key: cord-0956756-4w6rmkmx
authors: Parida, Pratap Kumar; Paul, Dipak; Chakravorty, Debamitra
title: The natural way forward: Molecular dynamics simulation analysis of phytochemicals from Indian medicinal plants as potential inhibitors of SARS‐CoV‐2 targets
date: 2020-09-24
journal: Phytother Res
DOI: 10.1002/ptr.6868
sha: 7286d872ede73f7090601176bdca1a57b3a9449e
doc_id: 956756
cord_uid: 4w6rmkmx

The pandemic COVID‐19 has become a global panic‐forcing life towards a compromised “new normal.” Antiviral therapy against SARS‐CoV‐2 is still lacking. Thus, development of natural inhibitors as a prophylactic measure is an attractive strategy. In this context, this work explored phytochemicals as potential inhibitors for SARS‐CoV‐2 by performing all atom molecular dynamics simulations using high performance computing for 8 rationally screened phytochemicals from Withania somnifera and Azadirachta indica and two repurposed drugs docked with the spike glycoprotein and the main protease of SARS‐CoV‐2. These phytochemicals were rationally screened from 55 Indian medicinal plants in our previous work. MM/PBSA, principal component analysis (PCA), dynamic cross correlation matrix (DCCM) plots and biological pathway enrichment analysis were performed to reveal the therapeutic efficacy of these phytochemicals. The results revealed that Withanolide R (−141.96 KJ/mol) and 2,3‐Dihydrowithaferin A (−87.60 KJ/mol) were with the lowest relative free energy of binding for main protease and the spike proteins respectively. It was also observed that the phytochemicals exhibit a remarkable multipotency with the ability to modulate various human biological pathways especially pathways in cancer. Conclusively we suggest that these compounds need further detailed in vivo experimental evaluation and clinical validation to implement them as potent therapeutic agents for combating SARS‐CoV‐2.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) pandemic has overwhelmed the world wide health sector (Zhu et al., 2020) . As of 28th June, 2020 there have been 9,843,073 confirmed cases of COVID-19, including 495,760 deaths (https:// covid19.who.int/). Moreover, according to World Health Organization reports-53% of the countries surveyed have partially or completely disrupted medical services for hypertension treatment; 49% for treatment for diabetes and diabetes-related complications; 42% for cancer treatment, and 31% for cardiovascular emergencies (WHO, 2020a) .

Hence, this viral pandemic is forcing us to a compromised "new normal" for survival. To overcome this illness many clinical trials on vaccine development and drug repurposing are underway (WHO, 2020b) .

Unfortunately, till now none of the vaccines have reached the global market as the disease cure. Thus, a prophylactic treatment regime needs to be developed using natural compounds as a promising alternative therapy for containing the spread of the virus. Phytochemicals All the authors have contributed equally to the paper. have been identified by computational drug development approaches to be effective against SARS-CoV-2 (Adem, Eyupoglu, Sarfraz, Rasul, & Ali, 2020; Chandel, Raj, Rathi, & Kumar, 2020; Gentile et al., 2020; Gonzalez-Paz et al., 2020; Khaerunnisa, Kurniawan, Awaluddin, Suhartati, & Soetjipto, 2020; Khan, Zia, Ashraf, Uddin, & Ui-Haq, 2020; Kumar, Dhanjal, Kaul, Wadhwa, & Sundar, 2020; McKee, Sternberg, Stange, Laufer, & Naujokat, 2020; Qamar ul, Alqahtani, Alamri, & Chen, 2020; Sharma & Kaur, 2020; Sun, Wong, & Guo, 2020) . For example, flavonoids have been reported to be active against many viruses, and are available as natural supplements at a daily dose ranging from 100 to 500 mg (Dai et al., 2019; McKee et al., 2020; Moghaddam et al., 2014) . However very few of them are undergoing clinical trials (WHO, 2020b) . Therefore, looking at their potential, it can be said that more research is required to understand the efficacy of phytochemicals in the treatment of COVID-19 by understanding their functional mechanism of inhibition by dynamically exploring their interactions within the target sites. Further, comparison of their binding modes with that of the repurposed drugs (undergoing clinical trials) will also shed light about their efficiency.

