Optimizing Hepatitis C virus NS5B Polymerase Inhibitors

描述(申请人提供)：该项目的总体目标是识别和开发丙型肝炎病毒NS5B聚合酶的选择性无毒抑制剂，并最终测试它们作为治疗慢性丙型肝炎的药物。在美国，至少有400万人患有慢性丙型肝炎，最近的NIH共识会议表明，这个数字实际上是这个数字的两到三倍。慢性丙型肝炎的高进展率(70-80%)、肝病(50%)以及慢性丙型肝炎的全球分布，使其成为发病率和死亡率的主要原因。丙型肝炎疫苗的生产是一项艰巨的挑战，应大力发展基于分子的丙型肝炎治疗药物。丙型肝炎病毒研究中的一个主要问题是开发模型系统来研究丙型肝炎病毒复制和识别有效的基于分子的疗法。我们已经建立了在大肠杆菌中表达和纯化mg量纯化的具有酶活性的NS5B聚合酶蛋白的方法。此外，NS5B聚合酶的晶体结构已经得到解决，基于细胞的强大复制子系统可以用于测试对丙型肝炎病毒RNA复制的抑制。这些进展使得虚拟筛选作为候选NS5B聚合酶抑制剂的小分子成为可能。该项目的具体目标是：1)表达和纯化足够数量的重组丙型肝炎病毒NS5B聚合酶，用于体外分析和晶体生长研究；2)测试其他已通过基于结构的虚拟筛选鉴定的化合物，以确定它们使用体外试验直接抑制丙型肝炎病毒NS5B聚合酶的能力。这些结果和目前的NS5B结构信息将被用于进一步设计、合成和测试先导化合物的修饰；3)进行NS5B、模板和抑制剂的共晶生长研究，目的是利用这种三元复杂结构来设计和优化NS5B聚合酶抑制剂。

DESCRIPTION (provided by applicant): The over-all objective of this project is identifying and developing selective nontoxic inhibitors of the hepatitis C virus NS5B polymerase and eventually testing them as therapeutics for chronic hepatitis C. At least four million people in the United States have chronic hepatitis C and the recent NIH Consensus Conference suggested that this number is actually two-three fold greater. The high rate of progression to chronic infection (70-80%), liver disease (>50%), and the worldwide distribution of chronic hepatitis C, makes it a major cause of morbidity and mortality. The production of HCV vaccines is a difficult challenge and an intensive development of molecular based HCV therapeutics should be pursued. A major problem in HCV research has been the development of model systems to study HCV replication and identifying effective molecular based therapeutics. We have developed methods for expressing in E. coli and purifying mg quantities of purified enzymatically active NS5B polymerase protein. In addition, crystal structures of NS5B polymerase have been solved and robust cell based replicon systems can be used to test for inhibition of HCV RNA replication. These developments permit virtual screening of small molecules as candidate NS5B polymerase inhibitors. The specific objectives of this project are: 1) to express and purify sufficient quantities of recombinant HCV NS5B polymerase for in vitro assays and crystal growth studies; 2) To test additional compounds, already identified by structure-based virtual screening, for their ability to directly inhibit the HCV NS5B polymerase using an in vitro assay. These results and current NS5B structural information will be used to further design, synthesize and test modifications of lead compounds; 3) To conduct co-crystal growth studies of NS5B, a template, and an inhibitor with the goal of using this ternary complex structure to design and optimize NS5B polymerase inhibitors.