key: cord-0696870-i0d4lxko
authors: Gurung, Arun Bahadur; Ali, Mohammad Ajmal; Lee, Joongku; Farah, Mohammad Abul; Al-Anazi, Khalid Mashay
title: Unravelling lead antiviral phytochemicals for the inhibition of SARS-CoV-2 M(pro) enzyme through in silico approach
date: 2020-05-22
journal: Life Sci
DOI: 10.1016/j.lfs.2020.117831
sha: 7b4c3e4a3a3c9c8712db2a3b46c12a89412ebe16
doc_id: 696870
cord_uid: i0d4lxko

A new SARS coronavirus (SARS-CoV-2) belonging to the genus Betacoronavirus has caused a pandemic known as COVID-19. Among coronaviruses, the main protease (M(pro)) is an essential drug target which, along with papain-like proteases catalyzes the processing of polyproteins translated from viral RNA and recognizes specific cleavage sites. There are no human proteases with similar cleavage specificity and therefore, inhibitors are highly likely to be nontoxic. Therefore, targeting the SARS-CoV-2 M(pro) enzyme with small molecules can block viral replication. The present study is aimed at the identification of promising lead molecules for SARS-CoV-2 M(pro) enzyme through virtual screening of antiviral compounds from plants. The binding affinity of selected small drug-like molecules to SARS-CoV-2 M(pro), SARS-CoV M(pro) and MERS-CoV M(pro) were studied using molecular docking. Bonducellpin D was identified as the best lead molecule which shows higher binding affinity (−9.28 kcal/mol) as compared to the control (−8.24 kcal/mol). The molecular binding was stabilized through four hydrogen bonds with Glu166 and Thr190 as well as hydrophobic interactions via eight residues. The SARS-CoV-2 M(pro) shows identities of 96.08% and 50.65% to that of SARS-CoV M(pro) and MERS-CoV M(pro) respectively at the sequence level. At the structural level, the root mean square deviation (RMSD) between SARS-CoV-2 M(pro) and SARS-CoV M(pro) was found to be 0.517 Å and 0.817 Å between SARS-CoV-2 M(pro) and MERS-CoV M(pro). Bonducellpin D exhibited broad-spectrum inhibition potential against SARS-CoV M(pro) and MERS-CoV M(pro) and therefore is a promising drug candidate, which needs further validations through in vitro and in vivo studies.

J o u r n a l P r e -p r o o f

Coronaviruses (CoVs) are positive-sense RNA enveloped viruses which derive their name from the crown-like spikes on their surface and they belong to Coronaviridae family. They are classified into four main subgroups-alpha, beta, gamma, and delta depending on their genomic structure [1] . Alpha-and beta coronaviruses cause respiratory infections in humans and gastroenteritis in other mammals [2, 3] . Likewise, the Middle East respiratory syndrome coronavirus (MERS-CoV) caused a disastrous pandemic in 2012 leading to 37% mortality [1] . All coronaviruses infecting humans usually known to have intermediate hosts such as bats or rodents [4] . Previous outbreaks of SARS-CoV and MERS-CoV involved civet cats and dromedary camels for their direct transmission to humans [1] . A new coronavirus caused an outbreak of the pulmonary disease in Wuhan (the capital of Hubei province in China) in December 2019 and has since spread across different parts of the world [5, 6] . Since its RNA genome shows about 82% identity to that of the SARS coronavirus (SARS-CoV), the new virus has been termed as SARS-CoV-2 [7] . However, both these viruses belong to the same clade of the genus Betacoronavirus [5, 6] . The SARS-CoV-2 caused a disease known as COVID-19. At the initial outbreak, cases were linked to the Huanan seafood and animal market in Wuhan but active human-to-human transmission caused exponential growth in the number of reported cases. The World Health Organization (WHO) confirmed the outbreak a pandemic on March 11, 2020. There have been more than 170,000 cumulative cases worldwide accounting for approximately 3.7% case-fatality rate as of March 15, 2020 [8] .

Due to the close similarity to SARS-CoV, the biochemical interactions and the pathogenesis of SARS-CoV-2 are highly likely to be similar [1] . The virus entry into the host cell is mainly mediated through the binding of the SARS spike (S) protein to the angiotensinconverting enzyme 2 (ACE-2) receptor on the cell surface [9] . Among coronaviruses, the main protease (M pro , also called 3CL pro ) has emerged as the best-described drug target [10] . The J o u r n a l P r e -p r o o f 4 polyproteins that are translated from the viral RNA are processed by this enzyme together with the papain-like protease(s) [11] . The M pro recognizes and acts remarkably on eleven cleavage sites typically Leu-Gln↓(Ser,Ala,Gly) on the large polyprotein 1ab (replicase 1ab) of approximately 790 kDa. Blocking the activity of this enzyme would help in inhibiting viral replication. There are no reported human proteases with a similar cleavage specificity and therefore, inhibitors against this enzyme are less probable to be toxic [8] . The three dimensional X-ray crystal structure of this enzyme in complex with α-ketoamide inhibitor 13b (O6K) was recently solved by Zhang et al. (2020) (PDB ID: 6Y2F) which offers an opportunity for structure-based drug design against the enzyme target. Understanding the relevance of the steady rise in the number of infected and death cases in recent time from COVID-19 and lack of effective therapeutic interventions such as drugs and vaccines, computer-aided drug design is an important strategy to be sought after. This rational based drug design will reduce the cost and time incurred in the drug discovery process. Structure-based drug design primarily relies on molecular docking to identify lead molecules against the target proteins from chemical libraries [12, 13] . Compared to the synthetic inhibitors plant based-drugs have less toxicity and much safer to use. The natural products such as traditional medicines and plant-derived compounds (phytochemicals) are the rich sources of promising antiviral drugs [14] . Around 44% of the approved antiviral drugs between 1981 and 2006 were derived from natural products [15] . The plant extracts have been extensively used and screened for drug molecules to evaluate theirs in vitro antiviral activities. Few examples of medicinal plants with proven antiviral activities include Phyllanthus amarus Schum. and Thonn which blocks human immunodeficiency virus (HIV) replication both in vitro and in vivo [16] ; Azadirachta indica Juss. (Neem) shows in vitro and in vivo inhibition properties against Dengue virus type-2 (DENV-2) [17] ; Geranium sanguineum L. significantly inhibits the replication of Herpes simplex virus type-1 and 2 (HSV-1 and HSV-2) in vitro [18] ; Acacia nilotica L. possesses activity against Hepatitis C virus (HCV) in vitro etc [19] .

