Small Molecule Inhibitors of Ebola Virus Polymerase Function

项目摘要 丝状病毒，包括埃博拉病毒和马尔堡病毒，是非节段的负义RNA 病毒（NNSV）导致严重的人类疾病。这些病毒作为新出现的病原体令人担忧， 潜在的生物恐怖主义威胁西非国家加强了其重要性和对公共卫生的影响， 疫情始于2014年冬季，已导致11，000多人死亡，并出口埃博拉病毒 病毒病到美国，英国和欧洲尽管过去一年在发展方面取得了进展， 由于缺乏有效的疫苗和治疗方法，目前的预防和治疗选择仍然有限。特别 缺乏有效的小分子抑制剂。抗丝状病毒药物的复杂开发是生物安全性 4级（BSL 4）遏制需要与活丝状病毒一起工作，这只在少数几个地点可用 国际吧由于对进入这类设施的调查员人数有很大限制， 在高通量环境中针对感染性病毒的抗病毒测试是有问题的。一种替代方法是 开发可在不产生感染性物质的情况下进行评估的特定病毒功能的测定。的 病毒RNA依赖性RNA聚合酶（RDRP）复合物是特别有前途的候选物。复杂 由病毒核蛋白（NP）、35KDa病毒蛋白（VP35）、VP30和大蛋白（L）组成， 复合物的酶成分和病毒编码的唯一酶。RDRP复合体是 是病毒mRNA表达和病毒基因组复制所必需的，因此是病毒生长所必需的。 抑制RDRP复合物将阻止病毒复制。Basler和Shaw实验室合作 为了优化用于384孔高通量筛选的微型基因组测定，其中功能性EBOV RDRP 通过将表达其四种组分的质粒转染到哺乳动物细胞中来重建复合物。 RDRP活性通过共表达编码RDRP的模型病毒RNA（微型基因组RNA）来测量。 报道基因，侧翼为适当的病毒衍生的顺式作用调节序列。该系统已 成功转移到Sanford Burnham Prebys医学发现研究所的合作者Sumit Chanda 其中进行了6，400个化合物的中试筛选。这一筛选产生了命中， BSL 4测试由罗伯特戴维在得克萨斯州生物医学研究所，并证明抑制埃博拉病毒 复制的我们建议利用这种测定和这种药物发现管道来鉴定新的小分子 埃博拉病毒聚合酶的抑制剂。我们还将开发其他与HTS兼容的微型基因组检测方法 基于与致命人类疾病相关的其他丝状病毒，包括本迪布焦埃博拉病毒， 马尔堡病毒，以识别和优先考虑具有泛丝状病毒活性的命中。微基因组测定和 丝状病毒BSL 4实验将确定作用机制，并与初始SAR研究一起优先考虑 为未来的发展而努力。这些研究的完成将大大增加潜在的 本发明的目的在于提供治疗性小分子，并提供对丝状病毒RDRP复合物的抑制的重要见解。

Project Summary Filoviruses, which include the ebolaviruses and marburgviruses, are non-segmented, negative-sense RNA viruses (NNSVs) that cause severe human disease. These viruses are of concern as emerging pathogens and as potential bioterrorism threats. Their importance and public health impact are reinforced by the West Africa epidemic that began in winter of 2014 and has resulted in more than 11,000 deaths and the export of Ebola virus disease to the U.S., the U.K. and Europe. Although the past year has seen progress toward development of effective vaccines and treatments, current prophylactic and treatment options remain limited. Particularly lacking are effective small molecule inhibitors. Complicating development of anti-filovirus drugs is the biosafety level 4 (BSL4) containment needed to work with live filoviruses, which is only available at a few locations worldwide. With substantial restrictions on the number of investigators who have access to such facilities, antiviral testing against infectious virus in a high throughput setting is problematic. An alternate approach is to develop assays of specific viral functions that can be assessed without generation of infectious materials. The viral RNA-dependent RNA polymerase (RDRP) complex is a particularly promising candidate. The complex consists of the viral nucleoprotein (NP), viral protein of 35KDa (VP35), VP30 and the large protein (L) which is the enzymatic component of the complex and the only enzyme encoded by the virus. The RDRP complex is required for viral mRNA expression and viral genome replication and is therefore essential for virus growth. Inhibition of the RDRP complex would arrest virus replication. The Basler and Shaw laboratories collaborated to optimize for 384-well high throughput screening a minigenome assay in which a functional EBOV RDRP complex is reconstituted by transfection of plasmids that express its four components into mammalian cells. RDRP activity is measured through the co-expression of a model viral RNA (minigenome RNA) that encodes a reporter gene flanked by the appropriate virus-derived cis-acting regulatory sequences. This system has been successfully transferred to collaborator Sumit Chanda at Sanford Burnham Prebys Medical Discovery Institute where a 6,400 compound pilot screen was performed. This screen yielded hits which were carried through to BSL4 testing by Robert Davey at Texas Biomedical Research Institute and demonstrated to inhibit Ebola virus replication. We propose to exploit this assay and this drug discovery pipeline to identify novel small molecule inhibitors of the Ebola virus polymerase. We will also develop additional HTS-compatible minigenome assays based on other filoviruses associated with deadly human disease, including Bundibugyo ebolavirus and Marburg virus, to identify and prioritize hits with pan-filovirus activity. A combination of minigenome assay and filovirus BSL4 experiments will define mechanisms of action, and together with initial SAR studies will prioritize hits for future development. The completion of these studies will significantly expand the number of potential therapeutic small molecules and provide significant insight into inhibition of the filovirus RDRP complex.