Chemical Genetics:Cellular Regulators of HCV (RMI)

化学遗传学：HCV 细胞调节因子 (RMI)

• 批准号: 6880174
• 负责人: RAYMOND T CHUNG
• 金额: $ 8.65万
• 依托单位: MASSACHUSETTS GENERAL HOSPITAL
• 依托单位国家: 美国
• 财政年份: 2004
• 资助国家: 美国
• 起止时间: 2004-09-30 至 2006-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6880174
• 关键词: bioassay; biomedical automation; biotechnology; cell cycle proteins; chemical genetics; combinatorial chemistry; hepatitis C; hepatitis C virus; high throughput technology; polymerase chain reaction; posttranslational modifications; protein quantitation /detection; protein structure function; robotics; technology /technique development; virus replication

DESCRIPTION (provided by applicant): Hepatitis C virus (HCV) infection is the most important cause of chronic liver disease in the US and a rapidly growing cause of hepatocellular carcinoma (HCC) worldwide. Our understanding of the cellular factors that regulate HCV replication has been limited by the paucity of tissue culture models supportive of viral replication. Two parallel developments now permit steps to be taken to comprehensively screen for cellular proteins that participate in the viral lifecycle. The first has been development of cell-based models of HCV replication, including one developed in our laboratory that is capable of sustained HCV (+) and (-) strand as well as protein synthesis. We have adapted an assay for HCV core protein production as a simple readout for this model. The second development has been the emergence of high throughput screens of large chemical libraries that permit identification of host proteins that regulate biological processes. We therefore propose to take steps toward development of a high-throughput chemical genetic screen to identify cellular regulators of HCV replication using our cell-based assay as a phenotypic readout. Preliminary data indicate that we can successfully convert our cell-based replication assay to the more tractable 96-well format and that replication is reproducibly regulated by interferon and dexamethasone. During this proposal period, we will (1) optimize miniaturization of our cell-based assay to the 96-well format; and (2) begin screening of chemical libraries in collaboration with the Institute for Chemistry and Cell Biology at Harvard Medical School using known biologically active compounds. Initial hits identified from primary screens will be confirmed using secondary biological assays, including real-time assays for HCV RNA. Ultimately, affinity approaches using small molecule "hits" as probes will be planned to identify cellular proteins that regulate HCV replication. The use of this assay system to screen large chemical libraries will have two important consequences in studies that emanate from the proposed planning study. First, it will serve as an antiviral discovery screen. Initial hits will then be analyzed by secondary and tertiary screens to discern structure-activity relationships. Second, discovery of hit compounds that positively or negatively regulate HCV replication will ultimately help lead to identification of novel cellular targets that can be exploited to further interrupt the viral lifecycle.

描述（由申请人提供）：丙型肝炎病毒（HCV）感染是美国慢性肝病的最重要原因，也是全球肝细胞癌（HCC）快速增长的原因。 由于缺乏支持病毒复制的组织培养模型，我们对调节 HCV 复制的细胞因素的理解受到限制。 现在，两项并行开发允许采取措施全面筛选参与病毒生命周期的细胞蛋白。 第一个是基于细胞的 HCV 复制模型的开发，包括我们实验室开发的能够持续 HCV (+) 和 (-) 链以及蛋白质合成的模型。 我们采用了 HCV 核心蛋白生产测定法作为该模型的简单读数。 第二个发展是大型化学库的高通量筛选的出现，其允许鉴定调节生物过程的宿主蛋白。 因此，我们建议采取措施开发高通量化学遗传筛选，以使用我们基于细胞的测定作为表型读数来识别 HCV 复制的细胞调节因子。 初步数据表明，我们可以成功地将基于细胞的复制测定转换为更容易处理的 96 孔格式，并且复制受到干扰素和地塞米松的可重复调节。 在此提案期间，我们将 (1) 将基于细胞的检测方法优化为 96 孔格式； (2) 与哈佛医学院化学和细胞生物学研究所合作，使用已知的生物活性化合物开始筛选化学库。 从初级筛选中确定的初步命中将使用二级生物测定进行确认，包括 HCV RNA 的实时测定。 最终，将计划使用小分子“命中”作为探针的亲和方法来鉴定调节 HCV 复制的细胞蛋白。 使用该测定系统来筛选大型化学库将对拟议的规划研究产生两个重要的影响。 首先，它将作为抗病毒发现筛选。 然后，初始命中将通过二级和三级屏幕进行分析，以辨别结构-活性关系。 其次，发现正向或负向调节 HCV 复制的命中化合物将最终有助于识别新的细胞靶标，这些靶标可用于进一步中断病毒生命周期。