RNA-dependent RNA Polymerase Assays for Biochemical Characterization and Antiviral Drug Discovery

用于生化表征和抗病毒药物发现的 RNA 依赖性 RNA 聚合酶测定

基本信息

批准号：
10164176
负责人：
Steven Gary Van Lanen
金额：
$ 41.75万
依托单位：
UNIVERSITY OF KENTUCKY
依托单位国家：
美国
项目类别：
财政年份：
2021
资助国家：
美国
起止时间：
2021-07-20 至 2024-06-30
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/10164176
关键词：
2019-nCoV Address American Antiviral Agents Biochemical Biochemistry Biological Assay COVID-19 assay COVID-19 outbreak COVID-19 pandemic COVID-19 therapeutics COVID-19 treatment Cessation of life Chemicals Clinical Clinical Trials Communicable Diseases Communities Complement Complex Coronavirus Coupled Data Detection Development Diphosphates Drug Design Drug Targeting Ebola virus Emergency Situation End Point Assay Enterovirus Enzymes Evaluation Event Experimental Designs FDA Emergency Use Authorization FDA approved Flavivirus Foundations Future Genetic Materials Genetic Transcription Goals In Vitro Influenza A Virus, H1N1 Subtype Influenza A virus Infrastructure Japan Libraries Life Liquid substance Mass Spectrum Analysis Mediating Methods Molecular Mutate Names National Institute of Allergy and Infectious Disease Natural Products Nucleotides Oligonucleotides Patients Poliomyelitis Polyacrylamide Gel Electrophoresis Polymerase Price Production Proteins RNA RNA Polymerase I RNA metabolism RNA-Directed RNA Polymerase Recombinants Recovery Reporting Research Resolution Resources Rhinovirus Structure System Testing Therapeutic Therapeutic Agents Time Vaccines Virus Virus Diseases West Nile virus Yellow Fever base catalyst combat cost drug discovery expectation flexibility high throughput screening in vivo influenzavirus inhibitor/antagonist interest intravenous injection novel nucleobase analog nucleoside analog pandemic disease pandemic influenza polymerization reconstitution remdesivir scaffold screening side effect tool tripolyphosphate

项目摘要

SARS-CoV-2 is a positive-sense, single-stranded RNA [(+)ssRNA] virus that relies on its RNA-dependent RNA polymerase (RdRp) for survival. Within the months of April and May, five groups have independently reported the production of recombinant SAR-CoV-2 RdRp and a preliminary activity assessment of this essential enzyme. Included in these studies was direct evidence that the triphosphate version of the nucleoside analogue remdesivir, which has received Emergency Use Authorization from the Food and Drug Administration to treat SARS-CoV-2, is incorporated in place of ATP into the growing RNA oligonucleotide, ultimately leading to chain termination. Other nucleobase and nucleoside analogues including EIDD-1931 and favipiravir, the latter of which has been approved in Japan to treat the (-)ssRNA influenza virus, have demonstrated promise as therapeutic agents against SARS-CoV-2 by likely interfering with RNA metabolism via inhibition or processing as alternative substrates for RdRp. This and other data suggest that RdRp is an excellent target for the discovery and development of novel SARS-CoV-2 therapeutics. However, one of the major bottlenecks in exploiting RdRp as a drug target is the relatively low throughput activity-based assays that are costly, prone to interference, and/or lack flexibility in the experimental design. The primary objective of this proposal is to develop a broadly applicable RdRp activity-based assay that will be used for antiviral drug discovery efforts. Our specific aim is to establish a novel, real-time assay using a five-enzyme coupled system with a colorimetric readout. The new assay will be directly compared to the traditional polyacrylamide gel electrophoresis and a liquid scintillation proximity end- point assay, and further validated with high resolution mass spectrometry. It is expected that, by accomplishing this aim, the assay will enable a thorough biochemical characterization of SARS-CoV-2 RdRp and, for the first time, enable the testing of synthetic compound and natural product libraries to identify inhibitors, alternative substrates, modulators, or effectors of SARS-CoV-2 RdRp activity in a high throughput screening format. Notably, the strategy implemented herein is expected to complement on-going structural-based anti-SARS-CoV-2 discovery efforts. Finally, the activity-based assay can be readily adapted for RdRp from other (+)ssRNA and (- )ssRNA viruses in an effort to identify therapeutics against a broad spectrum of pandemic-causing viruses.

SARS-CoV-2 是一种正义、单链 RNA [(+)ssRNA] 病毒，依赖于其 RNA 依赖性 RNA 聚合酶（RdRp）维持生存。 4 月和 5 月期间，有 5 个小组独立报告了重组 SAR-CoV-2 RdRp 的生产以及该必需酶的初步活性评估。这些研究中包括直接证据表明核苷类似物的三磷酸盐版本瑞德西韦（remdesivir）已获得美国食品和药物管理局的紧急使用授权，用于治疗 SARS-CoV-2 取代 ATP 并入不断生长的 RNA 寡核苷酸中，最终形成链终止。其他核碱基和核苷类似物，包括 EIDD-1931 和法匹拉韦，后者已在日本被批准用于治疗 (-)ssRNA 流感病毒，已显示出治疗前景对抗 SARS-CoV-2 的药物可能通过抑制或加工作为替代方法干扰 RNA 代谢 RdRp 的底物。该数据和其他数据表明，RdRp 是发现和研究的绝佳靶标。新型 SARS-CoV-2 疗法的开发。然而，利用 RdRp 的主要瓶颈之一是药物靶标是相对低通量的基于活性的测定，其成本昂贵、容易受到干扰和/或实验设计缺乏灵活性。该提案的主要目标是制定一个广泛适用的基于 RdRp 活性的测定，将用于抗病毒药物的发现工作。我们的具体目标是建立一个使用具有比色读数的五酶耦合系统进行新颖的实时测定。新的检测将是直接与传统的聚丙烯酰胺凝胶电泳和液体闪烁邻近末端进行比较点分析，并通过高分辨率质谱进一步验证。预计，通过完成为了实现这一目标，该检测将能够对 SARS-CoV-2 RdRp 进行彻底的生化表征，并且首次时间，能够测试合成化合物和天然产物库，以识别抑制剂、替代品以高通量筛选形式筛选 SARS-CoV-2 RdRp 活性的底物、调节剂或效应子。尤其，本文实施的策略预计将补充正在进行的基于结构的抗 SARS-CoV-2 发现努力。最后，基于活性的测定可以很容易地适应来自其他 (+)ssRNA 和 (-) ssRNA 的 RdRp )ssRNA 病毒，旨在确定针对广谱引起大流行的病毒的治疗方法。