key: cord-184744-oyc2djxk
authors: Parvez, Md Sorwer Alam; Azim, Kazi Faizul; Imran, Abdus Shukur; Raihan, Topu; Begum, Aklima; Shammi, Tasfia Saiyara; Howlader, Sabbir; Bhuiyan, Farhana Rumzum; Hasan, Mahmudul
title: Virtual Screening of Plant Metabolites against Main protease, RNA-dependent RNA polymerase and Spike protein of SARS-CoV-2: Therapeutics option of COVID-19
date: 2020-05-22
journal: nan
DOI: nan
sha: 
doc_id: 184744
cord_uid: oyc2djxk

Covid-19, a serious respiratory complications caused by SARS-CoV-2 has become one of the global threat to human healthcare system. The present study evaluated the possibility of plant originated approved 117 therapeutics against the main protease protein (MPP), RNA-dependent RNA polymerase (RdRp) and spike protein (S) of SARS-CoV-2 including drug surface analysis by using molecular docking through drug repurposing approaches. The molecular interaction study revealed that Rifampin (-16.3 kcal/mol) were topmost inhibitor of MPP where Azobechalcone were found most potent plant therapeutics for blocking the RdRp (-15.9 kcal /mol) and S (-14.4 kcal/mol) protein of SARS-CoV-2. After the comparative analysis of all docking results, Azobechalcone, Rifampin, Isolophirachalcone, Tetrandrine and Fangchinoline were exhibited as the most potential inhibitory plant compounds for targeting the key proteins of SARS-CoV-2. However, amino acid positions; H41, C145, and M165 of MPP played crucial roles for the drug surface interaction where F368, L371, L372, A375, W509, L514, Y515 were pivotal for RdRP. In addition, the drug interaction surface of S proteins also showed similar patterns with all of its maximum inhibitors. ADME analysis also strengthened the possibility of screened plant therapeutics as the potent drug candidates against SARS-C with the highest drug friendliness.

COVID-19 pandemic situation has tremendously turned the entire world into a place of horrible death tragedy. SARS-CoV-2, initially named as 2019 novel coronavirus (2019-nCoV) by the World Health Organization (WHO) is the causative agent of the recent serious respiratory complications resulting the COVID-19 pandemic [1, 2, 3, 4] . Though the symptoms of COVID-19 infection appear after an incubation period of approximately 5.2 days, the period from the onset of COVID-19 symptoms to death ranged from 6 to 41 days with a median of 14 days [5, 6] . It has already completed its world tour, and around 213 countries are now experiencing the deadly scene occurred by COVID-19 including 41, 52,670 infected patients and 2, 84,536 global death cases till 12th May, 2020 [7] . The scientific community is racing to explore the effective remedy against this severe health complications, but till to date there are no any potential therapeutics have been approved for clinical use [8] .

There have been few key proteins of SARS-CoV-2 that could be targeted as the vaccine or drug surface [9] . Similar to SARS and MERS, non-structural proteins (e.g. 3-chymotrypsin-like protease coronavirus main protease, papain-like protease, helicase, and RNA-dependent RNA polymerase), structural proteins (e.g. spike glycoprotein) and accessory proteins were investigated in the genome of SARS-CoV-2 where non-structural proteins constitute two-thirds of the entire genome [10] . Among the structural proteins, Nucleocapsid (N) protein is prerequisite for RNA genome assembly where Membrane (M) and Envelope (E) proteins are associated in viral assembly in the host environment [11] . Moreover, Spike (S) protein is mainly responsible for the viral entry into the host cell, and that is why spike protein is now being considered as a major therapeutic target for drug and vaccines against SARS-CoV-2 [12] . Again, The S protein interaction with the human ACE2 interface has been revealed at the atomic level, and the efficiency of ACE2 usage was found to be a main factor of coronavirus transmissibility in human to human [13] . On the contrary, coronavirus main protease (M pro) or 3C-like proteinase (3CLP) was reported for their ability to cleave the polyproteins into individual polypeptides that are required for replication and transcription [14] . The 3CLP is autocleaved initially from the polyproteins to become a mature enzyme leading the translation of the messenger RNA [15] . Then the 3CLP cleaves all the 11 downstream non-structural proteins. As 3CLP plays a vital role in the replication cycle of virus in the host, it has been reported as the attractive target against the human SARS virus [16] . RNA-dependent RNA polymerase, other key target protein of SARS-CoV-2 catalyses the synthesis of viral RNA possibly with the support of other non-structural proteins as co-factors [17, 18, 19] .

