Mechanical and Dynamical Regulation of Protein Kinases

蛋白激酶的机械和动态调节

• 批准号: 8199019
• 负责人: Christopher Lee McClendon
• 金额: $ 4.63万
• 依托单位: UNIVERSITY OF CALIFORNIA, SAN DIEGO
• 依托单位国家: 美国
• 财政年份: 2011
• 资助国家: 美国
• 起止时间: 2011-12-01 至 2014-11-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8199019
• 关键词: Active Sites; Address; Adverse effects; Agreement; Binding; Biological; Catalysis; Catalytic Domain; Cell physiology; Chemicals; Complex; Computer Simulation; Coupled; Coupling; Cyclic AMP-Dependent Protein Kinases; Data; Disease; Distal; Distant; Docking; Drug Delivery Systems; Drug Design; Entropy; Enzymes; Etiology; Evolution; Family; Goals; Homologous Gene; In Vitro; Kinetics; Lead; Lobe; Malignant Neoplasms; Maps; Mechanical Stress; Mechanics; Methods; Minor; Modeling; Molecular Conformation; Monitor; Motion; Mutate; Mutation; NMR Spectroscopy; Pathology; Pathway interactions; Pattern; Peptides; Pharmaceutical Preparations; Phosphorylation; Phosphotransferases; Population; Post-Translational Protein Processing; Protein Kinase; Regulation; Relative (related person); Relaxation; Resolution; Resources; Role; Sampling; Signal Transduction; Site; Solvents; Structure; Substrate Specificity; Testing; Thermodynamics; Time; Vertebral column; X-Ray Crystallography; base; computerized tools; design; flexibility; improved; in vitro Assay; insight; molecular dynamics; mutant; novel; research study; simulation; small molecule; supercomputer; tool; vector

DESCRIPTION (provided by applicant): Protein kinases are a family of key enzymes that regulate cellular function under healthy conditions and misregulate cellular function in diseased conditions, such as cancer. Protein kinases are complex, highly-regulated, and dynamic enzymes whose primary function is not to turn over substrate but rather to integrate biological signals to make targeted post-translational modifications (phosphorylation) in specific substrates. Since the catalytic core of kinases is structurally conserved across the whole family, kinase structure is not sufficient to provide precise control over activity and substrate specificity. Drug design against kinases is challenging because this conserved active site structure can lead to off-target binding, which takes drug away from the desired target and causes unforeseen side effects. In order to design novel drugs that target non-catalytic sites in the kinase and drugs that might operate by kinetic rather than thermodynamic control, it is imperative to understand how dynamics regulate function in kinases at mechanistic level, and how this dynamic regulation evolved. To predict how novel drugs might modulate kinase function, it is important to develop computational tools to observe how these dynamics are altered by perturbations such as mutations or substrate binding. To study functional kinase dynamics in detail, NMR spectroscopy will be combined with advanced conformational sampling methods that take advantage of commodity graphics processors to study slow motions relevant to catalysis. Markov State Model methods will be improved to interpret NMR chemical shifts and relaxation-dispersion data at an atomic level. A covariance of mechanical stress approach will be developed along with transfer entropy analysis in internal coordinates to identify mechanistic cause and effect in active site opening, the rate- limiting step in catalysis. Residues implicated by these analyses will be mutated, and the kinase's slow dynamics studied by NMR. The role of dynamics in substrate specificity will be studied by monitoring the substrate's effects on kinase dynamics - locally, and at distal substrate docking sites, using molecular dynamics simulations with and without substrate peptides. Dynamical changes caused by substrate peptide binding will be compared across multiple kinases to identify the conservation of dynamic coupling between the active site and a distal C-lobe substrate docking site, and to potentially guide design of novel drugs that could potentially have fewer off-target effects or that might alter a kinase's substrate specificity. PUBLIC HEALTH RELEVANCE: We will develop an approach that combines computational and experimental data to study how a signaling enzyme, protein kinase A, is regulated by dynamics. Our studies will help us understand how the kinase limits turnover under normal conditions and how mutations might cause pathogenic disregulation of kinase activity in disease. Mechanistic insights and tools developed in the course of this project are expected to be of general use in studying functionally-relevant conformational changes and dynamics and should be useful in guiding the discovery of novel small molecules targeting novel sites.

描述（由申请人提供）：蛋白激酶是一个关键酶家族，在健康状态下调节细胞功能，在疾病状态（如癌症）中调节细胞功能失调。蛋白激酶是一种复杂的、高度调控的动态酶，其主要功能不是翻转底物，而是整合生物信号，在特定底物中进行靶向翻译后修饰（磷酸化）。由于激酶的催化核心在整个家族中在结构上是保守的，因此激酶的结构不足以提供对活性和底物特异性的精确控制。针对激酶的药物设计具有挑战性，因为这种保守的活性位点结构可能导致脱靶结合，从而使药物远离预期的靶标并引起不可预见的副作用。为了设计针对激酶中非催化位点的新药，以及可能通过动力学而非热力学控制作用的药物，有必要了解动力学如何在机制水平上调节激酶的功能，以及这种动态调节是如何演变的。为了预测新药如何调节激酶功能，开发计算工具来观察这些动力学如何被突变或底物结合等扰动改变是很重要的。为了详细研究功能性激酶动力学，核磁共振波谱将与先进的构象采样方法相结合，利用商品图形处理器来研究与催化相关的慢动作。马尔可夫状态模型方法将得到改进，以在原子水平上解释核磁共振化学位移和弛豫-色散数据。机械应力的协方差方法将与内部坐标的传递熵分析一起发展，以确定活性位点打开的机械原因和影响，催化的限速步骤。这些分析所涉及的残基将发生突变，并通过核磁共振研究激酶的缓慢动力学。动力学在底物特异性中的作用将通过监测底物对激酶动力学的影响来研究-局部和远端底物对接位点，使用带和不带底物肽的分子动力学模拟。由底物肽结合引起的动态变化将在多个激酶之间进行比较，以确定活性位点和远端c叶底物对接位点之间的动态偶联性，并可能指导设计可能具有较少脱靶效应或可能改变激酶底物特异性的新药。