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
基本信息
- 批准号:10659068
- 负责人:
- 金额:$ 57.78万
- 依托单位:
- 依托单位国家:美国
- 项目类别:
- 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:
- 关键词:2019-nCoVAddressAntiviral AgentsBase Pair MismatchBase PairingBiochemicalBiological AssayCOVID-19COVID-19 pandemicCOVID-19 therapeuticsCOVID-19 treatmentCatalytic RNAClinical TrialsClinical effectivenessCombination Drug TherapyCombined Modality TherapyComplementComplexCoronavirusCryoelectron MicroscopyDNA-Directed DNA PolymeraseDataDevelopmentDiscriminationDrug usageEffectivenessEventExcisionExonucleaseFoundationsFutureGenomeGoalsHIVHIV/HCVHepatitis CHepatitis C virusHumanHydrolysisImmunityKineticsKnowledgeLaboratoriesLogicMeasurementMeasuresMethodsMitochondrial DNANucleotidesPharmaceutical PreparationsPlayPolymerasePolymersProbabilityProteinsRNARNA DegradationRNA primersRNA replicationRNA-Directed DNA PolymeraseRNA-Directed RNA PolymeraseResearchSARS coronavirusSamplingSiteSpecificityStructureTestingTherapeuticThermodynamicsTranslatingVaccinesViralVirus InhibitorsVirus ReplicationWorkWorkplaceacute infectionanalogbasecombatdesignexperienceimprovedinhibitorinnovationmutantnucleoside analognucleotide analogpolymerizationreconstitutionremdesivirsoundstructural determinantsvectorviral RNA
项目摘要
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/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。这项研究的理由是基于先前的经验
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被抑制。
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。我们还将使用冷冻EM,
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。
项目成果
期刊论文数量(11)
专著数量(0)
科研奖励数量(0)
会议论文数量(0)
专利数量(0)
Design and interpretation of experiments to establish enzyme pathway and define the role of conformational changes in enzyme specificity.
设计和解释实验以建立酶途径并定义构象变化在酶特异性中的作用。
- DOI:10.1016/bs.mie.2023.03.018
- 发表时间:2023
- 期刊:
- 影响因子:0
- 作者:Dangerfield,TylerL;Johnson,KennethA
- 通讯作者:Johnson,KennethA
Kinetics of elementary steps in loop-mediated isothermal amplification (LAMP) show that strand invasion during initiation is rate-limiting.
- DOI:10.1093/nar/gkac1221
- 发表时间:2023-01-11
- 期刊:
- 影响因子:14.9
- 作者:Dangerfield, Tyler L.;Paik, Inyup;Bhadra, Sanchita;Johnson, Kenneth A.;Ellington, Andrew D.
- 通讯作者:Ellington, Andrew D.
Substrate specificity and proposed structure of the proofreading complex of T7 DNA polymerase.
- DOI:10.1016/j.jbc.2022.101627
- 发表时间:2022-03
- 期刊:
- 影响因子:0
- 作者:Dangerfield TL;Kirmizialtin S;Johnson KA
- 通讯作者:Johnson KA
Structural basis for mismatch surveillance by CRISPR-Cas9.
- DOI:10.1038/s41586-022-04470-1
- 发表时间:2022-03
- 期刊:
- 影响因子:64.8
- 作者:Bravo JPK;Liu MS;Hibshman GN;Dangerfield TL;Jung K;McCool RS;Johnson KA;Taylor DW
- 通讯作者:Taylor DW
Expression and purification of tag-free SARS-CoV-2 RNA-dependent RNA polymerase in Escherichia coli.
大肠杆菌中无标记的SARS-COV-2 RNA依赖性RNA聚合酶的表达和纯化。
- DOI:10.1016/j.xpro.2021.100357
- 发表时间:2021-03-19
- 期刊:
- 影响因子:0
- 作者:Dangerfield TL;Huang NZ;Johnson KA
- 通讯作者:Johnson KA
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KENNETH ALLEN JOHNSON其他文献
KENNETH ALLEN JOHNSON的其他文献
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{{ truncateString('KENNETH ALLEN JOHNSON', 18)}}的其他基金
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 - 财政年份:2021
- 资助金额:
$ 57.78万 - 项目类别:
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 - 财政年份:2021
- 资助金额:
$ 57.78万 - 项目类别:
Correlating defects in mitochondrial DNA replication to physiology
将线粒体 DNA 复制缺陷与生理学相关联
- 批准号:
8860390 - 财政年份:2015
- 资助金额:
$ 57.78万 - 项目类别:
Correlating defects in mitochondrial DNA replication to physiology
将线粒体 DNA 复制缺陷与生理学相关联
- 批准号:
9412492 - 财政年份:2015
- 资助金额:
$ 57.78万 - 项目类别:
Correlating defects in mitochondrial DNA replication to physiology
将线粒体 DNA 复制缺陷与生理学相关联
- 批准号:
9206171 - 财政年份:2015
- 资助金额:
$ 57.78万 - 项目类别:
Dynamics of Hepatis C viral RNA-dependent RNA replication
丙型肝炎病毒 RNA 依赖性 RNA 复制的动力学
- 批准号:
8967146 - 财政年份:2014
- 资助金额:
$ 57.78万 - 项目类别:
Nucleotide selectivity and drug resistance by HIV reverse transcriptase
HIV逆转录酶的核苷酸选择性和耐药性
- 批准号:
7930581 - 财政年份:2009
- 资助金额:
$ 57.78万 - 项目类别:
Nucleotide selectivity and drug resistance by HIV reverse transcriptase
HIV逆转录酶的核苷酸选择性和耐药性
- 批准号:
8306332 - 财政年份:2009
- 资助金额:
$ 57.78万 - 项目类别:
Nucleotide selectivity and drug resistance by HIV reverse transcriptase
HIV逆转录酶的核苷酸选择性和耐药性
- 批准号:
8117771 - 财政年份:2009
- 资助金额:
$ 57.78万 - 项目类别:
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