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，干扰素/利巴韦林治疗有严重副作用的显著风险。因此，开发替代疗法至关重要。免疫抑制药物环孢素A （CsA）被报道在临床上对HCV感染有效。此外，最近的研究证明，非免疫抑制性CsA类似物，如NIM811、Debio 025和SCY-635，在体外和体内均以不依赖ifn的方式抑制HCV RNA复制。越来越多的证据表明，亲环蛋白（Cyps）对HCV生命周期很重要，表明Cyp抑制剂（CsA, Debio 025， NIM811或SCY- 635）通过作用于细胞内的Cyps，阻断HCV复制。最近的三项独立研究表明，HCV高度依赖亲环蛋白A （CypA）在人肝细胞中复制。我们发现CypA，而不是CypB， CypC和CypD，对HCV复制至关重要。我们证明了CypA的疏水性口袋，在那里CypA抑制剂结合，并控制CypA的异构酶活性，是HCV复制的关键。我们获得了几条线的证据，表明CypA分子群存在于蛋白酶抗性膜室中，类似于HCV复制的膜室。我们还发现CypA与这个隔室的关联并不依赖于HCV蛋白的存在。重要的是，我们发现CypA抑制剂会耗尽这一膜区室的CypA。从发生HCV复制的受保护内质网小球中CypA的耗尽，可能为CypA抑制剂的抗病毒作用机制提供了第一个线索。然而，在HCV复制过程中对CypA的分子需求，以及这类新型强效抗HCV药物（Cyp抑制剂）的抗病毒作用机制，都是完全不清楚的。通过体外筛选抗Cyp抑制剂的HCV突变体，研究将突变定位为两种非结构（NS）蛋白——NS5A和NS5B。综上所述，这些数据表明CypA、NS5A和NS5B之间存在一种对HCV复制至关重要的关系。为了验证这一假设，我们建议在本申请中进行一组旨在在分子水平上充分表征CypA-NS5A-NS5B相互作用的实验；分析丙型肝炎病毒对Cyp抑制剂耐药性的发展；研究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。这项应用的最终目的是提高我们对CypA在HCV生命周期中的作用的理解，以及这种新型有效的抗HCV药物- Cyp抑制剂介导的体外和体内阻断。

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. PUBLIC HEALTH RELEVANCE: An estimated 170 million people worldwide are chronically infected with Hepatitis C virus. 10-20% of these will develop cirrhosis and 1-5% will develop hepatocellular carcinoma. The only currently approved therapy is weekly injection with pegylated interferon and daily oral ribavirin for 6-12 months. The therapy is associated with severe side effects and results in sustained viral clearance in only 50% of all patients. There is therefore a high need to develop new therapies with better efficacy and tolerability. By demonstrating that cyclophilin (Cyp) inhibitors have potent efficacy against HCV, our recent clinical study opens a new line of opportunity to eradicate this prime human threat. The current proposal outlines a comprehensive series of experiments aimed at determining the mechanism of action of Cyp inhibitors that should lead to the identification of new targets for inhibition of HCV. The study of the roles of Cyp - the intracellular targets of the Cyp inhibitors - in HCV replication is novel and should lead to the identification of new host and viral targets for the development of innovative anti-HCV therapies.