Asymmetric Binding to NS5A by Daclatasvir (BMS-790052) and Analogs Suggests Two Novel Modes of HCV Inhibition

Symmetric, dimeric daclatasvir (BMS-790052) is the clinical lead for a class of picomolar inhibitors of HCV replication. While specific, resistance-bearing mutations at positions 31 and 93 of domain I strongly suggest the viral NS5A as target, structural mechanism(s) for the drugs’ activities and resistance remains unclear. Several previous models suggested symmetric binding modes relative to the homodimeric target; however, none can fully explain SAR details for this class. We present semiautomated workflows to model potential receptor conformations for docking. Surprisingly, ranking docked hits with our library-derived 3D-pharmacophore revealed two distinct asymmetric binding modes, at a conserved poly-proline region between 31 and 93, consistent with SAR. Interfering with protein–protein interactions at this membrane interface can explain potent inhibition of replication–complex formation, resistance, effects on lipid droplet distribution, and virion release. These detailed interaction models and proposed mechanisms of action will allow structure-based design of new NS5A directed compounds with higher barriers to HCV resistance.对称二聚体形式的达卡他韦（Daclatasvir, BMS-790052）是一类皮摩尔级丙型肝炎病毒（Hepatitis C Virus, HCV）复制抑制剂的临床先导化合物。尽管结构域I的31位与93位的耐药突变位点可明确指向病毒非结构蛋白5A（Non-structural protein 5A, NS5A）为该类药物的作用靶点，但此类药物的活性与耐药性的结构机制仍未阐明。此前已有多项模型提出了针对同二聚体靶点的对称结合模式，但均无法完全解释该类化合物的构效关系（Structure-Activity Relationship, SAR）细节。我们开发了半自动化工作流，用于建模分子对接所需的潜在受体构象。令人意外的是，通过基于化合物库构建的三维药效团对对接命中化合物进行排序后，我们发现了两种截然不同的不对称结合模式，该结合模式位于31位与93位之间的保守聚脯氨酸区域，与该类化合物的构效关系一致。在该膜界面处干扰蛋白质相互作用，可解释该类药物对复制复合物形成的强效抑制作用、耐药性、对脂滴（Lipid droplet）分布的影响以及病毒粒子（Virion）释放过程。这些详细的相互作用模型与提出的作用机制，将助力基于结构设计出具有更高HCV耐药屏障的新型NS5A靶向抑制剂。