key: cord-0823125-fz9h0soa
authors: Adegbola, Peter Ifeoluwa; Semire, Banjo; Fadahunsi, Olumide Samuel; Adegoke, Aanuoluwa Eunice
title: Molecular docking and ADMET studies of Allium cepa, Azadirachta indica and Xylopia aethiopica isolates as potential anti-viral drugs for Covid-19
date: 2021-04-10
journal: Virusdisease
DOI: 10.1007/s13337-021-00682-7
sha: d788257100bd4c72d14d4c457f430497f9a070f8
doc_id: 823125
cord_uid: fz9h0soa

Plants are repository of important constituents with proven efficacy against many human diseases including viral diseases. The antiviral activity of many plants including Azadirachta indica, Xylopia aethiopica and Allium cepa has been reported. The novel coronavirus disease is no exception among viral diseases in which plant compounds could serve as potent antagonist. Therefore, our study investigated the inhibitory potentials of Azadirachta indica and Xylopia aethiopica isolates against SARS-CoV-2 viral accessory proteins and the host serine protease. The protein data (SARS-CoV-2 Papain like protease (PLpro) (PDB: 6wx4), Chymotrypsin-like main protease (3CLpro) (PDB:6YB7), SARS-CoV nsp 12 (PDB: 6nus), Host cell protease (TMPRSS1) (PDB:5ce1) were obtained from the protein data bank (PDB), while the SDS format of each Ligands were obtained from Pubchem database. Molecular docking analysis was performed with Auto Dock Vina 1.5.6 and visualization of the interaction between the ligands and protein was done with discovery studio 2019. The ADMET prediction of pharmacokinetics and toxicity properties of the ligands was obtained using vNN Web Server. Our result showed that all the plant isolates demonstrated negative Gibb’s free energy, indicating good binding affinity for both the viral and host protein. Overall, twenty-three of the forty-seven isolates showed good binding affinity comparable with dexamethasone that was used as reference drug. Although many of the compounds have good binding affinity for the viral and host proteins, based on the ADMET prediction, only Azadironic acid, Nimbionone, Nimbionol and Nimocinol all from A. indica could serve as potential drug candidate with good pharmacokinetics and toxicity profile. This study provides an insight into potential inhibitors and novel drug candidates for SARS-CoV-2. Further studies will look forward into the wet laboratory validation of Azadironic acid, Nimbionone, Nimbionol and Nimocinol against corona virus disease. SUPPLEMENTARY INFORMATION: The online version contains supplementary material available at 10.1007/s13337-021-00682-7.

Plant remains a dependable resort for drug discovery. They are repository of important compounds with varied biological functions. Many pharmacological active drugs have their origin from medicinal plants [13, 46] . Among important plants with antiviral property are Azadirachta indica and Allium cepa. It has been reported that neem (A. indica) seed extracts exhibited inhibitory property against Duck viral enteritis [42] . In both in-vitro and in-vivo study, it showed inhibitory properties against Dengue virus type-2 [32] . Two polysaccharides isolated from the leaves inhibited Poliovirus type 1 [14] . Organosulfur constituents of A. cepa are important candidate in the antiviral properties [6, 23] . Onion extracts block attachment of new castle disease virus to host cell [18] and decrease the propagation of avian influenza virus in the embryonated chicken eggs [1] . In other words, these plants are promising source of antiviral drugs.

The novel coronavirus diseases, which up until now is a pandemic with no sight of relive from its incidence. The virus up until date has claimed 646,641 lives and more than 15,114,449 individuals have been infected globally [39] . The coronavirus disease is no exception among viral diseases in which plant compounds can serve as potent antagonist. Going by the genomic structure of the virus and the behavior in the human host, drugs could be developed to target the host cells protein involved in viral-host interaction or directly on the viral accessory proteins responsible for viral pathogenesis [41] . A number of research efforts are focused towards the discovery of potential drug candidates targeting one or more of the viral proteins involved in its virulence.

In this study, compounds previously identified in Allium cepa, Azadirachta indica and Xylopia aethiopica (Table 1) were docked against four target proteins of SARS-COV-2. The approach used in this study involved the identification of compounds that could directly target any of the proteins involved at different stages of viral pathogenesis or compounds with dual or multiple target potentials. In this regard, three of the proteins involved in viral RNA synthesis and replication (Papain-like protease (PLpro), 3Clike main protease (3CPLpro and RNA-dependent-RNA polymerase (RdRp), and host protein involved in viral entry were screened to identify potential inhibitors based on the formation of stable complex with ligands which exhibited more negative free energy of binding (DG) and low inhibition constant (Ki). The interaction of the natural compounds with the viral and host proteins was visualized in other to predict compounds that may inhibit the novel coronavirus and provide information on potential compounds for therapeutic development using computer aided study.

