Section of Oncology and Clinical Research, Leeds Institute of Molecular Medicine, St. James's University Hospital, Leeds, United Kingdom.
Hepatology. 2011 Jul;54(1):79-90. doi: 10.1002/hep.24371.
The hepatitis C virus (HCV) p7 ion channel plays a critical role during infectious virus production and represents an important new therapeutic target. Its activity is blocked by structurally distinct classes of small molecules, with sensitivity varying between isolate p7 sequences. Although this is indicative of specific protein-drug interactions, a lack of high-resolution structural information has precluded the identification of inhibitor binding sites, and their modes of action remain undefined. Furthermore, a lack of clinical efficacy for existing p7 inhibitors has cast doubt over their specific antiviral effects. We identified specific resistance mutations that define the mode of action for two classes of p7 inhibitor: adamantanes and alkylated imino sugars (IS). Adamantane resistance was mediated by an L20F mutation, which has been documented in clinical trials. Molecular modeling revealed that L20 resided within a membrane-exposed binding pocket, where drug binding prevented low pH-mediated channel opening. The peripheral binding pocket was further validated by a panel of adamantane derivatives as well as a bespoke molecule designed to bind the region with high affinity. By contrast, an F25A polymorphism found in genotype 3a HCV conferred IS resistance and confirmed that these compounds intercalate between p7 protomers, preventing channel oligomerization. Neither resistance mutation significantly reduced viral fitness in culture, consistent with a low genetic barrier to resistance occurring in vivo. Furthermore, no cross-resistance was observed for the mutant phenotypes, and the two inhibitor classes showed additive effects against wild-type HCV.
These observations support the notion that p7 inhibitor combinations could be a useful addition to future HCV-specific therapies.
丙型肝炎病毒 (HCV) p7 离子通道在感染性病毒产生过程中发挥着关键作用，代表着一个新的重要治疗靶点。其活性被结构不同的小分子类物质所阻断，对不同分离株 p7 序列的敏感性有所不同。虽然这表明了特定的蛋白质-药物相互作用，但缺乏高分辨率的结构信息妨碍了抑制剂结合部位的确定，其作用方式仍不清楚。此外，现有 p7 抑制剂的临床疗效不佳，这使得人们对其特定的抗病毒作用产生了怀疑。我们确定了两种 p7 抑制剂（金刚烷胺和烷基化脒基糖 (IS)）的作用模式所特有的耐药突变。金刚烷胺耐药是由 L20F 突变介导的，该突变已在临床试验中得到证实。分子建模表明，L20 位于膜暴露的结合口袋内，药物结合可防止低 pH 介导的通道开放。该外周结合口袋进一步通过一组金刚烷衍生物以及专门设计的与该区域具有高亲和力的分子进行了验证。相比之下，在基因型 3a HCV 中发现的 F25A 多态性赋予了 IS 耐药性，并证实这些化合物在 p7 三聚体之间插入，从而阻止了通道寡聚化。两种耐药突变都没有显著降低病毒在培养物中的适应性，这与体内发生的低遗传耐药屏障一致。此外，突变表型之间没有观察到交叉耐药性，两种抑制剂类对野生型 HCV 均表现出相加作用。
这些观察结果支持了这样一种观点，即 p7 抑制剂联合使用可能是未来 HCV 特异性治疗的有用补充。
