key: cord-0994159-p24yfl17
authors: Ram, Thrigulla Saketh; Munikumar, Manne; Raju, Vankudavath Naik; Devaraj, Parasannanavar; Boiroju, Naveen Kumar; Hemalatha, Rajkumar; Prasad, P.V.V.; Gundeti, Manohar; Sisodia, Brijesh S.; Pawar, Sharad; Prasad, G.P.; Chincholikar, Mukesh; Goel, Sumeet; Mangal, Anupam; Gaidhani, Sudesh; Srikanth, N.; Dhiman, K.S.
title: In silico evaluation of the compounds of the ayurvedic drug, AYUSH-64, for the action against the SARS-CoV-2 main protease
date: 2021-02-25
journal: J Ayurveda Integr Med
DOI: 10.1016/j.jaim.2021.02.004
sha: b93a2a5510ae60887f27341d8307bac6e78800e8
doc_id: 994159
cord_uid: p24yfl17

BACKGROUND: Outbreak of Corona Virus Disease in late 2019 (COVID-19) has become a pandemic global Public health emergency. Since there is no approved anti-viral drug or vaccine declared for the disease and investigating existing drugs against the COVID-19. OBJECTIVE: AYUSH-64 is an Ayurvedic formulation, developed and patented by Central Council of Research in Ayurvedic Sciences, India, has been in clinical use as anti-malarial, anti-inflammatory, anti-pyretic drug for few decades. Thus, the present study was undertaken to evaluate AYUSH-64 compounds available in this drug against Severe Acute Respiratory Syndrome-Corona Virus (SARS-CoV-2) Main Protease (M(pro); PDB ID: 6LU7) via in silico techniques. MATERIALS AND METHODS: Different molecular docking software’s of Discovery studio and Auto Dock Vina were used for drugs from selected AYUSH-64 compounds against SARS-CoV-2. We also conducted 100 ns period of molecular dynamics simulations with Desmond and further MM/GBSA for the best complex of AYUSH-64 with M(pro) of SARS-CoV-2. RESULTS: Among 36 compounds of four ingredients of AYUSH-64 screened, 35 observed to exhibits good binding energies than the published positive co-crystal compound of N3 pepetide. The best affinity and interactions of Akuammicine N-Oxide (from Alstonia scholaris) towards the M(pro) with binding energy (AutoDock Vina) of -8.4 kcal/mol and Discovery studio of Libdock score of 147.92 kcal/mol. Further, molecular dynamics simulations with MM-GBSA were also performed for M(pro)– Akuammicine N-Oxide docked complex to identify the stability, specific interaction between the enzyme and the ligand. Akuammicine N-Oxide is strongly formed h-bonds with crucial M(pro) residues, Cys145, and His164. CONCLUSION: The results provide lead that, the presence of M(pro)– Akuammicine N-Oxide with highest M(pro) binding energy along with other 34 chemical compounds having similar activity as part of AYUSH-64 make it a suitable candidate for repurposing to management of COVID-19 by further validating through experimental, clinical studies.

Coronavirus-2 (SARS-CoV-2), its RNA genome shares 82.30% and similar pathogenesis (host) of its identity with SARS coronavirus (SARS-CoV) [1, 4] . Ultimately, the disease became known as corona virus disease 2019 (COVID-19) (WHO, 2020). On January 30, 2020, WHO officially declared the COVID-19 epidemic a public health emergency of international concern, and on March 11, 2020, the outbreak was confirmed a pandemic (WHO, 2020).The SARS-CoV and SARS-CoV-2 shares the similar pathogenesis (host) due to similarities in their genome [1] .

Though several drug targets of coronaviruses have been identified, the main protease (M pro ), also known as 3-chymotrypsin-like protease (3CL pro ), has emerged as the bestdescribed drug target [5] . The M pro processes the large polypeptide, translated from the viral RNA, specifically at 11 splicing regions mostly Leu to Gln (Ser, Ala and Gly). These significant splicing regions non-homologous to human. Inhibiting enzyme activity might results in blocking viral pathogenesis, making it an attractive drug target for SARS-CoV-2.

Further, a mechanism-based peptide-like inhibitor (N3) has also been identified via computer-aided drug design, and then crystal structure of the M pro of SARS-CoV-2 in complex with this compound has been determined [6] .

