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）：丙型肝炎病毒感染了大约3％的世界人口，其中包括美国4-500万。慢性感染导致肝硬化患癌症，2007年，HCV的死亡率超过了HIV。预计成功治疗HCV感染将需要进行联合治疗，类似于当前的HIV感染治疗，并且HCV RNA依赖性RNA聚合酶（NS5B）的抑制剂将是该治疗方法的基础。 FDA最近批准了基于第一个直接抗病毒核苷类似物和新潜在的非核苷抑制剂（NNI）的新治疗方法。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，由于在体外的RNA合成效率低下，并且病毒聚合酶无法结合溶液中的双链RNA（引物/模板），并且通常认为NS5B的所有晶体结构都是不活跃的，因此，用于酶活性的生化筛选受到了RNA合成的效率低下而受到限制。当前的酶测定呈现缓慢的起始动力学和快速伸长的混合物，因此，不可能知道给定的药物是否抑制起始或伸长。缺乏有关结合亲和力和每类药物作用机理的定量数据。需要定量测定酶功能，以建立控制核苷酸掺入，延伸和核苷酸依赖性切除的动力学参数，我们最近显示的一种反应可以有效地去除核苷类似物。已经发现非核苷抑制剂（NNI）与聚合酶上至少四个不同位点结合。这些有关各种抑制剂的数据增加 关于不同NNI与不同酶位点结合的作用机理的重要问题。我们已经确定了有效形成和纯化的有效形成和纯化的高度延伸复合物的条件，这克服了NS5B催化复制的详细生化分析的主要障碍。在此提案中，我们将使用最先进的单个失误动力学方法来：（1）建立有关同源和非认知碱基对掺入的忠诚度和基线动力学参数； （2）检查核苷酸类似物的掺入，扩展和切除的动力学； （3）为每类非核苷抑制剂建立作用模式； （4）量化耐药性突变的影响。此外，氢/氘交换研究将揭示从非活性到活跃酶的过渡中酶柔韧性的变化，我们将尝试确定伸长率复合物的晶体结构。这项工作为理解控制RNA依赖性RNA聚合的结构/功能关系奠定了基础，目前正在研究的各种药物的作用机理以及耐药性的演变。