The computational drug repurposing method could allow the immediate search of potential antiviral therapy in case of re-emergence of viral infections as like as COVID-19 pandemic situation [20, 21, 22] .

Computational drug repurposing has already been used to identify promising drug candidates for other virus associated diseases like Dengue, Ebola, ZIKA, and influenza infections [23, 24] . Most importantly, the SARS-CoV-2 has shown evolutionary convergent relations with SARS-CoV and MERS-CoV, and the drug repurposing methods were also applied to SARS-CoV and MERS-CoV [25, 26, 27] . Hence, extensive in silico studies were performed to identify potential drug candidates, for example, Prulifloxacin, Bictegravir, Nelfinavir, and Tegobuvi, were identified as repurposing candidates against COVID-19 by looking for drugs with high binding capacity with SARS-CoV main protease [28] . Again, Nelfinavir, an HIV-1 protease inhibitor was also predicted to be a potential inhibitor of COVID-19 main protease by another computational-based study [29] .

However, secondary metabolites from plant origin are found to show effective defence mechanism against different deadly pathogens, and they have been widely used for conventional remedy to treat a wide range of human diseases since the ancient period of human civilization [30, 31, 32] . In this pandemic situation, researchers are trying find out the effective solution against COVID-19 where plant metabolites could be a promising wings for screening out potential drug candidates. Even few plant secondary metabolites have already been reported as effective against other coronaviruses [33, 34] . In the present study, a total of 117 plant based drug compound were screened out to check their potentiality for blocking the three important key proteins of SARS-CoV-2. The main protease proteins, RNA-dependent RNA polymerase and spike protein of SARS-CoV-2 were employed to molecular docking study with the repurposed drug candidates from plant origin for find out the better drug option towards the COVID-19 pandemic.

protein and acquisition of potential natural therapeutics PDB structures of SARS-CoV-2 main protease proteins (6LU7, 6Y2E), RNA-dependent RNA polymerase (6M71) and spike protein (6VYB) were retrieved from RCSB Protein Data Bank [35] .

Moreover, a total 117 plant based drugs were collected from PubChem database (Supplementary Table   1 ). Alpha-ketoamide (CID 6482451) were also retrieved from the PubChem database database (https://pubchem.ncbi.nlm.nih.gov/) of NCBI [36] .

Molecular docking is an effective approach for screening out potential therapeutics against specific drug-targets of deadly pathogens [37, 38] . The crystal structure of retrieved SARS-CoV-2 proteins (complexed with inhibitors) were refined by PyMOL v2.0 software [39] . Unwanted molecules i.e. water, ions, inhibitors were removed from the viral retrieved viral protein, and further employed to molecular docking experiment with 117 natural therapeutics. AutoDock Vina software [40] to analyse the binding affinity and interactive amino acids. Alpha-ketoamide, an inhibitor SARS-CoV-2 main protease protein were used as a positive control in this study [41] and also docked against the target proteins of SARS-CoV-2. The default parameters for grid box were set to 62 A° x 71 A° x 60 A° (x, y and z) and center -25.389 A° x 15.333 A° x 56.865 A° (x, y and z) to perform the action. Moreover, 2D ligand-protein interaction diagrams were generated by LigPlot+ find out the involved amino acids with their interactive position were identified in the docked complexes [42] . The ligand molecules' interactions with the viral proteins were visualize and analyzed by Discovery Studio [43] and PyMOL v2.0 software [44] .

