A High-Throughput Screen for Antiviral Inhibitors of the Alphavirus RNA Capping Enzyme

描述(申请人提供)：甲型病毒，包括委内瑞拉马脑炎(VEEV)和基孔肯雅(CHIKV)病毒，对世界各地的人类健康构成严重威胁。Veev已被开发为生物武器，并仍被认为是潜在的生物恐怖主义毒剂。2007年印度的一场CHIKV疫情导致数百万人患病，随后在非洲蔓延到欧洲。2013年12月，加勒比地区证实了CHIKV从蚊子传播到人类的情况，这表明CHIKV已经出现在西半球，美国正处于危险之中。尽管甲型病毒对人类健康构成威胁，但目前还没有针对任何甲型病毒感染的抗病毒治疗。没有可用的治疗方法的主要原因是缺乏高通量筛查(HTS)-适用于针对甲型病毒复制关键方面的检测。我们之前开发了黄病毒RNA封顶抑制剂的HTS检测方法，并使用这些检测方法来确定阻止黄病毒复制的分子，证明了化学抑制Virl RNA封顶有效地阻止了病毒复制。为了满足对甲型病毒特异性治疗的迫切需求，我们建议将我们的封顶抑制物方法应用于甲型病毒，并使用这一新的HTS筛选试验来识别甲型病毒RNA封顶的抑制剂。甲型病毒主要通过其nsP1 RNA封顶蛋白的作用来封顶它们的基因组。NsP1封帽酶与GTP结合，将GMP转移到RNA形成RNA帽。因此，特异性地模拟和阻断nsP1结合GTP的分子可能是病毒复制的有效抑制剂，就像我们对黄病毒所显示的那样。我们已经纯化了VEEV nsP1蛋白，并在HTS兼容的荧光偏振实验中证明了它具有酶活性并能够与GTP结合。该项目将利用这些结果，为VEEV和CHIKV nsP1蛋白建立可靠的HTS检测方法，使我们能够识别物种和属特异性的抗甲病毒抑制剂。目的1：优化VEEV和CHIKV nsP1蛋白的表达和纯化条件，建立筛选的最佳条件。目的2：筛选科罗拉多州立大学收藏的16,200种化合物，并用正交分析、二级生化分析和基于细胞的分析来表征筛选结果。该项目的一个关键组成部分是我们与科罗拉多州立大学药物发现中心的持续合作，该中心将提供关键的药物化学支持，帮助确定活性化学系列的优先顺序，并帮助简化我们的分析开发工作。该项目将使我们处于有利地位，将甲型病毒RNA封顶作为抗病毒治疗的有效靶点，并为随后的全面筛查和临床前开发工作奠定基础。

DESCRIPTION (provided by applicant): Alphaviruses, including Venezuelan Equine Encephalitis (VEEV) and Chikungunya (CHIKV) viruses, pose a serious threat to human health around the world. VEEV has been developed as a biological weapon and is still considered a potential bioterrorism agent. A CHIKV epidemic in India in 2007 sickened millions and subsequently spread through Africa and into Europe. In December 2013 CHIKV transmission from mosquitoes to humans was confirmed in the Caribbean, demonstrating that CHIKV has emerged in the Western Hemisphere and that the United States is at risk. Despite the dangers that alphaviruses pose to human health there is currently no antiviral treatment for any alphavirus infection. The major reason no treatments are available is the lack of high-throughput screening (HTS)-amenable assays that target critical aspects of alphavirus replication. We previously developed HTS assays for flavivirus RNA capping inhibitors and used the assays to identify molecules that block flavivirus replication, demonstrating that chemical inhibition of virl RNA capping effectively blocks viral replication. To address the pressing need for alphavirus-specific therapeutics, we propose adapting our capping inhibitor approach to alphaviruses and employing this novel HTS screening assay to identify inhibitors of alphavirus RNA capping. Alphaviruses cap their genomes primarily via the action of their nsP1 RNA capping protein. The nsP1 capping enzyme binds GTP and transfers a GMP to RNAs to form the RNA cap. Therefore, molecules that specifically mimic and block GTP binding by nsP1 may serve as potent inhibitors of viral replication, as we have shown for the flaviviruses. We have purified VEEV nsP1 protein and shown that it is enzymatically active and able to bind GTP in an HTS-amenable fluorescence polarization assay. This project will capitalize on these results and establish robust HTS assays for both VEEV and CHIKV nsP1 proteins, allowing us to identify both species- and genus-specific anti-alphavirus inhibitors. Aim 1: Optimize expression and purification parameters for VEEV and CHIKV nsP1 proteins and establish optimal parameters for screening. Aim 2: Screen 16,200 compounds housed at Colorado State University and characterize screening hits with orthogonal and secondary biochemical and cell-based assays. A key component of this project is our ongoing collaboration with the CSU Colorado Center for Drug Discovery, which will provide critical medicinal chemistry support, help prioritize active chemical series, and assist in streamlining our assay development efforts. This project will place us in an excellent position to establish alphavirus RNA capping as a potent target for antiviral therapeutics and set the stage for subsequent full-scale screening and preclinical development efforts.