Screening and development of anti-Ebola entry inhibitors

埃博拉病毒和马尔堡病毒属于丝状病毒科，可引起致命的出血热，其特征是广泛的组织破坏，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阐明进展受到阻碍，部分原因是安全性问题。目前，没有针对人类丝状病毒感染和发病机制的有效疫苗或治疗性治疗。此外，由于接种疫苗所需的最短时间为至少一个月并且丝状病毒爆发本质上是散发的事实，因此接种大部分人群以抵抗该疾病可能永远不完全实际或不理想。因此，鉴定和开发针对丝状病毒感染的有效抑制剂势在必行。在本申请中，我们证明了埃博拉糖蛋白（GP）可以有效地掺入到HIV病毒颗粒上，并且假型病毒粒子可以介导有效的病毒进入。更重要的是，在初步实验中，我们已经证明，该系统可以用作一种强大的检测方法，以筛选和鉴定可以阻止埃博拉病毒进入靶细胞的分子。本申请的总体目标是筛选和开发可以特异性抑制埃博拉病毒进入的分子。本申请将集中在以下三个具体目标：（1）通过迭代合成化学优化先导化合物。（2）使用高通量筛选（HTS）筛选和鉴定抑制埃博拉病毒进入的小化合物。埃博拉病毒进入抑制剂的验证和开发。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。在最初的提案“抗埃博拉病毒进入抑制剂的筛选和开发”（NIH UO 1 AI 7776701）中，我们提出了三个具体目标：（1）。通过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，和（3）.埃博拉病毒进入抑制剂的验证和开发。由于我们在原来的申请中提出了五年的研究计划，我们现在希望修改原来的申请范围，以反映较短的申请期限（两年而不是五年）。在此，我们将重点介绍当前应用程序中的主要修改，这些修改将在以下章节中详细说明：1.当前的申请将具有与原始申请中所述相同的三个具体目标（见上文），但拟议的实验将更加集中，以反映研究计划的较短持续时间。2.在最初的申请中，我们提议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，最初提议的动物实验极不可能在两年期间进行。我们要求完全取消拟议实验的这一部分，以及Viktor Volchkov博士在法国的小组每年75，000美元的费用。因此，当前应用不需要动物方案。3.在最初的申请中，我们提议在BSL-4设施中使用传染性埃博拉病毒测试和验证最有前途的进入抑制剂（目标3）。这些实验在当前的应用中非常重要，它们将与USAMRIID的丽莎汉斯莱博士和吉恩·奥林格博士合作进行。我们已经与这些埃博拉研究专家建立了合作协议（见所附协议）。然而，我们不要求这部分工作的任何预算，因为Hansley和Olinger博士有其他财政支持来进行拟议的实验。需要强调的是，由于其对人类的高致病性，埃博拉病毒被认为是一种严重的公共卫生威胁，并且是美国HHS公共卫生紧急医疗对策企业战略（HHS PHEMCE战略，联邦登记册，2007，72（53），1310913114）的组成部分。因此，当前的应用对国家安全具有重大意义。 1

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