High Throughput in vitro Hepatitis C Virus Screening

随着全球超过1.7亿人受到感染，丙型肝炎病毒(HCV)已成为一种重要的 公共卫生负担。不幸的是，目前还没有预防这种感染的疫苗，而且唯一的 批准的治疗有显著的毒副作用，只对部分患者有效。值得注意的是 由于缺乏强大的组织培养，了解丙型肝炎病毒感染的研究工作受到了阻碍 感染系统和小动物模型。然而，最近，我们和其他人开发了一个强大的 体外丙型肝炎病毒感染系统，这最终应该能够调查病毒感染的所有阶段。 鉴于迫切需要阐明这种病毒的生物学特性并确定替代的丙型肝炎病毒治疗方法 选项，此应用程序的目标是开发我们的体外丙型肝炎病毒感染系统，以实现高通量 潜在抗病毒化合物的筛选(HTS)。建议的基于细胞的感染分析将提供 与目前用于丙型肝炎病毒药物发现的HTS方法相比，有几个优点 因为它概括了整个病毒生命周期，并不将筛选限制在任何特定的 预定的病毒目标(S)。因此，这种HTS感染检测不仅有可能提供 药物发现工作的新线索，但也识别具有独特作用机制的新化合物 这可以作为研究丙型肝炎病毒感染的调查工具。 因此，这项研究有三个具体目标： 1)实现、优化和验证了一种符合HTS的基于FRET的NS3蛋白酶丙型肝炎病毒感染检测方法。 2)实施、优化和验证辅助屏幕，这些辅助屏幕可用于根据以下各项确定初始点击的优先顺序 特异性、有效性和毒性。 3)通过执行初步原则性筛选和验证来验证系统的高吞吐量 分析对目标的命中和/或动作机制。

With more than 170 million people infected worldwide, Hepatitis C Virus (HCV) has emerged as a significant public health burden. Unfortunately, there is no vaccine available to prevent this infection, and the only approved treatment has significant toxic side effects and is only effective in a subset of patients. Notably research efforts to understand HCV infection have been hindered by the lack of robust tissue culture infection systems and small animal models. Recently however, we and others have developed a robust in vitro HCV infection system, which should finally enable investigation of all stages of viral infection. In light of the immediate need to elucidate the biology of this virus and identify alternative HCV treatment option, the objective of this application is to develop our in vitro HCV infection system for high throughput screening (HTS) of potential antiviral compounds. The cell-based infection assay proposed would provide several advantages over the currently available HTS approaches being used for HCV drug discovery because it recapitulates the entire viral life cycle and does not restrict screening to any specific predetermined viral target(s). Thus, this HTS infection assay would have the potential to not only provide novel leads for drug discovery efforts, but also to identify new compounds with unique mechanisms of action that can be used as investigational tools to study HCV infection. Accordingly, this study has 3 Specific Aims: 1) Implement, optimize, and validate a FRET-based NS3 protease HCV infection assay amenable toHTS. 2) Implement, optimize, and validate secondary screens that can be used to prioritize initial hits based on specificity, efficacy, and toxicity. 3) Verify the high throughput capacity of the system by performing preliminary, proof-of-principle screens and analyzing hits to target and/or mechanism of action.