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

描述(申请人提供)：丙型肝炎病毒(丙型肝炎病毒)感染困扰着全球约1.7亿人，这些人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。目前还没有疫苗可以防止这种通过血液传播的病原体的传播，目前用于治疗患者的非特异性药物治疗对MOS常见的丙型肝炎病毒基因型的疗效有限。尽管在过去的十年中，在针对一些病毒蛋白的特定小分子抑制剂的开发方面取得了进展，但丙型肝炎病毒RNA复制的容易出错的性质表明，对这些抑制剂将产生耐药性。因此，使用多种针对丙型肝炎病毒生命周期不同方面的特定抑制剂的鸡尾酒疗法是未来治疗的谨慎策略，而且这种方法需要开发许多具有独特作用机制的特定抑制剂。尽管在病毒生命周期中扮演着重要的角色，但病毒NS2蛋白酶作为抗病毒靶标的活性令人惊讶地微乎其微。NS2是一种木瓜蛋白酶样半胱氨酸蛋白酶，它催化病毒多蛋白中NS2/NS3连接的自动催化切割，这种切割是必不可少的 用于生产性感染。使用一系列自主复制的工程丙型肝炎病毒复制子，我们已经开发出一种潜在的筛查，以确定以依赖于NS2活性的方式干扰病毒复制的化合物。尽管初步结果表明，这种检测的大格式版本可以识别ns2抑制剂，但在此之前还需要付出相当大的努力。 试验可以移入高含量的筛选环境。本申请针对公告PA-10-213，该计划专门针对最终进入NIH MLPCN筛查中心计划的分析开发。这项拟议项目的总体目标是将我们的NS2功能检测方法发展成适合高通量小分子筛选的检测方法，并开发适当的计数器和二级筛查方法，以最大限度地发挥未来筛查工作的影响。为此，我们提出了三个具体目标的一系列逻辑实验，以使我们的分析微型化和验证我们的分析，开发适合逻辑化合物分选计划的适当计数器筛选，并提供一个背景，从我们的筛选产生的工具化合物可能被用来了解丙型肝炎病毒生物学。 公共卫生相关性：该项目涉及优化丙型肝炎病毒NS2蛋白酶分析的研究工具，并应用这些工具开发该酶化学抑制剂的高通量筛选。NS2蛋白对丙型肝炎的复制是必不可少的，但人们对此知之甚少，抑制物化合物可能有助于确定该蛋白的功能，并有助于未来的人类治疗。

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.