Given the lack of any validated, approved therapeutic intervention, such as a drug or vaccine, the COVID-19 pandemic continues to spread around the world. In such circumstances, the concept of drug repositioning might be a cost-effective, time-efficient, and less labour-intensive option for development of possible therapeutic and/or prophylactic lead candidate(s) from existing traditional and/or approved drugs [7] . In comparison to synthetic inhibitors, plant based-drugs are known to have low toxicity and are much safer to use.

Natural products, such as traditional medicines and plant-derived compounds (phytochemicals) are considered rich sources of promising antiviral drugs [8] .

Natural products were the sources of approximately 44% of the approved antiviral drugs from 1981 to 2006 [9] . Recent studies have suggested that drug poisoning and severe to moderate adverse effects associated with hydroxychloroquine have been reported in diabetic and hypersensitive patients, in whom COVID-19 tends to affect severely. Further, hydroxychloroquine is also known for inhibition of pro-inflammatory cytokines which leads to Acute Respiratory Distress Syndrome (ARDS) [10] . Due to the severe adverse effects shown by these synthetic compounds, natural, non-synthetic, target specific drugs with minimal side effects are urgently required for prophylaxis and treatment of COVID-19 [11] .

Plant compounds and natural phyto-molecules are most economical and ideal for exploration to develop drugs through the drug discovery process [12] . Recent studies via integrated computational approaches have highlighted the repurposing of approved and known drugs against SARS-CoV-2 drug targets, either individually or in combination, to combat the virulence of COVID-19. Several plant compounds have been reported, using in silico approaches, as potential inhibitors of the M pro of SARS-CoV-2. Mimicked drugs of J o u r n a l P r e -p r o o f bictegravir, doultegravir, paritaprevir, and raltegravir have been studied against 3CLpro and 2'-OMTase through in silico approaches [13, 14] . Another study reported darunavir, remdesivir, and saquinavir, the natural compounds derivatives from coumarine and flavone which inhibits 3CLpro [13] [14] [15] . In another in silico study also shown that, Andrographis paniculata's compound Andrographolide, inhibits M pro of SARS-COV-2. Its good solubility, pharmacodynamic properties, target accuracy, and adherence to Lipinski's rule of five were also predicted using computational tools [11] .

The potent plant compounds nelfinavir, lopinavir, kaempferol, quercetin, luteolin-7glucoside, demethoxycurcumin, naringenin, apigenin-7-glucoside, oleuropein, curcumin, catechin, and epicatechin-gallate, have been shown to inhibit the M pro [12] . At the same time, several potential anti-SARS-CoV-2 lead compounds have been revealed in the medicinal plant library of traditional Chinese medicine compounds [16] . Another study identified oolonghomobisflavan-A from the tea plant as a potential inhibitor against M pro of SARS-CoV-2 [17] . In addition, a promising drug candidate from plant sources, bonducellpin D, has been reported to exhibit broad-spectrum inhibition potential against both the SARS-CoV M pro and the MERS-CoV M pro [18] . [19] .

To summarize the information provided in Table 1 , it is understood that all four ingredients of AYUSH-64 are characterized by tikta rasa (bitter taste) and therefore J o u r n a l P r e -p r o o f amapachak (able to digest the Ama or undigested form of food) and hence act as Jvaraghna (anti-pyretic). The combined effects of these herbs are jwarahara (able to relieve fever), sannipata jwarahara (able to relieve intermittent fevers), krimihara (wormicidal), jantuhara (anthelmintic/antimicrobial/antiviral), and shothahara (anti-inflammatory), rendering this compound a potent combination against conditions such as Influenza Like Illnesses having the symptoms of cough, cold, headache and fever. Several compounds found in the drug have been reported to exhibit several pharmacological activities including anti-malarial, anti-viral, anti-inflammatory, immunomodulatory etc ( Table 2 ).

