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）：蛋白质激酶是一个关键酶的家族，在健康条件下调节细胞功能并在患病（例如癌症）中不良细胞功能。蛋白激酶是复杂的，高度调节的和动态酶的，其主要功能不是要翻转底物，而是整合生物学信号以在特定底物中进行靶向的翻译后修饰（磷酸化）。由于激酶的催化核心在整个家庭中都是结构保守的，因此激酶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以导致脱靶结合，从而使药物脱离所需的靶标，并导致不可预见的副作用。为了设计新型药物，这些药物靶向激酶中的非催化位点以及可能通过动力学而不是热力学控制作用的药物，必须了解动力学如何在机械水平下调节激酶的功能，以及这种动态调节如何演变。为了预测新型药物如何调节激酶功能，重要的是要开发计算工具，以观察这些动力学如何通过扰动（例如突变或底物结合）改变。为了详细研究功能激酶动力学，NMR光谱将与先进的构象采样方法相结合，这些方法利用商品图形处理器来研究与催化有关的缓慢运动。马尔可夫州模型方法将得到改进，以解释原子水平的NMR化学位移和放松分散数据。机械应力方法的协方差将与内部坐标中的转移熵分析一起开发，以识别活动现场开放中的机理原因和效果，这是催化的速率限制步骤。这些分析与这些分析有关的残基将被突变，并且NMR研究了激酶的缓慢动力学。通过使用有或没有底物肽的分子动力学模拟，将研究动力学在底物特异性中的作用 - 在局部和远端底物对接位点上对底物对激酶动力学的影响进行研究。将在多种激酶之间进行比较由底物肽结合引起的动态变化，以确定活性位点和远端C-Lobe底物对接位点之间动态耦合的保存，并有潜在地指导新型药物的设计，而新药物的设计可能会更少具有偏外目标效果或可能会改变激酶的底物特异性。 公共卫生相关性：我们将开发一种结合计算和实验数据的方法，以研究信号传导酶（蛋白激酶A）如何受动力学调节。我们的研究将有助于我们了解激酶在正常条件下如何限制周转，以及突变如何导致疾病中激酶活性的致病性疏离。在该项目过程中开发的机械洞察力和工具预计将在研究功能相关的构象变化和动态方面普遍使用，并且对于指导发现针对新地点的新型小分子应该有用。