The emergence of drug-resistance-inducing mutations in Hepatitis C virus (HCV) coupled with genotypic heterogeneity has made targeting NS3/4A serine protease difficult. In this work, we investigated the mutagenic variations in the binding pocket of Genotype 3 (G3) HCV NS3/4A and evaluated ligands for efficacious inhibition. We report mutations at 14 positions within the ligand-binding residues of HCV NS3/4A, including H57R and S139P within the catalytic triad. We then modelled each mutational variant for pharmacophore-based virtual screening (PBVS) followed by covalent docking towards identifying a potential covalent inhibitor, i.e., cpd-217. The binding stability of cpd-217 was then supported by molecular dynamic simulation followed by MM/GBSA binding free energy calculation. The free energy decomposition analysis indicated that the resistant mutants alter the HCV NS3/4A-ligand interaction, resulting in unbalanced energy distribution within the binding site, leading to drug resistance. Cpd-217 was identified as interacting with all NS3/4A G3 variants with significant covalent docking scores. In conclusion, cpd-217 emerges as a potential inhibitor of HCV NS3/4A G3 variants that warrants further in vitro and in vivo studies. This study provides a theoretical foundation for drug design and development targeting HCV G3 NS3/4A.
Keywords: Hepatitis C virus; MD simulations; covalent inhibitor; drug resistance; pharmacophore-based virtual screening.