Identifying potent natural inhibitors of the hepatitis C virus NS3 protein using multiple computational and molecular...

The NS3 protein of hepatitis C virus is an appealing target for therapeutic research because of its notable role in hepatitis C virus (HCV) replication and immune evasion. We employed a comprehensive interdisciplinary strategy to discover new inhibitors of the NS3 protein, targeting all major HCV genotypes (1a-6a) and mutant strains, using a combination of a structural-based drug design approach with in vitro and molecular studies. Initially, 14 potential binders were identified from our in-house database of approximately 950 compounds via docking-based screening. Among 14, 4 inhibitors (C2, C4, C5, and C9) displayed remarkable pharmacokinetics and drug-like properties with good gastrointestinal absorption, no toxicity, and no allergy. Furthermore, comparative docking of these four inhibitors on drug-resistant NS3 mutants (R155K, A156T, and Y56H/D168A) revealed strong interactions between these molecules and the altered active sites, indicating their binding potential for mutant strains. Molecular dynamics simulations validated the remarkable impact of inhibitors binding on protein conformational dynamics. The MM-PBSA-based binding free energy estimation showed strong binding affinities of C2, C9, C4, and C5 (-23.313 ± 0.10, -21.106 ± 0.10, -15.922 ± 0.13, and -13.516 ± 0.11 kcal/mol, respectively) for NS3. Their inhibitory effects on the HCV NS3 gene and protein expression in HepG2 cell lines were evaluated. Real-time polymerase chain reaction (RT-PCR) and western blotting confirmed the stable integration and expression of the NS3 gene and protein, while immunofluorescence verified their subcellular location. These inhibitors effectively inhibited HCV replication with IC₅₀ of 3.51 ± 0.89, 4.37 ± 0.5, 6.67 ± 0.4, and 5.59 ± 1.4 μM. Compound C2 demonstrated the most potent antiviral properties with considerable inhibition of NS3 gene and protein expression. These compounds are promising candidates for future therapeutics, yielding their effectiveness across six major HCV genotypes and mutant strains. The broad applicability of our inhibitors is supported by the conserved nature of the catalytic domain across all six genotypes and mutant types. Further studies are needed to confirm their therapeutic potential.