Dopamine Transporter Structure and Function

描述(申请人提供)：几十年来，对治疗有用化合物的科学研究一直在进行中，以对抗精神刺激剂的滥用和成瘾，到目前为止还没有临床可用的药物。合理的、大脑受体蛋白引导的新型抗抑郁药物、注意力缺陷治疗药物和其他中枢神经系统作用药物的设计也是多巴胺转运体(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，主要负责可卡因和苯丙胺的奖赏/增强特性。该蛋白还与一系列中枢神经系统疾病有关。这项应用的长期目标是了解精神刺激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配体结合口袋。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3D DAT计算机模型的模板。这项建议的具体目标包括建立基于Leut的3-D DAT模型，并使用该模型通过DAT定点突变/药理学和新的DAT配体的交联来识别和表征对配体识别至关重要的DAT残基。这一发现将完善DAT分子模型，用于电子筛选结构文库，以寻找新的DAT配体。这些特定的目标将通过Leut和DAT分子建模来确定可能的配体结合口袋残基，通过定点DAT突变来解决这些靶点，用野生型或突变型DAT核酸转染哺乳动物细胞，分析转基因细胞对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半胱氨酸残基的可及性，以及在一系列新的DAT配体共价连接后对DAT蛋白水解物进行免疫化学分析。拟议实验的结果应该揭示可卡因如何通过DAT发挥作用的具体见解，以及关于DAT识别滥用潜力较小的药物的信息，如苯妥拉平和哌醋甲酯。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一个蓝图，这些药物可以削弱可卡因的作用，而不会反过来带来滥用和成瘾的可能性。此外，这些研究可能导致治疗其他与DAT相关的疾病，包括抑郁症、焦虑症、注意力缺陷多动障碍、发作性睡病、慢性疼痛和帕金森氏症。与公共健康相关：拟议的研究试图提供三维地图，说明多巴胺转运体(DAT)如何与其中枢神经系统活性抑制剂相互作用；这种地图将用于筛选在DAT发挥作用的新化合物的结构数据库。在这一过程中，包括特定的DAT蛋白成分如何有助于区分可卡因(高滥用潜力)和哌醋甲酯(低滥用潜力)的MAP细节可能被阐明。这种性质的发现为合理设计治疗DAT相关疾病的疗法打开了大门，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、发作性睡病、慢性疼痛和帕金森氏症。

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.