key: cord-0713255-th3wdcvi
authors: Halder, Pinku; Pal, Upamanyu; Paladhi, Pranab; Dutta, Saurav; Paul, Pallab; Pal, Samudra; Das, Debasmita; Ganguly, Agnish; IshitaDutta; SayarneelMandal; Ray, Anirban; Ghosh, Sujay
title: Evaluation of potency of the selected bioactive molecules from Indian medicinal plants with M(Pro) of SARS-CoV-2 through in silico analysis
date: 2021-05-21
journal: J Ayurveda Integr Med
DOI: 10.1016/j.jaim.2021.05.003
sha: d9afdea5c4c532fa73d9ea95920333de47e655b0
doc_id: 713255
cord_uid: th3wdcvi

BACKGROUND: The recent outbreak of novel SARs CoVid-2 across the globe and absence of specific drug against this virus lead the scientific community to look into some alternative indigenous treatments. India as a hub of ayurvedic and medicinal plants can shed light on its treatment using specific active bio-molecules from these plants. OBJECTIVES: Keeping our herbal resources in mind we were interested to inquire whether some phytochemicals from Indian spices and medicinal plants can be used as alternative therapeutic agents in contrast to synthetic drugs. MATERIALS AND METHODS: We used in-silico molecular docking approach to test whether bioactive molecules of herbal origin such as Hyperoside, Nimbaflavone, Ursolic acid, 6-gingerol, 6-shogaol& 6-paradol, Curcumin, Catechins&Epigallocatechin, α-Hederin, Piperine could bind and potentially block theM(pro)enzyme of Sars-CoV-2 virus. RESULTS: Ursolic acid showed the highest docking score (-8.7 kcal/mol) followed by Hyperoside (-8.6kcal/mol), α-Hederin (-8.5 kcal/mol) and Nimbaflavone (-8.0kcal/mol). Epigallocatechin, Catechins, and Curcumin also exhibited high binding affinity (Docking score -7.3, -7.1 and -7.1 kcal/mol) with the M(pro). Rest of the tested phytochemicals exhibited moderate binding and inhibitory effects. CONCLUSION: This finding provides a basis for biochemical assay on Sars-CoV-2 virus.

The SARS-CoV-2is a highly infectious virus for novel COVID-19 disease that causes outbreak recently in China in the December 2019 and rapidly spread to the other parts of the globe owing to its extreme contagious nature. Its initial symptoms include fever, dry cough, tiredness, aches or pains, diarrhoea, difficulty breathing etc. and in human body it probably settles through Angiotensin-converting enzyme 2 (ACE2) [1] receptor for entry into the host cell and the Transmembrane protease, serine 2 (TMPRSS2) for viral spike protein priming [2, 3] . The infection gradually took the shape of pandemic with extremely high mortality [4, 5] owing to lack of definite treatment regimen and medications against the virus as well as due to co-morbidity. Even the so called developed nations like U.S and European countries have failed to control the infection with their cutting edge medical technologies at very early phase. WHO has declared COVID-19 as a public health emergency of internationalconcern [6] .

The previous name of this betacoronavirus was 2019-nCoV. It was renamed as SARS-CoV-2 by the InternationalCommittee on Taxonomy of Viruses (ICTV) [7] . The genome of SARS-CoV-2 has been sequenced [8] . The whole genome sequence analysis of SARS-CoV-2 shows 96.2% similarity with batcoronavirus (SARSr-Co) [9, 10] , while shows low sequence identity with that of SARS-CoV (about 79%) or MERS-CoV (about 50%) [11, 12] .

The pandemic COVID-19 has triggered all researchers around the globe for rapid development of drugs and specific antiviral treatment strategies. Among all the SARS-CoV-2 targets the main protease (M pro , 3CL pro , nsp5) received major attention [13, 14] . Some alternative targets like spike protein (S), RNA-dependent RNA-polymerase (RdRp, nsp12), NTPase/helicase (nsp13) and papain-like protease (PL pro , part of nsp3) also reported in some J o u r n a l P r e -p r o o f literatures [15, 16] . The SARS-CoV-2 M pro is a 33.8 kDa enzyme which play a pivotal role in the cleavage of viral polyproteins (pp1a and pp1ab) in a in a site-specific ((L-Q|(S, A, G)) manner [17] , resulting in the release of functional replicase enzyme which is crucial for transcription and replication of the virus [18] [19] [20] . Other essential enzymes which are involve in replication process such as RdRp or nsp13 cannot fully function without this proteolytic action [13] , making M pro a key enzyme in viral replication cycle. As a result the inhibition of M pro could stop viral replication process and thus alleviate disease symptoms [21, 22] . For drug discovery against SARS-CoV-2 virus, M pro is one of the most attractive viral targets.

Some studies have already reported synthetic competitive inhibitors against SARS-CoV-2 M pro [17, 23, 24] but increase in substrate concentration often reduces the effectiveness of such inhibitors. Natural phytochemicals can provide safe and effective treatment by alleviating this limitation.