In this context, rational screening was employed in our previous work to identify effective phytochemicals from 55 Indian medicinal plants for the treatment of COVID-19 . The phytochemicals following drug likeness properties were screened from Withania somnifera and Azadirachta indica against 8 protein targets of SARS-CoV-2. The phytochemicals were also observed to possess multi-potency against various SARS CoV-2 targets . This corroborates with the report by Kumar et al. (2020) , where Withanone from Withania somnifera was shown to bind and stably interact with SARS-CoV-2 main protease. Therefore, from our previous work and recent reports, after analysing the binding potential of these phytochemicals with multiple SARS-CoV-2 targets, it becomes necessary to explore their multitarget binding affinity and stability by carrying out molecular dynamics simulations. The multi-potential binding ability of these phytochemicals is important because it can result in overcoming multi-drug resistance of very specific drugs designed for single targets (Kadioglu, Saeed, Johannes Greten, & Efferth, 2020) . It can also be mentioned here that phytochemicals are known to bind to multiple targets (Efferth & Koch, 2011) .

Among all the potential targets explored in our previous work and through literature survey, the spike glycoprotein was understood to be a lucrative target for drug development as it facilitates viral entry.

The receptor to which it has the highest affinity is human angiotensinconverting enzyme 2 (ACE2) (Y. Chen, Guo, Pan, & Zhao, 2020; Xu et al., 2020) . The spike/ACE2 interface has been illustrated at the atomic level, and the efficiency of ACE2 usage was found to be a key determinant of this virus transmissibility which makes spike protein the primary target of neutralising antibody, vaccine and drug development (Li, Li, Farzan, & Harrison, 2005; Tian et al., 2020) . Another important target is the SARS-CoV-2 main protease (M pro ). It can be targeted to prevent the virus replication within the cells. It has been reported that backbone and binding site conformations are conserved in M pro (Joshi et al., 2020) . Moreover, with its low similarity with human proteases, M Pro -inhibitors are found to be very less cytotoxic (Anand, Ziebuhr, Wadhwani, Mesters, & Hilgenfeld, 2003; Zhang et al., 2020) . Thus it can be said that SARS-CoV-2 spike (S) glycoprotein and main protease are crucial to the virulence and transmission of the virus. These attributes can be explored for drug development.

Therefore, in the present work, long timescale molecular dynamics simulations and calculation of free energy of binding along with biological pathway enrichment analysis were performed on the phytochemicals targeting the Spike glycoprotein and the main protease of SARS-CoV-2. Through this work, in light of the high infectivity rate of SARS-CoV-2 and lack of treatment per se, the experimental testing of phytochemicals from W. somnifera and A. indica as potential drugs against SARS-CoV-2 is being proposed.

High Performance Computing resources of Amazon EC2 G4 instances with the latest generation NVIDIA T4 GPUs were employed for carrying out computational work on Amazon Web Services cloud computing platform (https://aws.amazon.com) with the help of RONIN interface (https://ronin.cloud/) as an award granted by the COVID-19

High Performance Computing (HPC) Consortium for carrying out this work (https://covid19-hpc-consortium.org/projects). Multi GPU virtual machines with g4dn.12xlarge with 4 GPU instances and 48 virtual core CPU instances were used to carry out in total 1 μs molecular dynamics simulations. The performance obtained using this computational system configuration was 125 ns/day.

Phytochemicals from Indian medicinal plants were rationally screened by molecular docking against SARS-CoV-2 targets in our previous work . The eight top scoring phytochemicals that followed drug like properties-against SARS-CoV-2 spike glycoprotein (PDB ID: 6LZG) and the main protease (PDB ID: 6LU7) were analysed by all atom molecular dynamics (MD) simulations for 100 ns (Table 1) . Two repurposed drugs that had the lowest docking score against the spike protein and the main protease were also analysed by MD simulation as a control group. All the simulations were carried out with Gromacs 2020.2 software package (Lindahl, Hess, & van der Spoel, 2020) with AMBER99SB-ILDN force field. The force field parameters for the phytochemicals and the repurposed drugs were generated by ACPYPE (AnteChamber PYthon Parser interface) (Da Silva & Vranken, 2012) .

