Mechanical and Dynamical Regulation of Protein Kinases

蛋白激酶的机械和动态调节

基本信息

批准号：
8588944
负责人：
Christopher Lee McClendon
金额：
$ 3.58万
依托单位：
UNIVERSITY OF CALIFORNIA, SAN DIEGO
依托单位国家：
美国
项目类别：
财政年份：
2011
资助国家：
美国
起止时间：
2011-12-01 至 2014-07-11
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/8588944
关键词：
Active Sites Address Adverse effects Agreement Binding Biological Catalysis Catalytic Domain Cell physiology Chemicals Complex Computer Simulation Computing Methodologies 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 biophysical techniques computerized tools design flexibility improved in vitro Assay insight molecular dynamics mutant novel research study simulation small molecule supercomputer tool vector

项目摘要

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）：蛋白激酶是一个关键酶家族，其在健康条件下调节细胞功能，并在疾病条件（例如癌症）下错误调节细胞功能。蛋白激酶是复杂的、高度调控的动态酶，其主要功能不是翻转底物，而是整合生物信号以在特定底物中进行有针对性的翻译后修饰（磷酸化）。由于激酶的催化核心在整个家族中结构保守，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能导致脱靶结合，从而使药物远离所需靶标并导致不可预见的副作用。为了设计针对激酶中非催化位点的新药物和可能通过动力学而不是热力学控制起作用的药物，必须了解动力学如何在机械水平上调节激酶的功能，以及这种动态调节是如何演变的。为了预测新药物如何调节激酶功能，开发计算工具来观察突变或底物结合等扰动如何改变这些动力学非常重要。为了详细研究功能激酶动力学，核磁共振波谱将与先进的构象采样方法相结合，利用商用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 NMR 化学位移和弛豫色散数据。将开发机械应力方法的协方差以及内坐标中的传递熵分析，以识别活性位点开放（催化中的限速步骤）的机械原因和影响。这些分析涉及的残基将发生突变，并且通过 NMR 研究激酶的缓慢动力学。将通过使用有或没有底物肽的分子动力学模拟在局部和远端底物对接位点监测底物对激酶动力学的影响来研究动力学在底物特异性中的作用。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动态耦合的守恒性，并有可能指导新药物的设计，从而减少脱靶效应或可能改变激酶的底物特异性。