Defining the Translocation Mechanisms of SARS-CoV-2 nsp13 Helicase to Aid in Antiviral Development

定义 SARS-CoV-2 nsp13 解旋酶的易位机制以帮助抗病毒药物开发

• 批准号: 10490903
• 负责人: Martin McCullagh
• 金额: $ 43.45万
• 依托单位: OKLAHOMA STATE UNIVERSITY STILLWATER
• 依托单位国家: 美国
• 财政年份: 2021
• 资助国家: 美国
• 起止时间: 2021-09-17 至 2026-08-31
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10490903
• 关键词: 2019-nCoV; ATP Hydrolysis; ATP phosphohydrolase; Address; Antiviral Agents; Arginine; Attenuated Vaccines; Behavior; Binding; Biochemical; Biological Assay; COVID-19; Catalysis; Cessation of life; Characteristics; Communication; Coupled; Couples; Crystallization; Crystallography; Data; Dengue; Development; Elements; Enzyme Kinetics; Enzymes; Goals; Hydrolysis; In Vitro; Individual; Knowledge; Length; Ligand Binding; Ligands; Maps; Mechanics; Middle East Respiratory Syndrome; Modeling; Molecular; Molecular Conformation; Motion; Mutagenesis; Mutation; Nonstructural Protein; Nucleotides; Pathway Analysis; Personal Satisfaction; Persons; Phenotype; Play; Process; Proteins; Protocols documentation; RNA; RNA Binding; RNA Helicase; RNA Viruses; RNA-Protein Interaction; Reaction; Research Personnel; Resistance; SARS coronavirus; Site-Directed Mutagenesis; Structure; Structure-Activity Relationship; Subgroup; Techniques; Temperature; Testing; Vaccines; Viral; Viral Proteins; Virus; Virus Replication; Work; X-Ray Crystallography; analog; antiviral drug development; base; combat; enzyme mechanism; enzyme structure; experience; experimental study; health economics; helicase; holistic approach; improved; in silico; inhibitor; insight; molecular dynamics; molecular scale; multi-scale modeling; mutant; novel; quantum; rational design; resistance mutation; resistant strain; response; simulation; skills; targeted treatment; therapeutic development; tripolyphosphate; vaccine development; viral RNA

Project Summary SARS-CoV-2, the causative agent of COVID-19, has infected more than 103M people worldwide (February 2021) with more than 2.25M deaths, and represents a dire threat to the health and economic well-being of the entire world. Although vaccines seem to be effective against SARS-CoV-2, recent information regarding potential vaccine resistant strains highlights the importance of alternative strategies to combat this virus. The development of antiviral therapeutics on important mutation resistant viral proteins such as nsp13 is one such strategy. Improved knowledge of the molecular mechanisms utilized by nsp13 are necessary to rationally develop inhibitors. This project will address this deficiency utilizing an integrated multiscale modeling, protein crystallography, and biochemical approach to define how SARS-CoV-2 nsp13 helicase binds RNA and ATP substrates, transduces energy during ATP binding and hydrolysis, and changes conformation during ligand binding and catalysis. We propose the following: 1) Identification of molecular-level components of the RNA- binding and translocation mechanisms of nsp13. Preliminary all-atom molecular dynamics (aaMD) simulations of SARS-CoV-2 nsp13 have identified key protein-RNA interactions that will inform initial mutagenesis studies. Further simulation and protein crystallography will inform on the ATP-dependent protein-RNA interactions observed in the RNA cleft. Biochemical experiments will be performed to test the structure-function hypotheses generated by the structural-based approaches. 2) Identification of molecular-level features of the binding, hydrolysis and product release of ATP by nsp13. We have performed aaMD simulations of the SARS- CoV-2 nsp13 in all relevant substrate states. Soaked-in ATP and non-hydrolysable analogue protein crystallography will be performed to test these initial models. Subsequent quantum mechanical calculations will identify key components of the ATP hydrolysis reaction. Site-directed mutagenesis and well-established enzyme kinetics assays will be used to test effects predicted by these simulations. 3) Identification of allosteric networks in SARS-CoV-2 nsp13 that transduce energy from ATP binding and hydrolysis to perform RNA translocation. Utilizing network analyses of aaMD simulations, Motif V has been identified as a key allosteric contributor. Biochemical studies will be performed to verify that Motif V is necessary for nsp13 helicase function. Further work will be done to identify allosteric networks between additional components of the ATP pocket and RNA cleft identified in Aims 2 and 3. This work will produce unprecedented molecular-level insight into the translocation mechanism of SARS-CoV-2 nsp13 helicases. Key components of this mechanism represent new targets for antiviral development.

项目摘要 SARS-CoV-2是COVID-19的病原体，已感染全球超过1.03亿人（2月 2021年），死亡人数超过225万，对美国人民的健康和经济福祉构成严重威胁。 整个世界虽然疫苗似乎对SARS-CoV-2有效，但最近的信息表明， 潜在的疫苗耐药性菌株突出了对抗这种病毒的替代战略的重要性。的 对重要的突变抗性病毒蛋白如NSP 13的抗病毒治疗的开发就是这样一种 战略nsp 13所利用的分子机制的改进知识是必要的， 产生抑制剂。这个项目将解决这一不足，利用综合多尺度建模，蛋白质 晶体学和生物化学方法来定义SARS-CoV-2 nsp 13解旋酶如何结合RNA和ATP 底物，在ATP结合和水解过程中转换能量，并在配体结合和水解过程中改变构象。 结合和催化。我们提出以下建议：1）鉴定RNA的分子水平组分- nsp 13的结合和转运机制。初步的全原子分子动力学（aaMD）模拟 SARS-CoV-2 nsp 13的研究已经确定了关键的蛋白质-RNA相互作用，这将为初步突变研究提供信息。 进一步的模拟和蛋白质晶体学研究将有助于研究ATP依赖的蛋白质-RNA相互作用 在RNA裂缝中观察到。将进行生化实验以测试结构-功能 基于结构的方法产生的假设。2)分子水平特征的鉴定 nsp 13与ATP的结合、水解和产物释放。我们对SARS进行了aaMD模拟- 所有相关底物状态下的CoV-2 nsp 13。浸泡ATP和不可水解类似物蛋白 将进行晶体学以测试这些初始模型。随后的量子力学计算 将确定ATP水解反应的关键成分。定点突变和完善的 酶动力学分析将用于测试通过这些模拟预测的效果。3)别构药物的鉴别 SARS-CoV-2 nsp 13中的网络，其从ATP结合和水解中吸收能量以执行RNA 易位利用aaMD模拟的网络分析，基序V已被确定为关键的变构 贡献者。将进行生物化学研究以验证基序V对于nsp 13解旋酶是必需的 功能进一步的工作将做，以确定其他组件之间的ATP变构网络 目的2和3中鉴定的口袋和RNA裂缝。这项工作将产生前所未有的分子水平的洞察力 SARS-CoV-2 nsp 13解旋酶的易位机制。这一机制的关键组成部分 代表了抗病毒药物开发的新靶点。