Ullah Atta, Waqas Muhammad, Duran Shahid, Rehman Najeeb Ur, Rahman Sadeeq Ur, Abdellattif Magda H, Jan Afnan, Zubair Mohammad, Ahmed Mesaik M, Khan Ajmal, Halim Sobia Ahsan, Al-Harrasi Ahmed
Natural and Medical Sciences Research Center, University of Nizwa, Birkat-ul-Mouz 616, Nizwa, Sultanate of Oman.
Natural and Medical Sciences Research Center, University of Nizwa, Birkat-ul-Mouz 616, Nizwa, Sultanate of Oman; Department of Biotechnology and Genetic Engineering, Hazara University Mansehra, Mansehra, 2100, Pakistan.
Comput Biol Med. 2025 Sep;196(Pt C):110801. doi: 10.1016/j.compbiomed.2025.110801. Epub 2025 Aug 6.
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 IC 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.
丙型肝炎病毒的NS3蛋白因其在丙型肝炎病毒（HCV）复制和免疫逃逸中的显著作用，成为治疗研究中一个有吸引力的靶点。我们采用了一种全面的跨学科策略，结合基于结构的药物设计方法与体外和分子研究，来发现NS3蛋白的新型抑制剂，这些抑制剂针对所有主要的HCV基因型（1a - 6a）和突变株。最初，通过基于对接的筛选，从我们内部约950种化合物的数据库中鉴定出14种潜在的结合物。在这14种中，4种抑制剂（C2、C4、C5和C9）表现出显著的药代动力学和类药性质，具有良好的胃肠道吸收、无毒性且无过敏反应。此外，这四种抑制剂对耐药NS3突变体（R155K、A156T和Y56H/D168A）的比较对接显示，这些分子与改变的活性位点之间有强烈的相互作用，表明它们对突变株具有结合潜力。分子动力学模拟验证了抑制剂结合对蛋白质构象动力学的显著影响。基于MM - PBSA的结合自由能估计显示，C2、C9、C4和C5对NS3具有很强的结合亲和力（分别为 - 23.313 ± 0.10、 - 21.106 ± 0.10、 - 15.922 ± 0.13和 - 13.516 ± 0.11 kcal/mol）。评估了它们对HepG2细胞系中HCV NS3基因和蛋白表达的抑制作用。实时聚合酶链反应（RT - PCR）和蛋白质印迹法证实了NS3基因和蛋白的稳定整合与表达，而免疫荧光验证了它们的亚细胞定位。这些抑制剂以3.51 ± 0.89、4.37 ± 0.5、6.67 ± 0.4和5.59 ± 1.4 μM的IC有效抑制了HCV复制。化合物C2表现出最有效的抗病毒特性，对NS3基因和蛋白表达有显著抑制作用。这些化合物是未来治疗的有希望的候选物，在六种主要的HCV基因型和突变株中均有效。我们抑制剂的广泛适用性得到了所有六种基因型和突变类型催化结构域保守性质的支持。需要进一步研究来确认它们的治疗潜力。