Complex charges were neutralised with sodium and chloride ions. Simulation was conducted at temperature 300 K and under a pressure of 1 bar. Each system was minimised with 5,000 steps by steepest descent algorithm. Electrostatic interactions were calculated with Particle-Mesh-Ewald summation (PME) (Darden, York, & Pedersen, 1993) . The systems were equilibrated by 1 ns position restraint simulations of The phytochemicals (top docking scorers) were identified from a previous work to possess drug likeness properties . Source: The 2D images of the phytochemicals were obtained from PubChem Database (https://pubchem.ncbi.nlm.nih.gov/). 1,000 kJ mol −1 nm −2 in the NVT and NPT ensembles. Equilibrated systems were used to simulate a 100 ns no restraint production run. Post-MD analyses were performed, this includes root mean square deviation (RMSD), root mean square fluctuations (RMSF), the radius of gyration (Rg) and hydrogen bond occupancy.

Molecular Mechanics-Poisson Boltzmann Surface Area (MM-PBSA) was applied on snapshots obtained from MD trajectory to estimate the relative binding free energy ΔG b using the GROMACS tool g_mmpbsa (Baker, Sept, Joseph, Holst, & McCammon, 2001; Kumari et al., 2014) .

where b = binding free energy, v = vaccuum phase, s = solvation free energy which is the difference of solvation free energy values of complex, receptor and ligand:

where c = protein ligand complex, r = protein, l = ligand. The solvation free energy is composed of polar and non-polar energy computed as:

where p = polar energy, np = non − polar energy. The computed free energy of binding represents the affinity and stability of the compounds with the target proteins.

The principal component analysis (PCA) method was employed to understand the dynamical behaviour in the conformational space of SARS-CoV-2 spike glycoprotein and NSP5 bound to the phytochemicals and the repurposed drugs. PCA was performed using RStudio (RStudio Team, 2020) and Bio3d (Grant, Rodrigues, ElSawy, McCammon, & Caves, 2006) . The eigenvectors, eigenvalues and their projection along with the first two principal components were analysed (Al-Khafaji & Taskin Tok, 2020). PCA extracts the most variable dynamic motions in simulations, required for biological function (Amadei, Linssen, & Berendsen, 1993) . It was assumed that a stable drug bound complex will undergo less dynamic fluctuations due to 2.5 | Comparative pathway enrichment analysis between COVID-19 and the phytochemicals protein targets

The human protein targets of the phytochemicals used in this work were predicted from SwissTargetPrediction server (Gfeller et al., 2014) . The top 15 targets were retained for each phytochemical. After overlapping the targets and removal of redundancy it was possible to filter out 47 target proteins. The protein targets for human coronavirus were obtained by literature survey and a total of 172 targets were filtered after removal of redundancy (Chakrabarty, Das, Bulusu, & Roy, 2020; Zhou et al., 2020) . The targets have been presented in Table S3 . The gene set enrichment analysis web server, Enrichr was used for gene ontology (GO) enrichment which were summarised using REVIGO webserver (Kuleshov et al., 2016; Supek, Bošnjak, Škunca, & Šmuc, 2011) . The generated networks were visualized in Cytoscape v3.8 (Shannon et al., 2003) . The p-values computed using the Fisher exact test for pathway and database enrichment were performed for Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analysis) and VirusMint analysis using Enrichr (with p < .01).

VirusMint is database of interactions between viral and human pro-

Each of the best docked complexes of NSP5 with 4 phytochemicals (P1-P4) and 1 repurposed drug (PR5), obtained from our previous work, were subjected to 100 ns MD simulations. SARS-CoV-2 main protease (NSP5) consists domain I (residues 8-101) and II residues (102-184) with antiparallel β-barrel and domain III (residues 201-303) which is helical. A long loop (residues 185-200) connects domains III and II . The active site of NSP5 ( Figure 1A ) is located in between domains I and II with His41-Cys145 as catalytic dyad Yang et al., 2003) . Cys145, Gly143 and Ser144 also serves as the oxyanion hole Yang et al., 2003) .

His163, while Glu166 and Gln189 are also required for catalysis Yang et al., 2003) . Figure Figure 1D ).

Analysis of occupancy of hydrogen bonds (distance ≤4 Å) over 100 ns of the trajectories revealed that P4 and Gln189 in the connecting loop between domain II and domain III maintained~56% occupancy over 100 ns trajectory. Again the hydrogen bond occupancy of P4 was 28% with His41. PR5 maintained 41% with Gln189 and 18%

occupancy. The hydrogen bond occupancy was higher for P4 and PR5 than other phytochemicals with the active site residues (Table S1) .

