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

丙型肝炎病毒 NS2 蛋白酶高通量检测方法的开发

• 批准号: 8293899
• 负责人: TIMOTHY TELLINGHUISEN
• 金额: $ 34.65万
• 依托单位: SCRIPPS FLORIDA
• 依托单位国家: 美国
• 财政年份: 2012
• 资助国家: 美国
• 起止时间: 2012-02-15 至 2015-01-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8293899
• 关键词: Area; Biological Assay; Biology; Blood-Borne Pathogens; Calpain; Cell Count; Chemicals; Chronic; Cirrhosis; Development; Dimethyl Sulfoxide; Disease; Engineering; Environment; Excision; Flowcharts; Future; Gel; Generations; Genomics; Genotype; Goals; Grant; HIV-1; Hepatic; Hepatitis C; Hepatitis C virus; Human; Individual; Infection; Inflammation; Laboratories; Laboratory Research; Libraries; Life Cycle Stages; Liver; Liver diseases; Measurement; Methods; Monitor; Nature; Patients; Peptide Hydrolases; Pharmacotherapy; Poison; Polymerase; Polyproteins; Primary carcinoma of the liver cells; Property; Protease Inhibitor; Proteins; RNA replication; Reagent; Replicon; Research; Resistance; Role; Screening procedure; Series; Signal Transduction; Specificity; Staging; Testing; Therapeutic; Time; Toxic effect; Transactivation; Triage; United States National Institutes of Health; Vaccines; Validation; Viral; Viral Drug Resistance; Viral Genome; Viral Proteins; Virus; Virus Diseases; Virus Replication; assay development; base; high throughput screening; inhibitor/antagonist; method development; miniaturize; mutant; prevent; programs; protein function; research study; small molecule; tat Protein; therapeutic development; tool; transmission process; viral RNA

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.

描述（由申请人提供）：丙型肝炎病毒（HCV）感染困扰着全世界约 1.7 亿人，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。目前还没有疫苗可以预防这种血源性病原体的传播，而且目前用于治疗患者的非特异性药物疗法对于最常见的 HCV 基因型的疗效有限。尽管过去十年在针对多种病毒蛋白的特定小分子抑制剂的开发方面取得了进展，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出现。因此，使用多种特异性抑制剂针对 HCV 生命周期不同方面的基于鸡尾酒的治疗方法是未来治疗的谨慎策略，并且该方法需要开发大量具有独特作用机制的特异性抑制剂。尽管病毒 NS2 蛋白酶在病毒生命周期中发挥着重要作用，但其作为抗病毒靶标的活性却令人惊讶地少。 NS2是一种类似木瓜蛋白酶的半胱氨酸蛋白酶，催化病毒多蛋白中NS2/NS3连接处的自催化裂解，并且这种裂解是必需的 用于生产性感染。使用一系列工程化的自主复制 HCV 复制子，我们开发了一种潜在的筛选方法来识别以依赖 NS2 活性的方式破坏病毒复制的化合物。尽管初步结果表明该检测的大幅面版本可以识别 NS2 抑制剂，但在此之前还需要付出相当大的努力 测定可以转移到高内涵筛选环境中。该申请针对公告 PA-10-213，该计划专门针对最终访问 NIH MLPCN 筛选中心计划的分析开发。该拟议项目的总体目标是将我们的 NS2 功能测定开发为适合高通量小分子筛选的测定，并开发适当的计数器和二次筛选，以最大限度地提高未来筛选工作的影响。为此，我们针对三个具体目标提出了一系列逻辑实验，以小型化和验证我们的测定，开发适合逻辑化合物分类计划的适当计数器屏幕，并提供一个背景，在该背景下，我们的屏幕产生的工具化合物可用于理解 HCV 生物学。 公共健康相关性：该项目涉及丙型肝炎病毒 NS2 蛋白酶分析研究工具的优化，以及应用这些工具开发该蛋白酶化学抑制剂的高通量筛选。 NS2 蛋白对于丙型肝炎复制至关重要，但人们对其知之甚少，抑制剂化合物可能可用于确定该蛋白的功能以及有益于未来的人类治疗。