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

描述（由申请人提供）：丙型肝炎病毒（HCV）感染困扰着全球约1.7亿人，这些个体中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。目前还没有疫苗来预防这种血源性病原体的传播，并且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复制的易错性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基于鸡尾酒的治疗方法是未来治疗的谨慎策略，并且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。尽管病毒NS 2蛋白酶在病毒生命周期中起重要作用，但作为抗病毒靶标的活性令人惊讶地很小。NS 2是一种木瓜蛋白酶样半胱氨酸蛋白酶，其催化病毒多蛋白中NS 2/NS 3连接的自催化切割，并且这种切割是必需的 生产性感染使用一系列的工程自主复制HCV复制子，我们已经开发出一种潜在的筛选，以确定化合物破坏病毒复制的方式依赖于NS 2的活动。虽然初步结果表明，该检测的大格式版本可以识别NS 2抑制剂，但在此之前需要付出相当大的努力。 可以将分析移动到高内容筛选环境中。本申请针对公告PA-10-213，该计划专门针对最终进入NIH MLPCN筛查中心计划的检测开发。该项目的总体目标是将我们的NS 2功能检测开发为适合高通量小分子筛选的检测，并开发适当的计数器和二级筛选，以最大限度地提高未来筛选工作的影响。为此，我们提出了一系列的逻辑实验，在三个特定的目标，以验证和验证我们的检测，开发适当的计数器屏幕，适合一个逻辑的化合物筛选计划，并提供一个上下文，其中工具化合物，从我们的屏幕可能会被用来了解HCV生物学。 公共卫生相关性：该项目涉及丙型肝炎病毒NS 2蛋白酶分析的研究工具的优化，以及这些工具在开发该蛋白酶化学抑制剂的高通量筛选中的应用。NS 2蛋白是丙型肝炎复制所必需的，但了解甚少，抑制剂化合物可能有助于确定这种蛋白的功能，并作为未来的人类治疗药物。

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.