Screening and development of anti-Ebola entry inhibitors

埃博拉和马尔堡病毒属于丝状病毒科，可引起以广泛组织破坏为特征的致命性出血热，潜伏期为4-14天。出于安全考虑，这些病毒被指定为生物安全4级制剂。丝状病毒进入和发病机制的研究进展受到了一定程度上的安全问题的阻碍。目前还没有有效的疫苗或治疗方法来对抗丝状病毒在人体内的感染和发病机制。此外，由于疫苗接种所需的最短时间至少为一个月，而且丝状病毒的暴发是散发的，因此对大部分人口进行预防这种疾病的疫苗接种可能永远不完全实际或不可取。因此，鉴定和开发抗丝状病毒感染的有效抑制剂势在必行。在这个应用中，我们证明了埃博拉糖蛋白（GP）可以有效地结合到HIV病毒颗粒上，并且假型病毒粒子可以介导病毒的有效进入。更重要的是，在初步实验中，我们已经证明，该系统可以作为一种强大的检测方法，用于筛选和识别能够阻止埃博拉病毒进入目标细胞的分子。该应用程序的总体目标是筛选和开发能够特异性抑制埃博拉病毒进入的分子。本应用程序将重点关注以下三个具体目标：(1)通过迭代合成化学优化先导化合物。(2)利用高通量筛选（HTS）筛选和鉴定抑制埃博拉病毒进入的小化合物。(3)埃博拉病毒进入抑制剂的验证与开发。这些研究应确定和开发针对埃博拉病毒感染和疾病的有效进入抑制剂。在原始提案“筛选和开发抗埃博拉进入抑制剂”（NIH UO1 AI7776701）中，我们提出了三个具体目标：(1)。迭代合成化学优化先导化合物，(2)。筛选和鉴定抑制埃博拉病毒进入的小化合物，以及(3)。埃博拉病毒进入抑制剂的验证和开发。由于我们在原申请中提出了五年的研究计划，我们现在希望修改原申请的范围，以反映更短的申请时间（2年而不是5年）。在这里，我们将重点介绍当前应用程序中提出的主要修改，这些修改将在以下章节中详细说明：当前的申请将具有与原始申请相同的三个具体目标（见上文），但拟议的实验将更加集中，以反映较短的研究计划持续时间。2. 在最初的申请中，我们提出使用动物模型测试一系列埃博拉病毒进入抑制剂。然而，我们认为在两年内进行最初提议的动物实验是极不可能的。我们要求完全取消拟议实验的这一部分，以及每年为法国维克托·沃尔奇科夫博士小组提供75,000美元的转包合同。因此，目前的应用不需要动物实验。3. 在最初的申请中，我们建议在BSL-4设施中使用传染性埃博拉病毒测试和验证最有希望的进入抑制剂（Aim 3）。这些实验在当前的应用中很重要，他们将与博士合作进行。Lisa Hensley和Gene Olinger来自USAMRIID。我们与这些埃博拉研究专家建立了合作协议（见附件协议）。但是，我们不要求任何预算的这部分工作，因为博士。Hansley和Olinger有其他财政支持来进行拟议的实验。需要强调的是，由于埃博拉病毒对人类的高致病性，被认为是严重的公共卫生威胁，是美国卫生与公众服务部突发公共卫生事件医疗对策企业战略（HHS PHEMCE Strategy, Federal Register, 2007, 72(53), 1310913114）的组成部分。因此，目前的应用对国家安全具有重大意义。

Ebola and Marburg viruses belong to the family Filoviridae and can cause fatal hemorrhagic fevers characterized by widespread tissue destruction with an incubation period of 4-14 days. Because of the safety concerns, these viruses are designated as the biosafety level 4 agents. Progress in elucidation of the mechanisms of filoviral entry and pathogenesis has been hampered partly due to the safety concern. Currently there is no effective vaccine or therapeutic treatment against filoviral infection and pathogenesis in human. Further, it may never be entirely practical or desirable to vaccinate large portions of the population against this disease due to the facts that the minimal time required for vaccination is at least one month and that the filoviral outbreaks are sporadic in nature. Thus it is imperative to identify and develop potent inhibitors against filoviral infection. In this application, we demonstrate that Ebola glycoproteins (GP) can be efficiently incorporated onto HIV viral particles, and the pseudotyped virions can mediate efficent viral entry. More importantly, in the preliminary experiments, we have shown that this system can be used as a powerful assay to screen and identify molecules that can block Ebola entry to the target cells. The overall objective of this application is to screen and develop molecules which can specifically inhibit Ebola viral entry. This application will focus on the following three specific aims: (1) Optimization of lead compounds by iterative synthetic chemistry. (2)Screening and identification of small compounds which inhibit Ebola entry using high throughput screenings (HTS). And (3) Validation and development of Ebola entry inhibitors. These studies should identify and develop potent entry inhibitors against Ebola viral infection and disease. Summary In the original proposal "Screening and development of anti-Ebola entry inhibitors" (NIH UO1 AI7776701), we proposed three specific aims: (1). Optimization of the lead compounds by iterative synthetic chemistry, (2). Screening and identification of small compounds that inhibit Ebola entry, and (3). Validation and development of Ebola entry inhibitors. Since we proposed the five years of research plan in the original application, we now wish to modify the scope of the original application to reflect the shorter duration of the application (two years instead of five years). Here we will highlight the proposed major modifications in the current application and these changes will be elaborated in the following sections: 1. The current application will have the same three specific aims as stated in the original application (see above), but the proposed experiments will be more focused to reflect the shorter duration of the research plan. 2. In the original application, we proposed to test a series of Ebola entry inhibitors using animal models. However, we believe that it is highly unlikely that animal experiments as originally proposed will be performed during a two-year period. We request to remove that part of the proposed experiments altogether, and the subcontract of $75,000/per year for Dr. Viktor Volchkov's group in France. Therefore no animal protocol is needed for the current application. 3. In the original application, we proposed to test and validate the most promising entry inhibitors using infectious Ebola virus in a BSL-4 facility (Aim 3). These experiments are important in the current application, and they will be performed in collaboration with Drs. Lisa Hensley and Gene Olinger at USAMRIID. We have set up a collaboration agreement with these Ebola research experts (see the attached agreement). However, we do not request any budget for this part of work since Drs. Hansley and Olinger have other financial supports to carry out the proposed experiments. It is important to emphasize that due to its highly pathogenic nature to humans, Ebola virus is considered a serious public health threat, and it is an integral part in the US HHS Public Health Emergency Medical Contermeansures Enterprise Strategy (HHS PHEMCE Strategy, Federal Register, 2007, 72(53), 1310913114). Therefore, the current application has great implications for the national security. 1