Various pre-clinical studies were performed on AYUSH-64 to study its safety and efficacy aspects ( Table 3) . The major studies, namely anti-malarial property in albino mice, found safe and non-toxic in a dose of 500 mg/kg of body weight for 12 weeks (Anonymous, 1987) and potential antiviral activity of Chirakin (marketed name of AYUSH-64 by Zandu) against Chikungunya virus were investigated at the department of Molecular Virology Laboratory, Rajiv Gandhi Centre for Biotechnology, Thiruvananthapuram, by protection and plaque reduction assay. The efficacy of the compound was expressed in terms of activity index and selectivity index in protection assay and Vero cells were infected with Chikungunya Virus (CHIKV) and treated with an optimum concentration of Chirakin (25μg/ml). Chirakin treated cells and the reduction in virus yield was determined by plaque assay in Verocells. Chirakin shows antiviral activity against Chikungunya virus in this study.

Its activity was found better than ribavirin in protection assays, where as in plaque reduction assays, both perform almost equally with a 2 log10 reduction in virus number (CK Katiyar.

Technical note on Chirakin Tab -Unpublished report).

Attempts have also been made to clinically evaluate the compound for management of Microfilariasis [20, 21] effectiveness against P. vivax in ring stage than in gamete stage and also the mixed infection cases of P. vivax [22] decline in infectivity rate of malaria, double-J o u r n a l P r e -p r o o f blind study for comparative efficacy in comparison with chloroquine/primaquine in P. vivax malaria and lead candidate in the management of influenza like illnesses [23] (Table 4) .

Overall, the compound is considered to be safe and is prescribed widely by Ayurvedic experts for the effective management of malaria, joint pains, fever, and influenza like illnesses. In this in silico study,36 major compounds were analysed which are four ingredients of AYUSH-64 drug might be potent inhibitors against M pro of SARS-CoV-2 observed via two different docking strategies of AutoDock Vina and Discovery studio molecular docking tools.

In the drug discovery process for COVID-19 crisis, the development of novel drugs with potential interactions with therapeutic targets is of primal importance. Conventionally, promising-lead or drug identification is achieved by traditional or experimental highthroughput screening (HTS), which is time-consuming and cost-effective [24] . In contrast to the traditional drug discovery method (classical or forward pharmacology), the rational drug design is noted as efficient and economical [25] . In these contrasts, approach to identifying therapeutics is to repurpose approved compounds developed for other uses, by taking advantage of existing detailed information on human pharmacology and toxicology to enable rapid clinical trials and regulatory quick review. Furthermore, in silico drug design method is also known as reverse pharmacology, as the initial step is to identify promising drug targets (proteins/enzymes), after which they are used for screening of novel small-molecule inhibitors for the candidates [14, [26] [27] [28] . In the present study of in silico drug design method, Bank (PDB) [6] with the PDB ID of 6LU7 with crystal resolution 1.5 Ȧ for the molecular docking studies. Foremost, for the preparation of the protein Auto Dock Tool (ADT) used for removal of all HOH molecule, adding Kollman charges & polar hydrogen atoms, assigning hydrogen polarities. Further, Gasteiger charges were applied on prepared protein and protein structures file 6LU7.PDB converted to 6LU7.PDBQT [15] .

In BIOVIA Discovery Studio, complex of M pro -N3 was prepared by removing all the water molecules, ligands and hetro-atoms from the complex. To satisfy the valency, hydrogen atoms were added to each atom. The M pro structure was minimized by applying CHARMm vc41b1force field to remove the steric clashes between the atoms in order to get stable conformation.The 6LU7 amino acid residues, namely Thr24, Thr25, Thr26, Thr26, His41, Phe140, Leu141, Asn142, Gly143, Ser144, Cys145, His163, His164, Met165, Glu166, Pro168, His172, Arg188, Gln189, Thr190, Ala191, and Gln192 were found to be present within 4 Ȧ region of N3 peptide binding site [6] . Among the listed 22 amino acids, Gly143, His163, His164, Glu166 (2), Gln189, and Thr190 residues were found to be formed eight hydrogen bonds with native N3 peptide having bond lengths of 2.87, 2.37, 2.8, 2.98, 2.83, 2.89, and 2.85 respectively. In both the docking strategies, the grid box was generated around the 4Ȧ region of N3 peptide in the M pro of SARS-CoV2 [15] .

