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多种生理过程。两种主要的大麻素（CB）受体，命名为CB 1和CB 2，是由外源性天然大麻素（例如，植物大麻素δ 9-四氢大麻酚）和eCB，包括N-花生四烯酰乙醇胺（“花生四烯酰胺”）（AEA）和2-花生四烯酰甘油（2-AG）。CB受体和eCB生物合成和代谢酶的组织分布和整合活性是通过在空间和时间上限定eCB生物活性来实现稳态eCB信号传导的关键。CB 1主要存在于中枢神经系统中，其激活介导大多数CB精神和行为效应。在脑中以非常低的水平，CB 2主要在外周中由免疫活性细胞和造血细胞、破骨细胞和成骨细胞表达，并介导免疫应答、炎症、炎性和神经性疼痛以及骨重塑。eCB响应于各种刺激按需产生，并通过酶水解快速灭活：AEA，主要通过脂肪酸酰胺水解酶（FAAH），和2-AG，主要通过单酰基甘油脂肪酶（MGL）。内源性大麻素信号的变化伴随着各种生理和病理过程。过度活跃的CB 1传递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）。CB 1拮抗剂已作为减肥剂进入市场，尽管在临床上与副作用相关。通过小分子激动剂激活CB 2可能具有治疗疼痛和神经炎性疾病（例如，阿尔茨海默病和亨廷顿病）。这样的翻译应用使得在分子水平上对CB受体活化的机制有了透彻的理解，这对于旨在调节CB受体传递以获得治疗效果的药物发现是必不可少的。在没有晶体结构的情况下，CB 1和CB 2的同源性建模已经基于视紫红质的X射线晶体结构进行。A类GPCR的一般结构的特征在于细胞外定向的N-末端、细胞内羧基末端和跨越细胞膜并通过三个细胞外环和三个细胞质环连接的七个疏水跨膜1-螺旋的逆时针排列。与视紫红质类似，CB 2的活化已经被提出涉及跨膜（TM）螺旋3中的盐桥的破坏以及TM螺旋6和7的构象的改变。视紫红质还含有从TM螺旋7延伸的细胞质螺旋8（H8），但其参与其他GPCR（包括CB 2）的活化尚未充分确立。申请方假设螺旋6、7和8对CB 2激活至关重要。本提案中详细描述的实验旨在提供支持性实验证据，这些证据将通过使用CD和溶液以及固态NMR的组合来定义CB 2激活后这些螺旋所经历的结构变化。代表TM螺旋6和TM螺旋6-7/H8的适当标记的肽将在E.大肠杆菌，纯化，并在溶液中和定义的脂质环境（胶束，磷脂双层）中研究。一种新的，高亲和力的大麻能激动剂（AM 841）以前被证明与TM螺旋6中的CB 2半胱氨酸257特异性和共价反应，并激活受体将被用作亲和探针。分子建模将被应用于增强实验结果。由此产生的数据将形成未来工作的基础，以阐明CB激活中的其他CB 2螺旋结构域的参与，并为安全有效的CB 2选择性激动剂作为候选药物的设计提供信息。 公共卫生相关性：目前的建议将提供结构和动态信息的跨膜多肽的人CB 2受体。与配体接触的识别残基之间的特定取向和精确距离的知识，以及多肽-配体复合物的构象，将有助于优化大麻素受体的结合特性和配体的选择。因此，预计拟议的工作将提供具有治疗潜力的重要生物医学发现。

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.