Cyclophilins, Cyclophilin Inhibitors and Hepatitis C

亲环蛋白、亲环蛋白抑制剂和丙型肝炎

• 批准号: 8011692
• 负责人: PHILIPPE ANDRE GALLAY
• 金额: $ 47万
• 依托单位: SCRIPPS RESEARCH INSTITUTE, THE
• 依托单位国家: 美国
• 财政年份: 2010
• 资助国家: 美国
• 起止时间: 2010-01-15 至 2014-12-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8011692
• 关键词: Adverse effects; Antiviral Agents; Binding; Biological Assay; Cells; Chronic Hepatitis C; Cirrhosis; Clinical Research; Complex; Cyclophilin A; Cyclophilins; Cyclosporine; DNA-Directed RNA Polymerase; Data; Dependence; Development; Dose; Drug Industry; Genotype; Goals; Hepatitis C; Hepatitis C virus; Hepatocyte; Human; Immunosuppressive Agents; In Vitro; Injection of therapeutic agent; Interferons; Isomerase; Lead; Life Cycle Stages; Link; Maps; Mediating; Membrane; Molecular; Mutate; Mutation; Nonstructural Protein; Oral; Patients; Peptide Hydrolases; Pharmaceutical Preparations; Phosphorylation; Polymerase; Population; Primary carcinoma of the liver cells; Proteins; RNA replication; RNA-Directed RNA Polymerase; Recombinant Proteins; Replicon; Reporting; Research; Resistance; Ribavirin; Risk; Role; Series; Source; Specificity; System; Testing; Variant; Viral; Viral Proteins; Virus; Work; alternative treatment; analog; base; domain mapping; improved; in vitro Assay; in vivo; inhibitor/antagonist; innovation; mutant; novel; public health relevance; research study; resistance mechanism; success; viral RNA; yeast two hybrid system

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.

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，开发替代治疗方法至关重要。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。 公共卫生相关性：据估计，全世界有1.7亿人慢性感染丙型肝炎病毒。其中10-20%将发展为肝硬化，1-5%将发展为肝细胞癌。目前唯一批准的治疗是每周注射聚乙二醇干扰素和每日口服利巴韦林6-12个月。该疗法与严重的副作用有关，并且仅在所有患者中的50%中导致持续的病毒清除。因此，迫切需要开发具有更好疗效和耐受性的新疗法。通过证明亲环素（Cyp）抑制剂对HCV具有有效的疗效，我们最近的临床研究为根除这种主要的人类威胁开辟了一条新的途径。目前的提案概述了一系列旨在确定Cyp抑制剂作用机制的综合实验，这些实验应导致识别抑制HCV的新靶点。Cyp -Cyp抑制剂的细胞内靶点-在HCV复制中的作用的研究是新颖的，并应导致识别新的宿主和病毒靶点，用于开发创新的抗HCV疗法。