Identification and Analysis of Flavivirus Protease and RNA Helicase Inhibitors

黄病毒蛋白酶和 RNA 解旋酶抑制剂的鉴定和分析

• 批准号: 7134147
• 负责人: Radhakrishnan Padmanabhan
• 金额: $ 33.28万
• 依托单位: GEORGETOWN UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2006
• 资助国家: 美国
• 起止时间: 2006-06-01 至 2009-05-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7134147
• 关键词: Flaviviridae; RNA; endopeptidases; helicase; proteins; small molecule

DESCRIPTION (provided by applicant): The mosquito-borne flaviviruses include the NIAID list of Class A, B, or C emerging human pathogens such as Yellow fever virus (YFV), West Nile virus (WNV) and Dengue (DEN1-4) viruses that cause serious illnesses with considerable morbidity and mortality. These viruses, in addition to flu-like symptoms, can cause hemorrhagic fevers (YFV and DEN) or encephalitis and/or meningo-encephalitis in susceptible humans. One of the long term goals of this laboratory has been to develop antiviral therapeutics through understanding of key pathways in the viral life cycle using in vitro enzymatic assays that mimic those utilized by these viruses in their natural hosts. Two of the nonstructural proteins, NS3 and NS5, have multiple enzyme activities that are required for the virus life cycle. NS3, in association with NS2B, is a serine protease that is required for polyprotein processing, a key early event in the virus life cycle. Moreover, NS3 exhibits an RNA-stimulated NTPase, RNA helicase and the 5'-RNA triphosphatase (5'-RTPase) activities. The NTPase and 5'-RTPase activities are stimulated by interaction with NS5, the viral 5'-RNA methyltransferase and the RNA dependent RNA polymerase (RdRP). These enzyme activities are excellent targets for development of antiviral therapeutics. In Specific Aim 1, we propose test the hypothesis that small molecule compounds could potentially inhibit the interaction between the NS2B cofactor and the NS3 protease (NS3-pro) domain or by mimicking the substrate-protease interactions or both. To this hypothesis, we will screen approximately 175,000 compounds in the National Screening Laboratory using the in vitro protease assay. By using chemoinformatics and computer-assisted docking studies and the crystal structure of the DEN protease domain, we will select about 100 compounds with greater than or equal to 50% inhibition of (i) interaction between the NS2B cofactor-NS3-pro domain and (ii) interaction between the substrate-protease active site interaction by measuring the Kcat and Km values in the presence and absence of inhibitors. Further testing of selected compounds will be performed using cytotoxicity, viral infectivity and replicon-based assays. In Specific Aim 2, the hypothesis that small molecule compounds that could potentially inhibit NS3/NS5 interaction and block their individual functions will be tested. We will identify approximately 200 compounds using a similar strategy as in Aim 1 but by using the in vitro assay based on measurement of the Pi released from the hydrolysis of either NTP or the 5'-triphosphorylated RNA and the recently reported crystal structures of YFV and DEN2 RNA helicases and those of hepatitis C virus and bovine viral diarrhea virus (BVDV) RdRP for docking studies. Inhibitors of interaction between NS3-RNA, NS3-NTP, NS3-NS5 will be analyzed by immunoprecipitation, surface plasmon resonance and gel shift assays. Further testing of selected compounds will be performed using cytotoxicity, viral infectivity and replicon-based reporter assays. This proposed research is likely lead to identification of novel inhibitors that will be useful for co-crystallization and structure-function studies that could provide valuable insight into the nature of molecular interactions among flavivirus nonstructural proteins in the flavivirus life cycle.

描述（由申请人提供）：蚊媒黄病毒包括 NIAID 列表中的 A、B 或 C 类新兴人类病原体，例如黄热病病毒 (YFV)、西尼罗河病毒 (WNV) 和登革热 (DEN1-4) 病毒导致严重疾病，具有相当高的发病率和死亡率。这些病毒除了类似流感的症状外，还可在易感人群中引起出血热（YFV 和 DEN）或脑炎和/或脑膜脑炎。该实验室的长期目标之一是通过使用模拟这些病毒在其自然宿主中所利用的体外酶测定来了解病毒生命周期中的关键途径来开发抗病毒疗法。其中两种非结构蛋白 NS3 和 NS5 具有病毒生命周期所需的多种酶活性。 NS3 与 NS2B 一起是一种丝氨酸蛋白酶，是多蛋白加工所需的丝氨酸蛋白酶，多蛋白加工是病毒生命周期中的关键早期事件。此外，NS3 还具有 RNA 刺激的 NTPase、RNA 解旋酶和 5'-RNA 三磷酸酶 (5'-RTPase) 活性。 NTPase 和 5'-RTPase 活性通过与 NS5、病毒 5'-RNA 甲基转移酶和 RNA 依赖性 RNA 聚合酶 (RdRP) 的相互作用而受到刺激。这些酶活性是开发抗病毒疗法的极好靶标。在具体目标 1 中，我们建议测试以下假设：小分子化合物可能会抑制 NS2B 辅因子和 NS3 蛋白酶 (NS3-pro) 结构域之间的相互作用，或通过模拟底物-蛋白酶相互作用，或两者兼而有之。针对这一假设，我们将在国家筛选实验室使用体外蛋白酶测定法筛选大约 175,000 种化合物。通过使用化学信息学和计算机辅助对接研究以及 DEN 蛋白酶结构域的晶体结构，我们将选择约 100 种对 (i) NS2B 辅因子-NS3-pro 结构域和 ( ii) 通过测量存在和不存在抑制剂的情况下的 Kcat 和 Km 值来确定底物-蛋白酶活性位点之间的相互作用。将使用细胞毒性、病毒感染性和基于复制子的测定法对选定化合物进行进一步测试。在具体目标 2 中，将测试小分子化合物可能抑制 NS3/NS5 相互作用并阻断其各自功能的假设。我们将使用与目标 1 类似的策略来鉴定大约 200 种化合物，但使用基于 NTP 或 5'-三磷酸化 RNA 水解释放的 Pi 测量以及最近报道的 YFV 和 YFV 晶体结构的体外测定。 DEN2 RNA 解旋酶与丙型肝炎病毒和牛病毒性腹泻病毒 (BVDV) RdRP 的解旋酶用于对接研究。 NS3-RNA、NS3-NTP、NS3-NS5 之间相互作用的抑制剂将通过免疫沉淀、表面等离子共振和凝胶位移测定进行分析。将使用细胞毒性、病毒感染性和基于复制子的报告基因检测对所选化合物进行进一步测试。这项拟议的研究可能会导致新型抑制剂的鉴定，这些抑制剂将有助于共结晶和结构功能研究，从而为了解黄病毒生命周期中黄病毒非结构蛋白之间的分子相互作用的性质提供有价值的见解。