fpr than to fpr [ ] . wkymvm is by far the most potent peptide agonist for fpr , being able to activate fpr at picomolar concentrations in chemotaxis assays. wkymvm, a derivative of wkymvm with the substitution of l-methionine at the carboxyl terminus, becomes highly selective for fpr and is weakly agonistic for fpr [ ] . another peptide identified from library screen, mmk- (lesifrsllfrvm), is a highly selective chemotactic agonist for fpr [ , ] . cgen- a, a amino acids anti-inflammatory peptide obtained from a computational platform, was identified to be agonist for fpr and fpr [ ] . in another study, a tethered library was screened and a peptide with the sequence mmwll was identified as an fpr agonist [ ] . this peptide becomes -fold more potent when the first met is n-formylated, consistent with the preferential recognition of fmet-containing peptides by fpr . in a recent study [ ] , a series of aapeptides, a class of peptidomimetics based on n-acylated-n-aminoethyl amino acid residues, were designed and synthesized to mimic the structure and function of the prototypic tripeptide fmlf, the reference agonist of fpr . three of these fmlf-mimicking aapeptides were found effective in activating fpr-expressing cells in calcium mobilization assay, albeit at concentrations above µm. certain aapeptides were shown to be more efficacious than fmlf at a higher concentration ( µm). similar to fmlf, these aapeptides have receptor preference for fpr over fpr . in comparison with peptide/protein fpr agonists, the small molecule agonists screened from chemical library are more stable and highly selective for certain fpr members. these properties make them valuable tools for detailed characterization of fprs, which is important for a better understanding of the complex role of fprs in vivo. fprs are involved in a variety of signaling systems and their distribution and functions are not limited to the immune system. as a result, these small molecules screened by targeting fprs as agonists or antagonists are expected to serve as potentially useful agents in therapeutic treatment. the first reported non-peptide agonist for fpr from library screening is a quinazolinone derivative named quin-c [ ] . quin-c ( -butoxy-n-[ -( -methoxy-phenyl)- -oxo- , -dihydro- h-quinazolin- yl]-benzamide) was found highly selective for fpr as opposed to fpr . it was identified as a biased agonist, as it stimulated calcium mobilization through fpr but did not induce substantial neutrophil superoxide generation, even at concentrations up to µm. a later study revealed an anti-inflammatory role for quin-c in bleomycin-induced lung injury in a mouse model [ ] . the murine receptors for quin-c were determined to be mfpr and mfpr [ ] . competitive binding analysis showed that quin-c does not share the same recognition sites with formyl peptides in mfprs [ ] . given that quin-c is non-competitive with formyl peptides, it is interesting to surmise that quin-c may act at fpr as an ago-allosteric modulator. however, the precise mechanism for the agonistic activity of quin-c requires further examination. since the first synthetic non-peptide fpr agonist was reported, an increasing number of small-molecule agonists have been discovered in recent years [ ] . so far, a series of structurally divergent small-molecules with high-affinity have been identified as fpr ligands via cell-based assays for high throughput screening (hts), as well as structure-activity relationship (sar)-directed design and synthesis [ ] [ ] [ ] , ] . in general, the identification of new molecules relies on known structures, sar analysis and computer-aided design. using this strategy, around one hundred fpr agonists with high potency and clear selectivity have been identified and optimized in the reported research. based on the structures with varying scaffolds and sar-directed evaluation, these small compounds fall into at least groups: benzimidazoles, pyrazolones (e.g., compound ) , n-substituted benzimidazoles, pyridazin- ( h)-ones, chiral pyridazines, n-phenylureas, chiral -( h-indol- -yl)- -[ -( -nitrophenyl) ureido] propanamides, -(n-piperazinyl)acetamide derivatives, quinazolinones (exemplified by quin-c ), and other derivatives (reviewed by [ ] ). among these molecules, the most potent fpr -selective agonist was identified to be a polyphenylure derivative with an ec of nm in calcium flux assays and a ki of nm in binding assays. this compound, [ -phenyl- -((r)- -( -phenyl- -((s)- -( -phenylureido)propan- -yl)ureido)hexan- -yl)- -( -phenylbutyl)urea], coded as - is also the most potent synthetic non-peptide agonist for fpr known to date [ ] . in addition to - , other chemical agonists with high fpr specificity, such as the benzimidazole derivates [ ] and the pyridazinone-based compounds [ ] , have been described, although they act with ec above micromolar (> . µm). compared with fpr , fpr have more specific and potent agonists identified from screening of compound library and through rational design. currently, more than fpr -slective agonists have been reported to have an ec in the nanomolar range in functional assays, including three pyrazolone derivatives [ ] , an n-substituted benzimidazole derivative [ ] , an n-phenylurea derivative [ ] , a chiral n-phenylurea derivative [ ] , two benzodioxole derivatives of n-phenylureas [ ] , six chiral ureidopropanamido derivatives [ , ] , a phenylsubstituted urea compound [ ] , a compound with a unique -( -phenyl- -((phenylamino)methyl)- , , -triazol- -ylthio) acetamide scaffold [ ] , and a pyrrolidine bis-diketopiperazine derivative [ ] . as expected, there are numerous agonists with dual specificity (fpr /fpr ) (reviewed by [ ] ). noting that a pyrazolone derivative, designated in most publications as "compound ", was first identified as a fpr -specific agonist [ ] , but it was redefined to be a mixed fpr /fpr agonist in an independent screen recently [ ] . it is not clear for most of the reported small molecule agonists whether they interact with murine formyl peptide receptors since they are designed and screened to target human fprs and murine fprs have distinct properties compared to their human counterparts. lipoxin a (lxa ), derived from arachidonic acid, has highly potent anti-inflammatory and pro-resolving activities based on in vivo studies [ ] [ ] [ ] [ ] . this eicosanoid was reported to directly bind to fpr and a variant of mouse mfpr-rs [ , , , ] . lxa was shown to have a high affinity (k d of~ . nm) in binding to fpr -expressing chinese hamster ovary cells [ ] . despite the high-affinity binding, lxa is an atypical ligand for fpr as it cannot activate proinflammatory activities such as chemotaxis, enzyme release and ros production. lxa also fails to activate fpr -dependent signaling, such as calcium mobilization, in transfected cell lines [ ] [ ] [ ] . these findings do not support the original report that lxa could stimulate gtpase activity in fpr -expressing cho cells [ ] . it appears difficult to determine the role of lxa as an fpr agonist without establishing a standard in compound preparation. moreover, fpr specific antagonists, stereoselective analogs, antibodies, and transgenic approaches are required in further studies, and the interaction of lxa with other receptors such as the cannabinoid receptor cb [ ] , aryl hydrocarbon receptor (ahr) [ ] and estrogen receptor [ ] will have to be considered. resolvins, protectins and maresins are novel pro-resolving mediators that are biosynthesized from omega- fatty acids, including docosahexaenoic acid (dha) and eicosapentaenoic acid (epa). these lipid mediators block neutrophil recruitment, promote monocyte activation, and enhance macrophage phagocytosis. resolvin d , a biosynthetic product from dha, was reported to activate fpr and gpr , an orphan gpcr. similar with lxa , resolvin d cannot evoke calcium mobilization through fpr or gpr . the interaction of resolvin d with these gpcrs was examined by a gpcr-β-arrestin-coupled system [ ] . selective knock down or inhibition of fpr reduces the resolvin d response, such as macrophage phagocytosis [ ] and salivary epithelium migration [ ] . resolvin d and fpr are also implicated in pulmonary inflammation [ ] and obesity [ ] . oxidized low-density lipoprotein (oxldl), an atherogenesis associated molecule, was recently found to stimulate macrophages via fpr signaling [ ] . the oxldl-induced foam cell formation and tnf-α production could be inhibited by an fpr antagonist wrw , but not affected by an fpr antagonist. in