The protein data (SARS-CoV-2 Papain like protease (PLpro) (PDB: 6wx4), Chymotrypsin-like main protease (3CLpro) (PDB:6YB7), SARS-CoV nsp 12 (PDB: 6nus), Host cell protease (TMPRSS1) (PDB:5ce1) were obtained from the Protein Data Bank (PDB), which is a single worldwide depository of information about the 3D structures of large biological molecules, including proteins and nucleic acids (https://www.rcsb.org/structure). The SDS format of each Ligands were obtained from the Pubchem database, the world's largest free chemistry database (https://pubchem.ncbi.nlm.nih.gov) and taken to cactus online smiles translator (https://cactus.nci.nih.gov) for ligand download. Prior to docking analysis, interacting ligands, and water molecules were removed from the protein, thereafter saved in PDB format for docking analysis.

Docking of the proteins with the small molecule inhibitors was performed using Auto Dock Vina 1.5.6. On the Autodock tool, polar-H-atoms were first added to the proteins followed by Gasteiger charges calculation. The protein file was saved as pdbqt file and the grid dimensions were set. Docking calculations (Binding Affinity (DG) were then performed by using Vina folder [38] . Interactions between the ligand and proteins were visualized using discovery studio 2019.

PubChem database was applied to get the smiles structures of the natural compounds, and was further used for the ADMET prediction. The qualitative assessment of pharmacokinetics viz; absorption, distribution, metabolism, excretion and toxicity (ADMET) profile of selected compounds were predicted computationally by using vNN Web Server for ADMET Predictions (https://vnnadmet.bhsai. org).

Preventing viral synthesis and replication

Cysteine protease papain like protease (PLpro) recognizes a tetra peptide motif found in-between nsp1 and nsp2, nsp 2 and nsp 3, and nsp 3 and nsp 4 proteins that are essential for viral replication [17, 19, 34] . It has been reported that PLpro exhibits deubiquitination and de-ISGylation; thus blocking the interferon regulatory factor (IRF3) pathway [29, 31, 43] . Besides, it inhibits production of cytokines and chemokines responsible for host activation of innate immune response against virus [8, 12, 16] . Therefore, PLpro is not only essential in viral replication but also significant to antagonize host innate immunity [7, 27, 44] . Consequently, the protein is an important target in antiviral drug design against SARS-COV-2. Forty-seven compounds were docked against SARS-CoV-2 PLpro (PDB: 

Molecular docking and ADMET studies of Allium cepa, Azadirachta indica and Xylopia aethiopica… Interactions of the natural compounds with the amino acids at the catalytic site of CoV-2-PLpro was mediated via Our result showed the natural compounds with high binding affinity for PLpro and probable drug likeliness as revealed in Table 2 . Similar to Dexamethasone ADMET prediction revealed that Azadironic acid, Luteolin and Nimbionone among the other potent compounds showed no-toxicity potential viz; no drug induced liver injury (DILI), mutagenic (AMES) and cytotoxic likeliness. These compounds are as well non-inhibitor to drug metabolizing enzymes. It is important to note that, several of the compounds with anti-viral potentials were predicted to either have DILI, cytotoxicity and mutagenic (AMES) likeliness. For instance, Apigenin was predicted to be non-toxic but could be inhibitor of different isoforms of drug metabolizing enzymes, 3-deacetylsalanin, Cubebene, Kaempferol-3-O-rutinside, Meliacinin and Azadirachtin showed DILI likeliness. Isorhamnetin, Isorhamnetin 4-glucoside, Isohamnetin 3,4-diglucoside, Quercetin 3-glucoside, Quercetin 7,4-diglucoside and Quercetin 3,7,4-triglucoside were predicted to have mutagenic likeliness. Overall, similar to dexamethasone, our study showed that among the compounds with strong affinity for PLpro, only Azadironic acid, Nimbionone and Luteolin significant pharmacokinetic properties probable for drug development.