Although there are no approved drugs or vaccines for COVID-19, a number of clinical trials are in progress [25] . Lopinavir and Ritonavir, combined with Chinese herbal medicines, were used in preliminary clinical studies [26] .

Indian medicinal plants and spices are reach hub of ingredients which can be utilized for drug designing because of their high therapeutic values [27] . Previous docking study already reported phytochemicals such as Hyperoside and Nimbaflavone are good candidate drugs against Influenza virus strains [28] . A recent study already published high binding efficacy of Ursolic acid (Tulsi) against surface spike glycoprotein and RNA polymerase of SARS-CoV-2 virus [29] .Ajoene and Allicin (Garlic) shows strong virucidal activity against selected viruses including, herpes simplex virus type 1, herpes simplex virus type 2, parainfluenza virus type 3, vaccinia virus, vesicular stomatitis virus, and human rhinovirus type 2 [30] .Curcumin has diverse antiviral activity against dengue virus, herpes simplex virus, Zika and chikungunya virus [31] [32] [33] . Previous study reported that Catechins and Epigallocatechin form green tea J o u r n a l P r e -p r o o f leaves have profound antiviral effects [34] . A very recent in silico molecular docking study revealed that Piperine from Black pepper can act as a potent inhibitor of the antiviral enzymes of Dengue and Ebola viruses [35] .

Molecular docking is a computational technique, widely used for the study of molecular recognition, prediction of binding mode and binding affinity of complexes formed by two or more known structures. It has become a widely accepted tool for drug discovery [36] [37] [38] [39] [40] .

This high throughput technique can screened a variety of available drugs to identify potential drugs for novel diseases as well as to predict the adverse effects of noveldrugs in a very short time [41] [42] [43] [44] . Development of novel drugs is a time consuming process and generally several years ofwork are required for clinical approval [45] . Drug repositioning, also knownas repurposing, is an effective strategy to combat novel diseases caused by infectious agents thatspread rapidly [46] [47] [48] . Drugs thathave been approved for some disease, are safe for human use [49] , and only theireffectiveness against the disease of interest needs to be established [50] . In lifethreatening cases, where there is no alternative medicine or vaccine, such a drug repurposingstrategy is particularly attractive. However, clinical trials are necessary to ensure that suchtreatment is better than a placebo [51, 52] .

An in silico screening of herbal medicines for treatment of COVID-19 has also been reported [53] . Although several clinical trials are in progress to assess the potential effectofputative therapeutic agents, very limited data is available publicly regarding the invitro and in-vivoactivities of the drugs that are currently in clinical trials for treatment of COVID-19 [25] . Ithas been reported that Chloroquine phosphate shows anti COVID-19 activity [54] . Several clinical trials are assessing the potential of protease inhibitors such as Lopinavir andRitonavir that have been approved for treatment of other viral infections.

Lopinavir and Ritonavirwere identified in earlier studies to target the M pro of SARS virus.

In the present study we utilized the recently available high resolution experimental structure ofthe main protease of SARS-CoV-2 ( Fig.1 ) [55] , as the target formolecular docking based virtual screening. The predictions of this study will provide informationthat can be utilized for choice of candidate drugs for in vitro, in vivo and clinical trials. Ligand preparation has the following steps: addition of hydrogen atoms, removal of unwanted molecules, addition of all hydrogens, computation of Gasteiger charges, merging of non-polar hydrogens,generating ionization states at pH7, tautomers, geometric characteristics, and low-energy ring conformations. After preparation Ligand molecules were exported in PDBQT format for docking in PyRx 0.8 software.

The grid generation process was done in PyRx 0.8 software. It provided a square block at the active site of the protein for the accurate binding score with thermodynamic optimal energy.A grid box size of x = 27.9382928446, y = 28.2467551684 and z = 30.0038760533 Å points was generated to cover active Amino acid residues that are important for docking [55] .The grid was centered at x,y,z coordinates of -13.9467660792, 12.664485092, 68.4908850063. 

This study was done to identify possible compounds that can bind to themain protease, which may be used as a potential drug target for SARS-CoV-2. We have tested 15 bio-active J o u r n a l P r e -p r o o f compounds from Indian spices and medicinal plants that have been previously reported for their antiviral activity [28, 31, 34, [61] [62] [63] [64] [65] .These compounds can bind with the M pro with a docking score of -8.7 to -3.6 kcal/mol (Table 1) . Ursolic acid (Drugbank ID DB15588), compound of Tulsi, reported to have antiviral activity [29] , had highest docking score -8.7