fpr -expressing rbl- h cells, oxldl also stimulated significant calcium mobilization and chemotaxis [ ] . in another study, the mimetic peptide l- pa (table ) of apoa-i, a major component of circulating high-density lipoprotein (hdl), was reported to induce calcium flux and chemotaxis through fpr . l- pa is probably a biased agonist of fpr as it fails to induce superoxide generation in human neutrophils at a concentration up to µm [ ] . d- pa, the d-stereoisomer of l- pa, blocks l- pa signaling and induces chemotaxis but not calcium flux. it is unclear whether d- pa is an anti-inflammatory antagonist of fpr . it has been noted that apoa-i itself and apoe, another important component of hdl, are described as being anti-inflammatory, as they suppress chemotaxis of leukocytes in the airway. currently, there has been an increasing interest in a group of newly emerging molecules that modulate gpcr activity by entirely novel mechanisms. these molecules, known as pepducins, comprise a lipid moiety conjugated with a peptide that is derived from a sequence of the cytoplasmic loops or the c-terminal tail of the target gpcr [ , ] . with the facilitation of the lipid moiety, pepducins are thought to pass across plasma membrane and anchor in the cytosolic interface to activate or inhibit signaling of receptors [ ] . these lipidated peptides have been proposed as allosteric modulators, because the binding sites and action modes are different from those of conventional ligands that generally interact with extracellular domains of receptors [ , ] . despite that the 'allosteric modulation' mechanism is not yet fully clarified, several receptor-specific pepducins have been identified, including an fpr agonistic pepducin f pal . f pal is composed of palmitic acid linked to a -mer peptide with sequence identical to the third intracellular loop of fpr . f pal was found to activate fpr , like conventional fpr agonists, in phagocytes and stably transfected hl- cells [ , ] . a shorter variant f pal , with higher potency compared to f pal , was shown to act as a partial agonist for direct activation of fpr but as a full agonist for cross-talk triggered hpfr reactivation generated by paf receptor and atp receptor (p y r) [ , ] . it is noteworthy that pepducins are not always specific for their designated target receptor. for example, the pepducins p y palic and p y palic that contain sequences of the second and third intracellular loops of p y r, were identified to be fpr agonists and could activate neutrophils with responses inhibited by fpr antagonists but not p y r antagonist [ ] . in a recent study, a pepducin (ati- ) designed for cxcr , a chemokine receptor, was found to activate neutrophil functions through fpr -dependent signaling [ ] . the selectivity of pepducins for the fpr appears not entirely relying on the segment of the cognate receptor. specific sequence and amino acid are required for the action of fpr pepducins. it is also notable that pepducin derived from another intracellular loop has no effect [ ] and pepducin with an amino acid substitution lost all activity [ ] . more efforts are needed to delineate the mechanisms of action by which pepducins recognize and modulate fpr . of interest, although fpr and fpr share very similar sequence in the intracellular loops, there are no fpr specific pepducins so far. this suggests not only the divergence in the g protein signaling triggered from cytosolic domains of fpr and fpr , but also the presence of different forms (such as homo-or heterodimers) involved in the activation of these two major human fprs. however, this speculation needs verification. it also remains a question whether the pepducins mentioned above can interact with murine fprs. as fprs are potentially attractive drug target, molecules that inhibit cell responses induced by fpr agonists are thought to have therapeutic value for fpr-related diseases. these molecules, generally originating from natural peptides and secondary metabolites, can interact with fprs directly or interfere with their downstream signaling pathways. in addition to drug development, radio-labeled fpr antagonists can be used as tracking probes for in vivo imaging of infiltrating neutrophils [ , ] . the first fpr antagonists were obtained by modifying the amino termini of the classical tripeptide fmlf. early studies have shown that the n-formyl group is important for the formyl peptides to be recognized by fpr . freer et al. substituted this group with tert-butyloxycarbonyl group (t-boc) and found the resulting peptide (t-boc-mlf, boc- ) exhibiting fpr antagonistic properties [ ] . the switch from an agonist to an antagonist was also observed when other carbamates (such as iso-butyloxycarbonyl and benzyloxycarbonyl group) were used to replace the formyl group at the amino terminus [ ] . further c-terminal modification of iso-butyloxycarbonyl-mlf (i-boc-mlf) did not alter the antagonistic potency, implying the different roles of amino and carboxyl terminus in conferring the activities to these peptides. the t-butyloxycarbonyl analog of formylated peptide f-flflf (t-boc-flflf, boc- ) is another antagonistic peptide that potently inhibits neutrophil nadph-oxidase activity induced by fmlf [ , , ] . there is evidence that boc- and boc- begin to lose their receptor preference to fpr at high concentrations (> µm) and display inhibitory effects on fpr and even the complement component a (c a)-induced responses [ ] . thus, despite their fairly specific activity at low concentrations, these boc peptides may be classified as pan-antagonists for fprs. however, other disagree because receptor overlap was seen at high concentrations of all antagonists [ ] . in a recent study, optimization at the phe residues of boc- following ala-scanning led to a more potent fpr -selective antagonist [ ] . nevertheless, among the available formyl peptide-derived antagonists, boc- and boc- are still most potent and commonly used in experiments. chemotaxis inhibitory protein of s. aureus (chips) is another native protein ( . kda) with fpr antagonistic activity. the full length chips ( amino acids) was first reported to be an antagonist for both fpr and the c a receptor [ ] . however, the n-terminal peptides ftfepfptneeiesn and ftfepf (the first six residues) display only anti-fpr properties [ ] , suggesting a mechanism of s. aureus invasion through inhibiting fmlf-induced neutrophil chemotaxis. accordingly, neutralizing antibodies against chips may be useful for treating s. aureus infection [ ] given the fact that chips can bind to fpr with high affinity (k d~ nm) [ ] . in addition, another s. aureus-derived -amino acid protein flipr (fpr-like inhibitory protein), was reported to selectively inhibit the binding of and activation by fpr agonists [ ] . several peptides derived from viruses were also found inhibitory to fpr activation. these peptides include a retroviral p e-derived hexapeptide (ldllfl) that can inhibit fmlf-induced response in monocytes and granulocytes [ ] , and a coronavirus e-derived -mer peptide (etyikpwwvwl) that was identified as a potent antagonist of fpr with a k i of nm [ ] . additional peptides isolated from hiv- , hiv- , severe acute respiratory syndrome coronavirus and ebola virus were reported as fpr antagonists with lower affinities [ ] . it is interesting that n-formylation of etyikpwwvwl transforms this -mer peptide into an fpr agonist, which is consistent with the notion that modification of size and shape at the amino terminus can change the fpr agonistic/antagonistic property of some peptides. among the antagonistic cyclic peptides from natural sources, cyclosporin h (csh) is one of the most potent and specific fpr antagonists. csh and another cyclic undecapeptide csa are derived from peptide metabolites of the fungus tolypocladium inflatum [ , , ] . several cyclosporins were synthesized based on csh and sar analysis [ ] . competition assays conducted with radionuclide binding or n-acetyl-β-d-glucosaminidase release assay demonstrated that cyclosporin h and its derivatives are potent fpr antagonists with nanomolar ic values [ , ] . these compounds were shown to be specific to fpr only at low concentrations. the blocking effect on fpr signaling is also observed with csh at high concentrations (> . µm) [ ] . it was reported that csh could attenuate mouse acute inflammation evoked by cigarette smoking [ ] . another study revealed that administration of csh reduced opioid peptide secretion and analgesia associated with