These compounds from Azadirachta indica, Allium cepa and Xylopia aethiopica were also docked against the SAR-CoV-2 viral replicating protease; Chymotrypsin-like main protease (PDB: 6YB7). Chymotrypsin-like protease together with PLpro is involved in the proteolytic processing of viral polyprotein that are translated from the viral RNA [21] . It cleaves at not less than 11 conserved sites on the large polyprotein [20, 45] . The 3CLpro is an important drug target because of its important role in viral life cycle (Pillaiyar et al. [33] ). During its cleavage function on viral polyprotein, it has a unique recognition sequence (Leu-Gln; (Ser,Ala,Gly ;marks the cleavage site) that is not recognized by any known human protease [45] . Therefore, compounds capable of inhibiting the protein are unlikely to be toxic. The binding affinity of the compounds was between -3.9 kcal/mol (for s-propylcysteine) and -10.8 kcal/mol (for Quercetin 3,7,4-triglucoside) (Supplementary Table 2 ). To classify the compounds as potent, those with binding affinity C -8.0 kcal/mol were noted and regarded as having good binding affinity for 3CLpro. In this regard, twenty three of the compounds; Quercetin Table 2 ). The affinity of the derivatives for the viral protein increased with the number of glucoside chain. Visualization of interactions between the ligands and the protein showed that majority of the compounds with good binding affinity for the protein formed either Alkyl or Pi-Alkyl interaction with Ala 285 and/or Leu 286. The two residue are involved in the catalytic activity of the protease [28] and interaction between domain II and III of the main protease. 3-deacetylsalanin and Apigenin interacted with Leu 286 via alkyl and Pi-Alkyl interaction respectively. Asp 197, while it interacted with Leu 286 via Alkyl interaction. The ADMET prediction of the compounds showed that all the compounds have probable drug potentials relative to the standards, however, only Azadironic acid, Luteolin, Nimbionone, Nimbolide, Nimbionol, Nimocinol were predicted to have no toxicity likeliness ( Table 2) .

The 932 amino acid (a.a.) SARS-CoV nsp 12 (PDB: 6nus) was docked against the 47 natural compounds and Dexamethasone. The protein is a RNA-dependent RNA polymerase having its polymerase domain between a.a 398-919 [30] . It is a vital enzyme for coronavirus replication. In complex with nsp 7 and nsp 8 as co-factors, this protein is greatly stimulated to perform polymerase activity [2, 37] . Homology studies revealed that the SARS-COV nsp 12 shared several kinase catalytic residues with protein kinases [22] therefore suggesting potential nucleotide-binding site within the enzyme domain and nucleotidyl transfer [25] function. The detail understanding of the mechanism of activity of the SAR-COV RdRp is yet a big complex to be unraveled. Both the binding affinity and the inhibition constant were factors in the selection of the most potent of the natural compounds. Twenty-seven (27) (Table 2) . Azadironic acid, Buoebenone, Luteolin, Nimbionone, Nimbolide, Nimbionol and Nimocinol showed unique potentials as anti-viral drug with no toxicity likeliness.

The ligands were docked against the Hepsin serine protease (PDB: 5ce1), which is a host type II trans-membrane serine protease (TTSP), responsible for the activation cleavage for SARS-CoV spike [26] . The catalytic sites of diverse serine proteases linked to parainfluenza, influenza and coronavirus activation are structurally similar; therefore, active site inhibitors of this protein could have broad applicability against multiple respiratory viruses [24] . Twenty-three of the docked compounds showed good binding affinity with the protein similar to dexamethaxone. Compounds with binding affinity B -6.5 kcal/mol were included among the compounds (Supplementary Table 4 The ADMET prediction showed six of the compounds viz; Azadironic acid¸Luteolin, Nimbionone, Nimbolide, Nimbionol and Nimocinol together with Dexamethasone succeeded in all the ADMET prediction parameters (Table 2) . Overall, the results of the study indicated that some of the compounds have potentials for multiple target. For instance, Apigenin, Azadirachtin, Azadironic acid, 3-deacetylsalanin, Kaempferol-3-O-rutinside, Isorhamnetin, Isohamnetin 3,4-diglucoside, Luteolin, Nimbionone, Nimocinol, Nimbolide, Nimbanal, Meliacinin, Quercetin 3,4-diglucoside, Quercetin 3-diglucoside, Quercetin 4-diglucoside, Quercetin, Regorafenib, Rutin had good binding affinity for all the docked proteins. It is important to note that majority of this compounds are compounds already identified in either A indica (8) or A. cepa (11) .