Selected 36 compounds of AYUSH-64 were obtained from the PubChem database, as 3D coordinates of structure-data file (SDF) format, and each compound was converted to PDBQT file format with the help of PyMol and ADT tool, generating an input J o u r n a l P r e -p r o o f compound/ligand file for docking study in AutoDock Vina. In BIOVIA Discovery studio, 225 maximum conformations were generated for selected 36 compounds, and separate isomer conformations were also created within the threshold of 20.0 kcal/mol relative energy. The CHARMm force field (fast and accurate) was applied to minimize the compounds with 1000 steps of steepest descent (SD) algorithm. The root-mean-square deviation (RMSD) with <1.0 Å were considered as duplicates, and the higher specified RMSD value will reduce the number of output ligand poses and RMS gradient 0.001 [15] .

To predict the drug-likeness properties of selected36 AYUSH-64 compounds, in silico absorption, distribution, metabolism, excretion, and toxicity (ADME/T) prediction was carried out. We explored the ADME/T properties of the 36 AYUSH-64 selected compounds using the ADME/T Protocol in the Discovery Studio software package (Accelrys, San Diego, CA, USA). These examinations were exclusively founded on the substance structure of the particle. Some of the parameters that were calculated included, ADME 2Dimenctional Fast Polar Surface Area (ADME 2D FPSA), Atom-based Log P98 (ALogP98), Blood Brain Barrier (BBB), Cytochrome P4502D6 (CYP2D6), and Hepatotoxicity (HEPATOX).

The 

The first ranked docked molecular complex, AYUSH-64 with M pro selected for To assert constant Temperature at 300 K and Pressure of 1 atm throughout the molecular dynamics' simulation process, "Nose-Hoover thermostat algorithm [33] " and "Martyna-Tobias-Klein Barostat algorithm [34]" is used respectively. To affirm Coulombic long-range atomic interactions in the molecular docked complex throughout the molecular dynamic simulations process "Smooth Particle Mesh Ewald" method is used. To maintain the accuracy and tolerance of long-range interactions with the smaller value of 1e-9 is set for accurate computational implemented by SHAKE algorithm. The final production run was carried out for 100 ns, and the trajectory sampling was done at an interval of 1.0 ps [15, 30, 31] . Subsequently, in the MD simulations study, the binding free energy (ΔG bind ) for all simulated protein-ligand complexes were estimated by Desmond and Prime-molecular mechanics/generalized born surface area (MM-GBSA) method using thermal_mmgbsa.py script embedded in Schrödinger suite. 

Where

Where E complex , E protein , and E ligand are the minimized energies of the receptor-lead complex, receptor, and leads respectively

Where G solv(complex) , G solv(protein) , and G solv(ligand) are the solvation free energies of the complex, protein, and inhibitor, respectively

Where G SA (complex) , G SA (protein) , and G SA (ligand) are the surface area energies for the complex, protein and inhibitor, respectively

The simulations were carried out using the GBSA continuum model in Prime and every frame of the entire MD simulated trajectories were used, that is highly rigorous and more accurate approach to validate the docking and dynamics approaches in the study for calculating the binding free energy. Therefore, in the present study, all MD trajectory frames from start to end (~1000 frames) were selected to estimate the ΔG bind for proposed and native MD simulated complex.

Preferably, the distinguishing proof of the ideal docking and scoring blend will diminish the number of false positives and false negatives while ensuring optimal hit rates. It has accounted for enormous strategies for validating docking approach and scoring functions. through, re-docked with known co-crystal structure of 6LU7 with N3 peptide (Figure 1) . The re-docked results of RMSD revealed that, the AutoDock vina determined with 1.25Ȧ and Discovery studio has 1.02Ȧ. Also, 2D atomic coordination of the re-docked complexes was supplanted experimentally derived structure. Thus, these docking protocols was viewed good enough for replicating the docking results similar to co-crystal structure and consequently can be applied for further molecular docking analysis.

The SARS-CoV-2, M pro plays a significant role in RNA translation and is essential for viral replication. The three-dimensional structure of M pro (6LU7) is a monomer structure, consisting three major structural domains: domain I (residues 8-101), domain II (residues 102-184), which contribute an antiparallel β-barrel structure, and domain III (residues 201-303), which contains a five-fold anti-parallel α-helix cluster and is attached to long loop (residues 185 to 200) of domain II [6] . The amino acid of cysteine-Histidine dyadplays significant role in the maturation cleavage, which is located in the cleft between domains I and II the precursor M pro [6] . Moreover, M pro was non-homologous to human and it is an ideal anti-viral drug target for SARS-CoV-2 [15] . The co-crystal ligand of N3 peptide is located in the cleft region between domain I and II. The SARS-CoV-2 M pro shares highly (~99%) similarity and identity (100%) with binding site residues of SARS-CoV M pro [15] .

