key: cord-0889241-l433c4yl
authors: Nagar, Prinsa R.; Gajjar, Normi D.; Dhameliya, Tejas M.
title: In search of SARS CoV-2 replication inhibitors: Virtual screening, molecular dynamics simulations and ADMET analysis
date: 2021-12-15
journal: J Mol Struct
DOI: 10.1016/j.molstruc.2021.131190
sha: e62e085aa2d38fbe2f5aacb7ef555f26e7abf367
doc_id: 889241
cord_uid: l433c4yl

Severe acute respiratory syndrome has relapsed recently as novel coronavirus causing a life threat to the entire world in the absence of an effective therapy. To hamper the replication of the deadly SARS CoV-2 inside the host cells, systematic in silico virtual screening of total 267,324 ligands from Asinex EliteSynergy and BioDesign libraries has been performed using AutoDock Vina against RdRp. The molecular modeling studies revealed the identification of twenty-one macrocyclic hits (2–22) with better binding energy than remdesivir (1), marketed SARS CoV-2 inhibitor. Further, the analysis using rules for drug-likeness and their ADMET profile revealed the candidature of these hits due to superior oral bioavailability and druggability. Further, the MD simulation studies of top two hits (2 and 3) performed using GROMACS 2020.1 for 10 ns revealed their stability into the docked complexes. These results provide an important breakthrough in the design of macrocyclic hits as SARS CoV-2 RNA replicase inhibitor.

Novel coronavirus (CoV) has been recognized as a deadly respiratory pathogen and causative agent for severe acute respiratory syndrome coronavirus-2 (SARS CoV-2) or coronavirus disease 2019 (COVID- 19) [1] since its outbreak in the Hubei province of Wuhan, China in 2019. [2] [3] It has caused an unprecedented pandemic worldwide with the higher fatality rate than the previously known SARS CoV. The background of the virus traces back nearly two decades to a closely related previously emerged pathogen having genomic similarity of 75-80%. [4] The epidemiological study on the virus brings in to notice the first case to be reported on December 2019 in China, which was then observed with the aggressive or violent form by January 2019 with increasing number of cases transmitting in the other parts of the world. [5, 6] By January 2019, approximately eighteen countries were reported to be infected with the virus emerged in China led to the declaration as "Public Health Emergency of International Concern" by World Health Organization (WHO) on Mar 11, 2020. [7] As on Jul 9, 2021 there have been reported 185,291,530 confirmed cases with 4,010,834 fatality cases worldwide. [8] The entire world has to suffer from the huge numbers of challenges due to this global pandemic as all the vital sectors like health, education, economic, social, pharmaceutical, etc. have been widely affected.