Early assessments of pharmacokinetic and toxic properties are important step in drug discovery. Drug discovery is a risky, lengthy, and resource-intensive process, however, toxicity remains a hurdle, that has resulted in attrition of several drugs at the clinical trial stage and even already approved drugs [40] . Aside efficacy and toxicity, many drug development failures are imputable to poor pharmacokinetics and bioavailability [10, 9] . It is therefore imperative nowadays to design lead compounds with good pharmacokinetics and bioavailability and carefully select compounds during drug development to avoid late-stage attrition. In this study, forty-seven natural compounds and dexamethaxone were assessed for their pharmacokinetics and toxicity properties using in silico approach. According to the pharmacokinetic prediction in Table 2 , only Bornyl acetate has ability for blood brain barrier (BBB) penetration. All the compounds were predicted to be stable to the human liver microsomal (HLM). The human liver is the most important organ for drug metabolism. For a drug to achieve effective therapeutic concentrations in the body, the liver cannot metabolize it too rapidly. Otherwise, it would need to be administered at high doses, which are associated with high toxicity [36] . In metabolism, most of the compounds appeared to be none inhibitors to P450. However, Salannol acetate and 3-deacetylsalanin could be inhibitor to Cyp3A4, Apigenin could be inhibitor to Cyp1A2 and Cyp2C19, Quercetin and Isorhamnetin could be inhibitor to Cyp1A2 and Cyp2C9, Luteolin could be inhibitor to Cyp1A2, while Nimbanal and Nimbolide could be inhibitor to Cyp3A4. As earlier reported, inhibition of this protein could increase a compound in the plasma and decrease the clearance of its substrate [15] . P-glycoprotein (Pgp) is a critical determinant of the pharmacokinetic properties of drugs as it function to extracts foreign substances from the cell [3] . Cancer cells often overexpress Pgp, consequently increasing the efflux of chemotherapeutic agents from the cell and reducing the effective intracellular concentrations of such agents; multidrug resistance [5] . It is of great interest to identify if a compound is a substrate to Pgp (can be transported out of the cell) or inhibitor to Pgp (impair function). Majority of the compounds are Pgp substrate while some were predicted as both substrate and inhibitor. The toxicity prediction showed that of all the compounds, only Regorafenib and Verbenone were predicted to have cytotoxicity potential. Isohamnetin 3,4-diglucoside, Isorhamnetin 4-glucoside, Isorhamnetin, Quercetin 3,4-diglucoside, Quercetin 3,7,4-triglucoside, Quercetin 3-glucoside, Quercetin, Quercetin 7,4-diglucoside and Rutin could induce bacteria mutation (Ames) and therefore cause drug resistance [4] . The human ether-à-go-go-related gene (hERG) codes for a potassium ion channel involved in the normal cardiac repolarization activity of the heart [35] . Blockade of hERG function can cause long QT syndrome, which may result in arrhythmia and death [11] ; based on the prediction, all the compounds except Regorafenib have no cardio toxic potential. Some of the compounds could also induce liver injury (DILI) ( Table 2 ). Ten of the compounds predicted to potentiate mitochondria dysfunction (decrease MMP) was recorded as yes (Table 2 ). Finally, in Table 2 , the maximum recommended therapeutic dose (MRTD) for each compound was predicted and recorded as mg/day for an average adult with a body weight of 60 kg [36] .

In this study, the forty-seven isolates from Allium cepa, Azadirachta indica and Xylopia aethiopica that were docked against the viral and host proteins demonstrated negative binding affinity. Among the compounds, it appears Quercetin 3,7,4-triglucoside, Quercetin-7,4-diglucoside, Quercetin 3,4-diglucoside, Quercetin-4-glucoside, Isohamnetin-4-diglucoside, Quercetin-3-glucoside, Rutin, Isohamnetin 3,4-diglucoside, Kaempferol-3-O-rutinside, Nimbolide, Nimbanal, Quercetin, Apigenin, Luteolin and Regorafenib could target all the viral proteins studied by exhibiting good binding affinity and thus could serve as ideal inhibitors for SARS-CoV-2. Overall, although many of the compounds have good binding affinity for the viral and host proteins, our study indicated that only Azadironic acid, Nimbionone, Nimbionol and Nimocinol all from A. indica could serve as potential drug candidate with good pharmacokinetics and toxicity profile comparable with dexamethasone. Therefore, our study provides an insight into potential inhibitors and novel drug candidates for SARS-CoV-2. Further studies will determine the dynamics of the interactions of the potent compounds with the proteins.

Funding The authors received no funding from any source.

Conflict of interest The authors declare that they have no conflict of interest.

Consent for publication All authors read and approved the manuscript for application.

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