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.

项目摘要 药物滥用通过中唇多巴胺能途径诱导和促进多巴胺神经递质， 这修改了与奖励相关的行为，并与吸毒成瘾的发展有关。安装 有证据表明上瘾的个体在多巴胺的水平和信号上表现出严重的缺陷 D2受体（D2R），这有助于降低对天然增强剂的上瘾受试者的敏感性和 使它们容易受到更有效的药物刺激。因此，D2R提出了一个非常有希望的 在临床前和临床研究的支持下，药物滥用的治疗靶标。虽然D2R激动剂 正在对吸毒成瘾的治疗干预进行深入研究，他们的成功已经大大 由于依从性和功效差而受到阻碍，或与多巴胺能药物有关的相关副作用。 D2R上的结构信息不仅会揭示其信号传导机制，而且还提供了所需的信息 用于理性药物设计。 作为G蛋白偶联受体（GPCR）家族的成员，D2R臭名昭著地形成衍射质量 通过X射线晶体学确定高分辨率结构必不可少的晶体。这笔赠款 提案旨在为D2R的结构和功能研究开发一个全面，健壮的平台。到 实现这一目标，我们将解决三个主要目标：1）制定优化的协议以获得纯化 足以进行结晶尝试和功能研究的受体，2）使用稳定的受体 目标1以筛选最佳配体，并用一系列质量评估受体配体配合物的质量 控制分析和3）共结晶的受体配体复合物，以确定结构并设置受体 - 包含用于功能研究的纳米盘以及寻找高亲和力的纳米体或化合物的 d2r。该平台将涉及多个步骤，这些步骤紧密互连和通过向前和 向后反馈系统。 尽管本质上是高度挑战，但我们获得了一些可以表达的人类D2R结构 并在我们的实验室中纯化至每升生物量超过1 mg蛋白，这对于 实现随后的目标。这项研究的意义是多巴胺受体家族的多倍和 相关的药物滥用研究：1）获得对偏见激动剂的见解，2）揭示有关变构调制的信息， 3）屏幕广泛生产新的高亲和力配体D2R，4）表征D2R的机理 不同多巴胺受体亚家族之间的信号传导和配体选择，5）刺激基于结构的基于结构 药物设计，6）建立探索靶向D2R的配体的多疗法的平台，7） 为GPCR/G蛋白质复合信号研究铺平道路，并了解激活机制 未来。