Further, to extract the structural variations in details upon the binding of the phytochemicals and the repurposed drug, PCA on the Cα atom was performed. The first three Eigen vectors captured around~50% of the motions ( Figure S1 ). It is notable that the confor- 

The four best docked complexes (S1-S4) with phytochemicals following drug likeness properties were subjected to 100 ns MD simulations along with the best docked repurposed drug (SR5). The apo-protein of spike without ACE-2 receptor was also subjected to 100 ns MD simulations. Figure 3A illustrates the region of spike protein that interacts in comparison to the other phytochemicals ( Figure 3D ). Interestingly the compactness of SR5 decreases after 50 ns.

Analysis of occupancy of hydrogen bonds (distance ≤4 Å) over 100 ns of the trajectories revealed that the hydrogen bond occupancy was maximum for S4 followed by SR5 and the other phytochemicals (S1-S3). More number of residues were involved with hydrogen bonds with S4 in comparison to S1-S3 and SR5. Gln493, Ser494, Phe497, Tyr495, Tyr505, Asn501 of the RBD domain of spike protein were with >10% hydrogen bond occupancy with S4. Hydrogen bonding with Tyr495 was maintained for~53% of the trajectory, following by Tyr505 with~40% occupancy (Table S2 ). It can be said that the 

The pathway network based approach is a systems level understanding of the effect on the interconnected biological processes. The top 15 human protein targets for each phytochemical were retrieved from SwissTarget Database. The SARS-CoV-2 human protein targets were obtained by literature survey (Chakrabarty et al., 2020; Zhou et al., 2020) . Enrichr analysis of KEGG pathway enrichment for COVID-19 and the phytochemicals revealed that AGE-RAGE signalling pathway in diabetic complications and Human cytomegalovirus infection were common to both (Table S4) 

The whole gamut of computational methods is instrumental in designing highly specific inhibitors against important viral targets-offering scientifically sound basis for anti-viral drug discovery. Identification of potential inhibitors by rational screening is the first step towards the anti-viral inhibitor discovery process. The second step involves understanding the biophysical basis of inhibitor binding mechanism through molecular dynamics simulations. Currently the viral infection, COVID-19 is highly contagious with no effective therapy to combat the infection (Guan et al., 2020) . The high infectivity is due to its spike protein of SARS-CoV-2 that primes with human ACE2 receptor more tightly, by a factor of 10 to 15, than SARS-CoV spike protein . Another is involved in maintaining the viral life cycle, being an essential enzyme for viral replication (Huynh, Wang, & Luan, 2020) .

In this context previous research reported natural compounds like theaflavin, quercetin, withanone and caffeic acid phenethyl ester to be effective inhibitors of SARS-CoV-2 Lung et al., 2020; Zhang et al., 2020) . This corroborated with our previous work were 8 phytochemicals from Withania somnifera and RMSF is because such stabilities are essential to obtain good binding affinities (J. Chen & Shen, 2009; Doniach & Eastman, 1999; Dubey, Tiwari, & Ojha, 2013) . Enhancement in protein compactness on ligand binding can be elucidated by low values of radius of gyration. Again, large number of hydrogen bonds between ligand and important residues related to activity of protein signifies a strong binding affinity (Khan et al., 2020) . PCA is an essential dynamics (ED) approach-performed to reflect overall difference in motions in the protein on binding ligands in comparison to the apo-protein (Islam, Khan, Parves, Hossain, & Halim, 2019; Martens & Naes, 1992) . Like PCA, the protein residue atomic motions correlation and non-correlation can be computed with the help of DCCM (Hünenberger, Mark, & Van Gunsteren, 1995; McCammon, 1984 Leu455 was also reported to contribute favourably to ACE2 recognition (Shang et al., 2020) . Further we identified that hydrogen bonds between Withanolide R and NSP5 were stable with residues involved in the activity of NSP5 for example-His41 and Gln189. Similarly, Tyr495 and Tyr505 of the ACE2 binding region were involved with hydrogen bonding with the phytochemicals in the case of spike protein. These residues were also reported to be critical in binding to the ACE2 receptor (Chikhale et al., 2020) . Overall the phytochemicals with the lowest binding energy formed a more stable environment with the targets.

