key: cord-1001160-9ec36270
authors: Tiwari, Vishvanath
title: Denovo designing, retro-combinatorial synthesis, and molecular dynamics analysis identify novel antiviral VTRM1.1 against RNA-dependent RNA polymerase of SARS CoV2 virus
date: 2021-01-07
journal: Int J Biol Macromol
DOI: 10.1016/j.ijbiomac.2020.12.223
sha: 5f426510ec00ef42e11898550e843026a6a29aab
doc_id: 1001160
cord_uid: 9ec36270

A novel coronavirus disease (COVID-19) caused by SARS-CoV2 has now spread globally. Replication/transcription machinery of this virus consists of RNA-dependent RNA polymerase (nsp12 or RdRp) and its two cofactors nsp7 and nsp8 proteins. Hence, RdRp has emerged as a promising target to control COVID-19. In the present study, we are reporting a novel inhibitor VTRM1.1 against the RdRp protein of SARS CoV2. A series of antivirals were tested for binding to the catalytic residues of the active site of RdRp protein. In-silico screening, molecular mechanics, molecular dynamics simulation (MDS) analysis suggest ribavirin, and remdesivir have good interaction with the binding site of the RdRp protein as compared to other antiviral investigated. Hence, ribavirin and remdesivir were used for the denovo fragments based antiviral design. This design, along with docking and MDS analysis, identified a novel inhibitor VTRM1 that has better interaction with RdRp as compared to their parent molecules. Further, to produce a lead-like compound, retrosynthetic analysis, and combinatorial synthesis were performed, which produces 1000 analogs of VTRM1. These analogs were analysed by docking and MDS analysis that identified VTRM1.1 as a possible lead to inhibit RdRp protein. This inhibitor has a good docking score (−7.24 kcal/mol) and favourable binding energy (−79.88 kcal/mol) than remdesivir (−6.038; −72.07 kcal/mol) and bind at catalytic AS760 and Asp761 of the active site. The VTRM1.1 also interacts with RdRp in the presence of RNA primer and other cofactors. It was also seen that, VTRM1.1 do not have off-target in human. Therefore, the present study suggests a hybrid inhibitor VTRM1.1 for the RNA-dependent RNA polymerase of SARS CoV2 that may be useful to control infection caused by COVID-19.

A novel coronavirus disease caused by SARS-CoV2 [1] has now spread globally [2] . It has shifted its epicenter at different places across the globe and has now spread in more than 200 countries. On March 11, 2020 , the world health organization (WHO) declared this outbreak a pandemic. On Apr 15, 2020 there were > 15,00,00 cumulative cases globally and > 90,000 death with ~ 5% mortality rate in outcome cases. The main symptom of this disease includes fever, shortness of breath, cough, and gastrostomy complications. Coronavirus employs a multi-subunit replication/transcription machinery. Different non-structural proteins (nsp) produced as a cleavage product of ORF1a and ORF1b viral polyproteins [3] that are assemble to facilitate viral replication and transcription. A key component of this machinery is nsp12 also known as RNA-dependent RNA polymerase (RdRp), and responsible for RNA synthesis. RNA synthesis of nsp12 in SARS CoV is activated by two other non-structural proteins nsp7 and nsp8 [4] . In addition to that, this complex (nsp12/nsp7/nsp8) associates with nsp14 and has shown 3'-5' exoribonuclease (involved in replication fidelity) and RNA cap N-7-guanine methyltransferase activities (associated with 5'-RNA capping) in SARS-CoV [4] . This shows the importance of RdRp in the biology of SARS-CoV2. Hence, to control COVID-19 infection, there is an urgent need to find an inhibitor for RdRp. It is also reported that nsp12 (or RdRp) was considered as a primary target to control the infection of SARS-CoV by different inhibitors [5] . RdRp can be a potential therapeutic target to control the infection caused by SARS CoV2 as its human homolog of this nsp12 has not reported [6] therefore, therapeutic against nsp12 may be significant to control the COVID-19. The attempt has made to evaluate the FDA approved molecule like Ribavirin, Remdesivir, etc. as an inhibitor of RdRp [7] . Remdesivir, Ritonavir, and Lopinavir have been used to control other coronaviruses [8] . Remdesivir (GS-5734) is a broad-spectrum antiviral nucleotide prodrug with potent antiviral activity against diverse RNA viruses like SARS-CoV, MERS-CoV [9] .

to the catalytic residue of the active site.

