High Throughput in vitro Hepatitis C Virus Screening

描述（由申请人提供）：随着全球超过1.7亿人感染，丙型肝炎病毒（HCV）已成为一个重大的公共卫生负担。不幸的是，没有疫苗可用于预防这种感染，唯一批准的治疗方法具有显著的毒副作用，并且仅对一部分患者有效。值得注意的是，由于缺乏强大的组织培养感染系统和小动物模型，研究HCV感染的努力受到阻碍。然而，最近，我们和其他人已经开发了一个强大的体外HCV感染系统，这应该最终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和确定替代的HCV治疗选择，本申请的目的是开发我们的体外HCV感染系统，用于潜在抗病毒化合物的高通量筛选（HTS）。所提出的基于细胞的感染测定将提供优于目前可用的用于HCV药物发现的HTS方法的几个优点，因为它概括了整个病毒生命周期，并且不将筛选限制于任何特定的预定病毒靶标。因此，这种HTS感染检测不仅有可能为药物发现工作提供新的线索，而且还可以鉴定具有独特作用机制的新化合物，这些化合物可用作研究HCV感染的研究工具。 因此，本研究有3个具体目标： 1)实施、优化和验证基于FRET的NS 3蛋白酶HCV感染检测试剂盒，该试剂盒适用于HTS。 2)实施、优化和验证二次筛选，可用于根据特异性、疗效和毒性对初始命中进行优先排序。 3)通过执行初步的原理验证筛选并分析目标命中和/或作用机制，验证系统的高吞吐量能力。

DESCRIPTION (provided by applicant): 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 to HTS. 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.