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

SARS-CoV-2是一种依赖于RNA依赖的RNA的阳性单链RNA病毒 聚合酶(RdRp)的生存。在4月和5月的几个月里，五个小组独立地报告了 重组SAR-CoV-2 RdRp的制备及其活性的初步鉴定。 这些研究中包括的直接证据是核苷类似物的三磷酸版本 Redesivir，已获得食品和药物管理局的紧急使用授权治疗 SARS-CoV-2被掺入到不断增长的RNA寡核苷酸中，取代了ATP，最终导致链 终止。其他碱基和核苷类似物，包括EIDD-1931和法韦拉韦，后者 已经在日本被批准用于治疗(-)ssRNA流感病毒，已经显示出治疗的前景 可能通过抑制或加工替代干扰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是一个很好的发现和 SARS-CoV-2新型治疗药物的开发。然而，利用RdRp AS的主要瓶颈之一 药物靶标是相对低通量的基于活性的分析，其成本高、易受干扰和/或 实验设计缺乏灵活性。这项建议的主要目标是制定一种广泛适用的 基于RdRp活性的分析，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 新颖的，使用五酶偶联系统和比色读数的实时检测。新的化验方法将是 直接比较了传统的聚丙烯酰胺凝胶电泳法和近距离液体闪烁电泳法- 点样法，并用高分辨率质谱仪进一步验证。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，并首次 时间，使合成化合物和天然产物库能够测试以确定抑制剂、替代品 高通量筛选形式的SARS-CoV-2 RdRp活性的底物、调节物或效应物。值得注意的是， 预计在此实施的战略将补充正在进行的以结构性为基础的抗SARS-CoV-2 探索的努力。最后，基于活性的分析方法可以很容易地用于从其他(+)单链RNA和(-)单链RNA中提取RdRp )单链RNA病毒，以努力确定针对引起大流行的广泛病毒的治疗方法。

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.