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

描述（由申请方提供）：甲病毒，包括委内瑞拉马脑炎（VEEV）和基孔肯雅（CHIKV）病毒，对全世界的人类健康构成严重威胁。VEEV是作为一种生物武器开发的，仍然被认为是一种潜在的生物恐怖主义制剂。2007年，印度的CHIKV疫情导致数百万人患病，随后蔓延到非洲和欧洲。2013年12月，在加勒比地区证实了CHIKV从蚊子传播给人类，这表明CHIKV已经出现在西半球，美国处于危险之中。尽管甲病毒对人类健康构成危险，但目前还没有针对任何甲病毒感染的抗病毒治疗。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。 我们先前开发了黄病毒RNA加帽抑制剂的HTS测定，并使用该测定来鉴定阻断黄病毒复制的分子，证明了对病毒RNA加帽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剂的迫切需求，我们提出将我们的加帽抑制剂方法适应于甲病毒，并采用这种新型HTS筛选测定来鉴定甲病毒RNA加帽的抑制剂。甲病毒主要通过其nsP 1 RNA加帽蛋白的作用加帽其基因组。nsP 1加帽酶结合GTP并将GMP转移至RNA以形成RNA帽。因此，特异性模拟和阻断GTP与nsP 1结合的分子可以作为病毒复制的有效抑制剂，正如我们在黄病毒中所显示的那样。我们已经纯化了VEEV nsP 1蛋白，并表明它是酶活性的，能够结合GTP在HTS-适合的荧光偏振测定。该项目将利用这些结果，并建立针对VEEV和CHIKV nsP 1蛋白的稳健HTS检测，使我们能够鉴定种属特异性抗甲病毒抑制剂。 目的1：优化VEEV和CHIKV nsP 1蛋白的表达和纯化参数，并建立筛选的最佳参数。 目标二：筛选科罗拉多州立大学的16，200种化合物，并使用正交和二级生化和基于细胞的测定表征筛选命中。 该项目的一个关键组成部分是我们与CSU科罗拉多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，帮助优先考虑活性化学系列，并协助简化我们的检测开发工作。该项目将使我们处于一个非常有利的位置，将甲病毒RNA加帽作为抗病毒治疗的一个有效靶点，并为随后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.