Structure/Function of the CB2 Receptor Binding and G-protein Recognition Pockets

CB2 受体结合和 G 蛋白识别袋的结构/功能

• 批准号: 8035975
• 负责人: Xiang-Qun Xie
• 金额: $ 33.06万
• 依托单位: UNIVERSITY OF PITTSBURGH AT PITTSBURGH
• 依托单位国家: 美国
• 财政年份: 2010
• 资助国家: 美国
• 起止时间: 2010-04-01 至 2015-03-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8035975
• 关键词: Adenylate Cyclase; Adverse effects; Affinity; Agonist; Algorithms; Anti-Obesity Agents; Appetite Depressants; Autoimmune Diseases; Autoimmune Process; Binding; Binding Sites; Biochemical; Biological Assay; C-terminal; CNR2 gene; Cannabinoids; Cell Signaling Process; Chemicals; Chronic; Complication; Computer Simulation; Coupled; Coupling; Data; Depression and Suicide; Disease; Docking; Drug Delivery Systems; Drug Design; Enzymes; European; Event; Fluorescence; Future; G Protein-Coupled Receptor Genes; G Protein-Coupled Receptor Signaling; G-Protein-Coupled Receptors; GTP-Binding Proteins; Glioma; Goals; Guanosine Triphosphate Phosphohydrolases; Human; Immune; Immune system; Isotope Labeling; Length; Ligand Binding; Ligands; Light; Marketing; Mediating; Metabolism; Methods; Molecular; Mutagenesis; Mutation; Neurons; Outcomes Research; Pain; Patients; Pharmaceutical Preparations; Pilot Projects; Proteins; Publishing; Recombinants; Reporting; Research; Research Design; Resolution; Roentgen Rays; Signal Transduction; Site; Specificity; Structure; Structure-Activity Relationship; System; Techniques; Therapeutic; Therapeutic Studies; Transmembrane Domain; Validation; Work; base; cannabinoid receptor; design; drug discovery; endogenous cannabinoid system; experience; extracellular; interest; novel; pharmacophore; protein expression; public health relevance; receptor; receptor binding; receptor structure function; research study; rimonabant; three dimensional structure; tumor

DESCRIPTION (provided by applicant): The discovery of the endogenous cannabinoid (CB) system, i.e. the CB receptors (the subtypes CB1 and CB2), endogenous ligands and enzymes for CB ligand metabolism, has triggered intensive pharmacological research into the CB receptors and the therapeutic potential of cannabinergic ligands. The CB2 receptor is known to be involved in the signal transduction cascades in the immune system, and has a great therapeutic potential for developing CB2 drugs without CB1-related psychotropic side effects for treatment of chronic neuro-pain, neuronal disorders, autoimmune diseases, and gliomas and other tumors of immune origin, thereby benefiting the millions of patients who suffer from the autoimmune/immune-related diseases for which we have no cure. Over the years, however, structural and functional studies of CB receptors have focused on predicting structures by computer modeling, identification of the specific sites for ligand binding and G-protein coupling whereas studies on experiment 3D CB structures are limited, in particular for the CB2 receptor. To understand the molecular mechanism behind these pharmacological and biochemical events, it is important to characterize the contacting points and binding domains and then elucidate the specific CB2/ligand recognition and CB2/G-protein coupling mechanisms at molecular structural level. In our published and pilot studies, we have successfully investigated the structural and conformational features of several CB2 protein functional domains; CB ligand structures and active pharmacophoric features; and agonist/antagonist recognition sites in the CB2 receptor. However, many questions still remain about CB2 receptor structure-function relationship as well as CB2 ligands and G-protein recognition mechanisms. The objective of this proposal is to identify/characterize the key residues/functional domains and elucidate their 3D structures of recognition pockets important to agonist and antagonist binding as well as G-protein coupling recognitions in the CB2 receptor by the combined biophysical and biochemical approaches. Our long term goal is to understand, in structural and functional terms, the molecular mechanisms of human CB2 activation and G-protein cell signaling process in order to facilitate the structure-based design for novel CB2 ligands. Having completed the "proof-of-concept" research work, we propose the specific goals and in-depth research to three aims. Aim 1: Characterize the functional domains and key residues important to the CB2 ligand recognition and derive the structural determinants of the agonist/antagonist binding domains in the transmembrane and extra-cellular segments. Aim 2: Investigate and define the structural and functional features of the important CB2 intracellular segments and key residues involving G-protein coupling and intracellular cell signaling by the biophysical approaches developed and validated in Aim 1. Aim 3: Explore full-length CB2 receptor and confirm the key residues determined in Aims 1 and 2 and verify their importance to CB2 ligand recognition and G-protein coupling by functional binding assays and site-directed mutations of the native CB2 receptor. The elucidated CB2 agonist/antagonist recognition pockets and G-protein coupling domains will be further examined by our established computer modeling and receptor docking algorithms. Our proposed research and the outcomes will shed light onto a better understanding of CB2 structure/function and its mechanism of actions, and provide the structural bases for CB2-specific drug design in future. The techniques and methods developed from the proposed CB2 receptor research will also have a significant impact to other GPCRs. PUBLIC HEALTH RELEVANCE: This proposal is to identify and elucidate their 3D structures of recognition pockets important to agonist and antagonist binding as well as G-protein coupling recognitions in the cannabinoid CB2 receptor. The long-term benefits will allow us to better understand, in structural and functional terms, the molecular mechanisms of human CB2 activation and G-protein cell signaling process in order to facilitate structure-based design for novel CB2 chemical probes.