Unemployment, events cancellation, prohibited public places, declined foreign and domestic trades during the pandemic caused a huge fall in the global economy. [9, 10] The novel SARS CoV-2 viral pathogens responsible for the global pandemic have been derived from the previously emerged strain reported in 2002-03 which are suspected to be transmitted into humans through several animal hosts such bats, civets, pangolins and camels. [11] The clinical symptoms in the patients infected with coronaviruses may vary from moderate symptomatic to severe asymptomatic characteristics including headache, cough, cold, tiredness, aches, fever, loss of taste or smell, diarrhea, discoloration of finger or toes, breathing difficulties to dysfunction of organs. The viruses such as SARS and MERS are transmitted into humans through the direct or indirect exposure of the respiratory secretions. [12, 13] The large genome of the coronaviruses including 30 kb long nucleotide sequence has been comprised within the single stranded positive sense enveloped RNAs. [14] The crown like structural morphology of these viruses observed under electron microscope are Page 5 of 36 due to the club-like glycoproteins. [13] The CoV belongs to the β-coronaviruses among the four major genuses of the large family Nidovirales. [15] The critical insights into the viral pathogenesis and replication may shine a light towards the comprehensive understanding regarding the virus as presented in Figure 1 . The pathogenesis of corona virus begins with the fusion and attachment to the host receptor angiotensin converting enzyme-2 (ACE2) through its spike glycoprotein. Upon invasion and after the releasing its genomic material inside the host cell, the genome gets translated into several polypeptides followed by the translation into sixteen non-structural polyproteins (NSPs). The 3' end of the sub-genomic RNA gets transcribed into several structural proteins which are assembled on the endoplasmic reticulum (ER) and golgi body to release the viral progenies outside the plasma membrane. [16] Despite of current antiviral strategies including the anti-viral agents and vaccines to combat the deadly coronavirus, these could not prove themselves effective against the virulent disease. In order to generate the scopes for effective inhibitors of SARS CoV-2, several drugs have been repurposed and implemented to combat this ailment, though they have not shown any reliable results necessitating the demand for some potential inhibitors. [17, 18] In view of this fact, several vaccines containing live attenuated, RNA-DNA derived, viral vectored, protein contained and inactivated vaccines have been developed with positive results against the catastrophic virus, SARS CoV-2. [19] In contrary, the vaccines under phase III trials have also been noted with several side effects after the vaccination such as headache, fatigue, chills, diarrhoea, arthralgia, myalgia, redness, itching, swelling, etc. [20] To exemplify a nucleotide analog, remdesivir was declared under emergency use authorization by United States food and drug administration (USFDA) and was evaluated under in vivo and in vitro studies for the inhibition of viral replication inside the host body. [21] Despite of its efficacy, it has not been found effective solution to tackle the race for the inhibition of the deadly virus due to its impotency in patients with immunodeficiency and heart failure, hypotension, respiratory dysfunction, as well as renal impairment. [22] The conventional approach to the drug design and development may take plenty of resources such as cost, time and manpower with no guarantee to afford the desired candidates with efficacy and potency. Therefore, the computer assisted drug design (CADD) have been preferred over several years above the traditional approaches reducing the cost-burden by benefiting the feasibility and improved results by removing the gap between chemical and biological science. [23] Among these CADD tools, the use of molecular modeling should not be undermined as they provide significant interactions of identified hits against their biological targets to understand their mode of action. [24] [25] [26] [27] [28] Recently, we have performed in silico screening of significant phytochemicals against RNA-dependent RNA Polymerase (RdRp) and main protease (Mpro) using molecular docking followed by their druggability using ADMET assay and stability of their complexes using molecular dynamics (MD) simulations in search of effective SARS CoV-2 inhibitors. [29] Motivated by the potential of computational chemistry and in search of potent inhibitors of SARS CoV-2, herein we have performed the virtual screening of total 267,324 ligands from 2020-01 Asinex EiteSynergy (91,473) and BioDesign (175,851) libraries using AutoDock Vina against RdRp using molecular docking followed by their further assessment using rules for druggability, ADMET assay and MD simulations.

Xu et al. have reported the cryo-EM structure of the RdRp from SARS-CoV-2, cocrystallized with remdesivir having the resolution of 2.8 Å (PDB ID: 7BV2). [30, 31] In this complex, partial double-stranded RNA template was implanted into the central channel of the RdRp where remdesivir was covalently fused into the primer strand at the first replicated base pair and terminated the chain elongation. The core component of the viral replication complex is the non-structural proteins (nsp12) of the RdRp. [32, 33] 

The molecular docking has been performed against the selected protein (PDB ID: 7BV2) [30] using AutoDock Vina [34] Further, these hits have been found with better binding energy as compared to other promising RdRp inhibitors such as ribavirin (23) , penciclovir (24) , favipravir (25) , [36] molnupiravir (26) and sofosbuvir (27) . [37] Total twenty-one identified macrocyclic hits have been found to possess binding energy lesser than -12.5 Kcal/mol and presented in Table 1 and their 2D-possess with the formed interactions have been presented in Figure   S1 -S4 (see supporting informartion). The 3D-poses of identified ligands 2-7 bound at the active site of RdRp have been generated using Biovia Discovery Studio 2020 [38] and presented in Figure retained almost similar interactions including van der Waals bond (Lys500), π-π bond (Phe441, π-alkyl bond with Ala547 and Ile548), π-anion bond (Urd17 and Urd18 of chain P) and conventional HB (Ade14 of chain P and Ade11 of chain T, Figure 2d ). Compound 6

found to form conventional HB with Ade14, Gua16 and Ade19 of chain P, Ade13 of chain T, Lys500 and Asn497. It also formed C-H bond with Asn497, π-anion bond with Ade19 of chain P and π-alkyl bond with Ala547 and Urd18 of chain P (Figure 2e ). N-Methylsulfonyl piperidine substituted compound 7 interacted at the active site of RdRp by forming π-alkyl bond (Ala547, Ile548 and Ade13 of chain T), π-sulfur bond (Ade14 of chain P), π-anion bond (Urd17 and Urd18 of chain P), C-H bond (Lys500) and

conventional HB (Ade11 of chain T and Ade14 and 15 of chain P, Figure 2f ). In general, these interactions with the ligands have supported our hypothesis on the tight binding of identified ligands into the active site of protein. 2,4-Disubstituted pyridine derivative 8 has been found with binding energy of -12.8

