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

描述（由申请人提供）：甲病毒，包括委内瑞拉马脑炎（VEEV）和基孔肯雅（CHIKV）病毒，在全世界对人类健康构成严重威胁。VEEV已经发展成为一种生物武器，并且仍然被认为是一种潜在的生物恐怖主义制剂。2007年在印度发生的一次CHIKV流行病使数百万人患病，随后蔓延到非洲和欧洲。2013年12月，加勒比地区确认出现了由蚊子传播给人类的CHIKV病毒，这表明CHIKV病毒已在西半球出现，美国面临风险。尽管甲病毒对人类健康构成危险，但目前尚无针对甲病毒感染的抗病毒治疗方法。目前没有治疗方法的主要原因是缺乏针对甲病毒复制关键方面的高通量筛选（HTS）检测方法。我们之前开发了黄病毒RNA封盖抑制剂的HTS检测方法，并使用该检测方法鉴定了阻断黄病毒复制的分子，证明了病毒RNA封盖的化学抑制有效地阻断了病毒复制。为了解决对甲病毒特异性治疗的迫切需求，我们建议将我们的capping抑制剂方法应用于甲病毒，并采用这种新的HTS筛选试验来鉴定甲病毒RNA capping抑制剂。甲病毒主要通过其nsP1 RNA盖帽蛋白的作用来盖住它们的基因组。nsP1盖帽酶结合GTP并将GMP转移到RNA上形成RNA帽。因此，特异性模拟和阻断nsP1与GTP结合的分子可能作为病毒复制的有效抑制剂，正如我们在黄病毒中所展示的那样。我们纯化了VEEV nsP1蛋白，并在hts可适应的荧光偏振试验中证明它具有酶活性并能够结合GTP。该项目将利用这些结果，为VEEV和CHIKV nsP1蛋白建立强大的HTS检测，使我们能够识别物种特异性和属特异性抗α病毒抑制剂。目的1：优化VEEV和CHIKV nsP1蛋白的表达和纯化参数，建立筛选的最佳参数。目标2：筛选科罗拉多州立大学的16,200种化合物，并通过正交和二次生化和基于细胞的测定来表征筛选结果。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级，并协助简化我们的分析开发工作。该项目将使我们处于一个极好的位置，以建立甲病毒RNA封顶作为抗病毒治疗的有效靶点，并为后续的全面筛选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.