In the present study, the selected 36 compounds of AYUSH-64 were substituted (shows a similar inhibitor-binding mode) within the cleft between domain I and II, found to be having good docking score than the native substrate of N3 peptide (Table 5 and Figure 1A ). ADME/T properties prediction results are illustrated in Table 6 for drug-likenes. Two docking strategies, Akuammicine N-Oxide compound has best docking score, binding affinity and good ADME/T properties than the other selected 35 AYUSH-64 compounds and reference substrate of N3 peptide (Table 7 and Figure 2 ). The side chain residues, His41 (2) 

M pro is one of the best-characterized pan drug target for the coronavirus's family.

Along with M pro an essential enzyme for the endurance of virus, processing the polyproteins which are translated from the viral RNA [13] [14] [15] . During translation, the M pro exclusively cleaves at all 11 cleavage sites (after a glutamine residue) of polypeptide sequences on the large polyprotein named as 1ab (replicase 1-ab, ~790 kDa); Inhibiting the activity of this enzyme would block the viral replication. The cleavage site recognition sequence at most sites is Leu-Gln (Ser, Ala, Gly). To the best of our knowledge no human proteases with a similar cleavage site specificity are known, such inhibitors are unlikely to be toxic [13] [14] [15] .

In this present study, we performed different docking strategies of Discovery studio note that AYUSH-64 is a compound herbal preparation, which is already tested to some extent for its safety and efficacy for treating malaria, joint disorders and influenza like illness.

The currents in silico study provide a valid basis for its repurposing in the management of COVID-19.

This study revealed that, several compounds from an Ayurvedic drug are acting as J o u r n a l P r e -p r o o f DNA damage [66] . 4. Immuno-stimulatory: increase in hemagglutinating antibody titre and a change in delayed-type hypersensitivity [26] . 5. Two weeks after challenge with Pseudomonas aeruginosa, the Caesalpinia treated animals showed a significant bacterial clearance from the lungs, with less severe incidence of lung abscess [67] . 6. Exhibited activity against the vaccinia virus [68] . 7. in vivo experimental study, Neutrophil adhesion test, hemagglutinating antibody (HA) titre, delayed-type hypersensitivity (DTH) response, phagocytic activity and cyclophosphamide-induced myelosuppression were demonstrated to be positively activated pointing towards a promise in immunomodulation [26] . Table 3 . Preclinical pharmacological and toxicological /safety studies of AYUSH-64 and its ingredients

Preclinical pharmacological and toxicological /safety studies 1.

In albino mice, oral administration of AYUSH-64 at doses of 250-750mg/kg for five days exhibited significant anti-malarial property [67] .

The experimental studies of AYUSH-64 have shown that it was safe and non-toxic in a dose of 500 mg/kg of body weight for 12 weeks [67] . . Table 4 . Clinical studies of AYUSH-64

Clinical studies of AYUSH- 64 1 Clinically effective in cases of Microfilariasis [20] , [21] . 2 81% curative effect on Plasmodium vivax, drug is more effective in ring stage than in Gamete stage. In the cases of mixed infection of P.vivax & P.falciparum the curative effect was found to be 75% after longer therapy [69] . 3 Good effect in the management of microfilaraemia [70] . 4 In a study group of 4500 participants, AYUSH-64 was observed to be safe and nontoxic with good anti-malarial activity and also a major decline in the infectivity rate was noted by administering AYUSH-64 along with anti-mosquito measures, in various types of fevers [70] . 5

