In Silico Structure-Based Identification and Analysis of Viral Protease Inhibitors Targeting Chikungunya Virus Replication

Chikungunya virus (CHIKV) is a small spherical shaped mosquito-borne alphavirus. CHIKV infection is a major public health concern in many tropical and subtropical regions of the biosphere. It has single-stranded positive-sense RNA genome (11,811 nucleotides) that organises as 5′UTR-nsP1-nsP2-nsP3-nsP4-J-C-E3-E2-6K-E1-polyA-3′UTR. The non-structural precursor protein is proteolytically cleaved by non-structural protein 2 (nsp2) and this makes it a potential antiviral drug target. In the viral replication, nsP2 display three key roles as helicase, triphosphatase and protease. The C-terminal half of the nsP2 protein consists of two structural domains: a protease domain and a methyltransferase-like domain. These domains function together and are crucial for protease activity. Till date, there is no commercially available drug against CHIKV and launching a new drug into the market is a complicated and time-consuming process. Therefore, drug repurposing is a new but familiar approach to reduce the time and cost of drug discovery. Recently, crystal structure of CHIKV nsP2 protease domain (PDB ID: 4ZTB) was determined by our group. The mechanism of nsP2 protease function is related to deprotonation of the thiol group of cysteine, the catalytic dyad residue at the active site (Cys478 and His548). In this study, nsP2 protease active site has been targeted by virtual screening of compound libraries and molecular docking of potential inhibitory molecules. Based on the binding energies, hit molecules on the top were selected from Pharmacologically Active Compounds (LOPAC1280) library. Inhibitory molecules are being further analyzed by MD simulations to find the stable binding poses. Based on the analysis of docking and MD simulation compounds hve been selected, the protease inhibitory activity and antiviral efficacy have been tested for two of these molecules against CHIKV using cell-based in vitro assays. Based on kinetic studies, top compounds inhibited CHIKV nsP2 protease with inhibition constant (Ki) values of 36.5 M and 86.5 M. Interestingly, these two compounds inhibited CHIKV replication in Vero cells at concentrations much lower than their cytotoxic concentrations as validated by in vitro antiviral plaque assay experiments. Thus, structure-based approach to identify inhibitors is a very effective approach to identify and develop drug molecules against viral pathogens.