neutrophil activation during mycobacteria butyricum infection [ ] . csh and derivatives are anticipated to be useful in the treatment of airway, gastrointestinal tract and skin inflammatory disorders associated with neutrophil and eosinophil infiltration. an endogenous allergen uteroglobin (ug) was previously identified as an fpr antagonist. ug, known as clara cell kda protein (cc ) in humans, is an anti-inflammatory and anti-chemotactic protein that is constitutively expressed in mammalian airway epithelium. before molecular cloning of fpr, ug was found to inhibit fmlf-induced chemotaxis of monocytes and neutrophils [ ] . after molecular cloning of the fpr genes, the high affinity binding of ug to fpr was determined with a k d of nm in hek cells stably expressing fpr [ ] . the interaction of ug with fpr inhibits saa expression and saa-driven inflammation [ , ] . the activity of ug at fpr or other receptors has not been examined yet, thus the selectivity of this allergen is not fully understood. an additional natural peptide with antagonistic activity is a urokinase receptor-derived cyclic peptide. note that the linear form of this peptide ser-arg-ser-arg-tyr is actually a dual agonist for fpr and fpr [ ] . the cyclized srsry was reported to inhibit the binding of fluorescence labeled fmlf to fpr -expressing rbl- h cells and it inhibits fmlf-directed monocyte migration with a picomolar ic [ ] . however, the activity of cyclized srsry on fpr was not verified in the study. an extract of secondary metabolites from a marine bacillus sp. was also found to have fpr antagonistic properties [ ] . the precise molecule responsible for the inhibitory activity has not yet been identified. other endogenous non-cyclized peptides with antagonistic activity include spinorphin (lvvypwt) [ ] and sarpopeptate, an aurantiamide isolated from plants and fungi [ ] . spinorphin is an fpr antagonist [ , ] . sarpopeptate and its dipeptide derivatives were shown to have potent inhibitory effects on fmlf-stimulated neutrophil responses [ , ] and exhibit a receptor preference for fpr over fpr [ ] . however, the receptor specificity needs further identification because of the lack of data for binding analysis. in a ligand screen of hexapeptide libraries, several peptides were found to inhibit the binding of fpr agonist wkymvm. the peptide wrwwww (wrw ) is the most potent of these peptides with antagonistic activity against fpr agonists [ ] . in addition, wrw is the first fpr antagonist that can completely inhibit the activation of fpr by f l in fpr -transfected hek cells [ ] . it inhibits f l-induced response in mature monocyte-derived dendritic cells, which express fpr but not other fprs. recently, a cell permeable peptide of amino acids conjugated with a rhodamine group (rhob-qrlfqvkgrr, pbp ) was shown to have potent inhibitory activity on fpr . the peptide sequence of pbp is derived from pip -binding domains of the cytoskeletal protein gelsolin, and the rhodamine group is essential for the peptide to pass through plasma membrane and exert antagonistic activity. this rhodamine-linked decapeptide can completely inhibit ros production mediated by fpr but not by fpr [ ] . in a shorter form, rhob-qrlfqvg maintains potent antagonistic activity for fpr and displays the ability to partially inhibit fpr . since pbp peptides bind to the intracellular domain of receptors, the difference in length may reflect divergent structural features of the cytosolic side of fpr and fpr . it is notable that pbp is not an fpr -specific antagonist as it also affects non-fpr mediated signaling [ ] . the pepducin f pal , derived from a segment of the third intracellular loop of fpr , was found to inhibit fpr -mediated cellular response, but had no effect on fpr signaling [ ] . the fpr antagonistic property of f pal was confirmed by selective inhibition in the binding of a conventional fpr agonist (cy -wkymvm). this is not the first pepducin designed to target another gpcr but instead was found to hijack fpr ; however it is the first fpr pepducin with antagonistic activity. pam-(lys-βnspe) -nh , a lapidated α-peptide/β-peptoid oligomers was recently found to be a cross-species antagonist with selectivity for fpr and mfpr [ , ] . pam-(lys-βnspe) -nh belongs to a group of immunomodulatory host defense peptides (hdps), however, unlike most hdps, this synthetic hdp mimic is resistant to proteolysis. it was revealed that both the lipid and peptidomimetic portions are important for its immunomodulatory function. pam-(lys-βnspe) -nh can directly bind to fpr and prevent the binding of fluorescently labeled fpr -specific agonist (cy -wkymwm) but not the fpr -specific agonist (fitc-fnlfnyk) [ ] . the inhibitory potency of pam-(lys-βnspe) -nh is close to that of pbp . both fpr antagonists are membrane permeable and display a reversible inhibition pattern [ ] . despite the similarity, their action mechanisms appear divergent, because the rhodamine group in pbp cannot be exchanged for hexadecanoic acid in the hdp mimic peptide [ ] . it was suggested that pam-(lys-βnspe) -nh and its structural analogs are allosteric modulators of fpr , because they can inhibit neutrophil responses induced by the fpr pepducin f pal , which is assumed to act in a different way from conventional fpr agonists by anchoring to the intracellular loop of the receptor [ ] . however, like other fpr antagonists such as wrw , pbp and flipr were also found to inhibit f pal at fpr , an assumption requiring further verification. as fprs are potential drug targets, the search for novel fpr antagonists/inhibitors with high affinity and selectivity remains one of the fundamental aims of biomedical research in this field. the first reported fpr competitive antagonist is a non-steroidal anti-inflammatory drug (nsaid), sulfinpyrazone and its derivative , -diphenyl- -( -( -naphthyl)-propyl)- , -pyrazolidinedione (dnp) [ , ] . however, sulfinpyrazone is a low affinity (k i = µm) and non-specific ligand at fpr [ , ] . in recent years, numerous small molecule compounds have been discovered to have antagonistic/inhibitory effects using hts of either mixture-based combinatorial libraries or natural product-based drug designs combined with sar analysis and computational modeling. although there are hundreds of antagonistic/inhibitory small molecules in published reports, the fully characterized chemical antagonists with high potency (k i < µm) are no more than compounds after excluding those without binding analysis and/or agonistic activity assays. the h-chromone compound ( -hexyl- -methyl- -( -methyl- h-benzimidazol- -yl)- -oxo- hchromen- -yl acetate) was identified as the most potent fpr antagonist (k i~ nm) among the related synthetic and natural isoflavones. this isoflavone and analogs were found to suppress agonist-stimulated calcium flux and chemotaxis of neutrophils with ic values of . ~ µm [ ] . the competitive analysis showed that they are specific for fpr and did not inhibit fpr -, fpr -, cxcr -or murine mfpr -dependent responses. they have been also shown to be inactive as direct agonists of fpr /fpr and mfpr [ ] . a hederagenin smg- [ -o-( , -odi-acetylα-l-arabinopyranoside)-( → )-α-l-rhamnopyranosyl-( → )-α-l-arabinopyranoside], a saponin isolated from sapindus mukorossi, was reported to block binding of fluorescently labeled fpr ligand fnlfnyk (k i < µm) and inhibit fpr -mediated response in neutrophils and fpr -expressing hl- cells [ , ] . it has been verified that smg- has no direct agonist effects. in another study, a lignin pp- [( r, r)- -( , -dihydroxybenzyl)- -( , "-dimethoxybenzyl) butyrolactone], isolated from piper philippinum, was found to inhibit fmlf-induced neutrophil response and block fitc-fmlf binding to neutrophil with a k i of~ . µm. in the studies of a pharmacophore model of fpr antagonists and molecular docking, pp- displays impressive properties of fpr antagonism [ ] . however, it is difficult to consider pp- a competitive antagonist for fpr based on the experimental data because the antagonistic effect of pp- appears non-competitive and reversible [ ] . further studies are also necessary to address the specificity of this lignin. by screening combinatorial compound libraries, a pyrolidine bis-diketopiperazine-based compound - was recently identified as the most potent non-peptide fpr antagonist with a k i of nm and an ic of nm in calcium mobilization assays. the compound - [(r)- -(cyclohexylmethyl)- -( hydroxybenzyl)- -((r)- -((s)- -(((s)- -isopropyl- , -dioxopiperazin- -yl)methyl)pyrrolidin- -yl)- -(naphthalen- -yl)propan- -yl)piperazine- , -dione]) showed