Kcal/mol (Table 1) formed halogen interactions between chlorine atom present in it with Cyt15 and Ade14 of chain T (Figure 3d ). The replacement of pyrimidine-2,4(1H,3H)-dione-6-methyl in ligand 6 with pyridin-2-yl-acetamide in 12 has been resulted in almost similar binding energy (-12.8 and -12.7 kcal/mol, respectively) along the with formation of π-anion bond with

Ade19 of chain P, π-alkyl bond with Ala547 and Urd18 of chain P, conventional HB with Ade14, Ade15 and Ade19 belonging to chain P (Figure 3e ). Several π-alkyl interactions are found in docked complex of compound 13 with amino acids Phe441, Ile548, Lys500, nucleotides Urd17, Gua16 (chain P) and Ade11, Urd12 (chain T). Further, ligand 13

formed conventional HB with Lys545, Arg836 and Ade14 of chain T and π-anion bond with Urd18 of T chain and π-σ bond with Ala547 ( Figure 3f ). Compound 14, with binding energy of -12.6 kcal/mol, formed conventional HB with Ade19 of chain P and Ade11, Ade14 and Cyt15 of chain T, π-anion bond with Urd13 of chain P. Additionally, nucleotide Urd18 has found with π-π bond, π-sulfur bond and πalkyl bond with the ligand 14 ( Figure 4a ). Compound 15, a metamer of compound 9, formed completely dissimilar interactions in comparison with 9 such as C-H bond with Lys500, π-sulfur bond with Ade13 of T chain, π-alkyl bonds with Ala547 and Ile548, π-anion bonds with Urd17 and Urd18 from chain P and conventional HB with Asn496, Ade11 and Ade13 of chain T (Figure 4b ). Next, 2,3-dihydrobenzo[b][1,4]dioxin-6-sulfonyl derivative 16 found to form π-sulfur bond with Ade14 of chain T, π-alkyl bond with Ile548, π-anion bond with Urd17 and Urd18 of chain P and HBs with the amino acid Asn496 and nucleotides Ade15 and Ade19 from P chain and Ade13 and 14 from T chain (Figure 4c ). Ligands 20-22 have been observed with similar binding energy of -12.5 kcal/mol (Table 1) . Ligand 20 formed conventional HB and π-sulfur bond with Ade15 of chain P, C-H bonds with Lys500 and Ade14 of chain P, π-alkyl bonds with Ala547 and Ile548, π-anion bonds with Urd17 and 18 of chain P. One of the fluorine atom of phenyl ring interacted with Urd13 of chain P (Figure 5a ). Compound 21, being the positional isomer of 8, made πanion bond with Urd17 of chain P and conventional HB with Lys846, Arg858, and Ade11 of chain T and Ade14 of chain P. Additionally, two fluorine atoms of trifluoromethyl group in 21 were found to interact with Asp845 ( Figure 5b ). α-Naphthyl-1H-pyrrolidonone derivative 22 formed conventional HB with Ade15 of chain P, C-H bond with Asn496, πalkyl bonds with Ala547 and Urd18 of chain P and π-anion bonds with Arg836 and Ade19 of chain P (Figure 5c ) to accommodate on the active site of RdRp. 

