Inhibition of the Membrane Fusion Proteins of Flaviviruses and Alphaviruses

描述（由申请人提供）：黄病毒和甲病毒由蚊子和蜱虫传播，引起严重的人类和动物疾病，如脑炎和出血热。这些病毒包括许多潜在的生物恐怖主义制剂，属于A-C类病原体，如黄病毒登革热病毒、西尼罗河病毒、日本脑炎病毒和黄热病病毒，以及甲病毒委内瑞拉病毒、东部和西部马脑炎病毒。登革热病毒（DV）目前特别令人担忧，因为它已经在包括美国在内的100多个国家重新出现，成为地方病，现在是一个全球性的健康问题。据估计，每年有5000万至1亿例登革热病例和50万例更致命的并发症登革出血热病例，对人类健康和发展中国家的经济都产生重大影响。目前迫切需要针对黄病毒和甲病毒的抗病毒策略。黄病毒和甲病毒膜融合蛋白是II类病毒融合蛋白的成员。它们在结构上非常相似，并重新折叠成同源三聚体形式，介导病毒融合和感染。我们与F¿lix Rey博士合作，最近确定了来自塞姆利基森林病毒（SFV）的甲病毒融合蛋白的同源三聚体构象的结构。SFV同型三聚体结构与DV非常相似。以该结构为指导，我们开发了作为SFV和DV融合和感染特异性显性阴性抑制剂的蛋白质片段。基于这些信息，我们现在计划开发II类融合反应抑制剂的通用筛选。在目标1中，我们将建立并优化II类融合蛋白重折叠反应中蛋白质-蛋白质相互作用的体外方法。在目标2中，我们将使该分析适应高通量格式，并使用它来筛选我们机构以及NIH和东北生物防御联盟提供的肽和小分子文库。最终，这些抑制剂将成为抗病毒治疗的先导化合物，也是了解II类病毒融合反应的重要研究工具。黄病毒和甲病毒包括许多重要的人类病原体和潜在的生物恐怖主义威胁，如黄病毒登革热、西尼罗河、日本脑炎和黄热病病毒，以及甲病毒委内瑞拉、东部和西部马脑炎病毒。登革热病毒目前特别令人担忧，因为它已经在包括美国在内的100多个国家重新出现，成为地方病，现在是一个全球性的健康问题。本应用程序的重点是开发新的抗病毒策略，针对黄病毒和甲病毒，基于阻断参与病毒初始进入细胞的蛋白质的活性。

DESCRIPTION (provided by applicant): Flaviviruses and alphaviruses are spread by mosquito and tick vectors and cause severe human and animal illnesses such as encephalitis and hemorrhagic fever. These viruses include many potential bioterrorist agents that are category A-C pathogens, such as the flaviviruses dengue, West Nile, Japanese encephalitis and yellow fever viruses, and the alphaviruses Venezuelan, eastern, and western equine encephalitis viruses. Dengue virus (DV) is currently of particular concern as it has dramatically reemerged to become endemic in more than 100 countries including the US, and is now a global health problem. There are an estimated 50-100 million cases of dengue fever and 500,000 cases of the more lethal complication dengue hemorrhagic fever per year, with significant impact on both human health and the economies of developing countries. Antiviral strategies for the flaviviruses and alphaviruses are urgently needed. The flavivirus and alphavirus membrane fusion proteins are members of the class II virus fusion proteins. They are structurally very similar and refold to a homotrimer form to mediate virus fusion and infection. In collaboration with Dr. F¿lix Rey, we have recently determined the structure of the homotrimer conformation of the fusion protein from the alphavirus Semliki Forest virus (SFV). The SFV homotrimer structure is strikingly similar to that of DV. Using the structure as a guide, we have developed protein fragments that act as specific dominant-negative inhibitors of SFV and DV fusion and infection. Based on this information, we now plan to develop a general screen for inhibitors of class II fusion reactions. In aim 1 we will establish and optimize in vitro methods to follow the protein-protein interactions during the class II fusion protein refolding reaction. In aim 2, we will adapt this assay to a high throughput format, and use it to screen peptide and small molecule libraries available at our institution and through the NIH and Northeast Biodefense Consortium. Ultimately, such inhibitors will be lead compounds for antiviral therapy, and important research tools to understand the class II virus fusion reaction. Flaviviruses and alphaviruses include many important human pathogens and potential bioterrorist threats such as the flaviviruses dengue, West Nile, Japanese encephalitis and yellow fever viruses, and the alphaviruses Venezuelan, eastern, and western equine encephalitis viruses. Dengue virus is currently of particular concern as it has dramatically reemerged to become endemic in more than 100 countries including the US, and is now a global health problem. This application focuses on developing new antiviral strategies for the flaviviruses and alphaviruses, based on blocking the activity of the proteins involved in the initial entry of the virus into the cell.