Integrated computational approaches for identifying and evaluating phytochemical inhibitors against HCV NS3/4A...

Hepatitis C virus (HCV) remains a major global health concern, causing chronic hepatitis, cirrhosis, and hepatocellular carcinoma (HCC), with an estimated 185 million people affected worldwide. In the absence of an FDA-approved vaccine, the development of effective antiviral strategies remains essential. The HCV NS3-4A protease, which mediates cleavage of the viral polyprotein at specific downstream sites of NS3 and is essential for replication-complex formation, represents a key therapeutic target. In this study, approximately 1,480 phytocompounds were compiled from the IMPPAT database and previously reported in vitro studies and subjected to drug-likeness, toxicity, and biological activity screening using pkCSM, SwissADME, and PASS. Twenty compounds were subsequently prioritized and docked against WT, R155K, and D168Y NS3/4A protease structures. Among these, Ladanein (LAD) and Diosmetin (DMT) exhibited favourable binding across all protein variants. MD simulations indicated stable protein-ligand interactions, while MM/GBSA calculations revealed more favourable binding free energies for LAD than DMT across the WT and mutant complexes. Residue-wise energy decomposition identified more energetically favourable hotspot residues in LAD-bound complexes, whereas DMT exhibited higher overall hydrogen-bond occupancy. PCA and free-energy landscape analyses further demonstrated ligand-dependent alterations in collective conformational states induced by the R155K and D168Y substitutions. Overall, these findings computationally prioritize LAD as a promising candidate against WT and mutant HCV NS3/4A protease, warranting further in vitro and biochemical validation.