Dynamics of Hepatis C viral RNA-dependent RNA replication

丙型肝炎病毒 RNA 依赖性 RNA 复制的动力学

• 批准号: 8967146
• 负责人: KENNETH ALLEN JOHNSON
• 金额: $ 45.01万
• 依托单位: UNIVERSITY OF TEXAS AT AUSTIN
• 依托单位国家: 美国
• 财政年份: 2014
• 资助国家: 美国
• 起止时间: 2014-12-01 至 2018-11-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8967146
• 关键词: Address; Adverse effects; Affinity; Base Pairing; Binding; Biochemical; Biological Assay; Cell Line; Chemistry; Chronic; Clinic; Combined Modality Therapy; Complex; DNA-Directed DNA Polymerase; DNA-Directed RNA Polymerase; Data; Deuterium; Drug resistance; Effectiveness; Enzymes; Evaluation; Evolution; Excision; Foundations; Goals; Growth; HIV; HIV Infections; Health; Hepatitis C; Hepatitis C virus; Human; Hydrogen; In Vitro; Infection; Interferons; Kinetics; Liver Cirrhosis; Malignant Neoplasms; Malignant neoplasm of liver; Measurement; Methods; Mutation; Nucleosides; Nucleotides; Pharmaceutical Preparations; Pharmacologic Substance; Polymerase; Population; RNA; RNA Binding; RNA chemical synthesis; RNA primers; RNA replication; RNA-Directed RNA Polymerase; Reaction; Replicon; Research; Resistance; Ribavirin; Role; Site; Structure; Structure-Activity Relationship; System; Viral; Work; antiviral nucleoside analog; base; design; drug structure; effective therapy; enzyme activity; flexibility; hepatoma cell; inhibitor/antagonist; meetings; mortality; nucleoside analog; nucleoside inhibitor; nucleotide analog; polymerization; resistance mutation; standard of care; success; therapy development; tripolyphosphate; viral RNA

DESCRIPTION (provided by applicant): The hepatitis C virus infects approximately 3% of the world's population, including 4-5 million in the USA. Chronic infection leads to liver cirrhosis an cancer, and in 2007, HCV surpassed HIV in mortality rates. It is expected that successful treatment of HCV infections will require a combination therapy, analogous to current treatments for HIV infections, and that inhibitors of the HCV RNA-dependent RNA polymerase (NS5B) will be a cornerstone of that treatment. The FDA has recently approved a new treatment based upon the first direct antiviral nucleoside analog and new potential nonnucleoside inhibitors (NNI's) are currently in the pipeline. These pharmaceuticals have been developed by using screens based on subgenomic replicons that self- replicates in human hepatoma cell lines. However, biochemical screens for enzyme activity have been limited because of the inefficient de novo initiation of RNA synthesis in vitro and the inability of the viral polymerase to bind duplex RNA (primer/template) from solution, and it is commonly accepted that all crystal structures of NS5B are of an inactive state. Current enzyme assays present an unresolved mixture of slow initiation kinetics and fast elongation and, therefore, it is not possible to know whether a given drug inhibits initiation or elongation. Quantitative data on binding affinity and mechanism of action of each class of drugs are lacking. There is a need for a quantitative assay for enzyme function to establish the kinetic parameters governing nucleotide incorporation, extension and nucleotide-dependent excision, a reaction that we recently showed can effectively remove nucleoside analogs. Non-nucleoside inhibitors (NNI's) have been discovered that bind to at least four distinct sites on the polymerase. These data on various inhibitors raise important questions regarding the mechanisms of action of the different NNI's binding to distinct enzyme sites. We have established conditions for efficient formation and purification of an active, highly processive elongation complex, overcoming the major obstacle to detailed biochemical analysis of NS5B-catalyzed replication. In this proposal, we will use state of the art single turnover kinetic methods to: (1) Establish the fidelity and baseline kinetic parameters governing cognate and noncognate base pair incorporation; (2) Examine the kinetics of incorporation, extension and excision of nucleotide analogs; (3) Establish modes of action for each class of nonnucleoside inhibitors; and (4) Quantify the effects of drug resistance mutations. In addition, hydrogen/deuterium exchange studies will reveal changes in enzyme flexibility in the transition from inactive to active enzyme, and we will attempt to determine the crystal structure of the elongation complex. This work lays the foundation for understanding structure/function relationships governing RNA-dependent RNA polymerization, the mechanisms of action of various drugs currently being investigated, and the evolution of drug resistance.

描述（由申请人提供）：丙型肝炎病毒感染世界上约 3% 的人口，其中美国有 4-500 万人。慢性感染会导致肝硬化和癌症，2007 年，HCV 的死亡率超过了 HIV。预计 HCV 感染的成功治疗将需要联合疗法，类似于当前 HIV 感染的治疗，并且 HCV RNA 依赖性 RNA 聚合酶 (NS5B) 的抑制剂将成为该治疗的基石。 FDA 最近批准了一种基于第一种直接抗病毒核苷类似物的新疗法，目前正在研发新的潜在非核苷抑制剂 (NNI)。这些药物是通过使用基于在人肝癌细胞系中自我复制的亚基因组复制子的筛选来开发的。然而，由于体外 RNA 合成的从头起始效率低下以及病毒聚合酶无法结合溶液中的双链体 RNA（引物/模板），酶活性的生化筛选受到限制，并且普遍认为 NS5B 的所有晶体结构都处于非活性状态。目前的酶测定呈现出缓慢的起始动力学和快速的延伸的未解决的混合物，因此，不可能知道给定的药物是否抑制起始或延伸。缺乏每类药物的结合亲和力和作用机制的定量数据。需要对酶功能进行定量测定，以确定控制核苷酸掺入、延伸和核苷酸依赖性切除的动力学参数，我们最近证明该反应可以有效去除核苷类似物。已发现非核苷抑制剂 (NNI) 与聚合酶上至少四个不同位点结合。这些关于各种抑制剂的数据提出了 关于不同 NNI 与不同酶位点结合的作用机制的重要问题。我们已经建立了有效形成和纯化活性高持续性延伸复合物的条件，克服了对 NS5B 催化复制进行详细生化分析的主要障碍。在本提案中，我们将使用最先进的单转换动力学方法来：（1）建立控制同源和非同源碱基对掺入的保真度和基线动力学参数； (2) 检查核苷酸类似物的掺入、延伸和切除动力学； (3) 建立每类非核苷抑制剂的作用模式； (4) 量化耐药突变的影响。此外，氢/氘交换研究将揭示从非活性酶到活性酶的转变过程中酶灵活性的变化，并且我们将尝试确定延伸复合物的晶体结构。这项工作为理解控制RNA依赖性RNA聚合的结构/功能关系、目前正在研究的各种药物的作用机制以及耐药性的演变奠定了基础。