Ligand-Assisted Structural Studies of the Human Cannabinoid Receptor 2, Using NMR

描述(申请人提供)：内源性大麻素(ECB)信号系统有助于调节不同的生理过程。两种主要的大麻素受体，分别命名为CB1和CB2，是由外源天然大麻素(如植物大麻素Delta9-四氢大麻酚)和ECB刺激的A-G类蛋白偶联受体(GPCRs)，包括N-花生四氢乙醇胺(AEA)和2-花生四氢甘油(2-AG)。CB受体和ECB生物合成代谢酶的组织分布和整合活性是通过在空间和时间上界定ECB生物活性而实现动态平衡ECB信号的关键。CB1主要存在于中枢神经系统，它的激活介导了大多数CB的精神和行为效应。在脑组织中，CB2的表达水平很低，主要由免疫活性细胞和造血细胞、破骨细胞和成骨细胞表达，并介导免疫反应、炎症、神经性疼痛和骨重建。ECB是根据需要对各种刺激产生的，并通过酶解迅速失活：AEA，主要由脂肪酸酰胺水解酶(FAAH)和2-AG，主要由单酰甘油脂肪酶(MGL)。内源性大麻素信号的改变伴随着各种生理和病理过程。过度活跃的CB1传播与许多疾病状态有关，包括药物成瘾、物质滥用障碍、超重/肥胖和肥胖相关的心脏代谢风险(代谢综合征)。一种CB1拮抗剂已经作为减肥剂进入市场，尽管在临床上与不良反应有关。小分子激动剂激活CB2可能对疼痛和神经炎性疾病(例如阿尔茨海默病和亨廷顿病)具有治疗前景。这种翻译应用在分子水平上彻底了解了CB受体激活的机制，对于药物开发至关重要，目的是调节CB受体的传递以获得治疗收益。在没有晶体结构的情况下，基于视紫红质的X射线晶体结构，进行了CB1和CB2的同源模拟。A类GPCRs的一般结构特征是一个面向细胞外的N-末端，一个细胞内的羧基末端，以及七个跨越细胞膜的疏水性1-螺旋逆时针排列，并由三个细胞外和三个细胞质环连接。与视紫红质类似，CB2的激活被认为涉及跨膜(TM)螺旋3的盐桥的破坏以及TM螺旋6和7的构象改变。视紫红质也含有从TM螺旋7延伸出来的细胞质螺旋8(H8)，但它参与包括CB2在内的其他GPCR的激活还没有很好的确定。申请人假设螺旋6、7和8对CB2的激活是关键的。本提案中详细介绍的实验旨在提供支持的实验证据，这些证据将通过结合使用CD和溶液以及固态核磁共振来定义这些螺旋在CB2激活时经历的结构变化。代表TM螺旋6和TM螺旋6-7/H8的适当标记的多肽将在大肠杆菌中表达、纯化，并在溶液和特定的脂质环境(胶束、磷脂双层)中进行研究。一种新型的高亲和力大麻能激动剂(AM841)将被用作亲和探针，它能与TM螺旋6上的CB2半胱氨酸257特异地共价反应并激活受体。将应用分子模拟来增强实验结果。所得数据将为未来的工作奠定基础，以阐明其他CB2螺旋结构域参与CB激活，并为设计安全有效的CB2选择性激动剂作为候选药物提供信息。 公共卫生相关性：目前的提案将提供有关人类CB2受体跨膜多肽的结构和动态信息。了解与配体接触的已识别残基之间的特定方向和精确距离，以及多肽-配体复合体的构象，将有助于优化与大麻素受体的结合特性和配体的选择。因此，这项拟议的工作有望提供具有治疗潜力的重大生物医学发现。

DESCRIPTION (provided by applicant): The endocannabinoid (eCB) signaling system helps regulate diverse physiological processes. The two principal cannabinoid (CB) receptors, designated CB1 and CB2, are class-A G protein-coupled receptors (GPCRs) stimulated by exogenous natural cannabinoids (e.g., the plant cannabinoid delta9-tetrahydrocannabinol) and eCBs including N-arachidonoyl ethanolamine ("anandamide") (AEA) and 2-arachidonoylglycerol (2-AG). The tissue distribution and integrated activities of CB receptors and eCB biosynthetic and metabolizing enzymes are key to homeostatic eCB signaling by delimiting spatially and temporally eCB bioactivity. CB1 is predominantly found in the central nervous system, activation of which mediates most CB psychotropic and behavioral effects. At very low levels in brain, CB2 is expressed mainly in the periphery by immunocompetent and hematopoietic cells, osteoclasts, and osteoblasts and mediates immune responses, inflammation, inflammatory and neuropathic pain, and bone remodeling. eCBs are produced on-demand in response to various stimuli and are rapidly inactivated by enzymatic hydrolysis: AEA, primarily by fatty acid amide hydrolase (FAAH), and 2-AG, primarily by monoacylglycerol lipase (MGL). Changes in endocannabinoid signaling accompany various physiological and pathological processes. Hyperactive CB1 transmission has been implicated in a number of disease states including drug addiction, substance abuse disorders, overweight/obesity, and obesity-related cardiometabolic risk (metabolic syndrome). A CB1 antagonist has reached the market as a weight-loss agent, although associated in the clinic with adverse effects. Activation of CB2 by small-molecule agonists may hold therapeutic promise for pain and neuroinflammatory disorders (e.g., Alzheimer's and Huntington's diseases). Such translational applications make a thorough understanding of the mechanism of CB-receptor activation at the molecular level essential to drug discovery aimed at modulating CB receptor transmission for therapeutic gain. In the absence of their crystallographic structures, CB1 and CB2 homology modeling has been conducted based on the X-ray crystal structure of rhodopsin. The general architecture of class-A GPCRs has been characterized by an extracellularly oriented N-terminus, an intracellular carboxyl terminus, and a counterclockwise arrangement of seven hydrophobic transmembrane 1- helices spanning the cell membrane and connected by three extracellular and three cytoplasmic loops. By analogy with rhodopsin, activation of CB2 has been proposed to involve disruption of a salt bridge in transmembrane (TM) helix 3 as well as alterations in the conformation of TM helices 6 and 7. Rhodopsin also contains a cytoplasmic helix 8 (H8) which extends from TM helix 7, but its participation in the activation of other GPCRs, including CB2, is not well established. The applicant hypothesizes that helices 6, 7, and 8 are critical to CB2 activation. Experiments detailed in this proposal are designed to provide supporting experimental evidence that will define the structural changes these helices undergo upon CB2 activation by using a combination of CD and solution and solid-state NMR. Suitably labeled peptides representing TM helix 6 and TM helices 6-7/H8 will be expressed in E. coli, purified, and studied in solution and in defined lipid environments (micelles, phospholipid bilayers). A novel, high-affinity cannabinergic agonist (AM841) previously demonstrated to react specifically and covalently with CB2 cysteine 257 in TM helix 6 and activate the receptor will be used as affinity probe. Molecular modeling will be applied to augment the experimental results. The resulting data will form the basis for future work to elucidate the involvement of other CB2 helical domains in CB activation and inform the design of safe and effective CB2-selective agonists as drug candidates. PUBLIC HEALTH RELEVANCE: The current proposal will provide structural and dynamic information on the transmembrane polypeptides of the human CB2 receptor. Knowledge of the specific orientations and precise distances between identified residues in contact with the ligand, as well as the conformation of the polypeptide-ligand complex, will be helpful in optimizing the binding properties and selection of ligands to the cannabinoid receptor. Therefore, the proposed work is expected to provide significant biomedical findings with therapeutic potential.