Development of High Throughput Assays for the Hepatitis C Virus NS2 Protease

描述（由申请人提供）：全球约有1.7亿人感染丙型肝炎病毒（HCV），这些人体内的慢性病毒复制是肝脏炎症、肝硬化、肝细胞癌和许多肝外疾病状态的重要原因。目前还没有预防这种血源性病原体传播的疫苗，目前用于治疗患者的非特异性药物治疗对最常见的HCV基因型的疗效有限。尽管在过去十年中，针对许多病毒蛋白的特异性小分子抑制剂的开发取得了进展，但HCV RNA复制容易出错的性质表明，对这些抑制剂的耐药性将会发展。因此，使用针对HCV生命周期不同方面的多种特异性抑制剂的鸡尾酒治疗方法是未来治疗的谨慎策略，这种方法需要开发具有独特作用机制的许多特异性抑制剂。尽管在病毒生命周期中起着重要作用，但病毒NS2蛋白酶作为抗病毒靶点的活性却出奇地低。NS2是一种类似木瓜蛋白酶的半胱氨酸蛋白酶，它催化病毒多蛋白中NS2/NS3连接的自催化裂解，这种裂解是必不可少的

DESCRIPTION (provided by applicant): Hepatitis C virus (HCV) infection afflicts approximately 170 million people worldwide and chronic virus replication in these individuals is a significant cause of liver inflammation, cirrhosis, hepatocellular carcinoma, and a host of extra-hepatic disease states. No vaccine exists to prevent transmission of this blood borne pathogen, and the current non-specific drug therapy used to treat patients is of limited efficacy for the mos common HCV genotypes. Although progress has been made in the last decade in the development of specific small molecule inhibitors targeting a number of viral proteins, the error prone nature of HCV RNA replication suggests resistance to these inhibitors will develop. A cocktail-based therapeutic approach using multiple specific inhibitors targeting distinct aspects of the HCV life cycle is therefore a prudent strategy for future therapeutics, and this approach requires the development of numerous specific inhibitors with unique mechanisms of action. Despite an essential role in the virus life cycle, the viral NS2 protease has seen surprisingly litle activity as an anti-viral target. NS2 is a papain-like cysteine protease that catalyzes the auto-catalytic cleavage of the NS2/NS3 junction in the viral polyprotein, and this cleavage is essential for productive infections. Using a series of engineered autonomously replicating HCV replicons, we have developed a potential screen to identify compounds that disrupt viral replication in a manner dependent on NS2 activity. Although preliminary results suggest the large format version of this assay can identify NS2 inhibitors, considerable effort will be required before this assay can be moved into a high content screening environment. This application is directed towards announcement PA-10-213, a program specifically geared towards assay development for eventual access to the NIH MLPCN screening center program. The overall goal of this proposed project is to develop our assay for NS2 function into an assay appropriate for high throughput small molecule screening and develop appropriate counter and secondary screens to maximize the impact of future screening efforts. To this end we have proposed a series of logical experiments in three specific aims to miniaturize and validate our assay, develop appropriate counter screens that fit into a logical compound triaging plan, and provide a context in which tool compounds that result from our screen might be used to understand HCV biology. PUBLIC HEALTH RELEVANCE: This project involved the optimization of research tools for the analysis of the hepatitis C virus NS2 protease and the application of these tools to develop high throughput screens for chemical inhibitors of this protease. The NS2 protein is essential for hepatitis C replication, but poorly understood, and inhibitor compound may be useful in determining the functions of this protein as well as of benefit as future human therapeutics.