It can also be mentioned here that the therapeutic benefit of the phytochemicals is not limited to targeting spike and the main protease of SARS-CoV-2. In summary, these phytochemicals exhibit a remarkable multipotency with the ability to modulate various human biological processes and biochemical pathways. This was observed by the study of pathway enrichment by the phytochemicals and the SARS-CoV-2. in both the cases. Increased levels of advanced glycation end products (AGEs) especially carboxymethyllysine (CML), have been observed to be associated with atherosclerosis, and coronary artery disease and heart failure (Hegab, Gibbons, Neyses, & Mamas, 2012) . CMV is also a lung infection and HRCT findings and patterns are similar to COVID-19

infections (Cascella, Rajnik, Cuomo, Dulebohn, & Di Napoli, 2020) . The MAPK pathway enrichment can be justified because it is involved in release of pro-inflammatory cytokines such as IL-6 and has been implicated in acute lung injury and myocardial dysfunction (Grimes & Grimes, 2020) . It was reported that activation of p38 MAPK, is involved in replication of HCoV-229E and induction of overwhelming inflammation by SARS-CoV-2 (Grimes & Grimes, 2020; Kono et al., 2008) . Again clinical feature associated with COVID-19 like the SARS-CoV infection is pulmonary fibrosis and the associated lung failure. Phytochemicals targeting the R-SMAD binding pathway which has been reported to be involved in blocking apoptosis of SARS-CoVinfected host cells may result in decrease in fibrosis (Zhao, Nicholls, & Chen, 2008) . Similarly, the proteins targeted by the phytochemicals and SARS-CoV-2 were observed to be enriched in transition metal ion binding and metal ion binding have been reported to be an integral part of HCoV-229E viral protein (Chaturvedi & Shrivastava, 2005) . The pathways involved in DNA replication and protein transcription were also enriched in SARS-CoV-2 infection and by the phytochemicals. Further, pathways related to androgen receptor binding were enriched in COVID-19 and the phytochemicals. It was reported that cell entry of SARS-CoV-2 depends on binding of the viral spike (S) proteins to ACE2

and on S protein priming by TMPRSS2. Inhibition of TMPRSS2

(an androgen regulated gene) which is alleviated in prostate cancer patients may work to block or decrease the severity of SARS-CoV-2

infections (Montopoli et al., 2020) . Again the ubiquitin-proteasome system is a regulator of intracellular sorting and degradation of proteins.

Inhibition of the pathway was reported to severely impair coronavirus infection. The phytochemicals were observed to enrich this pathway similar to the SARS-CoV-2. Conclusively it can be said that the phytochemicals can regulate COVID-19 infection by also targeting these pathways along with the spike and the main protease of SARS-CoV-2.

In conclusion, this study reports the biophysical basis of interaction between eight phytochemicals and two repurposed drugs with spike protein and the main protease of SARS-CoV-2 by performing analysis of 100 ns molecular dynamics simulation trajectories. Overall, the simulation run confirms the stability and rigidity of the interactions of Withanolide R and 2,3-Dihydrowithaferin A from Withania somnifera to be the most potent phytochemical inhibitor for the main protease and the spike protein respectively. The energy calculations show the binding efficiency of these phytochemicals. Further, the important residues interacting with the phytochemicals were elucidated. Conclusively through this work experimental validation is being suggested to test the efficacy of these phytochemicals for the prevention and treatment of COVID-19.

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The natural way forward: Molecular dynamics simulation analysis of phytochemicals from Indian medicinal plants as potential inhibitors of SARS-CoV-2 targets

This work used resources services, and support provided via the COVID-19 HPC Consortium (https://covid19-hpc-consortium.org/), which is a unique private-public effort to bring together government, industry, and academic leaders who are volunteering free compute time and resources in support of COVID-19 research. We would also like to acknowledge Noor Enzymes Private Limited for providing us with the human resources needed to successfully complete this work.We would also like to specially thank Linda Hedges (HPC Solutions architect at Amazon Web Services), Tara Madhyastha (Principal research scientist at Amazon Web Services), Nathan Albrighton (Founder & CEO at RONIN) and Don Hancock (Co-Founder at RONIN) who provided us with timely guidance and assistance to complete this work.

No potential conflict of interest was reported by the authors.

All authors conceived, conducted and analysed the results of experiments equally.

https://orcid.org/0000-0002-8193-335XDipak Paul https://orcid.org/0000-0002-5664-4601Debamitra Chakravorty https://orcid.org/0000-0002-1520-0780