Ivermectin as a promising RNA-dependent RNA polymerase inhibitor and a therapeutic drug against SARS

Abstract

Purpose: COVID-19, caused by SARS-CoV2 virus is a contagious disease affecting millions of lives throughout the globe. Currently, there are no clinically approved drugs for SARS-CoV2 although some drugs are undergoing clinical trials. The present study investigates the binding property of ivermectin on four important drug targets, spike protein, RNA-dependent RNA polymerase, 3-chymotrypsin- and papain-like proteases of SARS-CoV2. Methods: The 3D structure of ivermectin along with known antiviral drug lopinavir, simeprevir and four nucleotides ATP, GTP, CTP, and UTP were downloaded from PubChem database. Crystal structures of proteins were downloaded from PDB database. PDB files were converted into pdbqt file using AutoDock tools. After proper processing and grid formation, docking was carried out in AutoDock vina. Furthermore, the co-crystallized RNA and its binding interactions with RdRp were studied using various visualization tools including Discovery studio. Results: Docking study showed that ivermectin is the best binding drug compared to lopinavir and simeprevir. The best binding interaction was found to be -9.7kcal/mol with RdRp suggesting potential inhibitor of the protein. Twenty-one amino acid residues of RdRp were found to interact with ivermectin including the catalytic residue Asp760. Furthermore, RNA-RdRp complex revealed that the catalytic active residues Ser759 and Asp760 of RdRp formed strong interactions with RNA chain. Binding of ivermectin in the active site of RdRp make clash with the nucleotides of RNA chain suggesting the possible inhibition of replication. Conclusions: The present study suggests ivermectin as a potential inhibitor of RdRp which may be crucial to combat the SARS-CoV2.


Ivermectin as a promising RNA-dependent RNA polymerase inhibitor and a therapeutic drug against SARS

Ligand Selection and Preparation

The 3D structure of ivermectin (Drugbank ID: DB00602) along with two antiviral drugs simeprevir (DB06290), and lopinavir (DB01601) from Drugbank database (https://www.drugbank.ca/). Four nucleotides ATP (PubChem CID: 5957), GTP (CID: 135398633), CTP (CID: 6176), and UTP (CID: 6133) were downoloaded from PubChem database. SDF files were converted into pdb file using PyMol software. All the PDB files of the ligands were processed and finally converted into .pdbqt file using AutoDock tool (Trott and Olson 2010).

Collection and Preparation of Proteins

Three-dimensional structures of S-protein (RBD) (PDB ID: 7BZ5), 3Clpro (PDB ID: 6M2N), Plpro (PDB ID: 7JN2), and RdRp (PDB ID: 6XQB) were downloaded from PDB database. The protein structures were cleaned by removing the water and other hetatms. Polar hydrogen atoms and Kollman charges were added to the structure and finally converted into .pdbqt file format for docking using AutoDock Tools.

Molecular Docking

After the ligand drugs and the target enzymes were prepared docking was carried out in AutoDock Vina (Trott and Olson 2010). The grid box parameters for docking of all the four proteins are presented in table 1. The docking algorithm was carried out by keeping the default exhaustiveness at 8. After docking, the pose scoring the lowest binding energy (kcal/mol) was selected and visualize in Discovery Studio.

COVID-19 pandemic has tremendously affected the health and wealth of global community. Speedy trial and approval of new drug is the only strategy to combat SARS-CoV2 along with social distancing. The present study revealed the structural inhibition of RdRp protein by ivermectin drug and therefore ivermectin may be a potential drug to combat COVID-19 and SARS-CoV2.

Discussion

SARS-CoV2 is a positive-sense single-stranded RNA virus. The entry of viral genome into the host body and its generation and invasion to the other cells is mediated by several proteins including spike proteins, 3chymotrypsin- and papain-like proteases. A polyprotein complex, RNA-dependent RNA-polymerase (RdRp) is a major protein in SARS-CoV2 that regulates viral replication (Yin et al. 2020). Inhibition of replication may be an important strategy to combat SARS-CoV2 and COVID-19. RdRp protein, therefore, is an important and potential therapeutic target of antiviral drugs. Compounds that bind effectively with the active site of the RdRp and inhibit the enzyme’s catalytic activity shall be an important aspect of controlling virus replication and generation. Several molecules and antiviral drugs have been screened by many researchers to find suitable molecule with high efficacy (Elfiky 2020; Babadaei et al. 2020; Touret et al. 2020). Ivermectin, a broad-spectrum antihelmintic drug has been recently established to be a potential inhibitor of SARS-CoV2 RdRp protein in in vitro study (Caly et al. 2020). It has also been opined that hydroxychloroquine and ivermectin may show effective result if administered simultaneously (Patri and Fabbrocini, 2020). Ivermectin is also known to inhibit DNA polymerase of pseudorabies virus (Lv et al. 2018). The present study revealed strong binding affinity of ivermectin to the RdRp protein. In association to surrounding key residues, Ser759, Aspertate760 and -761 forms the core catalytic site amino acid residues of RdRp in SARS-CoV2 (Yin et al. 2020). The present study observed that Ser759 form strong interaction with ivermectin and adenine-9 nucleotide as well. The study, however, did not find any interaction with catalytically active Asp760 and -761 residues of RdRp. Ornipressin, an FDA approved drug for vasoconstriction and liver cirrhosis, is reported to have strong interactions with eight amino acid residues of RdRp including the catalytic site residue Asp760 (Ahmad et al. 2020). In an in silico study, Yin et al. (2020) reported that remdesivir, a nucleoside analog used to inhibit the action of RNA polymerase in Ebola virus make phosphodiester bond with the 3’-OH group of the newly synthesized RNA strand as well as strong interaction with the Asp760 residue of SARS-CoV2 leading to the inhibition of replication. Ivermectin is not a nucleotide analog and therefore did not bind with the 3’OH- end of the leading strand. However, the study showed unfavourable interactions with the parental (template) RNA strand up to four nucleotides upstream of the replicating site. Binding of ivermectin to the active pocket of RdRp may prevent the catalytically favourable accommodation of the template RNA chain in the enzyme and hence inhibition of replication. The findings of the present study along with in vitro validation by earlier studies suggest the potentiality of ivermectin to be an effective inhibitor of SARS-CoV2 RNA-dependent RNA-polymerase.


Conclusion

COVID-19 pandemic has tremendously affected the health and wealth of global community. Speedy trial and approval of new drug is the only strategy to combat SARS-CoV2 along with social distancing. The present study revealed the structural inhibition of RdRp protein by ivermectin drug and therefore ivermectin may be a potential drug to combat COVID-19 and SARS-CoV2.


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