no agonistic activity at concentrations up to µm [ ] . a quinazolinone derivative was previously reported to suppress fpr agonist-stimulated inflammatory responses in vitro and in an in vivo model [ ] . this quin-c related quinazolinone, namely quin-c , was proven to have no agonist activity. it could displace [ i]wkymvm binding to fpr with an estimated k i of . µm. it did not inhibit the binding of [ h]fmlf to fpr at concentrations up to µm, indicating a high preference for fpr over fpr [ ] . quin-c was described as an fpr -specific antagonist, albeit its activity at other inflammation-related receptors (such as c ar and cxcr) has not been fully examined. additional fpr -specific antagonists with an -phenylimidazo[ , -a]pyrimidine scaffold were discovered through the use of a high-throughput hypercyt flow cytometric platform [ ] [ ] [ ] [ ] . they were reported to competitively bind to fpr with k i values of . , . and . µm. the subsequent functional analysis revealed no direct agonistic effects in one of the most potent compounds [ ] . more information and analysis are provided in recent reviews [ , ] . in summary, future studies of binding properties and off-target effects are needed to determine the bona fide antagonists among compounds that have been identified to suppress fpr agonist-stimulated cell responses in functional activity assays. moreover, most of the antagonists mentioned above have been characterized as human ligands, and their potency and specificity for the murine fprs remain to be determined. despite the lack of crystal structures, efforts in understanding fpr structure-function relationship has been propelled with approaches of molecular modification (including construction of receptor chimera and site-directed mutagenesis) and computational docking studies. several clusters of key residues have been identified to be crucial for the interaction between fprs and various modulators with highly divergent profiles and properties. mutational studies have confirmed the individual functions of some of these residues in fpr-ligand interaction [ ] . using receptor chimeras constructed by domain swapping between fpr /fpr [ ] and fpr /c ar [ ] , early studies demonstrated that the first, second and third extracellular loops and adjacent membrane regions seem to be important for recognition of fmlf. this approach was also taken to study chemotaxis mediated by fprs [ ] . following these studies, point mutations were introduced into both fprs, resulting in the identification of multiple charged amino acids that are important for the interaction with fmlf [ ] [ ] [ ] [ ] . these non-contiguous residues include arg , lys , arg , arg , and asp in fpr (figure ). among them, lys in the second transmembrane segment (tm ) and asp in tm were proven essential for the high-affinity binding of fmlf [ , ] . it is suggested that binding of fmlf to fpr disrupts the electrostatic interaction between lys and asp , which might contribute to the receptor activation [ , , ] (figure ) . the lack of a 'salt bridge' formed by lys/asp in fpr (becoming met and asn in corresponding positions) is responsible for the low affinity binding of fmlf and most formyl peptide, although it cannot explain the relatively effective recognition by formyl peptides of other compositions and lengths. arg , arg , and asp in fpr (figure ). among them, lys in the second transmembrane segment (tm ) and asp in tm were proven essential for the high-affinity binding of fmlf [ , ] . it is suggested that binding of fmlf to fpr disrupts the electrostatic interaction between lys and asp , which might contribute to the receptor activation [ , , ] (figure ) . the lack of a 'salt bridge' formed by lys/asp in fpr (becoming met and asn in corresponding positions) is responsible for the low affinity binding of fmlf and most formyl peptide, although it cannot explain the relatively effective recognition by formyl peptides of other compositions and lengths. figure ). reproduced from [ ] with permission. using a similar approach of site-directed mutagenesis aided by computer modeling, another charged residue asp in fpr (gly in fpr ) was found to be crucial for the interaction of fpr with certain formyl peptides that contain a positively charged c-terminal residue (figure ), such as residues referenced in the manuscript are boxed. conserved side-chains between both receptors are shown in black, and major differences in amino acids are highlighted in green for n-formyl peptide binding to fpr ) or gray (for n-formyl peptide binding to fpr ). b and c, molecular electrostatic potential on the inner surface of the binding cavity of a computational model of fpr (b) and fpr (c), viewed from top of the receptors. fpr and fpr were modeled using the structure of the cxcr chemokine receptor as template. arrows show key positive area in fpr due to arg and lys , and negative area in fpr due to asp sequence alignment and location of positive selection sites in fpr , fpr and mfpr (see also figure ). reproduced from [ ] with permission. using a similar approach of site-directed mutagenesis aided by computer modeling, another charged residue asp in fpr (gly in fpr ) was found to be crucial for the interaction of fpr with certain formyl peptides that contain a positively charged c-terminal residue (figure ), such as lys in fmlfk [ ] . in this case, the formation of 'salt bridge' seems still important because the d g substitution of fpr alone failed to restore the potency of fmlf to the level in fpr [ ] . studies have also shown that longer formyl peptides with α-helical and amphipathic properties favor fpr over fpr [ , , ] . a simple explanation is that the binding pocket of fpr is larger and deeper than the relatively shallow and narrow one in fpr , thus providing an added advantage in accommodating longer peptides. to understand the structural basis for the selective activation of fpr by pro-resolving modulators, chimeric receptors were constructed between fpr and other receptors, including fpr and blt . the study using these chimeras demonstrated that tm and adjacent regions of fpr are essential for the recognition of lxa . in contrast, the extracellular loops were required for high affinity binding for pro-inflammatory peptide agonists such as mmk and the mhc peptides. in addition, conserved n-glycosylation sites in fpr (asn and asn ) were also implicated in the interaction with these peptides but not with lxa [ , ] . in another study, a computational model of fpr -annexin peptide interaction was built and it suggested that the transmembrane portions (tm , tm , tm , tm ) of the binding pocket of fpr is important to optimal binding of the n-terminal residue in ac- qawf , the core structure of active annexin peptides [ ] . using the same set of chimeric fpr /fpr receptor [ ] stably expressed in hek cells [ ] , the n-terminal region and the second extracellular loop of fpr were identified as required for annexin a -mediated signaling [ ] . chimeric receptors were also constructed to dissect the mechanisms of action by which cell-penetrating lipopeptides (e.g., pepducins and pbp ) use unconventional access to modulate fpr . it was suggested that the pepducins f pal and f pal act primarily through the third intracellular loop of fpr . the third intracellular loops of fpr and fpr differ in only two amino acids. however, a substitution of these amino acids from fpr to fpr did not affect the selectivity of these pepducins [ ] . another study focused on pbp , an antagonist against fpr agonists, suggested that neither the third intracellular loop nor the presumed signaling cytoplasmic tail of fpr alone is responsible for the specific inhibition of pbp [ ] . clearly, the current knowledge on the action mode and binding sites of these lipopeptides is still very limited. perhaps new strategies should be exploited to understand the structure-function relationship of fpr in association with these allosteric modulators before crystal structures become available. the relatively shallow and narrow one in fpr , thus providing an added advantage in accommodating longer peptides. to understand the structural basis for the selective activation of fpr by pro-resolving modulators, chimeric receptors were constructed between fpr and other receptors, including fpr and blt . the study using these chimeras demonstrated that tm and adjacent regions of fpr are essential for the recognition of lxa . in