The PDB structure of RNA-dependent RNA polymerase (RdRp) was taken from RCSB (PDB number 6m71, resolution 2.9Å). This PDB structure has four chains [10] including nsp12 (chain A), nsp7 (chain C), nsp8 (chain B and chain D). The nsp12 consists of the thumb, palm, and finger domain ( Figure 1 ). The PDB structure showed that the active site of RdRp domain involves residues like Asp618 of the motif A; Ser759, Asp760, and Asp761 of motif C. It was also seen that Asp618 Asp760 and Asp761 are involved in the binding with Mn 2+ ; Arg555, Val557, Asp623, Thr680, Ser682, Asn691, and Asp760 are involved in binding of UDP and Remdesivir [10] . Hence, these residues Asp618, Ser759, Asp760, Asp761, Asp623, Arg555, Val557, Ser682, Asn691, Thr680 were used for receptor grid generation. This receptor gird was used to screen antiviral molecule targeting RdRp.

The antiviral molecules were selected based on the currently available literature search and their efficacy on the SARS-CoV2. The selected antiviral drugs in the present study were remdesivir, ribavirin, favipiravir, ritonavir, umifenovir, hydroxychloroquine, ascorbate, oseltamivir, tenofovir, and acetyl-serine. The ligand preparation of these antiviral molecules has resulted in the 87 tautomers, and prepared ligands were investigated for its interaction with the binding site of RdRp.

We have performed docking studied in HTVS, SP, and XP mode. Docking results suggested that among all the antiviral molecules investigated, ribavirin (G-score, -6.109kcal/mol) and remdesivir (G-score, -6.038kcal/mol) have shown better interaction with the active site of Table ST1 Ribavirin involves Arg553, Arg555, Thr556, Ser682, Arg624, Asp623, Cys622, Lys621,

All the antivirals were undergone molecular mechanics analysis with generalized born and surface area solvation (MM-GBSA) methods to calculate the Gibbs free energy of binding.

The result of this analysis is listed in table 1. It was found that all the molecules investigated, have favourable Gibbs free energy change for binding to RdRp. Antivirals were first filtered by docking scores, followed by Gibbs free energy if their docking scores were similar. Based on docking and binding free energy results, remdesivir and ribavirin are selected for the further study. As these two molecules are nucleotide analog, hence the interaction of different NTP and dNTPs with the RdRp was also monitored. The result showed that Remdesivir has better binding energy as compared to native nucleotides (ATP, GTP, CTP, UTP), while ribavirin has better binding energy than NTPs like ATP (Supplementary Table ST2 ). which has the best docking (G-score, -7.953kcal/mol). This docking score was found to be higher than their parent molecules (Table 1 ) and named as 'VTRM1', and chemically Figure 2E and 2F). To further confirm the interaction, the MDS analysis of the binding site of the RdRp protein was performed. The result showed that RMSD of RdRp protein (in ligand complex state) was found to be <2Å, and ligand's RMSD is less than <4.8Å, that showed stable complex ( Figure 3A ). RMSF analysis showed that most of the protein (except some part) has the RMSF <2Å that showed stable protein conformation ( Figure 3B ). Further, the interaction between VTRM1 and RdRp protein was investigated and found that it involves Asp452, Arg553, Thr556, Asp618, Tyr619, Lys621, Cys622, Asp623, Arg624,

(2R,3R,4S,5R)-2-{4-aminopyrrolo[2,1-f][1,2,4]triazin-7-yl}-5-({[(S)-({3-[(2R,3S,4R,5S)-5- {4-aminopyrrolo [2,1-f][1,2,4]triazin-7-yl}-3,4-dihydroxyoxolan-2-yl]-2- oxopropyl}amino)(phenoxy)phosphoryl] oxy}methyl)-3,4-dihydroxyoxolane-2-carbonitrile (

Journal Pre-proof Asp760, Asp761, Lys798, Glu811, and at least nine contacts always exist more than 30% simulation time ( Figure 3D ).

