Structure and Function of Dopamine Receptors

多巴胺受体的结构和功能

• 批准号: 9317762
• 负责人: Wei Liu
• 金额: $ 18.32万
• 依托单位: ARIZONA STATE UNIVERSITY-TEMPE CAMPUS
• 依托单位国家: 美国
• 财政年份: 2017
• 资助国家: 美国
• 起止时间: 2017-03-15 至 2019-02-28
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/9317762
• 关键词: Address; Adherence; Adverse effects; Affect; Affinity; Agonist; Applications Grants; Architecture; Area; Baculovirus Expression System; Behavior; Binding; Biochemical; Biological; Biological Assay; Biological Process; Biomass; Biophysics; Brain; C-terminal; Clinic; Clinical Research; Complex; Crystallization; Data; Development; Disease; Dopamine; Dopamine D2 Receptor; Dopamine Receptor; Drug Addiction; Drug Design; Drug abuse; Engineering; Euphoria; Exhibits; Family; Feedback; Follow-Up Studies; Future; G-Protein-Coupled Receptors; GTP-Binding Proteins; Generations; Goals; Human; Individual; Laboratories; Length; Libraries; Ligands; Lipid Bilayers; Lipids; Medicine; Membrane; Minor; Molecular; Molecular Conformation; Mutation; N-terminal; Nature; Pathway interactions; Pharmaceutical Preparations; Pharmacology; Phase; Play; Proteins; Protocols documentation; Quality Control; Receptor Signaling; Recruitment Activity; Research; Research Institute; Resolution; Rewards; Role; Sampling; Scaffolding Protein; Signal Pathway; Signal Transduction; Site; Structure; System; Therapeutic; Therapeutic Intervention; X-Ray Crystallography; base; design; drug development; improved; insight; large scale production; medical schools; member; mimetics; mutant; nanobodies; nanodisk; neurotransmission; novel; pre-clinical; protein complex; protein expression; protein purification; receptor; reconstitution; reinforcer; screening; success; targeted treatment; therapeutic target; thermostability

Project Summary Drug abuse induces and facilitates dopamine neurotransmission through the mesolimbic dopaminergic pathway, which modifies reward-related behaviors and is associated with the development of drug addiction. Mounting evidence suggests that addicted individuals exhibited significant deficits on the level and signaling of dopamine D2 receptor (D2R), which contribute to decreased sensitivity of addicted subjects to natural reinforcers and predispose them to more potent drug stimulation of euphoria. Therefore, D2R presents a highly promising therapeutic target for drug abuse, as supported by both pre-clinical and clinical studies. Although D2R agonists are being intensively studied as therapeutic intervention for drug addiction, their success have been greatly hampered due to poor adherence and efficacy, or associated side effects related to dopaminergic medications. Structural information on D2R will not only reveal its signaling mechanisms, but also provide required information for rational drug design. As a member in the G protein-coupled receptor (GPCR) family, D2R is notorious to form diffraction-quality crystals that are essential for the determination of high-resolution structures by X-ray crystallography. This grant proposal aims to develop a comprehensive and robust platform for structural and functional studies of D2R. To achieve this goal, we will address three major aims: 1) develop optimized protocols for obtaining purified receptors in quantities sufficient for crystallization attempts and functional studies, 2) use stable receptors from Aim 1 to screen for optimal ligands and assess the quality of receptor-ligand complexes with an array of quality- control analyses, and 3) co-crystallize receptor-ligand complexes for structure determination and set up receptor- containing nanodiscs for functional studies as well as searching for high-affinity nanobodies or compounds for D2R. This platform will involve multiple steps that are closely interconnected and looped through a forward and backward feedbacks system. Although highly challenging in nature, we have acquired a few human D2R constructs that could be expressed and purified to more than 1 mg protein per liter of biomass in our laboratory, which is essential for the accomplishment of subsequent aims. The significance of this study is multi-fold on dopamine receptor family and related drug abuse studies: 1) gain insights into biased agonism, 2) reveal information on allosteric modulations, 3) screen extensively to produce new high-affinity ligands for D2R, 4) characterize the mechanisms of D2R signaling and ligand selection between different dopamine receptor subfamilies, 5) stimulate structure-based drug design, 6) establish the platform for exploring the polypharmacology of the ligands that target D2R, and 7) pave the road for GPCR/G protein complex signaling studies and understanding the activation mechanism in the future.

项目概要 药物滥用通过中脑边缘多巴胺能途径诱导并促进多巴胺神经传递， 它改变与奖励相关的行为并与毒瘾的发展有关。安装 有证据表明，成瘾者的多巴胺水平和信号传导存在显着缺陷 D2 受体 (D2R)，导致成瘾者对天然强化物的敏感性降低， 使他们容易受到更有效的欣快感药物刺激。因此，D2R 是一种非常有前途的 药物滥用的治疗目标，得到临床前和临床研究的支持。尽管 D2R 激动剂 作为毒瘾的治疗干预措施正在深入研究，其成功已取得了巨大的成功 由于依从性差和疗效不佳，或多巴胺能药物相关的副作用而受到阻碍。 D2R的结构信息不仅可以揭示其信号机制，还可以提供所需的信息 进行合理的药物设计。 作为 G 蛋白偶联受体 (GPCR) 家族的一员，D2R 因形成衍射质量而臭名昭著 对于通过 X 射线晶体学测定高分辨率结构至关重要的晶体。这笔补助金 该提案旨在开发一个全面且强大的平台，用于 D2R 的结构和功能研究。到 为了实现这一目标，我们将解决三个主要目标：1）开发优化的方案以获得纯化的 受体的数量足以进行结晶尝试和功能研究，2）使用来自 目标 1 筛选最佳配体并通过一系列质量评估受体-配体复合物的质量- 控制分析，以及 3) 共结晶受体-配体复合物以进行结构测定并建立受体- 包含用于功能研究的纳米圆盘以及寻找高亲和力纳米体或化合物 D2R。该平台将涉及多个步骤，这些步骤通过前向和循环紧密互连和循环。 后向反馈系统。 尽管本质上极具挑战性，但我们已经获得了一些可以表达的人类 D2R 构建体 并在我们的实验室中纯化至每升生物量超过 1 毫克蛋白质，这对于 后续目标的实现。这项研究对于多巴胺受体家族具有多重意义 相关药物滥用研究：1）深入了解偏向激动，2）揭示变构调节信息， 3) 广泛筛选以生产新的 D2R 高亲和力配体，4) 表征 D2R 的机制 不同多巴胺受体亚家族之间的信号传导和配体选择，5）基于刺激结构 药物设计，6) 建立探索靶向 D2R 的配体多药理学的平台，以及 7) 为 GPCR/G 蛋白复合物信号研究和了解激活机制铺平道路 未来。