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: 10452645
• 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/10452645
• 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; 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.

项目摘要/摘要 尽管有效的疫苗有很大的希望，可以打击Covid-19，但仍有紧迫的需求需要发展 如果疫苗无法提供保护性免疫，则直接作用抗病毒药物治疗急性 感染，以及可能逃避现有疫苗的未来冠状病毒菌株。 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标，因为病毒RNA依赖性抑制剂 聚合酶构成了抗病毒药物联合疗法的基石，用于成功治疗HIV和 丙型肝炎病毒感染。 Remdesivir是Gilead开发的核苷酸类似物，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发展更有效，毒性较小的发展 针对SARS COV-2 RDRP的药物。这项研究的理由是基于先前的经验 证明对病毒的核苷酸掺入和切除的精确测量 聚合酶/外核酸酶直接转化为了解病毒RNA复制，并可以指导设计 强大的测定法以找到有效的抑制剂。动力学分析将基于单个周转率快速运动 聚合的测量以提供明确的结果以定义核苷酸的机械基础 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进行治疗 基于对掺入动力学的分析相对于切除动力学的分析，以量化 校对外切酶。具体而言，这项研究的目的是量化核苷酸的动力学 使用单个失误动力学分析合并，以建立 RNA复制。平行研究将建立抑制核苷酸的动力学和机械基础 类似物。我们还将包括对校对外切酶的动力学的广泛表征以定义 管理不匹配的碱基对和核苷酸类似物的规则。我们也将使我们的冷冻 基于我们的生化知识的样品，以获得与remdesivir的聚合酶的结构 与外切核酸酶合并并与RDRP合并。这些研究具有创新性，因为它们利用了 PI开发的单个失误动力学分析和全局数据拟合的最先进的方法 建立聚合酶特异性的动力学和热力学基础，以揭示歧视的基础 针对核苷酸类似物。没有其他实验室将这种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指向结构/活性更有效抑制剂的媒介 关系研究。这项工作非常基于PI的先前工作，并基于解释的初步数据 动力学基础Remdesivir在与ATP竞争中的有效性。拟议的研究将大大 促进我们理解COV RNA复制的机制和动力学，并提供声音定量 基础查找直接针对病毒复制作用的抑制剂。这项研究具有发挥钥匙的强大潜力 在开发直接作用抗病毒药物中的作用，以对抗SARS COV-2和未来的冠状病毒。