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，是一种无节段的负义rna。 导致严重人类疾病的病毒(NNSV)。这些病毒作为新出现的病原体和 作为潜在的生物恐怖主义威胁。西非加强了它们的重要性和对公共卫生的影响 始于2014年冬季的疫情，已导致11,000多人死亡，埃博拉病毒的出口 病毒病传播到美国、英国和欧洲。尽管在过去的一年里，在发展方面取得了进展 在有效疫苗和治疗方面，目前的预防和治疗选择仍然有限。尤其是 缺乏有效的小分子抑制剂。使抗丝病毒药物的开发复杂化的是生物安全性 处理实时丝状病毒所需的4级(BSL4)遏制，仅在少数地点可用 全世界。由于调查人员进入此类设施的人数受到很大限制， 在高通量环境中对传染性病毒进行抗病毒测试是有问题的。另一种方法是 开发特定病毒功能的分析，可以在不产生传染性物质的情况下进行评估。这个 病毒RNA依赖的RNA聚合酶(RDRP)复合体是一个特别有前途的候选者。情结 由病毒核蛋白(NP)、35 KDa病毒蛋白(VP35)、VP30和大蛋白(L)组成。 该复合体的酶成分，也是病毒编码的唯一酶。RDRP复合体是 这是病毒表达和病毒基因组复制所必需的，因此对病毒的生长是必不可少的。 抑制RDRP复合体将阻止病毒复制。巴斯勒和肖的实验室合作 优化384孔高通量筛选EBOV RDRP功能基因的微型基因组分析方法 复合体是通过将表达其四个组分的质粒导入哺乳动物细胞而重组的。 RDRP活性是通过模型病毒RNA(微型基因组RNA)的共表达来测量的，该RNA编码 报告基因两侧有适当的病毒衍生的顺式作用调控序列。这一制度一直是 成功转到桑福德·伯纳姆普雷比斯医学发现研究所的合作者Sumit Chanda 其中进行了6,400个复合导航筛查。这个屏幕产生的点击量一直延续到 德克萨斯生物医学研究所的罗伯特·戴维进行的BSL4测试证明可以抑制埃博拉病毒 复制。我们建议利用这种分析和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兼容的微型基因组分析方法 基于与致命人类疾病有关的其他丝状病毒，包括本迪布乔埃博拉病毒和 马尔堡病毒，以识别和优先命中泛丝状病毒活性。微基因组分析和联合检测技术的应用 丝状病毒BSL4实验将确定作用机制，并将与初步的SAR研究一起确定优先顺序 未来发展的热门话题。这些研究的完成将大大扩大潜在的 治疗小分子，并为抑制丝状病毒RDRP复合体提供重要的洞察力。

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.