Structural and pharmacological dynamics of KCNQ2: A cryoEM and biophysics study

KCNQ2 的结构和药理学动力学：冷冻电镜和生物物理学研究

• 批准号: 9900049
• 负责人: Michael Clark
• 金额: $ 5.05万
• 依托单位: UNIVERSITY OF CHICAGO
• 依托单位国家: 美国
• 财政年份: 2018
• 资助国家: 美国
• 起止时间: 2018-04-01 至 2021-03-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/9900049
• 关键词: Anticonvulsants; Antidepressive Agents; Antipsychotic Agents; Arachidonic Acids; Attention deficit hyperactivity disorder; Behavior; Binding; Biochemical; Biochemistry; Biological Assay; Bipolar Disorder; Brain; Calmodulin; Calorimetry; Cell membrane; Cells; Charge; Clinical; Complex; Coupled; Coupling; Cryoelectron Microscopy; Data; Dependence; Detergents; Development; Drug Screening; Drug Targeting; Entropy; Environment; Fluorescence; Future; Genes; Human; Image; In Vitro; Ion Channel; Ion Channel Gating; Ion Channel Protein; Learning Disabilities; Link; Lipids; Liposomes; Major Depressive Disorder; Maps; Measures; Membrane Potentials; Mental disorders; Micelles; Modeling; Molecular; Molecular Conformation; Movement; Mus; Muscarinic Acetylcholine Receptor; Muscarinics; Mutation; Nervous system structure; Neurons; Pharmaceutical Preparations; Pharmacology; Pharmacology Study; Phosphatidylinositol 4,5-Diphosphate; Physiological; Play; Polyunsaturated Fatty Acids; Potassium Channel; Predisposition; Preparation; Proteins; Protons; Reporting; Research; Resolution; Rest; Role; Schizophrenia; Signal Transduction; Site; Structure; Technical Expertise; Techniques; Testing; Thermodynamics; Titrations; Work; Xenopus; Zinc; autism spectrum disorder; base; biophysical analysis; cholinergic; design; drug development; drug discovery; enthalpy; experimental study; high throughput screening; human data; improved; inhibitor/antagonist; insight; mutant; nanodisk; neuronal excitability; novel; novel therapeutics; patch clamp; programs; reconstitution; resilience; screening; small molecule; structured data; therapeutic target; voltage

PROJECT SUMMARY / ABSTRACT In the brain, KCNQ2 – KCNQ5 co-assemble to form the M-channel, which regulates neuronal excitability and is a high-impact therapeutic target in several mental disorders, including Major Depressive Disorder, Schizophrenia, Bipolar Disorder and Attention-Deficit Hyperactivity Disorder. Physiologically, the M-channel is responsible for dynamic control of the resting membrane potential. Cholinergic signaling through muscarinic receptors results in depletion of plasma membrane PIP2, which in turn results in closure of the M-channel. A direct PIP2 – channel interaction is required to open the pore domain of KCNQ channels, however the structural basis of this interaction is unknown. This work will determine the activated-state structure of human KCNQ2 bound to calmodulin (CaM) and PIP2 in a lipid environment, using cryo electron microscopy. In a general sense, this structure will expand the understanding of ion channel activation mechanism. In a specific sense, the structure of activated KCNQ2-CaM will be a valuable asset to structure-guided development of KCNQ2 activators, targeting the above-mentioned mental disorders. In addition to structural studies, this proposal seeks to characterize the pharmacology of KCNQ2, elucidating the state-dependent thermodynamics and cooperativity of known KCNQ2 small molecule activators using isothermal titration calorimetry. The interaction enthalpies and entropies measured will report on how these small molecule activators engage their target, KCNQ2, and can inform future drug development efforts to improve the potency of these compounds. Finally, this project will develop and validate direct, in vitro, high-throughput assays for KCNQ2 activators and inhibitors. The assays will be based on a standard liposome flux fluorescence assay (LFFA) that be adapted to study KCNQ2 without and with PIP2 in order to test activators and inhibitors, respectively. These assays will be validated with known KCNQ2 activators and inhibitors, and will significantly expand the current arsenal of KCNQ2 drug screening techniques. In support of these proposed studies, robust biochemical preparations of KCNQ2-CaM and KCNQ1-CaM have been developed, and preliminary cryoEM data on KCNQ1-CaM have been collected. Additionally, the applicant has developed technical expertise in cryoEM while engaged in studies of an archaeal potassium channel, and has conducted LFFAs for a human proton channel. The structural, functional and pharmacological studies proposed here will yield insights into the molecular mechanisms of neuronal function, as well as directly inform future drug development efforts.

项目总结/摘要 在大脑中，KCNQ 2-KCNQ 5共同组装形成M通道，其调节神经元兴奋性， 是几种精神疾病的高影响力治疗靶点，包括重度抑郁症， 精神分裂症，双相情感障碍和注意力缺陷多动障碍。从生理学上讲，M通道是 负责动态控制静息膜电位。胆碱能信号通过毒蕈碱 受体的释放导致质膜PIP 2的消耗，这又导致M通道的关闭。一 需要直接的PIP 2通道相互作用来打开KCNQ通道的孔域，然而， 这种相互作用的结构基础是未知的。这项工作将确定人类的激活态结构 KCNQ 2结合钙调蛋白（CaM）和PIP 2在脂质环境中，使用冷冻电子显微镜。中 从一般意义上讲，这种结构将扩大对离子通道激活机制的理解。以特定 从某种意义上说，激活的KCNQ 2-CaM结构将是结构导向开发的宝贵资产， KCNQ 2激活剂，针对上述精神障碍。除了结构研究， 一项提案试图描述KCNQ 2的药理学特征，阐明状态依赖热力学 和已知KCNQ 2小分子活化剂的协同性。的 测量的相互作用熵和熵将报告这些小分子活化剂如何与它们的 目标，KCNQ 2，并可以告知未来的药物开发工作，以提高这些化合物的效力。 最后，该项目将开发和验证KCNQ 2激活剂的直接，体外，高通量测定， 抑制剂的该试验将基于标准脂质体通量荧光试验（LFFA），适用于 研究KCNQ 2与PIP 2，以测试激活剂和抑制剂分别。这些试验将 用已知的KCNQ 2激活剂和抑制剂进行了验证，并将显着扩大目前的药物库， KCNQ 2药物筛选技术。为了支持这些拟议的研究， KCNQ 2-CaM和KCNQ 1-CaM已经被开发出来，KCNQ 1-CaM的初步cryoEM数据已经被 被收集。此外，申请人在从事冷冻EM的同时， 研究古细菌钾通道，并进行了人类质子通道的LFFA。的 结构，功能和药理学研究提出这里将产生深入的分子 神经元功能的机制，以及直接告知未来的药物开发工作。