The drug surface hotspot of SARS-CoV-2 proteins were identified by analysing the docked structures of each protein with the top most natural therapeutics by LigPlot+, PyMOL v.2.0 and Discovery Studio software. Binding patterns of Azobechalcone, Rifampin, Isolophirachalcone, Tetrandrine, Fangchinoline with SARS-CoV-2 proteins and the results were allowed for the comparative structural analysis of screened natural therapeutics. Furthermore, interactions of Alpha-ketoamide with the studied proteins were also investigated.

The ADME (Absorption, Distribution, Metabolism and Excretion) properties of top drug candidates were analysed by SwissADME server [45] . The pharmacokinetics, drug-likeness property and medicinal chemistry were assessed [46] . Default parameters were used to evaluate various physiochemical parameters (Molar Refractivity, Molecular weight, TPSA), lipophilicity (Log Po/w (WLOGP), Log Po/w (MLOGP), Log Po/w (XLOGP3), Log Po/w, (SILICOS-IT), Log Po/w (iLOGP), Consensus Log Po/w), pharmacokinetics parameters (Log Kp; skin permeation) and water solubility of the probable drug candidates [47] . The inhibition effects of these natural therapeutics with different cytochromes P450s (CYP2C9, CYP2C19 CYP1A2, CYP3A4, CYP2D6) were also studied.

In addition, admetSAR and OSIRIS Property Explorer were employed to evaluate the toxic or undesired effects (i.e. mutagenicity, tumerogenecity) of the compounds [48, 49, 50] .

All of the retrieved natural therapeutics were employed for molecular docking against MPP, RdRp and Spike protein of SARS-CoV-2 (Supplementary Table 2 ). The scoring function of AutoDock Vina was utilized to predict the interaction between the ligands (therapeutics) and the proteins. The top five inhibitors for each protein was identified based on their free binding energy. Results showed that Rifampin had the highest negative binding energy (-16.3 kcal/mol) among top MPP inhibitors (Table   1 ). Azobechalcone (-14.6 kcal/mol), Isolophirachalcone (-13 kcal/mol), Amentoflavone (-12.8 kcal/mol) and Cepharanthine (-12.7 kcal/mol) docked with the MPP of SARS-CoV-2 were also exhibited topmost place with a higher negative binding energy (<-12.7 kcal/mol) as well (Table 1) .

While interacting with RdRp of SARS-CoV-2, the most negative binding energy was scored by Azobechalcone (-15.9 kcal /mol) following by Rifampin (-15.6 kcal/mol), Tetrandrine (-13.9 kcal/mol), Biflavone (-13.7 kcal/mol), Biflavone (-13.7 kcal/mol) ( Table 2) . Moreover, Azobechalcone, Rifampin, Isolophirachalcone, Fangchinoline and Tetrandrine were found to be top most natural inhibitors for the spike protein of SARS-CoV-2. Azobechalcone required lowest energy (-14.4 kcal/mol) to interact with the spike protein, while Rifampin, Isolophirachalcone, Fangchinoline and Tetrandrine scored -13.7 kcal/mol , -12.8 kcal/mol, -12.6 kcal/mol and -12.5 kcal/mol respectively (Table 3) . However, Azobechalcone, Rifampin, Isolophirachalcone, Tetrandrine and Fangchinoline were found to be most effective inhibitory natural compounds when the docking results were compared for all three SARS-CoV-2 proteins (Figure 2 and Table 4 ). All of these plant based natural inhibitors required minimum energy (not more than -12.3 kcal/mol) to interact with the studied protein molecules.

The docking pattern and interacting amino acid residues with their respective position were analyzed to unravel the binding sites of studied SARS-CoV-2 proteins. Rifampin were involved with the amino acid H41, N142, S144, C145, H163, M165, E166, D187, R188, Q189 of MPP of SARS-CoV-2 ( Figure 2 ). The position of H41, C145, and M165 were also crucial for the binding of Amentoflavone However, an additional residue T430 were involved in case of Isolophirachalcone (Table 3) . P426, D428, T430, P463, F464 and F515 were also critical for binding pattern of Rifampin and SARS-CoV-2 spike protein.

