High throughput screening and drug discovery for antagonists of the Ebola VP40 protein assembly

项目总结 这份R43提案响应了RFA PA-22-176关于分析开发和 解决抗病毒药物开发中未得到满足的需求进行化学探针筛选 通过创新的高含量小分子筛查治疗埃博拉患者 用于埃博拉VP40基质蛋白的拮抗剂。需要一种速效疗法。 它独立于免疫系统，以基本的病毒蛋白为靶标。这样的一个 新的治疗方法有望提高感染的人的存活概率 埃博拉疫情的热点地区。我们选择EBOV VP40是因为它绝对是 在细胞膜上组装EBOV颗粒所需的，能够萌发病毒- 分离表达的类颗粒(VLP)与VP40蛋白-蛋白质相互作用 结构域已经确定。进一步指导此应用程序的是我们的 发布的试点屏幕通过演示建立了概念验证 埃博拉病毒VP40蛋白-蛋白相互作用对双效抗菌素的可及性 EBOV VP40病毒粒子组装和病毒的小分子拮抗剂 复制机器。鉴于这一成功，并由于预期的并发症 双重目标化合物固有的功效和MOA研究，我们的特定目标 建议筛选一个包含约123,000个小分子化合物的文库以识别拮抗剂 VP40在细胞膜上的积聚，以形成VLP并从细胞中释放 通过全自动、定量和高含量的化验。化验结果已经被 为了量化小分子对荧光分子细胞分布的影响而进行的审查 VP40在293T细胞中表达。通过qHTS和Display验证命中率为剂量依赖 根据他们对VP40的绝对需求，低细胞毒性将进一步优先考虑 VP40非依赖性抗病毒作用机制的研究 微型基因组分析。先导化合物也将因其有利的ADMET而优先考虑 配置文件。我们提出的关键路径预期识别2-4个剂量依赖的VP40- 具有强大抗病毒活性的选择性拮抗剂，未来显示低细胞毒性 药物化学与临床前发展。

PROJECT SUMMARY This R43 proposal answers the call of the RFA PA-22-176 for assay development and chemical probe screening by addressing the unmet need for development of antiviral treatments for Ebola patients through an innovative high-content small molecule screen for antagonists of the Ebola VP40 matrix protein. There is a need for a fast-acting therapy that is independent of the immune system and targets essential viral proteins. Such a novel therapeutic is anticipated to enhance the survival probability for infected people in hot zones of an Ebola outbreak. We chose to target EBOV VP40 because it is absolutely required for EBOV particle assembly at the cell membrane, is capable of budding virus- like particles (VLPs) when expressed in isolation and VP40 protein-protein interaction domains have been structurally determined. Further guiding this application is our published pilot screen which established proof-of-concept by demonstrating the accessibility of Ebola VP40 protein-protein interactions to sangivamycin, a dual acting small molecule antagonist of both EBOV VP40 assembly of virions and the viral replication machinery. Given this success and due to anticipated complications with efficacy and MOA studies inherent to compounds with dual targets, our Specific Aims propose to screen a library of ~123,000 small molecule compounds to identify antagonists of VP40 accumulation at the cell membrane for VLP formation and release from cells through a fully automated, quantitative, and high-content assay. The assay has been vetted to quantify the effect of small molecules on the cellular distribution of a fluorescent VP40 expressed in 293T cells. Hits validated as dose-dependent by qHTS and displaying low cytotoxicity will be further prioritized based on their absolute requirement for VP40 in antiviral mechanism of action through counter screening with the VP40- independent minigenome assay. Lead compounds also will be prioritized by their favorable ADMET profiles. Our proposed critical path anticipates identifying 2-4 dose-dependent, VP40- selective antagonists with potent antiviral activity that display low cytotoxicity for future medicinal chemistry and preclinical development.