A double-blind study demonstrated comparative efficacy of AYUSH-64 to the chloroquine /primaquine as the standard modern control in sixty cases of P. Vivax malaria [70] . 6 In a prospective, open-label, nonrandomized, single group, single-centre pilot study with pre-test and post-test design one-week intervention of 'AYUSH-64' in a dose of 3gms/day effectively helped to recover from Influenza Like Illnesses, and also the quicker return to normal life with reduced frequency of usage of acetaminophen/ antihistaminic. No adverse effects were found during the study [71] 7 A pilot study done on 112 participants produced 97.14% result with prophylactic treatment in P.F. Malaria [72] . a 0, 1, 2, 3, 4, and 5 denote extremely low, very low but possible, low, good, optimal, and too soluble, respectively. b 0, 1, 2, 3, and 4 denote very high, high, medium, low, and undefined, respectively. c 0 and 1 represent nontoxic and toxic, respectively. d 0, 1, 2, and 3 denote good absorption, moderate absorption, low absorption, and very low absorption, respectively. e 0 and 1 denote noninhibitor and inhibitor, respectively. f 0, 1, and 2 indicate <90% binding, ≥90% binding, and ≥95% binding, respectively. 

Sars-cov-2 and coronavirus disease 2019: What we know so far

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A pneumonia outbreak associated with a new coronavirus of probable bat origin

Genomic characterisation and epidemiology of 2019 novel coronavirus: implications for virus origins and receptor binding

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Structure of Mpro from SARS-CoV-2 and discovery of its inhibitors

Repurposing host-based therapeutics to control coronavirus and influenza virus

Antiviral properties of phytochemicals

Natural products as sources of new drugs over the last 25 years

Could Chloroquine / Hydroxychloroquine Be Harmful in Coronavirus Disease 2019 (COVID-19) Treatment?

Andrographolide As a Potential Inhibitor of SARS-CoV-2 Main Protease: An In Silico Approach

Potential Inhibitor of COVID-19 Main Protease ( M pro ) from Several Medicinal Plant Compounds by Molecular Docking Study

Identification of chymotrypsin-like protease inhibitors of SARS-CoV-2 via integrated computational approach

Targeting SARS-CoV-2: a systematic drug repurposing approach to identify promising inhibitors against 3C-like proteinase and 2′-O-ribose methyltransferase

GS-5734) as a therapeutic option of 2019-nCOV main protease -in silico approach

Structural basis of SARS-CoV-2 3CLpro and anti-COVID-19 drug discovery from medicinal plants

Identification of bioactive molecules from tea plant as SARS-CoV-2 main protease inhibitors

Unravelling lead antiviral phytochemicals for the inhibition of SARS-CoV-2 Mpro enzyme through in silico approach

New Delhi: Central Council for Research in Ayurveda and Siddha

Effect of ayush-64 and saptaparnaghana vati on microfilaraemia

An epidemiological survey on microfilaraemia in the villages around bhubaneswar with therapeutic effect of ayush-64

A double blind clinical trial with ayush-64 an ayurvedic drug in p. vivax malaria

AYUSH 64, a polyherbal Ayurvedic formulation in Influenza like Illness: results of a pilot study

Structure-based virtual screening for drug discovery: A problem-centric review

Recent advances in computer-aided drug design

Modeling, molecular docking, probing catalytic binding mode of acetyl-CoA malate synthase G in Brucella melitensis 16M

High throughput docking for library design and library prioritization

Discovery of potential lumazine synthase antagonists for pathogens involved in bacterial meningitis: In silico study

In silico design of small peptides antagonist against leptin receptor for the treatment of obesity and its associated immune-mediated diseases

How does a drug molecule find its target binding site?

The Nose-Hoover thermostat

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Induces protection against DNA and membrane damage

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Screening of Indian plants for biological activity: I

A CLINICAL TRIAL OF AYUSH 64 (A CODED ANTIMALARIAL MEDICINE) IN CASES OF MALARIA

an Epidemiological Survey on Microfilaraemia in the Villages Around Bhubaneswar With Therapeutic Effect of Ayush-64

AYUSH 64, a polyherbal Ayurvedic formulation in Influenza like Illness: results of a pilot study

An indigenous herbal formulation to combat Falciparum Malaria

 [45] .

activity [46] . [47] . 4. Enhances DNA repair capacity [48] . [49] . 6. Potent antiplasmodial activity against P. falciparum [50, 51] .

Katuki (Picrorhiza kurroa Royle ex. Benth) [52] . [53, 54] . 3. Protection against red blood cell (RBC) haemolysis and