To produce lead-like compounds, retrosynthetic analysis, and combinatorial synthesis was Figure 5A ). RMSF analysis showed that most of the protein has the RMSF <3Å that showed stable protein conformation ( Figure 5B ). Interaction between VTRM1.1 and RdRp protein involves at least nine contacts that always exist more than 30% simulation time ( Figure 6A) and involves Arg553, Asp618, Lys621, Asp623, Arg624, Asp760, Asp761, Trp800, Glu811 amino acid residues ( Figure 6B) . The ligand has a small deviation (RMSD <2.5 Å) during the simulation with reference to its conformation at the start of the simulation ( Figure 6C ).

Ligand has only one intramolecular hydrogen bonding till 18ns after that there is no intramolecular bonding, which further suggests stable conformation of ligands. The van der Waals surface area (MolSA), solvent accessible surface area (SASA), polar surface area (PSA) also supported the good interaction between ligand and RdRp ( Figure 6C ).

In figure 7A , the top panel shows the total number of specific contacts of protein with ligand throughout the trajectory. The bottom panel shows interacting residues of protein with ligand in each trajectory frame ( Figure 7A ). This confirms a good interaction of VTRM1.1 with catalytically active amino acid residues like Asp760, Asp761, Asp623 of RdRp. In figure 7B ,

In addition to this, ADMET analysis of all the selected lead molecules, i.e., VTRM1, VTRM1.1, and VTRM1.2 were performed, and their results are shown in Table 2 . The comparative analysis showed that although VTRM1 has the best docking score but it has bad ADMET properties that make it a weak lead. The VTRM1.1 has better ADMET parameters and has the docking and binding energy very close to VTRM1; hence VTRM1.1 is suggested as a possible inhibitor for RdRp of SARS CoV2. Table ST3 ) that further supports the biological significance of the lead molecule.

Presence of lead's off-targets in human may reduce the efficacy and produce the side effect.

Hence, human off-targets of VTRM1.1 was predicted using Swiss Target Prediction. The result showed that the designed lead VTRM1.1 do not possess any off-targets in human. This further enhances the significance of VTRM1.1 as a possible lead against the RdRp.

SARS-CoV2 has emerged as a lethal pathogen causing a global pandemic. No specific FDA approved treatment is available for COVID-19. Some non-specific treatments like remdesivir, ribavirin, etc. [11] are used. Ribavirin is a guanosine analog that has been reported to have multiple mechanisms of action [12] and has also shown to have an inhibitory effect against SARS-coV2 [13] . Remdesivir (GS-5734) is currently used with lopinavir against COVID-19 without knowing its specific targets in SARS-CoV2 [14] . The essential components of the replication/transcription machinery of SARS CoV2 are RNA-dependent RNA polymerase (nsp12 or RdRp) along with its two cofactors nsp7 and nsp8. In addition to this, this complex nsp12/nsp7/nsp8 binds with nsp14, which then modulates replication fidelity and 5'-RNA capping in SARS-CoV [4] . Human homologs of nsp12 have not reported [6] , hence, RdRp can be a potential therapeutic target to control the infection caused by SARS CoV2. In the present study, a novel inhibitor VTRM1.1 targeting to RdRp was J o u r n a l P r e -p r o o f Journal Pre-proof designed using denovo designing, retrosynthetic analysis, and combinatorial synthesis, and molecular dynamic simulation.

