Identification and Analysis of Flavivirus Protease and RNA Helicase Inhibitors

描述（由申请人提供）：蚊媒黄病毒包括NIAID列出的A、B或C类新发人类病原体，如黄热病病毒（YFV）、西尼罗河病毒（WNV）和登革热（DEN1-4）病毒，它们会导致严重疾病，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利用体外酶法模拟这些病毒在其自然宿主中使用的酶法，通过了解病毒生命周期中的关键途径来开发抗病毒治疗方法。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是一种丝氨酸蛋白酶，是多蛋白加工所必需的，多蛋白加工是病毒生命周期的一个关键早期事件。此外，NS3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治疗药物的良好靶点。在Specific Aim 1中，我们提出验证小分子化合物可能潜在地抑制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有的假设。根据这一假设，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，我们将通过测量存在和不存在抑制剂时的Kcat和Km值，选择约100种抑制大于或等于50%的化合物(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的分析对选定的化合物进行进一步测试。在Specific Aim 2中，将测试小分子化合物可能抑制NS3/NS5相互作用并阻断其个体功能的假设。我们将使用与Aim 1类似的策略鉴定大约200种化合物，但使用基于NTP或5'-三磷酸化RNA水解释放Pi的体外测定，以及最近报道的YFV和DEN2 RNA解旋酶的晶体结构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之间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移位分析。对选定化合物的进一步检测将使用细胞毒性、病毒感染性和基于复制的报告基因测定。这项研究可能会导致鉴定出新的抑制剂，这些抑制剂将对共结晶和结构-功能研究有用，可以为黄病毒生命周期中黄病毒非结构蛋白之间分子相互作用的本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.