The most potent MPP, RdRp and spike protein inhibitors (Azobechalcone, Rifampin, Isolophirachalcone, Tetrandrine and Fangchinoline) were investigated in the spheres of physicochemical parameters, lipophilicity, pharmacokinetics and water solubility (Table 5) .

Lipophilicity, partition coefficient between n-octanol and water (log Po/w) were also calculated by using five widely available predictive models (XLOGP3, WLOGP, MLOGP, SILICOS-IT, iLOGP).

GI absorption was lower for all the drug candidates. Azobechalcone, Rifampin, Isolophirachalcone, Tetrandrine and Fangchinoline molecules had no repressive action with the P450 (CYP) isoforms (CYP1A2, CYP2C19, CYP2C9, CYP2D6, CYP3A4). Moreover, no permeant BBB exists among the protein inhibitors of MPP, RdRp and Spike. The compounds sowed water solubility from moderate to high level i.e. 1.30e-16 mg/ml, 1.50e-02 mg/ml, 1.18e-10 mg/ml, 9.78e 09 mg/ml, 4.61e-08 mg/ml, respectively ( Table 5 ). The toxicity analysis of these inhibitors showed that there were no carcinogenic effect and organ toxicity. However, Rifampin, Tetrandrine and Fangchinoline were slightly positive in terms of mutagenesis though Azobechalcone and Isolophirachalcone inhibitors were completely negative. Among the top five inhibitors Azobechalcone was listed in the acute oral toxicity category 2 and rest of them were listed in the category 3.

Global pandemic caused by SARS-CoV-2 has become a major concern due to its excessive infection rate and lethality [51, 52, 53, 54] . Despite huge research regarding the pathogen, no drugs or vaccine has proven satisfactory to combat infections caused by SARS-CoV-2 [55, 56] . Several investigational drugs exist, however none of these could treat the patients unquestionably. Moreover, lack of rapid detection procedures made SARS-CoV-2 diagnosis troublesome [57] . Computational approach and drug repurposing strategies hold promise to face such challenges caused by SARS-CoV-2. Hence, in the present study, attempts were taken to suggest probable drug candidates by checking the efficacy of natural inhibitors to inhibit the key proteins of SARS-CoV-2.

The race against the COVID-19 pandemic has allowed the drug repurposing through virtual for finding drugs that could be used for the treatment of COVID-19. Recent studies prioritized MPP inhibitors of SARS-CoV-2 i.e. alpha-ketoamide, Hydroxy, Remdesivir, Chloroquine and Favipiravir to evaluate their potency as drug [58, 59, 60] . Several in silico approach was also adopted to screen putative drug candidates against SARS-CoV-2 [61, 62] . However, all these experiments used either main protease proteins or RNA-dependent RNA polymerase of SARS-CoV-2 as probable drug targets. In this study, we screened potential natural therapeutics against SARS-CoV-2 MPP, RdRp and spike protein by molecular docking approach. Here, Rifampin, Azobechalcone and Azobechalcone were determined as top most drug candidates as they interacted with SARS-CoV-2 MPP, RdRp and spike protein with lowest negative binding energy had the highest negative binding energy (-16.3 kcal/mol, -15.9 kcal /mol and -14.4 kcal/mol respectively. However, comparative analysis revealed the superiority of 5 drug candidates i.e. Azobechalcone, Rifampin, Isolophirachalcone, Tetrandrine, Fangchinoline against SARS-CoV-2 ( Table 4 ). The common drug surface hotspots were analyzed along with modeling of pharmacophore which is very important step for drug discovery. Three amino acid residues i.e. H41, C145, and M165 played the crucial role for the interaction of MPP with its inhibitors (i.e. Rifampin, Amentoflavone, Cepharanthine) (Table 1) .

