Kinetic and structural basis for SARS-CoV-2 RNA-dependent RNA polymerase specificity and inhibition

项目总结/摘要 尽管有效的疫苗对抗COVID-19的希望很大，但仍迫切需要开发 在疫苗不能提供保护性免疫的情况下，直接作用的抗病毒药物，用于治疗急性 感染，以及未来可能逃避现有疫苗的冠状病毒株。SARS冠状病毒（CoV- 2)RNA依赖性RNA聚合酶（RdRp）是一个有吸引力的靶点，因为病毒RNA依赖性RNA聚合酶（RdRp）的抑制剂是一种有效的靶点。 聚合酶形成成功治疗HIV的抗病毒药物联合疗法的基石， 丙型肝炎病毒感染Remdesivir是吉利德开发的一种核苷酸类似物， 在临床试验阶段这项研究的长期目标是促进开发更有效、毒性更小的 针对SARS CoV-2 RdRp的药物。这项研究的基本原理是基于以前的经验 证明了通过病毒的核苷酸掺入和切除的动力学的准确测量 聚合酶/核酸外切酶直接翻译理解病毒RNA复制，并可以指导设计 找到有效抑制剂的强大检测方法。动力学分析将基于单周转快速动力学 聚合的测量以提供确定性结果来定义核苷酸聚合的机理基础。 选择性。我们的工作假设是，可以鉴定出有效的核苷酸类似物，并且其治疗作用可以通过以下方式实现： 基于掺入动力学相对于通过酶切的切除动力学的分析， 校对核酸外切酶。具体来说，本研究的目的是量化核苷酸的动力学 使用单周转动力学分析进行合并，以建立该方法的机制和整体保真度。 RNA复制平行研究将建立核苷酸抑制的动力学和机理基础 类似物我们还将包括校对核酸外切酶的动力学的广泛表征，以定义 管理错配碱基对和核苷酸类似物的去除的规则。我们还将使用cryoEM 基于我们的生物化学知识获得Remdesivir聚合酶的结构 将RdRp与核酸外切酶结合。这些研究是创新的，因为它们利用了 PI开发的最先进的单周转动力学分析和全球数据拟合方法， 建立聚合酶特异性的动力学和热力学基础，以揭示区分的基础 针对核苷酸类似物。没有其他实验室将这样的标准应用于这个重要的问题。而且这 定量分析提供了指向结构/活性上更有效的抑制剂的准确矢量 关系研究。这项工作是建立在PI先前工作的基础上的，并基于解释 Remdesivir与ATP竞争的有效性的动力学基础。这项研究将大大 推进我们对CoV RNA复制机制和动力学的理解，并提供一个合理的定量 找到直接对抗病毒复制的抑制剂的基础。这一研究具有很强的潜力， 在开发直接作用的抗病毒药物以对抗SARS CoV-2和未来的冠状病毒方面发挥作用。

Project Summary/Abstract Although there is much hope for an effective vaccine to combat COVID-19, a pressing need remains to develop direct acting antivirals in the event that vaccines fail to provide protective immunity, for the treatment of acute infections, and for future coronavirus strains that might evade existing vaccines. The SARS coronavirus (CoV- 2) RNA-dependent RNA polymerase (RdRp) is an attractive target because inhibitors of viral RNA-dependent polymerases form the cornerstone of antiviral drug combination therapy for successful treatment of HIV and hepatitis C virus infections. Remdesivir, a nucleotide analog developed by Gilead, is already showing promise in clinical trials. The long-term goal of this research is to facilitate the development of more effective, less toxic drugs directed against the SARS CoV-2 RdRp. The rationale for this research is based on prior experience demonstrating that accurate measurements of the kinetics of nucleotide incorporation and excision by the viral polymerase/exonuclease translates directly to understanding viral RNA replication and can guide the design of robust assays to find effective inhibitors. Kinetic analysis will be based on single turnover rapid-kinetic measurements of polymerization to provide definitive results to define the mechanistic basis for nucleotide selectivity. Our working hypothesis is that an effective nucleotide analog can be identified and its therapeutic potential quantified based on analysis of the kinetics of incorporation relative to the kinetics of excision by the proofreading exonuclease. Specifically, the aims of this research are to quantify the kinetics of nucleotide incorporation using single turnover kinetic analysis in order to establish the mechanism and overall fidelity of the RNA replication. Parallel studies will establish the kinetic and mechanistic basis for inhibition for nucleotide analogs. We will also include extensive characterization of the kinetics of the proofreading exonuclease to define the rules governing removal of mismatched base pairs and nucleotide analogs. We will also us cryoEM with samples based on our biochemical knowledge to obtain structures of the polymerase with Remdesivir incorporated and of the RdRp with the exonuclease. These studies are innovative in that they take advantage of the most advanced methods of single turnover kinetic analysis and global data fitting developed by the PI to establish the kinetic and thermodynamic basis for polymerase specificity to reveal the basis for discrimination against nucleotide analogs. No other lab is applying such standards to this important problem. Moreover, this quantitative analysis provides an accurate vector pointing toward more effective inhibitors in structure/activity relationship studies. The work is soundly based the the PI's prior work and on preliminary data explaining the kinetic basis for the effectiveness of Remdesivir in competing with ATP. The proposed research will significantly advance our understanding the mechanism and kinetics of CoV RNA replication and provide a sound quantitative basis to find inhibitors acting directly against viral replication. This research has a strong potential to play a key role in the developing direct acting antiviral drugs to combat SARS CoV-2 and future coronaviruses.