contrast, the extracellular loops were required for high affinity binding for pro-inflammatory peptide agonists such as mmk and the mhc peptides. in addition, conserved n-glycosylation sites in fpr (asn and asn ) were also implicated in the interaction with these peptides but not with lxa [ , ] . in another study, a computational model of fpr -annexin peptide interaction was built and it suggested that the transmembrane portions (tm , tm , tm , tm ) of the binding pocket of fpr is important to optimal binding of the n-terminal residue in ac- qawf , the core structure of active annexin peptides [ ] . using the same set of chimeric fpr /fpr receptor [ ] stably expressed in hek cells [ ] , the n-terminal region and the second extracellular loop of fpr were identified as required for annexin a -mediated signaling [ ] . due to the lack of crystal structures for fprs, homology models and molecular docking analysis based on the mutagenesis/chimera studies represent an alternative approach for explaining ligand binding and receptor activation. the current homology models of fpr receptors (including fpr and fpr ) are generally created by using the crystal structures of two classes of receptors: ( ) the bovine rhodopsin receptor, that has a relatively low sequence identity to fprs (~ %) and was selected as a template because of its higher resolution ( . Å) [ ] ; ( ) the cxcr -based templates, usually cxcr alone, which has a higher sequence identity with fprs ( %) but has a lower resolution ( . Å) compared to that of rhodopsin [ ] . a dual template strategy (cxcr and µor opioid receptor) was developed in a recent docking study focused on fpr and non-peptide ligands [ ] . despite questions about the low similarity of rhodopsin receptor with fprs, several interesting points have been made by using the homology models based on this receptor. the homology model derived from the rhodopsin template shows that the fpr ligand binding site comprises two channels, two cavities, and the bottom [ ] . several amino acids are found to be involved in the interaction of fpr with agonists, including asn , thr , arg , tyr , and thr [ , , , ] . docking studies with fpr non-peptide antagonists (including the bile acids dca and cdca) suggested that thr , tyr , and thr are involved in the formation of three stable h-bond interactions with dca and cdca in the docked pose [ , ] . a pharmacophore model based on best docking poses of four fpr antagonists including csh described three anchor points: two acceptors for h-bonding and one hydrophobic point [ , , ] . in comparison with the agonists, fpr antagonists are characterized as containing more oh groups, which can serve as h-bond donors and/or acceptors upon binding to fpr [ ] . however, the three-point model was further proved to be not only fitted by fpr antagonists but also by fpr agonists [ , ] . thus, this model may be helpful in prediction for structures of fpr ligands without discriminating agonists and antagonists. in another study, a cxcr -based homology model of fpr suggested an important role of water molecule in discrimination between fpr agonists and antagonists [ ] . in a comparative model, the potent antagonist boc- was found to interact with fpr by forming h-bonds with arg and lys , similarly to the interaction between fmlf and fpr . however, boc- directly forms an h-bond with asp while water molecule mediates h-bonding between the fmlf carbonyl group and asp [ ] . the rhodopsin-based model with five fpr agonists described the binding pocket of fpr as dumb-bell shaped, with a large cavity and a small cavity linked by a narrow channel. the proposed model comprises three sub-pockets, the pharmacophore sub-pocket i (within the small cavity) surrounded by positive residues, and the hydrophobic sub-pockets ii and iii (within the large cavity) surrounded by polar residues [ ] . the fpr -specific peptide agonist wkymvm fits well with this model by occupying all three sub-pockets, with the n-terminal indole moiety located in sub-pocket i in the best docked pose [ ] . in another docking simulation, residues his , val , asp , leu , and trp were identified as being critical for agonist binding [ ] . in a more recent study, comparative docking analysis was performed on a homology model of fpr that used cxcr and µor as a dual template [ ] . the model proposed that three hydrophobic clusters are important in binding non-peptide ligands, including agonists and antagonists. the first cluster is formed by tm and tm while his is crucial in the interaction with docked ligands. the second cluster is located between tm and tm with phe as the key residue in binding to aromatic groups of docked compounds. the third cluster is buried in the protein consisting of residues phe , val , phe and phe [ ] . the docking model suggested that the proper orientation is required for penetration into the binding site: the peptide ligands are placed upright while non-peptide ligands prefer a more horizontal position. these findings have yet to be substantiated by mutational studies. still, the differences in binding modes between antagonists and agonists to fpr are not fully understood. to date, there are no reports describing modeling of fpr and mouse mfprs. the fprs have evolved to be a class of receptors that not only recognize bacteria-derived formyl peptides but also ligands with drastically different structures, including non-formyl peptides of microbial origins, endogenous peptides and synthetic small molecules. because of these features, the fprs are highly 'promiscuous' in terms of ligand recognition. how fprs recognize these different ligands remains unclear at present without available crystal structures. molecular docking and computer simulation approaches combined with site-directed mutagenesis, however, have provided clues to our understanding of how formyl peptides interact with fpr and to a lesser extent, fpr . over a very long period of time, adaptive evolution has occurred in fprs, especially fpr , with positive selection for receptor interaction with the bona fide or orthosteric ligands. indeed, several residues involved in h-bonding with amino acids in formyl peptides are located within the predicted binding sites undergoing positive selection, suggesting that human fpr is under selection pressure for its binding of formyl peptides. as for other fpr agonists as well as for fpr , the picture remains unclear. there is no doubt that some agonists for the fprs have been in existence only recently and they are likely candidates for allosteric or ago-allosteric modulators of the fprs. with respect to fpr , it apparently has diverged from the evolutionary path taken by fpr , resulting in a different recognition profile including binding of longer formyl peptides and a wide range of endogenous and microbial peptides. a better understanding of the mechanisms by which fprs interact with their ligands will help to sort out agonists and antagonists that are crucial for the biological functions of these receptors, and to speed up identification of therapeutically important molecules. author contributions: h.q.h. and r.d.y. initiated and designed the study, collected the literature and wrote the manuscript. the authors declare no conflict of interest. the following abbreviations are used in this manuscript: formyl peptide receptors: a promiscuous subfamily of g protein-coupled receptors controlling immune responses international union of basic and clinical pharmacology. lxxiii. nomenclature for the formyl peptide receptor (fpr) family n-formylmethionyl peptides as chemoattractants for leucocytes mitochondrial n-formylmethionyl proteins as chemoattractants for neutrophils basic characteristics of the neutrophil receptors that recognize formylated peptides, a danger-associated molecular pattern generated by bacteria and mitochondria development of small molecule non-peptide formyl peptide receptor (fpr) ligands and molecular modeling of their recognition new development in studies of formyl-peptide receptors: critical roles in host defense synthesis and use of a novel n-formyl peptide derivative to isolate a human n-formyl peptide receptor cdna the human n-formylpeptide receptor. characterization of two cdna isolates and evidence for a new subfamily of g-protein-coupled receptors isolation of a cdna that encodes a novel granulocyte n-formyl peptide receptor a structural homologue of the n-formyl peptide receptor. characterization and chromosome mapping of a peptide chemoattractant receptor family