描述（由申请人提供）：内源性大麻素（CB）系统（即CB受体（亚型CB 1和CB 2）、内源性配体和CB配体代谢酶）的发现引发了对CB受体和大麻能配体治疗潜力的深入药理学研究。已知CB 2受体参与免疫系统中的信号转导级联，并且具有开发用于治疗慢性神经疼痛、神经元病症、自身免疫性疾病和神经胶质瘤以及其他免疫源性肿瘤的没有CB 1相关精神病副作用的CB 2药物的巨大治疗潜力，从而使数百万患有我们无法治愈的自身免疫/免疫相关疾病的患者受益。然而，多年来，CB受体的结构和功能研究主要集中在通过计算机建模预测结构，识别配体结合和G蛋白偶联的特异性位点，而对实验3D CB结构的研究有限，特别是对于CB 2受体。为了理解这些药理学和生物化学事件背后的分子机制，重要的是表征接触点和结合结构域，然后在分子结构水平上阐明特异性CB 2/配体识别和CB 2/G蛋白偶联机制。在我们发表的和试点研究中，我们已经成功地研究了几个CB 2蛋白功能域的结构和构象特征; CB配体结构和活性药效特征;以及CB 2受体中的激动剂/拮抗剂识别位点。然而，关于CB 2受体的结构与功能的关系、CB 2配体和G蛋白识别机制仍存在许多问题。本提案的目的是通过生物物理和生物化学相结合的方法来鉴定/表征CB 2受体中的关键残基/功能结构域，并阐明其对激动剂和拮抗剂结合以及G蛋白偶联抑制重要的识别口袋的3D结构。我们的长期目标是从结构和功能上了解人CB 2激活和G蛋白细胞信号传导过程的分子机制，以促进基于结构的新型CB 2配体的设计。在完成了概念验证研究工作后，提出了具体目标，并对三个目标进行了深入研究。目标1：表征对CB 2配体识别重要的功能结构域和关键残基，并推导跨膜和细胞外片段中激动剂/拮抗剂结合结构域的结构决定簇。目标二：研究和定义重要的CB 2细胞内片段和关键残基的结构和功能特征，涉及G-蛋白偶联和细胞内细胞信号转导的生物物理方法开发和验证的目的1。目标3：探索全长CB 2受体并确认目标1和2中确定的关键残基，并通过功能性结合试验和天然CB 2受体的定点突变验证其对CB 2配体识别和G蛋白偶联的重要性。阐明CB 2激动剂/拮抗剂识别口袋和G蛋白偶联结构域将进一步检查我们建立的计算机建模和受体对接算法。我们的研究成果将有助于更好地了解CB 2的结构/功能及其作用机制，并为未来CB 2特异性药物设计提供结构基础。从拟议的CB 2受体研究中开发的技术和方法也将对其他GPCR产生重大影响。 公共卫生相关性：这个建议是确定和阐明他们的三维结构的识别口袋重要的激动剂和拮抗剂的结合，以及G-蛋白偶联大麻素CB 2受体的抑制。长期的好处将使我们能够更好地了解，在结构和功能方面，人CB 2激活和G蛋白细胞信号传导过程的分子机制，以促进基于结构的新型CB 2化学探针的设计。