Dopamine Transporter Structure and Function

描述（由申请人提供）：在对抗精神兴奋剂滥用和成瘾方面，对治疗有用化合物的科学研究已经进行了几十年，迄今为止还没有临床可用的药物。以脑受体蛋白为指导，合理设计新型抗抑郁药、注意力缺陷治疗剂和其他中枢神经系统作用药物，也是多巴胺转运体（DAT）研究领域尚未实现的目标。一个主要的障碍是关于质膜DAT蛋白的结构和功能的有限信息。药理学和行为学研究表明，DAT是大脑受体，主要负责可卡因和安非他明的奖励/强化特性。该蛋白还与许多中枢神经系统疾病有关。这项应用的长期目标是了解精神兴奋剂和其他中枢神经系统活性抑制剂如何与DAT相互作用，这需要在氨基酸残基水平上绘制DAT配体结合袋。这种水平的分辨率现在可能成为可能，因为最近发表的同源LeuT转运体的晶体结构可以作为创建3-D DAT计算机模型的模板。本提案的具体目标是创建一个基于leut的3-D DAT模型，并使用该模型通过DAT位点定向突变/药理学和新型DAT配体的交联来识别和表征DAT残基对配体识别的关键。该研究结果将完善DAT分子模型，用于在寻找新型DAT配体的过程中进行结构文库的计算机筛选。这些特定目标将通过LeuT和DAT分子模型来解决，以确定可能的配体结合口袋残基，通过位点定向DAT诱变来解决这些目标，用野生型或突变型DAT核酸转染哺乳动物细胞，分析转染细胞对DAT配体的结合和摄取。通过免疫印迹和半胱氨酸烷基化实验评估突变转运蛋白的细胞表面表达和引入的半胱氨酸残基的可及性，以及在一系列新的DAT配体共价附着后对DAT蛋白水解物进行免疫化学分析。拟议实验的结果将揭示可卡因如何通过DAT发挥作用的具体见解，以及DAT识别诸如苯托品和哌醋甲酯等滥用可能性较小的药物的信息。在氨基酸残基水平上阐明区分滥用和非滥用底物和抑制剂的机制，可能为合理设计药物提供蓝图，这些药物既能减弱可卡因的作用，又不会产生滥用和成瘾的可能性。此外，这些研究可能会导致其他与dat相关的疾病的治疗方法，包括抑郁症、焦虑症、注意缺陷多动障碍、嗜睡症、慢性疼痛和帕金森病。公共卫生相关性：拟议的研究旨在提供三维地图，说明多巴胺转运体（DAT）如何与其cns活性抑制剂相互作用；这些图谱将用于筛选结构数据库，寻找在DAT中起作用的新化合物。在此过程中，地图细节包括特定的DAT蛋白质成分如何有助于区分，例如可卡因（高滥用潜力）和哌醋甲酯（低滥用潜力）可能会得到阐明。这一性质的发现为合理设计dat相关疾病的治疗方法打开了大门，这些疾病包括药物滥用和成瘾、抑郁症、焦虑症、注意力缺陷多动障碍、嗜睡症、慢性疼痛和帕金森病。

DESCRIPTION (provided by applicant): A scientific search for therapeutically useful compounds in combating psychostimulant abuse and addiction has been in progress for decades, with no clinically available agents to date. Rational, brain receptor protein-guided design of novel antidepressants, attention deficit therapeutics and other CNS-acting agents is also an unfulfilled goal of the dopamine transporter (DAT) research field. A major hindrance has been the limited information on the structure and function of the plasma membrane DAT protein. Pharmacological and behavioral studies indicate that the DAT is the brain receptor chiefly responsible for the reward/reinforcing properties of cocaine and the amphetamines. The protein is further implicated in a host of CNS disorders. The long-term objective of this application is to understand how psychostimulant and other CNS-active inhibitors interact with the DAT, which entails mapping DAT ligand binding pockets at the level of the amino acid residue. This level of resolution may now be a possibility, as the recently published crystal structure of the homologous LeuT transporter can serve as a template for creation of 3-D DAT computer models. The specific aims of this proposal entail creation of a LeuT-based 3-D DAT model and use of the model to identify and characterize, via DAT site-directed mutagenesis/pharmacology and crosslinking of novel DAT ligands, the DAT residues key to ligand recognition. The findings will refine the DAT molecular model for in silico screening of structural libraries in the search for novel DAT ligands. These specific aims will be addressed by LeuT and DAT molecular modeling to identify likely ligand binding pocket residues, addressing these targets via site-directed DAT mutagenesis, transfecting mammalian cells with wildtype or mutant DAT nucleic acids, assaying transfected cells for binding and uptake of DAT ligands, assessment of cell surface expression and accessibility of introduced cysteine residues of the mutant transporter proteins via immunoblotting and cysteine alkylation experiments, and immunochemical analysis of DAT proteolysates following covalent attachment of a series of novel DAT ligands. Results from the proposed experiments should reveal specific insights as to how cocaine exert its effects via the DAT, as well as information on DAT recognition of drugs with less abuse potential such as benztropine and methylphenidate. Elucidating at the amino acid residue level the mechanisms of discriminating abused and non-abused substrates and inhibitors may provide a blueprint for rational design of medications that blunt cocaine actions without in turn carrying the potential for abuse and addiction. Additionally, these studies may lead to therapeutics for other DAT-related conditions including depression, anxiety disorders, attention deficit hyperactivity disorder, narcolepsy, chronic pain and Parkinson's disease. PUBLIC HEALTH RELEVANCE: The proposed studies seek to provide 3-dimensional maps illustrating how the dopamine transporter (DAT) interacts with its CNS-active inhibitors; such maps will be used to screen structural databases for novel compounds that function at the DAT. Along the way, map details including how specific DAT protein components contribute to discrimination between, for example, cocaine (high abuse potential) and methylphenidate (low abuse potential) may be elucidated. Findings of this nature open the door to rational design of therapeutics for DAT-related conditions that include substance abuse and addiction, depression, anxiety disorders, attention deficit hyperactivity disorder, narcolepsy, chronic pain and Parkinson's disease.