mapping of genes for the human c a receptor (c ar), human fmlp receptor (fpr), and two fmlp receptor homologue orphan receptors (fprh , fprh ) to chromosome cloning of a cdna encoding a receptor related to the formyl peptide receptor of human neutrophils adaptive evolution of formyl peptide receptors in mammals potential role of the formyl peptide receptor-like (fprl ) in inflammatory aspects of alzheimer's disease bacterial lipopolysaccharide selectively up-regulates the function of the chemotactic peptide receptor formyl peptide receptor in murine microglial cells interferon and granulopoiesis signatures in systemic lupus erythematosus blood role of formyl peptide receptor-like (fprl /fpr ) in mononuclear phagocyte responses in alzheimer disease leukocyte chemoattractant receptor fpr may accelerate atherogenesis formyl peptide receptors at the interface of inflammation, angiogenesis and tumor growth impaired antibacterial host defense in mice lacking the n-formylpeptide receptor formylpeptide receptors are critical for rapid neutrophil mobilization in host defense against listeria monocytogenes reduced fear memory and anxiety-like behavior in mice lacking formylpeptide receptor n-formylpeptides induce two distinct concentration optima for mouse neutrophil chemotaxis by differential interaction with two n-formylpeptide receptor (fpr) subtypes. molecular characterization of fpr , a second mouse neutrophil fpr identification of formyl peptides from listeria monocytogenes and staphylococcus aureus as potent chemoattractants for mouse neutrophils functional characterization of three mouse formyl peptide receptors serum amyloid a is a chemotactic agonist at fpr , a low-affinity n-formylpeptide receptor on mouse neutrophils amyloid-beta induces chemotaxis and oxidant stress by acting at formylpeptide receptor , a g protein-coupled receptor expressed in phagocytes and brain f l, a peptide derived from heme-binding protein, chemoattracts mouse neutrophils by specifically activating fpr , the low-affinity n-formylpeptide receptor identification and characterization of an endogenous chemotactic ligand specific for fprl characterization of fpr-rs , an atypical member of the mouse formyl peptide receptor gene family formyl peptide receptor-like proteins are a novel family of vomeronasal chemosensors formyl peptide receptors are candidate chemosensory receptors in the vomeronasal organ aspirin-triggered -epi-lipoxin a (lxa ) and lxa stable analogues are potent inhibitors of acute inflammation: evidence for anti-inflammatory receptors characterization of two new members of the formyl peptide receptor gene family from s mice intravascular danger signals guide neutrophils to sites of sterile inflammation circulating mitochondrial damps cause inflammatory responses to injury intravital imaging of neutrophil recruitment reveals the efficacy of fpr blockade in hepatic ischemia-reperfusion injury the isolation and partial characterization of neutrophil chemotactic factors from escherichia coli further studies on the structural requirements for synthetic peptide chemoattractants purification and identification of formyl-methionyl-leucyl-phenylalanine as the major peptide neutrophil chemotactic factor produced by escherichia coli acute colitis produced by chemotactic peptides in rats and mice chemotactic peptide-induced acute colitis in rabbits guinea pig ileum motility stimulation elicited by n-formyl-met-leu-phe (fmlf) involves neurotransmitters and prostanoids chemotactic peptides. mechanisms, functions, and possible role in inflammatory bowel disease increased neutrophil receptors for and response to the proinflammatory bacterial peptide formyl-methionyl-leucyl-phenylalanine in crohn's disease defective polymorphonuclear leukocyte formyl peptide receptor(s) in juvenile periodontitis formyl-methionyl-leucyl-phenylalanine causes bronchoconstriction in rabbits haematological effects of inhalation of n-formyl-methionyl-leucyl-phenylalanine in man hemodynamic and metabolic effects of intravenous formyl-methionyl-leucyl-phenylalanine (fmlp) in rabbits endotoxin augments hemodynamic and metabolic effects of formyl-methionyl-leucyl-phenylalanine (fmlp) in rabbits structural changes of the ligand and of the receptor alters the receptor preference for neutrophil activating peptides starting with a formylmethionyl group human mitochondria-derived n-formylated peptides are novel agonists equally active on fpr and fprl , while listeria monocytogenes-derived peptides preferentially activate fpr mitocryptide- , a neutrophil-activating cryptide, is a specific endogenous agonist for formyl-peptide receptor-like a proinflammatory peptide from helicobacter pylori activates monocytes to induce lymphocyte dysfunction and apoptosis a seven-transmembrane, g protein-coupled receptor, fprl , mediates the chemotactic activity of serum amyloid a for human phagocytic cells structural mechanism of serum amyloid a-mediated inflammatory amyloidosis amyloid (beta) activates a g-protein-coupled chemoattractant receptor, fpr-like- endogenous lipid-and peptide-derived anti-inflammatory pathways generated with glucocorticoid and aspirin treatment activate the lipoxin a receptor annexin and its bioactive peptide inhibit neutrophil-endothelium interactions under flow: indication of distinct receptor involvement ll- , the neutrophil granule-and epithelial cell-derived cathelicidin, utilizes formyl peptide receptor-like (fprl ) as a receptor to chemoattract human peripheral blood neutrophils, monocytes, and t cells cross-talk between fmlp and vitronectin receptors triggered by urokinase receptor-derived srsry peptide urokinase induces basophil chemotaxis through a urokinase receptor epitope that is an endogenous ligand for formyl peptide receptor-like and -like the neurotoxic prion peptide fragment prp( - ) is a chemotactic agonist for the g protein-coupled receptor formyl peptide receptor-like proinflammatory proteases liberate a discrete high-affinity functional fprl (ccr ) ligand from ccl vip differentially activates beta integrins, cr , and matrix metalloproteinase- in human monocytes through camp/pka, epac, and pi- k signaling pathways via vip receptor type and fprl a peptide with unique receptor specificity: stimulation of phosphoinositide hydrolysis and induction of superoxide generation in human neutrophils utilization of two seven-transmembrane, g protein-coupled receptors, formyl peptide receptor-like and formyl peptide receptor, by the synthetic hexapeptide wkymvm for human phagocyte activation the synthetic peptide trp-lys-tyr-met-val-met-nh specifically activates neutrophils through fprl /lipoxin a receptors and is an agonist for the orphan monocyte-expressed chemoattractant receptor fprl identification of surrogate agonists for the human fprl- receptor by autocrine selection in yeast apolipoproteins and apolipoprotein mimetic peptides modulate phagocyte trafficking through chemotactic activity activation of human monocytes by a formyl peptide receptor -derived pepducin the leukocyte chemotactic receptor fpr , but not the closely related fpr , is sensitive to cell-penetrating pepducins with amino acid sequences descending from the third intracellular receptor loop identification of a human cdna encoding a functional high affinity lipoxin a receptor a novel nonpeptide ligand for formyl peptide receptor-like identification of novel small-molecule agonists for human formyl peptide receptors and pharmacophore models of their recognition new 'chemical probes' to examine the role of the hfprl (or alxr) receptor in inflammation potent hfprl (alxr) agonists as potential anti-inflammatory agents formyl peptide receptor and dual agonist inhibits human neutrophil chemotaxis by the induction of chemoattractant receptor cross-desensitization synthesis and pharmacological evaluation of new pyridazin-based thioderivatives as formyl peptide receptor (fpr) agonists synthesis, enantioresolution, and activity profile of chiral -methyl- , -disubstituted pyridazin- ( h)-ones as potent n-formyl peptide receptor agonists -( h-indol- -yl)- -[ -( -nitrophenyl)ureido]propanamide enantiomers with human formyl-peptide receptor agonist activity: molecular modeling of chiral recognition by fpr novel -( h-indol- -yl)- -[ -( -methoxyphenyl)ureido]propanamides as selective agonists of human formyl-peptide receptor selective agonists and antagonists of formylpeptide receptors: duplex flow cytometry and mixture-based positional scanning libraries n-terminal residues of the