Mechanical and Dynamical Regulation of Protein Kinases

蛋白激酶的机械和动态调节

• 批准号: 8409855
• 负责人: Christopher Lee McClendon
• 金额: $ 4.92万
• 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/8409855
• 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 Design; Drug Targeting; 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.

描述（由申请人提供）：蛋白激酶是在健康状况下调节细胞功能和在疾病状况（如癌症）下误调节细胞功能的关键酶家族。蛋白激酶是复杂的、高度调节的和动态的酶，其主要功能不是翻转底物，而是整合生物信号以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结构保守，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是具有挑战性的，因为这种保守的活性位点结构可能导致脱靶结合，这会使药物远离所需的靶标并导致不可预见的副作用。为了设计靶向激酶非催化位点的新型药物和可能通过动力学而不是热力学控制来操作的药物，必须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进化的。为了预测新药如何调节激酶功能，重要的是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所改变。为了详细研究功能性激酶动力学，NMR光谱学将与先进的构象采样方法相结合，该方法利用商品图形处理器来研究与催化相关的慢动作。将改进马尔可夫状态模型方法，以在原子水平上解释NMR化学位移和弛豫色散数据。将沿着与内坐标中的转移熵分析一起开发机械应力方法的协方差，以识别活性位点开放（催化中的速率限制步骤）中的机械原因和影响。这些分析所涉及的残基将被突变，并且激酶的缓慢动力学通过NMR研究。通过监测底物对激酶动力学的影响，将研究底物特异性中动力学的作用-局部和远端底物对接位点，使用有和无底物肽的分子动力学模拟。将在多种激酶中比较由底物肽结合引起的动态变化，以鉴定活性位点和远端C端半叶底物对接位点之间的动态偶联的保守性，并潜在地指导可能具有更少脱靶效应或可能改变激酶的底物特异性的新型药物的设计。