Cyclophilins, Cyclophilin Inhibitors and Hepatitis C

描述(申请人提供)：目前治疗慢性丙型肝炎病毒感染的方法包括大剂量干扰素联合利巴韦林。然而，成功率仍然很低(约50%的接受治疗的患者取决于基因)。此外，干扰素/利巴韦林治疗存在严重副作用的重大风险。因此，开发替代疗法是至关重要的。据报道，免疫抑制药物环孢素A(CsA)在临床上对丙型肝炎病毒感染有效。此外，最近的研究提供了证据表明，非免疫抑制CsA类似物，如NIM811，Debio 025和SCY-635，在体外和体内都以非干扰素依赖的方式抑制丙型肝炎病毒的RNA复制。越来越多的证据表明，亲环素(Cyps)对丙型肝炎病毒的生命周期很重要，这表明Cyp抑制剂(CsA、Debio 025、NIM811或SCY-635)通过作用于细胞内的CyP，阻止丙型肝炎病毒的复制。最近的三项独立研究表明，丙型肝炎病毒高度依赖亲环素A(CypA)在人肝细胞中复制。我们发现CypA，而不是CypB，CypC和CypD对丙型肝炎病毒的复制是关键的。我们证明了CypA的疏水口袋是丙型肝炎病毒复制的关键，它与Cyp抑制剂结合，并控制CypA的异构酶活性。我们获得了几条证据，证明一群CypA分子居住在一个与丙型肝炎病毒复制相似的抗蛋白酶的膜室中。我们还发现，CypA与这个间隔的联系并不依赖于丙型肝炎病毒蛋白的存在。重要的是，我们证明了Cyp抑制剂耗尽了CypA的这个膜室。在发生丙型肝炎病毒复制的地方，受保护的内质网球体中CypA的缺失，可能为Cyp抑制剂的抗病毒作用机制提供第一个线索。然而，丙型肝炎病毒复制中对CypA的分子需求以及这类新型有效的抗丙型肝炎病毒药物--CyP抑制剂--的抗病毒作用机制完全不清楚。通过在体外选择抗Cyp抑制剂的丙型肝炎病毒突变体，研究将突变定位为两种非结构(NS)蛋白-NS5A和NS5B。总之，这些数据表明，CypA、NS5A和NS5B之间存在着对丙型肝炎病毒复制至关重要的关系。为了验证这一假设，我们建议在这一应用中进行一系列实验，目的是在分子水平上充分表征CypA-NS5A-NS5B的相互作用；分析丙型肝炎病毒对Cyp抑制剂的耐药性的发展；以及调查Cyp抑制剂对丙型肝炎病毒复制复合体的组成和聚合酶活性的影响。这一应用的最终目的是提高我们对CypA在丙型肝炎病毒生命周期中的作用的理解，以及对这类新型高效抗丙型肝炎病毒药物--Cyp抑制剂介导的体外和体内阻断的了解。

DESCRIPTION (provided by applicant): Current therapy for patients with chronic HCV infection includes high doses of IFN in combination with ribavirin. However, success rates remain low (around 50% of treated patients depending on genotype). Furthermore, the IFN/ribavirin treatment carries a significant risk of serious side effects. Thus, the development of alternative treatments is essential. The immunosuppressive drug cyclosporine A (CsA) was reported to be clinically effective against HCV infection. Moreover, recent studies provided evidence that non- immunosuppressive CsA analogs such as NIM811, Debio 025 and SCY-635 inhibit HCV RNA replication both in vitro and in vivo in an IFN-independent manner. A growing body of evidence suggests that cyclophilins (Cyps) are important for the HCV life cycle, suggesting that Cyp inhibitors (CsA, Debio 025, NIM811 or SCY- 635), by acting on intracellular Cyps, block HCV replication. Three independent studies recently demonstrated that HCV highly relies on cyclophilin A (CypA) to replicate in human hepatocytes. We showed that CypA, but not CypB, CypC and CypD, is critical for HCV replication. We demonstrated that the hydrophobic pocket of CypA, where Cyp inhibitors bind, and which control the isomerase activity of CypA, is critical for HCV replication. We obtained several lines of evidence that a population of CypA molecules resides in a protease- resistant membrane compartment similar to that where HCV replicates. We also found that the association of CypA with this compartment does not depend on the presence of HCV proteins. Importantly, we showed that Cyp inhibitors deplete this membrane compartment of CypA. The depletion of CypA from the protected ER spherules where HCV replication occurs, may provide the first hint for the mechanism of antiviral action of Cyp inhibitors. However, the molecular requirements for CypA in HCV replication as well as the antiviral mechanisms of action of this novel class of potent anti-HCV agents - the Cyp inhibitors - are completely obscure. By selecting Cyp inhibitor-resistant HCV mutant's in vitro, studies mapped mutations into two nonstructural (NS) proteins - NS5A and NS5B. Altogether these data suggest the existence of a relationship between CypA, NS5A and NS5B that is critical for HCV replication. To test this hypothesis, we propose in this application to conduct a set of experiments aimed at fully characterizing CypA-NS5A-NS5B interactions at a molecular level; analyzing the development of HCV resistance to Cyp inhibitors; and investigating the effect of Cyp inhibitors on the composition and polymerase activities of HCV replication complexes. The ultimate goal of this application is to improve our understanding of the role of CypA in the HCV life cycle and of the in vitro and in vivo block mediated by this novel class of potent anti-HCV agents - the Cyp inhibitors.