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.

项目总结/文摘