In the present study, we have screened small drug-like molecules from a dataset of phytochemicals possessing antiviral activities using drug-like filters and toxicity studies. The 

The information about a set of thirty-eight phytochemicals from medicinal plants with antiviral activities was retrieved through literature search [14] . The summary of the selected phytochemicals (class, plant source and antiviral activity) is provided in Suppl. Table 1 

The phytochemicals were screened based on physicochemical properties obeying Lipinski's rule of five (ROF) filters [23] and further tested for in silico toxicity studies such as mutagenicity, tumorigenicity, reproductive effects and irritancy. The physicochemical properties of the phytochemicals were determined using DataWarrior program version 4.6.1 (Sander et al., 2015) . Table 1 .

The sequence percentage identity of the SARS-CoV-2 M pro to SARS-CoV M pro and MERS-CoV M pro was determined using a standard protein Basic Local Alignment Search (BLASTp) tool (https://blast.ncbi.nlm.nih.gov/Blast.cgi?PAGE=Proteins). Multiple sequence alignment of the three sequences were performed using CLUSTAL W algorithm [26] . Pairwise structural clustering of SARS-CoV-2 M pro , SARS-CoV M pro and MERS-CoV M pro was analyzed using UCSF Chimera tool [27] .

To check the suitability of molecular docking parameters and algorithm to reproduce the native binding poses, a redocking experiment was performed using the co-crystal compound. 

where ∆G is the binding energy in kcal/mol, R is the universal gas constant (1.987 calK −1 mol −1 ) and T is the temperature (298.15 K)

A stable complex is formed between a protein and ligand which exhibits more negative free energy of binding and low K i indicates high potency of an inhibitor [29, 30] . The hydrogen bonds and hydrophobic interactions between the compounds and the target enzymes were studied using

A total of 38 bioactive phytochemicals possessing antiviral activities were selected for the study.

These compounds were chosen based on the previous reports of their potent antiviral effects against various pathogenic viruses such as adenovirus, influenza virus, respiratory syncytial virus, human cytomegalovirus, herpes simplex virus, poliovirus, varicella-zoster virus etc.

(Suppl. Table 1 ). The compound set consists of different classes of phytochemicals including active flavonoids (N=14), active organic acids (N=5), active alkaloids (N=5), active essential oils (N=3), active stilbenes (N=6) and other phytoconstituents (N=5). The three-dimensional structures the compounds were modelled and optimized. A list of these phytochemicals is enumerated in Table 2 . These optimized structures were used further for virtual screening and molecular docking studies.

From a total of 38 phytochemicals, 10 compounds (four active flavonoids, two active alkaloids, two active essential oils and two other phytoconstituents) were found to be orally bioactive with J o u r n a l P r e -p r o o f 8 respect to ROF criterion (Molecular weight (MW) ≤500, cLogP (partition coefficient between noctanol and water) ≤5, number of hydrogen bond donors (HBD) ≤5 and number of hydrogen bond acceptors (HBA) ≤10 [23] ) without any significant toxicity issues such as being nonmutagenic, non-tumourigenic, non-irritant and no adverse effects on reproductive health (Table   3 ). These drug-like compounds were further taken for molecular docking studies. The drugattrition rate in preclinical and clinical trials is quite high due to the poor pharmacokinetic studies and therefore initial screening of these drug-like molecules can increase the chances of passing through the clinics. His163, His164, Met165, Pro168, Asp187, Gln189, Thr190 and Gln192) ( Figure 3D ).

Interestingly, the residues His41 and Cys145 which form catalytic dyad residues are also found interacting with the inhibitor. Thus all the three lead molecules have better binding affinity to SARS-CoV-2 M pro compared to the standard inhibitor. (N=17) ( Figure 4D ). It also shows good binding to MERS-CoV M pro which involves seven hydrogen bonds with Cys145, Ser147, Cys148, Gln167 and Glu169 and hydrophobic interactions via residues Met25, Thr26, Leu27, His41, Phe143, Leu144, Gly146, His166, Met168, Leu170, Ala171, Gln192, Val193, His194 and Gln195 (N=15) ( Figure 5D ).

The binding energies and inhibition constants of the phytochemicals with the SARS-CoV-2 M pro enzyme were compared with that of a set of twelve FDA approved antiviral drugs-a) Viral 

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List of Abbreviations ACE-2 :Angiotensin Converting Enzyme 2 BLAST: Basic Local Alignment Search cLogP: Partition Coefficient Between N-Octanol And Water HBA: Hydrogen Bond Acceptor HBD: Hydrogen Bond Donor MERS-CoV: Middle East Respiratory Syndrome Coronavirus Mpro: Main Protease MW: Molecular weight PDB: Protein Data Bank RMSD: Root Mean Square Deviation ROF: Rule of five SARS-CoV: Severe Acute Respiratory Syndrome Coronavirus

LigPlot analysis for top three lead compounds along with the control against SARS

83 Å)

91 Å)