Repurposing of RdRp Inhibitors against SARS-CoV-2 through Molecular Docking Tools

Department of Pharmaceutical Chemistry, ISF College of Pharmacy, Ferozepur G.T. Road, Moga-142 001, Punjab, India; Research Scholar, Department of Pharmaceutical Sciences & Technology, MRSPTU, Bathinda-151001, Punjab, India; Department of Pharmaceutics, ISF College of Pharmacy, Ferozepur G.T. Road, Moga-142 001, Punjab, India; Department of Chemistry, Maharishi Markandeshwar (Deemed to be University), Mullana-133207, Haryana, India


INTRODUCTION
Coronaviruses (CoVs) are a family of +RNA viruses that infect a wide range of vertebrates, including humans causing severe acute respiratory syndrome (SARS) and various other respiratory infections [1]. CoV-19 belongs to the beta type of coronavirus family. Beta coronavirus genome encodes several proteins, which include glycosylated spike protein (S), angiotensin-converting enzyme 2 (ACE2) and nonstructural proteins, including RNA-dependent RNA polymerase (RdRp), coronavirus main protease (3CLpro), and papainlike protease (PLpro) [2][3][4]. All the proteins have significant roles throughout the viral progression cycle [5]. RdRp is an enzyme that is responsible for the replication of viral RNA from an RNA template [6]. Due to its central role in the replication of viruses, RdRp is a significant and attractive target for drug development and design against SARS-CoV-2 infections [7][8]. Currently, there is no drug or vaccine that has been approved against SARS-CoV-2. Although some drugs/vaccines have reached the clinical trial stages, but it will take 12 to 18 months to be launched in the market. In such a condition, drug repurposing approach is promising in *Address correspondence to this author at the Department of Pharmaceutical Chemistry, ISF College of Pharmacy, Ferozepur G.T. Road, Moga-142001, Punjab, India; Tel: 086288-85561; E-mail: bhatiarohit5678@gmail.com drug discovery. Drug repurposing is a technique for utilization of the therapeutic value of an existing drug by focusing on infections other than that for which it was initially proposed [9]. Remdesivir, ribavirin, sofosbuvir and galidesivir have been focused on by many research groups for drug repurposing [10][11][12][13][14]. These four drugs have already been approved by the FDA against different viral infections ( Table  1). These drugs have excellent potential of inhibiting RdRp, which ultimately interferes with viral RNA replication and protein synthesis. In a recent report, Elfiky et al., carried out molecular docking studies of these four drugs on SARS-HCoV RNA dependent RNA polymerase (RdRp) (PDB ID:6NUR) because it was the most sequelogous solved structure (97.08% sequence identity) to SARS-CoV-2 RdRp [15]. In the presented work, we carried out molecular docking studies on the recently released crystal structure of SARS-CoV-2 (PDB Id:7BTF) [16] utilizing the drug repurposing approach.

Preparation of Protein Structure
The 3D crystal structure of SARS CoV-2 RdRp (PDB ID: 7BTF) was obtained from RCSB-PDB (http:/www.rcsb. org/pdb) pdb format with the resolution of 2.95 Å [23]. Binds to viral RNA polymerase and causes structural changes in it leading to premature termination of RNA strand

Hepatitis C, Ebola virus infection
The protein cavity was created by using Mo-lecular Operating Environment (MOE) software (License provided by Chemical Computing Group) by following a series of steps involving the addition of hydrogens/polar hydrogens, deletion of waters and ligands, site finder, isolation of atoms and extending nearby up to 4.5 Å. The prepared protein cavity has been depicted in Fig. (1).

Preparation of Ligands
The 2D structures of all the four drugs were drawn in Chem Biodraw 15.0 and were saved as "mol" files. Further, these were energy minimized by selecting force field MMFF94x, Austin model 1 (AM 1) with a gradient value of 0.0001 kcal/mol. The energy minimized structures were saved as mdb.mol files.

Docking and Validation Protocol
Docking of prepared ligands was carried out on RdRp cavity using MOE 2019.0102 software. The process involved the selection of docking site, ligand site, selection of mdb file and Run. The validation of the docking protocol was carried out by redocking of a ligand over internal ligand and RMSD value was calculated. The 2D and 3D binding poses of ligands with the receptor were saved as JPEG files for further analysis.

RESULTS AND DISCUSSION
Molecular docking was performed to study the inhibitory potential of remdesivir, ribavirin, sofosbuvir and galidesivir on SARS-CoV-2 RNA dependent RNA polymerase enzyme using MOE software. The various interaction points of these drugs with the target protein, along with the corresponding distances have been summarized in Table 2. All four drugs revealed excellent interactions at very short distances. The main amino acid residues involved in binding are Asp623, Cys622, Asn691, Ser681, Arg555 & 553, Thr556, Asp452, Arg623 & 624.
Remdesivir revealed three hydrogen bonding interactions at very short distances with a docking score -8.99. Fig. (2) represents the 2D interactions, interaction distances and the 3D embedding of remdesivir within the receptor pocket.
Ribavirin also revealed three hydrogen bonding interactions at very short distances with a docking score -8.82. Fig.  (3) represents the 2D interactions, interaction distances and the 3D embedding of ribavirin within the receptor pocket.
Sofosbuvir interacted through five hydrogen bonds at distances less than 2.2Å with a docking score -8.67. Fig. (4) represents the 2D interactions, interaction distances and the 3D embedding of sofosbuvir within the receptor pocket.
Galidesivir interacted through four hydrogen bonds at distances less than 2.5Å with a docking score of -8.91. It also revealed one arene-cation interaction between its pyrimidine ring and Arg555 residue. Fig. (5) represents the 2D interactions, interaction distances and the 3D embedding of sofosbuvir within the receptor pocket.
The docking protocol was validated by re-docking of ligands and RMSD values were found to be less than 2. An overlay of re-docked ligands has been depicted in Fig. (6).

CONCLUSION
The current in silico study on the previously approved four drugs was carried out to predict binding pattern to SARS-CoV-2 RdRp. Docking studies of these drugs have been reported against SARS-HCoV RdRp, but not reported for SARS-CoV RdRp. Docking scores and hydrogen bond interactions were predicted through molecular docking against PDB ID: 7BTF. All the four drugs revealed almost comparable docking scores and excellent interactions with the amino acid residues inside the receptor pocket. The drugs displayed, mainly, hydrogen bonding interactions and arenecation interactions. The primary aim of this study was to predict the therapeutic potentials of previously approved drugs against the corona virus (SARS-CoV-19) through drug repurposing utilizing molecular docking tools. It is anticipated that in the future research, in vitro and in vivo testing is desirable to experimentally validate the SARS-CoV-2 RdRp inhibitory activity of these drugs.

ETHICS APPROVAL AND CONSENT TO PARTICI-PATE
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HUMAN AND ANIMAL RIGHTS
No Animals/Humans were used for studies that are base of this research.

CONSENT FOR PUBLICATION
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AVAILABILITY OF DATA AND MATERIALS
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FUNDING
None.