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

描述（由申请人提供）：内源性大麻素（CB）系统，即CB受体（CB1和CB2亚型），内源性配体和用于CB配体代谢的酶的发现，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。众所周知，CB2受体参与免疫系统的信号转导级联反应，开发无cb1相关精神药物副作用的CB2药物具有巨大的治疗潜力，可用于治疗慢性神经痛、神经元疾病、自身免疫性疾病、胶质瘤和其他免疫源性肿瘤，从而使数百万患有自身免疫性/免疫相关疾病的患者受益。然而，多年来，对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，确定配体结合和g蛋白偶联的特定位点，而对实验三维CB结构的研究有限，特别是对CB2受体的研究。为了了解这些药理和生化事件背后的分子机制，重要的是表征接触点和结合域，然后在分子结构水平上阐明CB2/配体识别和CB2/ g蛋白偶联机制。在我们发表的和初步研究中，我们已经成功地研究了几个CB2蛋白功能域的结构和构象特征；CB配体结构及活性药理特征；以及CB2受体的激动剂/拮抗剂识别位点。然而，CB2受体的结构-功能关系以及CB2配体和g蛋白的识别机制仍存在许多问题。本研究的目的是通过生物物理和生物化学相结合的方法，鉴定/表征CB2受体中激动剂和拮抗剂结合以及g蛋白偶联识别的关键残基/功能域，并阐明其识别袋的三维结构。我们的长期目标是在结构和功能方面了解人类CB2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CB2配体设计。在完成“概念验证”的研究工作后，我们提出了具体目标和深入研究的三个目标。目的1：表征对CB2配体识别重要的功能域和关键残基，并推导跨膜和细胞外段激动剂/拮抗剂结合域的结构决定因素。目标2：通过在目标1中开发和验证的生物物理方法，研究和定义涉及g蛋白偶联和细胞内细胞信号传导的重要CB2细胞内片段和关键残基的结构和功能特征。目的3：探索全长CB2受体，确认目标1和目标2中确定的关键残基，并通过功能结合试验和天然CB2受体的定点突变验证其对CB2配体识别和g蛋白偶联的重要性。我们建立的计算机模型和受体对接算法将进一步研究已阐明的CB2激动剂/拮抗剂识别口袋和g蛋白偶联结构域。我们的研究和结果将有助于更好地了解CB2的结构/功能及其作用机制，并为未来设计CB2特异性药物提供结构基础。从提议的CB2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。

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.