The identified macrocyclic hits have been analyzed for the several physicochemical properties ( Table 2) Thus, most of the hits may have potential to be a better drug molecule in comparison with remdesivir (1, Table 2 ). Rules for the drug-likeliness such as Lipinski's rule of 5 (Ro5), Ghose rule, Veber's rule, Egan's rule and Muegge's rule have been also studied to ensure the drug-likeliness of Figure 6 . According to the rule of 5, the molecule can serve as better drug candidate with the suitable properties including MW (≤500 Da), HBD (≤5), HBA (≤10), LogP (≤5) and RB (≤5) [42] . Not more than one violation of these parameters will reduce the druggability of the molecule. Most of the hits (except 8, 11, 13, 18, 21 and 22 ) have found to meet the desired properties suggesting their candidature for the anti-viral drug. As per the Veber's rule, compound should have the polar surface area less than 140 Å 2 to have the better oral bioavailability [43] . None of hits to be the good drug candidate [46] and all the hits meet the suggested properties. However, as compared to Remdesivir, the identified hits may have potential to act as a good drug candidate in the future as they obey the rules for druggability and drug-likeness. Further, we analyzed the additional parameters to predict the metabolism, excretion and toxicity profile of remdesivir and these hits ( Table 4 ). Most of the molecules inhibited respectively. The blue circles indicate inhibition of P-glycoprotein.

To study the stability of ligand into the binding site of protein, molecular dynamics (MD) simulations provide the better understanding of ligand through several statistical parameters. [48, 49] Several scientific literatures support the reliability and satisfactory stability for the MD run (≤ 10 ns) through in silico endeavor. [50] [51] [52] [53] [54] [55] In a similar line of approach, the hit complexes obtained from the evaluation of ADMET and drug-likeliness properties, were subjected for MD simulations using GROMACS 2020.1 to assess the stability of the stability of the ligands (2) (3) in the active site of docked complex at various time points up to 10 ns. [56, 57] Next, we subjected the complex of compound 2 to MD simulation studies and the graphical representation of plots of statistical parameters has been presented in Figure 8 . The electrostatic (coulombic short-range, Coul-SR) and van der Waals/hydrophobic (LJ-SR) energies have been represented in Table 5 for the complexes of identified hits 2 and 3

with RdRp. These complexes have been observed to be stabilized by the van der Waals or hydrophobic interactions significantly over the electrostatic or coulombic interactions as evident from the higher energies of LJ-SR over Coul-SR. 

In fine, the in silico based virtual screening of total 267,324 ligands from 2020-01

Asinex 

The multi-ligand molecular docking was performed using the prepared protein using AutoDock Vina to reveal the binding affinity and interactions of the ligands with RdRp protein (PDB ID: 7BV2). [30] All the ligands (267,324) were docked on the site using the optimized grid box and ten poses per ligand were generated. The docked pose of remdesivir was compared with the co-crystallized inhibitor remdesivir to validate the present docking protocol. The docking interactions of hits were visualized and analyzed using Biovia

Discovery studio 2020. [38] The 2D interactions of docked compounds with RdRp have been presented in supporting information ( Figures S1-S4 ).

Various physicochemical properties like molecular weight, number of hydrogen bond donors, number of hydrogen bond acceptors, oil to water partition co-efficient and number of rotational bonds of the selected hits and remdesivir (1) were studied using Swiss ADME [39] and pkCSM. [40, 41] Several rules such as Lipinski's rule of 5, Ghose rule, Veber's rule, Egan's rule and Muegge's rule were applied to the selected hits using the SwissADME to ensure their druggability and to identify numbers of violated parameters.

The ADMET properties like water solubility, molar refractivity, topological polar surface area, CaCO2 cell permeability, intestinal absorption, volume of distribution, fraction unbound, total renal clearance, hepatotoxicity and ability to inhibit the P-glycoprotein were also studied using pkCSM.

Two molecules (2 and 3) , obtained from the manual analysis of the hits found from the results of molecular docking and ADMET analysis, were selected for MD simulation using GROningen MAchine for Chemical Simulations (GROMACS) 2020.1 [56, 57] software. CHARMM36 (Chemistry at Harvard Macromolecular Mechanics) as an all atom force field [60] and CHARMM General Force Field (CGenFF) server to retrieve the topology of (2 and 3) were used. [61, 62] After solvation (TIP3P water model), neutralization (Na + and Clions), equilibration [canonical (NVT) and isobaric-isothermic (NPT) ensemble for 100 picoseconds], the complexes were subjected to molecular dynamics run for 10 ns. The equilibrium of trajectories the systems was found satisfactorily equilibrated as evident from the plots of temperature, pressure and energy equilibria of the system ( Figures S5 and S6 , see supporting information).

The authors declare no competing financial interest. 

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