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

项目摘要/摘要 尽管对抗新冠肺炎的有效疫苗希望很大，但仍有迫切需要开发 疫苗未能提供保护性免疫的情况下的直接作用抗病毒药物，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(冠状病毒- 2)依赖于RNA的RNA聚合酶(RdRp)是一个很有吸引力的靶标，因为病毒的抑制物依赖于RNA 聚合酶是成功治疗艾滋病毒和艾滋病的抗病毒药物联合疗法的基石 丙型肝炎病毒感染。由Gilead开发的核苷酸类似物Remdesivir已经显示出希望 在临床试验中。这项研究的长期目标是促进开发更有效、毒性更低的 针对SARS CoV-2 RdRp的药物。这项研究的理论基础是基于先前的经验。 证明了对病毒的核苷酸掺入和切除动力学的准确测量 聚合酶/核酸外切酶直接转化为了解病毒RNA复制，并可以指导设计 找到有效的抑制剂的可靠检测方法。动力学分析将基于单周转的快速动力学 聚合反应的测量，以提供明确的结果来确定核苷酸的机理基础 选择性。我们的工作假设是，可以确定一种有效的核苷酸类似物，并对其进行治疗 电势的量化是基于对掺入动力学的分析相对于由 校对核酸外切酶。具体地说，这项研究的目的是量化核苷酸的动力学 公司使用单一周转动力学分析，以建立机制和整体保真度 RNA复制。平行研究将建立抑制核苷酸的动力学和机制基础 类比。我们还将包括校对核酸外切酶动力学的广泛表征，以定义 去除不匹配的碱基对和核苷酸类似物的规则。我们还将使用冷冻EM 根据我们的生化知识获取带有Remsivir的聚合酶的结构 并将RdRp与核酸外切酶结合。这些研究具有创新性，因为它们利用了 最先进的单次周转动力学分析和全局数据拟合方法 建立聚合酶专一性的动力学和热力学基础以揭示鉴别的基础 对抗核苷酸类似物。没有其他实验室将这样的标准应用到这个重要的问题上。此外，这一点 定量分析提供了一个准确的指向结构/活性更有效的抑制剂的载体 关系研究。这项工作是建立在PI之前的工作和解释 雷米西韦与三磷酸腺苷竞争有效性的动力学基础。拟议中的研究将显著地 加深对冠状病毒RNA复制机制和动力学的认识，为进一步研究冠状病毒RNA复制提供合理的定量依据 寻找直接作用于病毒复制的抑制剂的基础。这项研究具有很强的潜力发挥关键作用 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.