chemotaxis inhibitory protein of staphylococcus aureus are essential for blocking formylated peptide receptor but not c a receptor uteroglobin suppresses allergen-induced th differentiation by down-regulating the expression of serum amyloid a and socs- genes cyclosporin h is a potent and selective formyl peptide receptor antagonist. comparison with n-t-butoxycarbonyl-l-phenylalanyl-l-leucyl-l-phenylalanyl-l-leucyl-l-phenylalanine and cyclosporins a, b, c, d, and e cyclization of the urokinase receptor-derived ser-arg-ser-arg-tyr peptide generates a potent inhibitor of trans-endothelial migration of monocytes identification of peptides that antagonize formyl peptide receptor-like -mediated signaling structural characterization and inhibitory profile of formyl peptide receptor selective peptides descending from a pip -binding domain of gelsolin a neutrophil inhibitory pepducin derived from fpr expected to target fpr signaling hijacks the closely related fpr instead the proteolytically stable peptidomimetic pam-(lys-betanspe) -nh selectively inhibits human neutrophil activation via formyl peptide receptor the peptidomimetic lau-(lys-betanspe) -nh antagonizes formyl peptide receptor expressed in mouse neutrophils antagonism of human formyl peptide receptor (fpr ) by chromones and related isoflavones pharmacological characterization of a novel nonpeptide antagonist for formyl peptide receptor-like structural determinants for the interaction of formyl peptide receptor with peptide ligands community-associated mrsa: what makes them special? human formyl peptide receptor senses highly pathogenic staphylococcus aureus formyl peptide receptor-mediated proinflammatory consequences of peptide deformylase inhibition in staphylococcus aureus receptor-dependent and -independent immunomodulatory effects of phenol-soluble modulin peptides from staphylococcus aureus on human neutrophils are abrogated through peptide inactivation by reactive oxygen species t /dp , a synthetic leucine zipper-like domain of the hiv- envelope gp , attracts and activates human phagocytes by using g-protein-coupled formyl peptide receptors t /dp , an ectodomain peptide of human immunodeficiency virus type gp , is an activator of human phagocyte n-formyl peptide receptor a synthetic peptide derived from human immunodeficiency virus type gp downregulates the expression and function of chemokine receptors ccr and cxcr in monocytes by activating the -transmembrane g-protein-coupled receptor fprl /lxa r n , a synthetic n-terminal heptad repeat domain of the hiv- envelope protein gp , is an activator of human phagocytes activation of the chemotactic peptide receptor fprl in monocytes phosphorylates the chemokine receptor ccr and attenuates cell responses to selected chemokines the hiv- cell entry inhibitor t- potently chemoattracts neutrophils by specifically activating the n-formylpeptide receptor basophils infiltrate human gastric mucosa at sites of helicobacter pylori infection, and exhibit chemotaxis in response to h. pylori-derived peptide hp( - ) promotion of formyl peptide receptor -mediated neutrophil chemotactic migration by antimicrobial peptides isolated from the centipede scolopendra subspinipes mutilans serum amyloid a induces il- secretion through a g protein-coupled receptor, fprl /lxa r local expression of the serum amyloid a and formyl peptide receptor-like genes in synovial tissue is associated with matrix metalloproteinase production in patients with inflammatory arthritis opposing regulation of interleukin- and nf-kappab responses by lipoxin a and serum amyloid a via the common lipoxin a receptor differential production of leukotriene b or prostaglandin e by wkymvm or serum amyloid a via formyl peptide receptor-like serum amyloid a opposes lipoxin a( ) to mediate glucocorticoid refractory lung inflammation in chronic obstructive pulmonary disease serum amyloid a mediates human neutrophil production of reactive oxygen species through a receptor independent of formyl peptide receptor like- endogenous acute phase serum amyloid a lacks pro-inflammatory activity, contrasting the two recombinant variants that activate human neutrophils through different receptors serum amyloid a isoforms display different efficacy at toll-like receptor and formyl peptide receptor an il- r/il- circuit regulates epithelial serum amyloid a to promote local effector th responses th cell induction by adhesion of microbes to intestinal epithelial cells pleiotropic roles of formyl peptide receptors beta amyloid peptide (abeta ) is internalized via the g-protein-coupled receptor fprl and forms fibrillar aggregates in macrophages humanin, a newly identified neuroprotective factor, uses the g protein-coupled formylpeptide receptor-like- as a functional receptor n-formylated humanin activates both formyl peptide receptor-like and an annexin n-terminal peptide activates leukocytes by triggering different members of the formyl peptide receptor family neutrophil nadph-oxidase activation by an annexin ai peptide is transduced by the formyl peptide receptor (fpr), whereas an inhibitory signal is generated independently of the fpr family receptors mouse cathelin-related antimicrobial peptide chemoattracts leukocytes using formyl peptide receptor-like /mouse formyl peptide receptor-like as the receptor and acts as an immune adjuvant fprl -mediated induction of superoxide in ll- -stimulated imr human fibroblast ll- inhibits serum amyloid a-induced il- production in human neutrophils human endogenous antibiotic ll- stimulates airway epithelial cell proliferation and wound closure the pro-inflammatory peptide ll- promotes ovarian tumor progression through recruitment of multipotent mesenchymal stromal cells leucine leucine- uses formyl peptide receptor-like to activate signal transduction pathways, stimulate oncogenic gene expression, and enhance the invasiveness of ovarian cancer cells the expression of beta-defensin- , and ll- induced by candida albicans phospholipomannan in human keratinocytes leukotriene b /antimicrobial peptide ll- proinflammatory circuits are mediated by blt and fpr /alx and are counterregulated by lipoxin a and resolvin e the fibrinolytic receptor for urokinase activates the g protein-coupled chemotactic receptor fprl /lxa r identification of fam d as a new endogenous chemotaxis agonist for the formyl peptide receptors distinct signaling cascades elicited by different formyl peptide receptor (fpr ) agonists the synthetic peptide trp-lys-tyr-met-val-d-met inhibits human monocyte-derived dendritic cell maturation via formyl peptide receptor and formyl peptide receptor-like synthetic peptide mmk- is a highly specific chemotactic agonist for leukocyte fprl a novel peptide agonist of formyl-peptide receptor-like (alx) displays anti-inflammatory and cardioprotective effects tethered ligand library for discovery of peptide agonists design, synthesis and characterization of fmlf-mimicking aapeptides characterization of quin-c for its anti-inflammatory property in a mouse model of bleomycin-induced lung injury stable formyl peptide receptor agonists that activate the neutrophil nadph-oxidase identified through screening of a compound library a non-peptide receptor inhibitor with selectivity for one of the neutrophil formyl peptide receptors, fpr molecular docking of -(benzimidazol- -ylthio)-n-phenylacetamide-derived small-molecule agonists of human formyl peptide receptor further studies on -arylacetamide pyridazin- ( h)-ones: design, synthesis and evaluation of , -disubstituted analogs as formyl peptide receptors (fprs) agonists high-throughput screening for small-molecule activators of neutrophils: identification of novel n-formyl peptide receptor agonists gastrin-releasing peptide/neuromedin b receptor antagonists pd , pd , and related analogs are potent agonists of human formyl-peptide receptors lipoxins and aspirin-triggered -epi-lipoxins are the first lipid mediators of endogenous anti-inflammation and resolution the lipoxin receptor alx: potent ligand-specific and stereoselective actions in vivo lipoxins: update and impact of endogenous pro-resolution lipid mediators resolution of inflammation: state of the art, definitions and terms identification, cloning, and functional characterization of a murine lipoxin a receptor homologue gene lipoxin a( ) metabolites/analogues from two commercial sources have no effects on tnf-alpha-mediated priming or activation through the neutrophil formyl peptide receptors what formyl peptide receptors, if