Cyclophilins, Cyclophilin Inhibitors and Hepatitis C

描述（由申请人提供）：目前慢性HCV感染患者的治疗包括高剂量IFN联合利巴韦林。然而，成功率仍然很低（约50%的治疗患者取决于基因型）。此外，IFN/利巴韦林治疗具有严重副作用的显著风险。因此，开发替代疗法至关重要。免疫抑制剂环孢素A（CsA）被报道在临床上有效地对抗HCV感染。此外，最近的研究提供了非免疫抑制性CsA类似物如NIM 811、Debio 025和SCY-635在体外和体内以不依赖IFN的方式抑制HCV RNA复制的证据。越来越多的证据表明，亲环蛋白（Cyps）对HCV的生命周期很重要，这表明Cyp抑制剂（CsA，Debio 025，NIM 811或SCY- 635）通过作用于细胞内Cyps，阻断HCV的复制。最近三项独立的研究表明，HCV高度依赖亲环素A（CypA）在人类肝细胞中复制。我们发现CypA，而不是CypB，CypC和CypD，是HCV复制的关键。我们证明了CypA的疏水口袋，其中Cyp抑制剂结合，并控制CypA的异构酶活性，是HCV复制的关键。我们获得了几条证据，表明CypA分子群体存在于蛋白酶抗性膜隔室中，类似于HCV复制的隔室。我们还发现CypA与该隔室的关联不依赖于HCV蛋白的存在。重要的是，我们发现Cyp抑制剂耗尽了CypA的这种膜室。CypA从HCV复制发生的受保护的ER小球中的耗尽可能为Cyp抑制剂的抗病毒作用机制提供了第一个提示。然而，CypA在HCV复制中的分子要求以及这类新型强效抗HCV药物-Cyp抑制剂-的抗病毒作用机制完全不清楚。通过体外筛选Cyp受体耐药的HCV突变体，研究将突变定位到两个非结构（NS）蛋白-NS 5A和NS 5 B。总之，这些数据表明CypA、NS 5A和NS 5 B之间存在关系，这对HCV复制至关重要。为了验证这一假设，我们在本申请中提出进行一组实验，旨在在分子水平上充分表征CypA-NS 5A-NS 5 B相互作用;分析HCV对Cyp抑制剂耐药性的发展;并研究Cyp抑制剂对HCV复制复合物的组成和聚合酶活性的影响。本申请的最终目标是提高我们对CypA在HCV生命周期中的作用以及由这类新型强效抗HCV药物-Cyp抑制剂介导的体外和体内阻断的理解。

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.