PDB structure of RdRp was retrieved from the RCSB, and the grid was prepared at an active site that binds with metal and nucleoside-diphosphate (NDP). The docking, binding energy calculation, and molecular dynamic simulation calculation have shown that ribavirin and remdesivir have good interactions with the active site of RdRp. Various in-vitro experimental data suggest that the ribavirin has shown an inhibitory effect on SARS-CoV2 [13] . Similarly, remdesivir effectivity inhibits the 2019-nCoV in-vitro [15] by delayed chain termination and inhibits RNA synthesis [16] . Recently, it is also seen that it incorporate into the primer stand at the first replicated base pair and terminates chain elongation [17] . An in-silico study on the modeled RdRp has also shown that the ribavirin and remdesivir has the best interaction with RdRp among the approved antiviral investigated [18] To find a novel inhibitor, we have used denovo fragment-based drug design using FDA approved antiviral molecules ribavirin and remdesivir that showed direct interaction with the active site of RdRp of SARS CoV2. A total of 93 fragments were generated from these two molecules. They were docked to the binding site of RdRp, and best-docked fragments were manually analyzed of its interaction with RdRp. The selected fragments were joined via denovo fragment-based drug design. This approach joined the different fragments based on the structural similarity to the active molecules and their scaffolds, hence used to navigate a considerable chemical space [19] . The hybrid antiviral was further confirmed for its interaction with the active site of RdRp using the XP-mode of docking and molecular dynamics simulations. This select VTRM-1 as a possible denovo synthesized hybrid molecule that can interact with RdRp and involves Asp618, Asp760, Asp761 amino acid residue that is important for the catalysis of this protein [10] . VTRM1 has many ADMET parameters outside its permissible limit; hence, we have modified it.

Optimization of lead required the synthesis and validation of thousands of lead's analogs before clinical candidate nomination, which takes a longer time. Further, to explore possible improvement in the VTRM1, we have used in-silico based retrosynthetic analysis, combinatorial synthesis, and MDS analysis to design an improved new antiviral molecule.

RNA-depended RNA polymerase as per our published protocol [21] . The docking score predicts the binding affinity between the target protein and particular pose of ligand docked.

Waals energies. The virtual screening was performed in three modes of docking i.e., HTVS, SP, and XP mode [22] . 

MDS was performed using Desmond modules of the Schrodinger 2019-4 as per published methods [24] using the OPLS3e force field. The system was built for the protein-ligand complex using the TIP3P solvent model; sodium ion was added to make charge-neutral, 0.15M NaCl was added to make the system close to the natural system. The simulation was run for 5ns (or 25ns for VTRM1.1.-RdRp complex), with 5ps trajectory recording intervals.

System energy was set to be 1.2, and the ensemble class used was NPT. The simulation was set to run at 300k at 1.01325bar. The option to relax the system before simulation was selected. The simulated system was analysed for the simulation interaction diagram.

De-novo fragment-based drug designing is a newly emerged approach [19] . 

To produce synthetically tractable lead-like compounds, retrosynthetic analysis, and combinatorial synthesis were performed for the denovo synthesised VTRM.1 as per the published method using PathFinder [25] . PathFinder can incorporate more than 100 reactions like C-C bond formation that are required for the molecular scaffolds and drug discovery [26] . A total of 100 number of pathways are investigated to produce 1000 products. The top 10% product that is similar to the input molecule VTRM.1 are selected for further analysis.

The selected 1000 products were analysed by docking, and molecular dynamics simulations analysis to select the antiviral lead.

The ADMET analysis was performed that identified absorption distribution, metabolism, excretion, and toxicity of the leads molecule using QikProp as per published protocol [27] .

The interaction of lead with RdRp in the presence of cofactors has a biological relevance, hence the PDB structure of RdRp complexed with primer RNA, cofactors, and remdesivir was retrieved from RCSB (PDB number 7BV2, resolution 2.5Å). The remdesivir was removed from the complex, and the structure was pre-processed, optimised, minimised to 0.3RMSD using the OPLS_2005 force field. The residues Asp618, Ser759, Asp760, Asp761, Asp623, Arg555, Val557, Ser682, Asn691, Thr680 are used for receptor grid generation.

This receptor gird was used to validate the selected leads.

The human off-targets of the designed lead was predicted using Swiss Target Prediction [28] using the published protocol [29].

[29] V. Tiwari, K. Meena, M. Tiwari, Differential anti-microbial secondary metabolites in different ESKAPE pathogens explain their adaptation in the hospital setup, Infection, Genetics and Evolution 66 (2018) 57-65. 

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