(kcal/mol) than others (Table 1) , forms three Hydrogen bonds (H-bonds) with Thr24, Leu141*, Ser144* residues of M pro (Fig.2) . Hyperoside and Nimbaflavone were predicted to have a docking score of -8.6 and -8.0 kcal/mol ( (Fig.2) . Natural compounds of Ginger, 6-gingerol (PubChem ID 442793), 6-shogaol (PubChem ID 5281794) and 6-paradol (PubChem ID 94378) were predicted to have a docking score of -5.8, -5.8 and -5.7 kcal/mol (Table 1) . 6gingerol forms five interacting H-bonds with Arg188*, Gln189, Thr190*, Gln192 residues, 6shogaol forms four interacting H-bonds with three residues of SARS-CoV-2 M pro (Arg188*, Thr190*, Gln192), whereas 6-paradol exhibited five interacting H-bonds with Glu166, Arg188*, Thr190*, Gln192 residues of viral M pro (Fig.2) . Curcumin (DrugBank ID DB11672), active compound of Turmeric, had docking score of -7.1 kcal/mol (Table 1) forms four H-bonds with two interacting residues of M pro (Gly143*, Ser144*) (Fig.2) . Two main naturally occurring compounds of Tea plants,Catechins (PubChem ID 1203) and

Epigallocatechin (DrugBank ID DB03823) predicted to have a docking score of -7.1 kcal/mol and -7.3 kcal/mol respectively (Table 1) .Catechins forms two H-bonds with Thr26, Gln189 residues ( Fig.2 ) of viral M pro whereas Epigallocatechin exhibited seven interacting H-bonds with Leu141*, Ser144*, Cys145, His163* residues of M pro (Fig.3) . α-Hederin J o u r n a l P r e -p r o o f (PubChemID73296), had a significantly higher docking score of -8.5kcal/mol (Table 1) , forms four interacting H-bonds with His163*, Glu166, Gln189 residues of SARS-CoV-2 M pro (Fig.3) . Echinocystic acid diacetate (PubChem ID 476534), a triterpenoid saponin compound, had a docking score prediction -6.7kcal/mol (Table 1) , exhibited single interacting H-bond with Glu166 residue of M pro (Fig.3) . Two main natural compounds of Garlic, Ajoene (PubChem ID 5386591) and Allicin (DrugBank ID DB11780) were predicted to have a docking score of -4.1kcal/mol and -3.6 kcal/mol (Table 1) . Allicin forms two interacting Hbonds with Ser144*, Cys145 residues of M pro whereas Ajoene doesn't form any H-bond with SARS-CoV-2 M pro (Fig.3) . Piperine (DrugBank ID DB12582), a naturally occurring bioactive compound of Black pepper, had a docking score prediction -6.8 kcal/mol (Table 1) .

Piperine forms three interacting H-bonds with Thr25, Ser144*, Cys145 residues of viral M pro (Fig.3) . Docking results were validated by Webina 1.0.2 web server using the same docking parameters. Not much significant difference was observed between binding affinity predictions by PyRx 0.8 and Webina 1.0.2 web server by Durrant lab (Table 3) .

We selected all 15 ligands to simulate using MDWeb web portal [60] . Figure 4B shows B-factor fluctuation per residue of protein and all protein-ligand complexes. From general trend of change point of view B-factor fluctuation of protein-ligand complexes are nearly similar to that of protein backbone; however, some residues show higher flexibility. As shown in Figure 4B , B-factor values were fluctuated between 5Å 2 and 15 Å 2 with an average of 9 Å 2 . For more details show our supplementary 

effective interaction with all the tested. These residues can be targeted for potential drug designing to block SARS-CoV-2 M pro . In addition we compared ( Table 2) the binding capacity of these tested bio-active compounds with widely used popular drugs against SARS-CoV-2 infection across the world. These widely used drugs are quercetin, remdesivir, PF-00835231. Interestingly, three of our tested compounds namely ursolic acid, hyperoside, αhederin exhibited stronger binding and inhibitory potential against SARS-CoV-2 M pro in compare to quercetin, remdesivir, PF-00835231.

Our study suffers from some potential limitations. It would have been better to use GROMACS full package for entire molecular dynamics simulationanalyses. We did not use MM/PBSA and MM/GBSA program for calculating free binding energy calculation of each bio-active compound. We could not determine the inhibitory effects in term of IC50 value for each of these tested drug. Moreover, our finding needs wet lab experimental validation.

In present study, we have selected 15 bio-active compounds that are found among CoV2 infection. Our study will help the researcher to carry similar analyses for other drugs and ayurvedic bio-active agents and all these would contribute effectively to win the battle against SARC-CoV2 infections.

The work is financially supported by Department of Science and Technology, Government of West Bengal, India, Grant no. SG/WBDST/S&T 1000114/2016

The authors declare that there are noconflicts of interest. Table   Table 1 . Summary of all 15 bioactive compounds screened against SARS-CoV-2M pro with their respective source, binding energy, interacting residues and inhibition constant. 

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The infrastructural support and instrumental facilities were provided by UGC-UPE II, DST-