Azobechalcone, the top scorer among RdRp inhibitors, Again, the position of F368, L371, L372, A375, W509, L514, Y515 were vital for binding of RdRP with Tetrandrine, Biflavone and Fangchinoline (Table 2 ). Most importantly, the binding patterns of spike protein with Isolophirachalcone, Fangchinoline and Tetrandrine were significantly similar. This study revealed the possibility of these amino acids to efficiently interact with drugs, though requires validation in wet lab trials. ADME analysis of top drug candidates reveled no undesirable consequences by these compounds. Various physico-chemical parameters, lipophilicity, pharmacokinetics properties and water solubility were determined (Table 5 ). In addition, no BBB permeant were identified among the top most inhibitors of MPP, RdRp and spike protein. The consequence of association of the natural inhibitory drugs of three key proteins with the cytochrome P450 (CYP) suggests that no substantial inhibition can occur. However, toxicity analysis revealed that Rifampin, Tetrandrine and Fangchinoline can be slightly mutagenic, though there was no possibility for organ toxicity.

The results suggest that Azobechalcone, Rifampin, Isolophirachalcone and Tetrandrine, Fangchinoline could be an option to treat SARS-CoV-2 infections. However, the study employed various computational approaches to screen the potent natural therapeutics and does not involve in-vivo assay.

Currently investigational drugs of SARS-CoV-2 are under immense experimental evaluation. Therefore, we suggest clinical trials for the experimental validation of our findings.

COVID-19 pandemic situation is going to be a worst condition throughout the world. Rapid detection and social distancing are being encouraged at this stage, but we need to search for immediate therapeutic options and effective vaccine candidates for battling this serious health crisis. Drug repurposing approaches could screened out the already approved drugs for reusing against any serious causative agents that are causing health complications. Plant metabolites based repurposed drug molecules could be a promising options against SARS-CoV-2. In the present study, five plantr based therapeutics such as Azobechalcone, Rifampin, Isolophirachalcone, Tetrandrine and Fangchinoline were suggested for potential inhibitors for the Main Protease protein, RNA dependent RNA polymerase and Spike protein of SARS-CoV-2 by using molecular docking based virtual screening study. The study initiated the window towards the thinking of plant based therapy against COVID-19, though extensive research and wet lab validation needs to make it usable for patient. 

A novel coronavirus from patients with pneumonia in China

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

Clinical features of patients infected with 2019 novel coronavirus in Wuhan, China. The lancet

Epidemiological and clinical characteristics of 99 cases of 2019 novel coronavirus pneumonia in Wuhan, China: a descriptive study. The Lancet

Bat origin of a new human coronavirus: there and back again

A data-driven analysis in the early phase of the outbreak. International journal of infectious diseases

An interactive web-based dashboard to track COVID-19 in real time. The Lancet infectious diseases

Discovering drugs to treat coronavirus disease 2019 (COVID-19). Drug discoveries & therapeutics

Research and development on therapeutic agents and vaccines for COVID-19 and related human coronavirus diseases

Quantitative structure-activity relationship and molecular docking revealed a potency of anti-hepatitis C virus drugs against human corona viruses

From SARS to MERS, thrusting coronaviruses into the spotlight

Homologous recombination within the spike glycoprotein of the newly identified coronavirus may boost cross-species transmission from snake to human

Structure of SARS coronavirus spike receptor-binding domain complexed with receptor

Substrate specificity profiling and identification of a new class of inhibitor for the major protease of the SARS coronavirus

Design of wide-spectrum inhibitors targeting coronavirus main proteases

Antiviral drugs specific for coronaviruses in preclinical development. Current opinion in virology

Structure of the SARS-CoV nsp12 polymerase bound to nsp7 and nsp8 co-factors

The potential chemical structure of anti-SARS-CoV-2 RNA-dependent RNA polymerase

SARS-CoV-2 RNA dependent RNA polymerase (RdRp) targeting: An in silico perspective

Network-based drug repurposing for novel coronavirus 2019-nCoV/SARS-CoV-2. Cell discovery

Potential inhibitors against papain-like protease of novel coronavirus (SARS-CoV-2) from FDA approved drugs

Drug Repurposing in Antiviral Research: A Current Scenario

Screening of commercial cyclic peptide conjugated to HIV-1 Tat peptide as inhibitor of N-terminal heptad repeat glycoprotein-2 ectodomain Ebola virus through in silico analysis

