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

SARS-CoV-2 RNA 依赖性 RNA 聚合酶特异性和抑制的动力学和结构基础

• 批准号: 10278189
• 负责人: KENNETH ALLEN JOHNSON
• 金额: $ 57.78万
• 依托单位: UNIVERSITY OF TEXAS AT AUSTIN
• 依托单位国家: 美国
• 财政年份: 2021
• 资助国家: 美国
• 起止时间: 2021-07-16 至 2026-06-30
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10278189
• 关键词: 2019-nCoV; Address; Antiviral Agents; Base Pair Mismatch; Base Pairing; Biochemical; Biological Assay; COVID-19; COVID-19 pandemic; COVID-19 therapeutics; COVID-19 treatment; Catalytic RNA; Clinical Trials; Clinical effectiveness; Combination Drug Therapy; Combined Modality Therapy; Complement; Complex; Coronavirus; Cryoelectron Microscopy; DNA-Directed DNA Polymerase; Data; Development; Discrimination; Drug usage; Effectiveness; Event; Excision; Exonuclease; Foundations; Future; Genome; Goals; HIV; HIV/HCV; Hepatitis C; Hepatitis C virus; Human; Hydrolysis; Immunity; Kinetics; Knowledge; Laboratories; Logic; Measurement; Measures; Methods; Mitochondrial DNA; Nucleotides; Pharmaceutical Preparations; Play; Polymerase; Probability; Proteins; RNA; RNA Degradation; RNA primers; RNA replication; RNA-Directed DNA Polymerase; RNA-Directed RNA Polymerase; Research; SARS coronavirus; Sampling; Site; Specificity; Structure; Structure-Activity Relationship; Testing; Therapeutic; Thermodynamics; Translating; Vaccines; Viral; Virus Inhibitors; Virus Replication; Work; acute infection; analog; base; combat; design; experience; improved; inhibitor/antagonist; innovation; mutant; nucleoside analog; nucleotide analog; polymerization; reconstitution; remdesivir; sound; vector; viral RNA

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.

项目概要/摘要 尽管人们对研制出一种有效的疫苗来对抗 COVID-19 抱有很大希望，但仍迫切需要开发 在疫苗无法提供保护性免疫力的情况下，直接作用抗病毒药物，用于治疗急性感染 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（SARS 冠状病毒） 2）RNA依赖性RNA聚合酶（RdRp）是一个有吸引力的靶标，因为病毒RNA依赖性抑制剂 聚合酶构成了成功治疗艾滋病毒和艾滋病毒的抗病毒药物联合疗法的基石 丙型肝炎病毒感染。瑞德西韦 (Remdesivir) 是吉利德 (Gilead) 开发的一种核苷酸类似物，已显示出前景 在临床试验中。这项研究的长期目标是促进开发更有效、毒性更低的药物 针对 SARS CoV-2 RdRp 的药物。这项研究的基本原理是基于先前的经验 证明可以准确测量病毒核苷酸掺入和切除的动力学 聚合酶/核酸外切酶可直接理解病毒 RNA 复制并指导设计 稳健的检测以寻找有效的抑制剂。动力学分析将基于单周转快速动力学 聚合测量提供明确的结果来定义核苷酸的机制基础 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及其治疗方法 基于相对于切除动力学的掺入动力学分析来量化潜力 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掺入，以建立机制和整体保真度 RNA复制。平行研究将建立核苷酸抑制的动力学和机制基础 类似物。我们还将包括校对核酸外切酶动力学的广泛表征，以定义 管理去除不匹配碱基对和核苷酸类似物的规则。我们还将使用冷冻电镜 基于我们的生化知识的样品，以获得瑞德西韦聚合酶的结构 并入 RdRp 和核酸外切酶。这些研究的创新之处在于它们利用了 PI 开发的最先进的单周转动力学分析和全局数据拟合方法 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问题。而且，这 定量分析提供了一个准确的向量，指向更有效的结构/活性抑制剂 关系研究。这项工作完全基于 PI 之前的工作以及解释该问题的初步数据 瑞德西韦与 ATP 竞争有效性的动力学基础。拟议的研究将显着 增进我们对 CoV RNA 复制机制和动力学的理解，并提供可靠的定量方法 寻找直接作用于病毒复制的抑制剂的基础。这项研究具有发挥关键作用的强大潜力 在开发直接作用抗病毒药物以对抗 SARS CoV-2 和未来冠状病毒方面发挥着重要作用。