Identification of Ebola Virus Entry Inhibitors

描述(申请人提供)：埃博拉病毒(EBOV)导致非洲周期性爆发严重的病毒性出血热，感染患者的死亡率很高。EBOV可用于生物恐怖主义行为，因为它具有高度传染性和雾化形式的稳定性。它被疾病控制和预防中心(CDC)列为A类生化武器制剂。目前，还没有FDA批准的疫苗或抗病毒药物对人类感染EBOV有效。然而，疫苗只能部分有效地控制EBOV暴发或生物恐怖袭击，因为EBOV感染的快速发展为发展获得性免疫提供了很少的机会。因此，迫切需要对暴露后或生物恐怖袭击做出反应。EBOV感染是由病毒膜糖蛋白(GP)介导的病毒膜与宿主细胞膜的融合引起的。EBOV gp和宿主细胞表面受体分子之间的这种选择性相互作用对于感染的启动和建立是必不可少的。因此，阻断EBOV进入其靶细胞将是有利的，因为这将导致在其生命周期的早期抑制病毒的传染性。我们的目标是发现和开发抗EBOV感染的小分子进入抑制剂。考虑到EBOV感染的侵袭性及其极端的毒力，需要生物安全4级遏制设施来研究该病毒。为了规避这些严格的生物危害条件，我们将产生EBOV伪型病毒作为替代模型来研究EBOV的附着/进入。这个模型系统允许研究病毒进入细胞而不复制病毒生命周期的其他方面，因此，可以在生物安全二级设施中进行。在第一阶段，我们将开发一种利用EBOV伪型病毒来测量病毒感染的高通量筛选(HTS)方法。这项试验将用于筛选“MicroBiotix Inc.”储存库中结构多样化的小分子文库，以确定有效的EBOV进入抑制剂。抑制物将在二次EBOV假型病毒空斑试验和传染性EBOV空斑试验中进一步证实其抗病毒活性。还将对它们进行哺乳动物细胞毒性评估。在第二阶段，我们将通过合理的药物设计计划来开发最有希望的支架，并将在动物模型中测试先导化合物的有效性和毒性。毒性最小的活性最高的化合物将进入IND使能研究(第三阶段)。因此，这一阶段应用的具体目标是：1.建立高通量筛选方法，用于选择EBOV附着/进入的抑制剂；2.筛选多样化的化合物文库“MicroBiotix Inc.”S文库，以鉴定和确认EBOV的选择性抑制剂；3.EBOV抑制剂的确认；以及4.基于光谱、细胞毒性和机制的优先筛选HITS。第一阶段工作的最终结果将是确定特定于EBOV进入抑制的HITS。

DESCRIPTION (provided by applicant): Ebola virus (EBOV) causes periodic outbreaks of severe viral hemorrhagic fevers in Africa with high mortality rates in infected patients. EBOV can be used in acts of bio-terrorism because of its highly infectious nature and stability in aerosolized form. It is classified as a Category A bioweapons agent by the Centers for Disease Control and Prevention (CDC). Currently, there is no FDA approved vaccine or antiviral drug that is effective against EBOV infections in humans. However, vaccines will only be partially effective in controlling an EBOV outbreak or bioterrorist attack since rapid progression of EBOV infection offers little opportunity for developing acquired immunity. Therefore, there is a critical need for to respond to postexposure or bioterrorist attack. EBOV infection is initiated by the fusion between viral and host cell membranes, which is mediated by the viral membrane glycoprotein (GP). This selective interaction between EBOV GP and host cell surface receptor molecules is essential for the initiation and establishment of the infection. Therefore, blocking of EBOV entry into its target cell will be advantageous since this will lead to suppression of viral infectivity early in its life cycle. Our objective is to discover and develop small molecule entry inhibitors against EBOV infection. Considering the aggressive nature of EBOV infection, and its extreme virulence, biosafety level 4 containment facilities are required to study the virus. To circumvent these stringent biohazard conditions, we will generate EBOV pseudotype virus as surrogate model to study EBOV attachment/entry. This model system allows the study of viral entry into the cell without replicating other aspects of the viral life cycle and therefore, can be performed in a biosafety level 2 facility. In Phase I, we will develop a high-throughput screening (HTS) assay utilizing the EBOV pseudotype virus to measure virus infection. This assay will be used to screen libraries of structurally diverse small molecules in "Microbiotix Inc's" repository, to identify potent inhibitors of EBOV entry. Inhibitors will be further confirmed for antiviral activity in a secondary EBOV pseudotype virus plaque assay, and infectious EBOV plaque assay. They will also be assessed for mammalian cytotoxicity. In Phase II, we will progress the most promising scaffolds through a rational drug design program, and lead compounds will be tested for efficacy and toxicity in animal models. The most active compound, with the least toxicity, will advance to IND enabling studies (Phase III). Therefore the specific aims of this Phase I application are to: 1. Develop a high-throughput screening (HTS) assay for selecting inhibitors of EBOV attachment/entry; 2. Screen a diverse compound library "Microbiotix Inc."s repository to identify and confirm selective inhibitors of EBOV; 3. EBOV inhibitor confirmation; and 4. Prioritize screening hits based on spectrum, cytotoxicity and mechanism. The end result of Phase 1 work will be identification of hits specific for EBOV entry inhibition.