Middle East respiratory syndrome and severe acute respiratory syndrome: current therapeutic options and potential targets for novel therapies

Design of efficient computational workflows for in silico drug repurposing. Drug Discovery Today

Computational Methods for Drug Repurposing

Therapeutic Drugs Targeting COVID-19 Main Protease by High-Throughput Screening

Nelfinavir was predicted to be a potential inhibitor of 2019-nCov main protease by an integrative approach combining homology modelling, molecular docking and binding free energy calculation

The global importance of plants as sources of medicines and the future potential of Chinese plants. InDrug discovery and traditional Chinese medicine

Plant Secondary Metabolites of Antiviral Properties a Rich Medicinal Source for Drug Discovery: A Mini Review

The role of natural products in modern drug discovery. Anais da Academia Brasileira de Ciências

Plant-derived natural products in drug discovery and development: an overview

Medicinal and therapeutic potential of herbs and plant metabolites/extracts countering viral pathogens-current knowledge and future prospects

The RCSB protein data bank: integrative view of protein, gene and 3D structural information

Database resources of the national center for biotechnology information

Virtual screening for HIV protease inhibitors: a comparison of AutoDock 4 and Vina

Main Protease Inhibitors and Drug Surface Hotspot for the Treatment of COVID-19: Drug Repurposing and Molecular Docking Approach

Pymol: An open-source molecular graphics tool. CCP4 Newsletter on protein crystallography

Benchmarking sets for molecular docking

Jalview Version 2-a multiple sequence alignment editor and analysis workbench

Crystal structure of SARS-CoV-2 main protease provides a basis for design of improved αketoamide inhibitors

LigPlot+: multiple ligand-protein interaction diagrams for drug discovery

Interaction of α-cyperone with human serum albumin: Determination of the binding site by using Discovery Studio and via spectroscopic methods

Pharmacophore modeling for COX-1 and-2 inhibitors with LigandScout in comparison to Discovery Studio. Future medicinal chemistry

SwissADME: a free web tool to evaluate pharmacokinetics, drug-likeness and medicinal chemistry friendliness of small molecules. Scientific reports

Bioavailability prediction of phytochemicals present in Calotropis procera

VITRO ADME STUDIES OF TUG-891, A GPR-120 INHIBITOR USING SWISS ADME PREDICTOR. Journal of Drug Delivery and Therapeutics

P0899: Preclinical profile of the pan-genotypic HCV NS3/4A protease inhibitor GS-9857

Development and Marketing of INCIVEK (Telaprevir

Combination with PEGylated Interferon and Ribavirin. HCV: The Journey from Discovery to a Cure: Volume I

Can we contain the COVID-19 outbreak with the same measures as for SARS

What we know so far: COVID-19 current clinical knowledge and research

SARS-CoV-2 and COVID-19: The most important research questions

Immune responses in COVID-19 and potential vaccines: Lessons learned from SARS and MERS epidemic

Sensitivity of chest CT for COVID-19: comparison to RT-PCR

Discovering drugs to treat coronavirus disease 2019 (COVID-19). Drug discoveries & therapeutics

COVID-19: a recommendation to examine the effect of hydroxychloroquine in preventing infection and progression

Remdesivir as a possible therapeutic option for the COVID-19. Travel medicine and infectious disease

Chloroquine and hydroxychloroquine as available weapons to fight COVID-19

Favipiravir versus arbidol for COVID-19: a randomized clinical trial

Anti-HCV, nucleotide inhibitors, repurposing against COVID-19. Life sciences

Drug repurposing for coronavirus (COVID-19): in silico screening of known drugs against coronavirus 3CL hydrolase and protease enzymes

Authors would like to acknowledge the Faculty of Biotechnology and Genetic Engineering, SylhetAgricultural University for the technical support of the project.

This research did not receive any specific grant from funding agencies in the public, commercial, or not-for-profit sectors.

Authors declare that they have no conflict of interests.