Inhibition of the Membrane Fusion Proteins of Flaviviruses and Alphaviruses

描述（由申请人提供）：黄病毒和甲病毒通过蚊子和蜱媒传播，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剂，它们是A-C类病原体，如登革热、西尼罗河、日本脑炎和黄热病病毒，以及委内瑞拉、东部和西部马脑炎病毒。登革热病毒（DV）目前特别令人担忧，因为它在包括美国在内的100多个国家急剧重新出现，成为地方病，现在是一个全球性的健康问题。据估计，每年有5 000万至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产生重大影响。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，并重折叠成同源三聚体形式以介导病毒融合和感染。在与博士合作. F <$lix Rey，我们最近已经确定了来自甲病毒Semliki森林病毒（SFV）的融合蛋白的同源三聚体构象的结构。SFV同源三聚体结构与DV的结构惊人地相似。使用的结构作为指导，我们已经开发出的蛋白片段，作为SFV和DV融合和感染的特异性显性负抑制剂。基于这些信息，我们现在计划开发II类融合反应抑制剂的一般筛选。目的一：建立和优化体外方法，跟踪II类融合蛋白复性过程中蛋白质间的相互作用。在目标2中，我们将使该测定适应高通量形式，并使用它来筛选我们机构以及NIH和Northeast Biodefense Consortium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，以及了解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，因为它已在包括美国在内的100多个国家重新出现并成为地方病，现在是一个全球性的健康问题。该应用程序的重点是开发新的抗病毒策略的黄病毒和甲病毒，基于阻断参与病毒进入细胞的初始进入的蛋白质的活性。

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.