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

使用 NMR 进行人大麻素受体 2 配体辅助结构研究

• 批准号: 8015565
• 负责人: ELVIS K TIBURU
• 金额: $ 18.85万
• 依托单位: NORTHEASTERN UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2010
• 资助国家: 美国
• 起止时间: 2010-02-01 至 2013-01-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8015565
• 关键词: 2-arachidonylglycerol; Adverse effects; Affinity; Agonist; Alzheimer' s Disease; Architecture; Behavioral; Binding; Biochemical; Bone remodeling; Boston; Brain; CNR2 gene; Cannabinoids; Cell membrane; Cells; Clinic; Complementary DNA; Complex; Coupling; Cysteine; Cytoplasm; DNA Sequence Rearrangement; Data; Data Aggregation; Detergents; Disease; Drug Addiction; Drug or chemical Tissue Distribution; Endocannabinoids; Environment; Enzymes; Escherichia coli; Ethanolamines; Event; Future; G-Protein-Coupled Receptors; GTP-Binding Proteins; Hematopoietic; Homology Modeling; Human; Huntington Disease; Hydrolysis; Immune response; Immunocompetent; Inflammation; Knowledge; Label; Length; Ligands; Link; Lipids; Marketing; Mass Spectrum Analysis; Mediating; Medical; Membrane; Metabolic syndrome; Micelles; Molecular; Molecular Conformation; Molecular Models; Monoacylglycerol Lipases; Neuraxis; Obesity; Osteoblasts; Osteoclasts; Overweight; Pain; Pain management; Pathologic Processes; Peptides; Phospholipids; Physiological Processes; Plants; Property; Proteins; Receptor Activation; Relative (related person); Research; Rhodopsin; Risk; Roentgen Rays; Signal Transduction; Sodium Chloride; Solutions; Stimulus; Structure; Study models; Substance abuse problem; System; Techniques; Tetrahydrocannabinol; Therapeutic; Universities; Weight-Loss Drugs; Work; anandamide; base; cannabinoid receptor; design; drug candidate; drug discovery; extracellular; fatty acid amide hydrolase; inflammatory neuropathic pain; membrane model; molecular modeling; novel; polypeptide; professor; protein structure; public health relevance; receptor; research study; response; small molecule; solid state nuclear magnetic resonance; transmission process

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.

描述（由申请人提供）：内源性大麻素（ECB）信号系统有助于调节各种生理过程。指定为CB1和CB2的两个主要大麻素（CB）受体是由外源性天然大麻素（例如，植物大麻素Deltabinoid deltabinoid deltahyarahabinol）和ECB刺激的蛋白质偶联受体（GPCR），以及包括N- arachidonoylelaylaylaylaylaylaylalamine（Anandanolamine）（Aanandanolamine）（AANANDANOMIDEN）（AANANDANOMIDEN）（“ aman）（”） 2-芳基烯丙基甘油（2-AG）。 CB受体以及欧洲央行的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划定欧洲央行生物活性的稳态欧洲信号传导的关键。 CB1主要在中枢神经系统中发现，其激活介导了大多数CB精神和行为效应。在大脑水平非常低的情况下，CB2主要由免疫能力和造血细胞，成骨细胞以及成骨细胞和成骨细胞表达，并介导免疫反应，炎症，炎症，神经性疼痛以及骨骼重塑。 ECB是针对各种刺激的按需产生的，并通过酶水解迅速灭活：AEA，主要是由脂肪酸酰胺水解酶（FAAH）和2-AG灭活，主要由单酰基甘油脂肪酶（MGL）。内源性大麻素信号的变化伴随着各种生理和病理过程。多动态CB1传播与许多疾病状态有关，包括吸毒，滥用药物，超重/肥胖和与肥胖相关的心脏代谢风险（代谢综合征）。 CB1拮抗剂已成为减肥药，尽管在诊所中与不良影响有关。小分子激动剂对CB2的激活可能会对疼痛和神经炎症性疾病（例如，阿尔茨海默氏症和亨廷顿疾病）持有治疗诺言。这种翻译应用使对药物发现必不可少的CB受体激活的机制有透彻的了解，旨在调节CB受体传播以获得治疗增益。在没有晶体学结构的情况下，CB1和CB2同源性建模是基于Rhodopsin的X射线晶体结构进行的。 A类GPCR的一般体系结构的特征是细胞外定向的N末端，细胞内羧基末端以及七个疏水性跨膜1-横跨细胞膜并通过三个细胞外和三个细胞外和三个细胞质层的逆时针排列。 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.申请人假设螺旋6、7和8对CB2激活至关重要。本提案中详细介绍的实验旨在提供支持的实验证据，以通过使用CD和溶液和固态NMR的组合来定义这些螺旋在CB2激活时发生的结构变化。代表TM螺旋6和TM螺旋6-7/H8的适当标记的肽将在大肠杆菌中表达，在溶液和确定的脂质环境（胶束，磷脂双层）中进行纯化和研究。一种新型的高亲和力大麻能激动剂（AM841）先前证明是在TM螺旋6中与CB2半胱氨酸257特异性反应，并激活受体将用作亲和力探针。分子建模将用于增强实验结果。最终的数据将构成未来工作的基础，以阐明其他CB2螺旋结构域参与CB激活，并告知安全有效的CB2选择性激动剂作为候选药物。 公共卫生相关性：当前的建议将提供有关人CB2受体跨膜多肽的结构和动态信息。了解与配体接触的确定残基之间的特定方向和精确距离以及多肽配体配合物的构象，将有助于优化与大麻素受体的结合特性和选择配体的选择。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。

项目成果

Expression, Purification, and Monitoring of Conformational Changes of hCB2 TMH67H8 in Different Membrane-Mimetic Lipid Mixtures Using Circular Dichroism and NMR Techniques.

使用圆二色性和 NMR 技术表达、纯化和监测不同膜模拟脂质混合物中 hCB2 TMH67H8 的构象变化。

• DOI: 10.3390/membranes7010010
• 发表时间: 2017
• 期刊: Membranes
• 影响因子: 4.2
• 作者: Tiburu,ElvisK;Zhuang,Jianqin;Fleischer,HeidimarieNA;Arthur,PatrickK;Awandare,GordonA
• 通讯作者: Awandare,GordonA