Structure Based Design of Dengue Virus Fusion Inhibitors

描述（由申请人提供）：超过一半的世界人口面临感染登革热病毒的风险，登革热病毒是黄病毒家族的一种蚊子传播的成员，由四种不同的血清型组成。每年大约发生5000万至1亿例感染，造成约50万例危及生命的登革出血热或登革休克综合征。由于登革热感染的发病率增加和地理分布的扩大，它被认为是一种新出现的疾病，并被NIAID确定为a类优先病原体。尽管与登革热感染相关的疾病负担很大，但目前还没有批准的疫苗或抗病毒药物。该项目的目标是发现和开发抗病毒药物，通过阻断病毒生命周期中的细胞进入步骤来抑制登革热病毒感染。宿主细胞的登革热感染是由包膜蛋白介导的，这是一种II类病毒融合蛋白，负责病毒附着并触发病毒与宿主细胞膜之间的融合。夏威夷生物技术公司以纯化的原生形式表达了登革热病毒的包膜蛋白。作为与哈佛医学院Stephen Harrison实验室的一个合作项目，最近已经解决了融合前和融合后状态下登革热-2包膜蛋白的三维结构。这些结构揭示了包膜蛋白上抑制融合过程的两个潜在药物靶点。一个目标是存在于病毒颗粒的天然包膜中的疏水性口袋，另一个目标是存在于参与膜融合后期的蛋白质融合中间形式中的通道。这些靶点将通过虚拟药物筛选过程中的分子模型进行检查，以找到潜在的抑制剂化合物，然后进行实验测试。纯化的包膜蛋白在体外适当条件下进行融合过程的构象重排。这将形成基于荧光的分析方法，可用于识别融合抑制剂的发展基础。还将开发一种基于细胞的登革热包膜介导膜融合试验，用于测试候选抑制剂。通过评估代表融合过程中不同阶段的两个不同分子靶点，并探索几种不同的检测设计，我们打算在I期结束时选择最佳途径，以在II期发现有效的登革热和其他黄病毒抗病毒药物。

DESCRIPTION (provided by applicant): Over half of the world population is at risk for infection by dengue virus, a mosquito borne member of the Flavivirus family that consists of four distinct serotypes. Approximately 50 to 100 million infections occur annually resulting in an estimated 500,000 cases of life threatening dengue hemorrhagic fever or dengue shock syndrome. Due to the increased incidence and spreading geographic distribution of dengue infection, it is considered to be an emerging disease and is identified by NIAID as a category A priority pathogen. Despite the significant disease burden associated with dengue infection there is presently no approved vaccine or antiviral drug. The goal of this project is to discover and develop antiviral drugs that inhibit dengue virus infection by blocking the cell entry step in the virus life cycle. Dengue infection of host cells is mediated by the envelope protein, a class II viral fusion protein responsible for virus attachment and for triggering fusion between the virus and host cell membranes. Hawaii Biotech has expressed the envelope proteins of dengue virus in purified native form. The 3-dimensional structures of the dengue-2 envelope protein in both a pre and a post fusion state have recently been solved as a collaborative project with Stephen Harrison's laboratory at Harvard Medical School. The structures reveal two potential drug target sites on the envelope protein for inhibitors of the fusion process. One target is a hydrophobic pocket present in the native envelope of the virus particle, and the other is a channel present in the fusion intermediate form of the protein involved in the late stages of membrane fusion. These targets will be examined by molecular modeling through a process of virtual drug screening to find potential inhibitor compounds that will then be tested experimentally. The purified envelope protein undergoes the conformational rearrangements of the fusion process in vitro under appropriate conditions. This will form the basis for development of fluorescence based assay methods that can be used to identify fusion inhibitors. A cell based assay for dengue envelope mediated membrane fusion will also be developed for testing of inhibitor candidates. By evaluating two distinct molecular targets that represent different stages in the fusion process, and by exploring several different assay designs, we intend to be in a position at the conclusion of Phase I to select the best path to discovery of effective antiviral drugs for dengue and other Flaviviruses during Phase II.