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

• 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
• 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

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）的生存。在4月和5月，五个团体独立报告说， 重组SAR-CoV-2 RdRp的生产和这种必需酶的初步活性评估。 这些研究中包括的直接证据表明，核苷类似物的三磷酸版本 Remdesivir已获得食品和药物管理局的紧急使用许可， SARS-CoV-2取代ATP掺入到生长的RNA寡核苷酸中，最终导致链 终止其他核碱基和核苷类似物包括EIDD-1931和法匹拉韦，后者 在日本已被批准用于治疗（-）ssRNA流感病毒， 通过抑制或加工替代可能干扰RNA代谢的抗SARS-CoV-2药物 RdRp的底物。这一数据和其他数据表明，RdRp是一个很好的发现目标， 开发新型SARS-CoV-2治疗药物。然而，利用RdRp作为 药物靶标是相对低通量的基于活性的测定，其昂贵，易于干扰，和/或 实验设计缺乏灵活性。本建议的主要目的是制定一个广泛适用的 将用于抗病毒药物发现工作的基于RdRp活性的测定。我们的具体目标是建立一个 使用具有比色读数的五酶偶联系统的新型实时测定。新的检测方法将 直接与传统的聚丙烯酰胺凝胶电泳和液体闪烁接近端相比， 点测定，并进一步用高分辨率质谱法验证。预计，通过实现 为此，该检测方法将能够对SARS-CoV-2 RdRp进行彻底的生物化学表征， 时间，使合成化合物和天然产物库的测试，以确定抑制剂，替代 底物，调节剂，或效应的SARS-CoV-2 RdRp活性的高通量筛选格式。值得注意的是， 本文实施的策略有望补充正在进行的基于结构的抗SARS-CoV-2 探索的努力。最后，基于活性的测定可以容易地适用于来自其他（+）ssRNA和（-）ssRNA的RdRp。 ）ssRNA病毒，旨在寻找针对广泛的流行病病毒的治疗方法。