any, are triggered by compound and lipoxin a ? heterologously expressed formyl peptide receptor (fpr /alx) does not respond to lipoxin a( ) anti-inflammatory lipoxin a is an endogenous allosteric enhancer of cb cannabinoid receptor lipoxin a : a new class of ligand for the ah receptor lipoxin a is a novel estrogen receptor modulator resolvin d binds human phagocytes with evidence for proresolving receptors resolvin d prevents tnf-alphamediated disruption of salivary epithelial formation aspirin-triggered resolvin d reduces mucosal inflammation and promotes resolution in a murine model of acute lung injury resolvin d and resolvin d govern local inflammatory tone in obese fat oxidized low-density lipoprotein-induced foam cell formation is mediated by formyl peptide receptor insider access: pepducin symposium explores a new approach to gpcr modulation turning receptors on and off with intracellular pepducins: new insights into g-protein-coupled receptor drug development a pepducin derived from the third intracellular loop of fpr is a partial agonist for direct activation of this receptor in neutrophils but a full agonist for cross-talk triggered reactivation of fpr a pepducin designed to modulate p y r function interacts with fpr in human neutrophils and transfers atp to an nadph-oxidase-activating ligand through a receptor cross-talk mechanism data on human neutrophil activation induced by pepducins with amino acid sequences derived from β ar and cxcr . data brief localization of radiolabeled chemotactic peptide at focal sites of escherichia coli infection in rabbits: evidence for a receptor-specific mechanism a novel neutrophil-specific pet imaging agent: cflflfk-peg- cu selective inhibition of n-formylpeptide-induced neutrophil activation by carbamate-modified peptide analogues antibiotics and peptides with agonist and antagonist chemotactic activity cyclosporin h, boc-mlf and boc-flflf are antagonists that preferentially inhibit activity triggered through the formyl peptide receptor development of potent antagonists for formyl peptide receptor based on boc-phe-d-leu-phe-d-leu-phe-oh chemotaxis inhibitory protein of staphylococcus aureus binds specifically to the c a and formylated peptide receptor characterisation of receptor binding by the chemotaxis inhibitory protein of staphylococcus aureus and the effects of the host immune response a new staphylococcal anti-inflammatory protein that antagonizes the formyl peptide receptor-like an immunosuppressive retrovirus-derived hexapeptide interferes with intracellular signaling in monocytes and granulocytes through n-formylpeptide receptors peptides derived from hiv- , hiv- , ebola virus, sars coronavirus and coronavirus e exhibit high affinity binding to the formyl peptide receptor cyclosporins: structure-activity relationships for the inhibition of the human fpr formylpeptide receptor the immunosuppressant cyclosporin a antagonizes human formyl peptide receptor through inhibition of cognate ligand binding genetic ablation of the fpr gene confers protection from smoking-induced lung emphysema in mice mycobacteria attenuate nociceptive responses by formyl peptide receptor triggered opioid peptide release from neutrophils inhibition of phagocyte chemotaxis by uteroglobin, an inhibitor of blastocyst rejection sodin-semrl, s. uteroglobin, a possible ligand of the lipoxin receptor inhibits serum amyloid a-driven inflammation bioactive secondary metabolites of a marine bacillus sp. inhibit superoxide generation and elastase release in human neutrophils by blocking formyl peptide receptor . molecules the endogenous opioid spinorphin blocks fmet-leu-phe-induced neutrophil chemotaxis by acting as a specific antagonist at the n-formylpeptide receptor subtype fpr two trypanocidal dipeptides from the roots of zapoteca portoricensis (fabaceae) inhibitory effects of spinorphin, a novel endogenous regulator, on chemotaxis, o -generation, and exocytosis by n-formylmethionylleucyl-phenylalanine (fmlp)-stimulated neutrophils constituents and bioactive principles of polygonum chinensis design and synthesis of new n-(fluorenyl- -methoxycarbonyl) (fmoc)-dipeptides as anti-inflammatory agents design and synthesis of tryptophan containing dipeptide derivatives as formyl peptide receptor antagonist trp-arg-trp-trp-trp-trp antagonizes formyl peptide receptor like -mediated signaling receptor-directed inhibition of chemotactic factor-induced neutrophil hyperactivity by pyrazolon derivatives. definition of a chemotactic peptide antagonist the interaction of , -pyrazolidinedione drugs with receptors for f-met-leu-phe on human neutrophil leukocytes: a study of the structure-activity relationship. can inhibition of human platelet cyclo-oxygenase activity by sulfinpyrazone and three of its metabolites high-throughput screening with hypercyt flow cytometry to detect small molecule formylpeptide receptor ligands the hederagenin saponin smg- is a natural fmlp receptor inhibitor that suppresses human neutrophil activation antagonism of human formyl peptide receptor with natural compounds and their synthetic derivatives r, r)- -( , -dihydroxybenzyl)- -( , -dimethoxybenzyl)butyrolactone suppresses fmlp-induced superoxide production by inhibiting fmlp-receptor binding in human neutrophils high-content screening: flow cytometry analysis biomolecular screening of formylpeptide receptor ligands with a sensitive, quantitative, high-throughput flow cytometry platform a novel fluorescent cross-reactive formylpeptide receptor/formylpeptide receptor-like hexapeptide ligand duplex high-throughput flow cytometry screen identifies two novel formylpeptide receptor family probes multiple domains of the n-formyl peptide receptor are required for high-affinity ligand binding. construction and analysis of chimeric n-formyl peptide receptors species and subtype variants of the n-formyl peptide chemotactic receptor reveal multiple important functional domains identification of functional domains in the formyl peptide receptor-like for agonist-induced cell chemotaxis the ligand binding site of the formyl peptide receptor maps in the transmembrane region identification of an n-formyl peptide receptor ligand binding domain by a gain-of-function approach human formyl peptide receptor function role of conserved and nonconserved charged residues characterization of the binding site on the formyl peptide receptor using three receptor mutants and analogs of met-leu-phe and met-met-trp-leu-leu identification of a ligand binding site in the human neutrophil formyl peptide receptor using a site-specific fluorescent photoaffinity label and mass spectrometry ligand-specific regulation of the extracellular surface of a g-protein-coupled receptor linking agonist binding to histamine h receptor activation insight into structure-function relationship in phenol-soluble modulins using an alanine screen of the phenol-soluble modulin (psm) alpha peptide peptide length and folding state govern the capacity of staphylococcal beta-type phenol-soluble modulins to activate human formyl-peptide receptors or activation of lipoxin a( ) receptors by aspirin-triggered lipoxins and select peptides evokes ligand-specific responses in inflammation the annexin i sequence gln( )-ala( )-trp( )-phe( ) is a core structure for interaction with the formyl peptide receptor annexin a interaction with the fpr /alx receptor: identification of distinct domains and downstream associated signaling crystal structure of rhodopsin: a g protein-coupled receptor structures of the cxcr chemokine gpcr with small-molecule and cyclic peptide antagonists non-peptide ligand binding to the formyl peptide receptor fpr -a comparison to peptide ligand binding modes integration of virtual screening with high-throughput flow cytometry to identify novel small molecule formylpeptide receptor antagonists pharmacophore model for bile acids recognition by the fpr receptor c-and n-terminal residue effect on peptide derivatives' antagonism toward the formyl-peptide receptor identification of novel formyl peptide receptor-like agonists that induce macrophage tumor necrosis factor alpha production the role of water in activation mechanism of human n-formyl peptide receptor (fpr ) based on molecular dynamics simulations stimulation of human formyl peptide receptors by calpain inhibitors: homology modeling of receptors and ligand docking simulation this article is an open access article distributed under the terms and conditions of the creative commons attribution (cc by) license