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

甲病毒 RNA 加帽酶抗病毒抑制剂的高通量筛选

基本信息

批准号：
8963432
负责人：
Brian Geiss
金额：
$ 37.18万
依托单位：
COLORADO STATE UNIVERSITY
依托单位国家：
美国
项目类别：
财政年份：
2014
资助国家：
美国
起止时间：
2014-12-01 至 2017-11-30
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/8963432
关键词：
Address Africa Alphavirus Alphavirus Infections Antiviral Agents Arthralgia Biochemical Biological Biological Assay Bioterrorism Caribbean region Cells Chemicals Chikungunya virus Chronic Collaborations Colorado Country Culicidae Development Disease Disease Outbreaks Enzymes Epidemic Europe Evaluation Exonuclease Family Flavivirus Fluorescence Polarization Foundations GTP Binding Genome Genomics Guanosine Guanosine Monophosphate Guanosine Triphosphate Health Home environment Housing Human In Vitro India Infection Lead Libraries Medical Molecular Monitor National Institute of Allergy and Infectious Disease Nucleotides Patients Performance Pharmaceutical Chemistry Positioning Attribute Process Proteins Protocols documentation Public Health RNA RNA Caps RNA Viruses RNA replication Reaction Recombinants Risk Series Small RNA Staging Structure Supportive care Testing Therapeutic Time Toxic effect Translations United States Universities Venezuelan Equine Encephalitis Virus Venezuelan Equine Encephalomyelitis Viral Viral Genome Viral Pathogenesis Virus Virus Diseases Virus Replication Work assay development base biodefense chikungunya drug discovery guanylyltransferase high throughput screening improved in vivo inhibitor/antagonist interest mRNA guanylyltransferase mortality mutant novel novel strategies pathogen pre-clinical prevent scaffold screening tool transmission process viral RNA weapons

项目摘要

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.

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，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，并且仍然被认为是潜在的生物恐怖主义制剂。 2007 年，印度爆发了一场 CHIKV 疫情，导致数百万人患病，随后蔓延至非洲和欧洲。 2013年12月，加勒比海地区证实了CHIKV通过蚊子传播给人类，这表明CHIKV已在西半球出现，美国也面临风险。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。没有可用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加帽抑制剂的HTS测定法，并使用该测定法来识别阻断黄病毒复制的分子，证明病毒RNA加帽的化学抑制有效地阻断了病毒复制。为了满足对甲病毒特异性治疗的迫切需求，我们建议将我们的加帽抑制剂方法应用于甲病毒，并采用这种新型 HTS 筛选试验来鉴定甲病毒 RNA 加帽的抑制剂。甲病毒主要通过 nsP1 RNA 加帽蛋白的作用来加帽其基因组。 nsP1 加帽酶结合 GTP 并将 GMP 转移到 RNA 上以形成 RNA 帽。因此，特异性模拟和阻断 nsP1 与 GTP 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证明的那样。我们纯化了 VEEV nsP1 蛋白，并表明它具有酶活性，并且能够在适合 HTS 的荧光偏振测定中结合 GTP。该项目将利用这些结果，为 VEEV 和 CHIKV nsP1 蛋白建立强大的 HTS 检测方法，使我们能够鉴定物种特异性和属特异性的抗甲病毒抑制剂。目标 1：优化 VEEV 和 CHIKV nsP1 蛋白的表达和纯化参数，并建立最佳筛选参数。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细胞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科罗拉多州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有利地位，将甲病毒RNA加帽确立为抗病毒治疗的有效靶点，并为随后的全面筛选和临床前开发工作奠定基础。