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）：α病毒，包括委内瑞拉马脑炎（VEEV）和基孔肯尼亚（Chikv）病毒，对世界各地的人类健康构成了严重威胁。 Veev是作为生物武器开发的，仍然被认为是潜在的生物恐怖剂。 2007年印度的Chikv流行病使数百万，随后蔓延到非洲并进入欧洲。 2013年12月，加勒比海地区证实了从蚊子到人类的Chikv传播，表明Chikv已在西半球出现，并且美国处于危险之中。尽管α病毒对人类健康构成构成危险，但目前尚无任何抗病毒治疗任何α病毒感染。没有可用治疗的主要原因是缺乏高通量筛选（HTS） - 针对α病毒复制的关键方面的无典型测定法。我们以前开发了用于黄病毒RNA限制抑制剂的HTS测定法，并使用这些测定来鉴定阻断黄素复制的分子，这表明化学抑制VIRL RNA限值有效地阻断了病毒复制。为了满足对α病毒特异性治疗剂的紧迫需求，我们提议将限制抑制剂方法适应α病毒，并采用这种新型的HTS筛选测定法以鉴定α病毒RNA限额的抑制剂。 α病毒主要通过其NSP1 RNA封端蛋白的作用限制其基因组。 NSP1上限酶结合GTP并将GMP传输到RNA以形成RNA盖。因此，正如我们所显示的那样，NSP1特别模仿和阻断GTP结合的分子可以作为病毒复制的有效抑制剂。我们已经纯化了VEEV NSP1蛋白，并表明它具有酶活性，并且能够结合HTS无荧光极化测定法中的GTP。该项目将利用这些结果，并为VEEV和CHIKV NSP1蛋白质建立强大的HTS分析，从而使我们能够鉴定物种和特异性抗阿尔非恶毒抑制剂。 AIM 1：优化VEEV和CHIKV NSP1蛋白的表达和纯化参数，并建立用于筛选的最佳参数。 AIM 2：屏幕16,200化合物位于科罗拉多州立大学，并用正交和继发生化和基于细胞的测定法进行筛选。该项目的关键组成部分是我们与科罗拉多州科罗拉多州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，有助于优先考虑主动化学系列，并帮助简化我们的测定开发工作。该项目将使我们处于一个极好的位置，以建立α病毒RNA限额作为抗病毒疗法的有效目标，并为随后的全尺度筛查和临床前开发工作奠定了基础。