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

描述（由申请人提供）：内源性大麻素（eCB）信号系统有助于调节多种生理过程。两种主要的大麻素受体，CB1和CB2，是a类G蛋白偶联受体（gpcr），受外源性天然大麻素（例如，植物大麻素delta9-tetrahydrocannabinol）和eCBs（包括n-花生四烯醇乙醇胺（“anandamide”）（AEA）和2-花生四烯醇甘油（2-AG））的刺激。CB受体和CB生物合成和代谢酶的组织分布和综合活性是通过空间和时间划分CB生物活性的稳态eCB信号传导的关键。CB1主要存在于中枢神经系统，其激活介导大多数CB1的精神和行为作用。CB2在大脑中的表达水平非常低，主要通过免疫能力细胞和造血细胞、破骨细胞和成骨细胞在外周表达，介导免疫反应、炎症、炎症性和神经性疼痛以及骨重塑。eCBs在各种刺激下按需产生，并通过酶水解迅速失活：主要由脂肪酸酰胺水解酶（FAAH）和主要由单酰基甘油脂肪酶（MGL）水解的AEA。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CB1传递与许多疾病状态有关，包括药物成瘾、药物滥用障碍、超重/肥胖和肥胖相关的心脏代谢风险（代谢综合征）。一种CB1拮抗剂已作为减肥剂进入市场，尽管在临床中存在不良反应。小分子激动剂激活CB2可能对疼痛和神经炎性疾病（如阿尔茨海默病和亨廷顿病）具有治疗前景。这样的翻译应用使得在分子水平上对CB受体激活机制的透彻理解对于旨在调节CB受体传递以获得治疗增益的药物发现至关重要。在没有它们的晶体结构的情况下，基于视紫红质的x射线晶体结构对CB1和CB2进行了同源性建模。a类gpcr的总体结构具有细胞外取向的n端、细胞内羧基端和7个跨膜1-螺旋的逆时针排列，这些1-螺旋横跨细胞膜，由3个细胞外环和3个细胞质环连接。与视紫红质类似，CB2的激活涉及跨膜（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。视紫红质也含有从TM螺旋7延伸而来的细胞质螺旋8 (H8)，但其参与其他gpcr的激活，包括CB2，尚未得到很好的证实。申请人假设螺旋6、7和8对CB2激活至关重要。本提案中详细的实验旨在提供支持性的实验证据，通过使用CD和溶液以及固态核磁共振的组合来定义这些螺旋在CB2活化后所经历的结构变化。代表TM螺旋6和TM螺旋6-7/H8的适当标记肽将在大肠杆菌中表达，纯化，并在溶液和确定的脂质环境（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）先前被证明与CB2半胱氨酸257在TM螺旋6中特异性共价反应并激活受体，将被用作亲和探针。将应用分子模型来增强实验结果。所得到的数据将为未来阐明其他CB2螺旋结构域参与CB激活的工作奠定基础，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。

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.