Impact of membrane composition on cholecystokinin receptor structure and function

膜成分对胆囊收缩素受体结构和功能的影响

基本信息

批准号：
10541873
负责人：
LAURENCE J MILLER
金额：
$ 46.16万
依托单位：
MAYO CLINIC ARIZONA
依托单位国家：
美国
项目类别：
财政年份：
2022
资助国家：
美国
起止时间：
2022-01-01 至 2025-11-30
项目状态：
未结题

来源：
https://reporter.nih.gov/project-details/10541873
关键词：
Acute Address Affect Afferent Neurons Affinity Agonist Binding Biology Cell membrane Cholecystokinin Cholecystokinin Receptor Cholesterol Chronic Clinical Medicine Clinical Trials Collaborations Complex Coupling Cryoelectron Microscopy Data Development Disease Docking Drug Receptors Drug Targeting Environment Event Exhibits Face Fluorescence G-Protein-Coupled Receptors GTP-Binding Proteins Heterotrimeric GTP-Binding Proteins Hormones Investigation Kinetics Knowledge Lateral Ligand Binding Ligands Link Lipids Mediating Membrane Membrane Lipids Methodology Molecular Molecular Conformation Obesity Occupations Peptides Persons Pharmaceutical Preparations Pharmacology Photoaffinity Labels Physiological Population Prevention Process Public Health Refractory Regulation Research Personnel Resistance Resolution Satiation Scaffolding Protein Series Signal Transduction Sincalide Site Stimulus Structure System Texture Therapeutic Therapeutic Effect Toxic effect Work antagonist dieting drug development drug discovery experience extracellular feeding genetic regulatory protein insight molecular site mutant negative affect novel obese person obesity treatment patient subsets receptor receptor function recruit response screening side effect skills structural biology therapeutic target trafficking

项目摘要

PROJECT SUMMARY/ABSTRACT Cholecystokinin acts on type 1 receptors (CCK1Rs) present on vagal afferent neurons to regulate satiety, important in prevention and treatment of obesity. While CCK1R agonists can acutely reduce feeding, such agents with high potency and long duration of action tend to be associated with side effects and potential toxicity not tolerated for chronic therapy of healthy people. It is also now clear that a subset of the population is refractory to effects of CCK agonists, due to impact of membrane cholesterol on receptor conformation and dysfunctional stimulus-activity coupling, likely negatively affecting previous clinical trials. Our long term objective is to target this receptor in a safer and more effective way. The underlying hypothesis is that lack of mechanistic understanding of interplay between disease states (obesity) and receptor function, and under- appreciation of novel modes of GPCR drug targeting are key barriers to realizing the therapeutic potential of CCK1R drugs. Efforts will focus on acquiring molecular understanding of how the membrane environment affects CCK1R structure and function, and utilizing these insights to pursue opportunities for allosteric modulation to correct negative impact, and for ligand-directed bias to sculpt signaling and regulatory responses to activators of this therapeutic target. We will utilize a strong, well-established collaboration, reflecting the highly complementary skills and experience of Drs. Miller and Sexton. Aim 1 provides a coherent assessment of signaling and regulatory events initiated by stimulation of CCK1R with orthosteric agonists and allosteric drugs, and impact of membrane lipids on these events. This provides the opportunity to link distinct ligand pharmacologies to impact effector engagement, regulatory protein recruitment, control of G protein efficacy, and receptor sequestration and trafficking, as well as how allosteric cooperativity between sites of molecular interaction can modify these events. This aim also includes quantification of kinetics of agonist ligand binding and the events of the G protein cycle that are interdependent, yielding aberrant stimulus-activity coupling with high binding affinity and reduced signaling at CCK1R in elevated cholesterol. Aim 2 explores the physical basis for this process, focusing on the site of lipid interaction outside the receptor helical bundle, and its impact on the mode of natural peptide ligand docking at the ectodomain of this receptor. These studies will utilize focused chimeric CCK1R:CCK2R constructs, as well as a series of site-directed mutants, and photoaffinity labeling and fluorescence probing. Aim 3 provides a structural framework for understanding aberrant CCK stimulus-activity coupling at CCK1R, utilizing our recent structural breakthroughs in determination of active- state structures of agonist-occupied CCK1R in complex with heterotrimeric G proteins using cryo-EM. These studies provide a unique paradigm for the investigation of potential drug targets that are affected by the composition of the plasma membrane in which they reside.

项目概要/摘要胆囊收缩素作用于迷走神经传入神经元上的 1 型受体 (CCK1R) 来调节饱腹感对于预防和治疗肥胖很重要。虽然 CCK1R 激动剂可以急剧减少进食，此类高效且作用持续时间长的药物往往会产生副作用和潜在的副作用。健康人的长期治疗无法耐受毒性。现在也清楚的是，人口的一个子集是由于膜胆固醇对受体构象的影响，对 CCK 激动剂的作用无效功能失调的刺激-活动耦合，可能会对之前的临床试验产生负面影响。我们的长期目标是以更安全、更有效的方式靶向该受体。基本假设是缺乏对疾病状态（肥胖）和受体功能之间相互作用的机制理解，以及对 GPCR 药物靶向新模式的认识是实现 GPCR 治疗潜力的关键障碍 CCK1R药物。努力的重点是获得对膜环境如何进行分子理解。影响 CCK1R 结构和功能，并利用这些见解来寻求变构机会调节以纠正负面影响，并通过配体定向偏差来塑造信号传导和监管反应该治疗靶点的激活剂。我们将利用强大、完善的合作，反映博士的技能和经验高度互补。米勒和塞克斯顿。目标 1 提供连贯的评估由正构激动剂和变构刺激 CCK1R 引发的信号传导和调节事件药物，以及膜脂对这些事件的影响。这提供了连接不同配体的机会影响效应器参与、调节蛋白募集、G蛋白功效控制的药理学，和受体隔离和运输，以及分子位点之间的变构协同作用如何交互可以修改这些事件。该目标还包括激动剂配体结合动力学的量化和 G 蛋白循环的事件是相互依赖的，产生与在胆固醇升高时，CCK1R 具有高结合亲和力并减少信号传导。目标 2 探索物理该过程的基础，重点关注受体螺旋束外部脂质相互作用的位点及其影响关于天然肽配体对接在该受体的胞外域的模式。这些研究将利用聚焦嵌合 CCK1R:CCK2R 构建体，以及一系列定点突变体和光亲和力标记和荧光探测。目标 3 提供了理解异常 CCK 的结构框架 CCK1R 的刺激-活动耦合，利用我们最近在确定活性- 使用冷冻电镜观察激动剂占据的 CCK1R 与异三聚体 G 蛋白复合物的状态结构。这些研究为研究受药物影响的潜在药物靶标提供了独特的范例它们所在的质膜的组成。