Identification and Analysis of Flavivirus Protease and RNA Helicase Inhibitors

描述(申请人提供)：蚊媒黄病毒包括NIAID A、B或C类新出现的人类病原体，如黄热病病毒(YFV)、西尼罗河病毒(WNV)和登革热(DEN1-4)病毒，这些病毒会导致严重的疾病，具有相当高的发病率和死亡率。除了流感样症状外，这些病毒还可以引起出血热(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体外酶分析来了解病毒生命周期中的关键途径来开发抗病毒疗法，这种方法模仿这些病毒在其自然宿主中所利用的那些途径。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结合，是一种丝氨酸蛋白酶，是多蛋白加工所必需的，是病毒生命周期中的一个关键早期事件。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的活性受NS5、病毒5’-RNA甲基转移酶和RNA依赖的RNA聚合酶(RdRP)相互作用的刺激。这些酶的活性是开发抗病毒治疗药物的极佳靶点。在特定的目标1中，我们提出了一种假设，即小分子化合物可以潜在地抑制NS2B辅因子和NS3蛋白(NS3-PRO)结构域之间的相互作用，或者通过模仿底物-蛋白酶相互作用或两者兼而有之。根据这一假设，我们将在国家筛选实验室使用体外蛋白酶试验筛选大约175,000个化合物。通过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，我们将通过测量有无抑制剂时的Kcat和Km值，筛选出大约100个化合物，它们对(I)NS2B辅因子-NS3-Pro结构域之间的相互作用和(Ii)底物-蛋白酶活性部位之间的相互作用具有大于或等于50%的抑制作用。将使用细胞毒性、病毒感染性和基于复制子的分析对选定的化合物进行进一步测试。在具体目标2中，将测试可能抑制NS3/NS5相互作用并阻断其单独功能的小分子化合物的假设。我们将使用与目标1中类似的策略鉴定大约200个化合物，但使用基于NTP或5‘-三磷酸化RNA水解释放的PI的体外实验，以及最近报道的YFV和DEN2RNA解旋酶以及丙型肝炎病毒和牛病毒性腹泻病毒(BVDV)RdRP的晶体结构进行对接研究。NS3-RNA、NS3-NTP、NS3-NS5相互作用的抑制剂将通过免疫沉淀、表面等离子体共振和凝胶位移分析来分析。将使用细胞毒性、病毒感染性和基于复制子的报告分析对选定的化合物进行进一步测试。这项拟议的研究可能会导致识别新的抑制剂，这些抑制剂将有助于共结晶和结构功能研究，这些研究可能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。

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.