Dopamine Transporter Structure and Function

描述（由申请人提供）：几十年来，对治疗上有用的化合物在对抗精神兴奋剂滥用和成瘾方面的科学研究一直在进行，迄今为止还没有临床可用的药物。合理的，脑受体蛋白指导设计的新型抗抑郁药，注意力缺陷治疗和其他中枢神经系统作用剂也是多巴胺转运蛋白（DAT）研究领域尚未实现的目标。一个主要的障碍是有限的信息的结构和功能的质膜DAT蛋白。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。该蛋白质还涉及许多CNS病症。本申请的长期目标是了解精神兴奋剂和其他CNS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。这种分辨率水平现在可能是一种可能性，因为最近发表的同源LeuT转运蛋白的晶体结构可以作为创建3-D DAT计算机模型的模板。该提案的具体目标需要创建基于LeuT的3-D DAT模型，并使用该模型通过DAT定点诱变/药理学和新型DAT配体的交联来鉴定和表征配体识别的关键DAT残基。这些发现将完善DAT分子模型，用于在寻找新型DAT配体的过程中对结构库进行计算机筛选。这些特定目的将通过LeuT和DAT分子建模来解决，以鉴定可能的配体结合口袋残基，通过定点DAT诱变来解决这些靶标，用野生型或突变体DAT核酸转染哺乳动物细胞，测定转染细胞的DAT配体结合和摄取，通过免疫印迹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接近性，半胱氨酸烷基化实验，以及在共价连接一系列新型DAT配体后DAT蛋白水解产物的免疫化学分析。从拟议的实验结果应该揭示具体的见解，可卡因如何发挥其作用，通过DAT，以及DAT识别药物的滥用潜力较低，如苯扎托品和哌甲酯的信息。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，这些药物会减弱可卡因的作用，而不会反过来导致滥用和成瘾。此外，这些研究可能导致其他DAT相关疾病的治疗，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：拟议的研究试图提供三维地图，说明多巴胺转运蛋白（DAT）如何与其CNS活性抑制剂相互作用;这样的图谱将用于筛选在DAT处起作用的新化合物的结构数据库。沿着这条路，包括特异性DAT蛋白组分如何有助于区分，例如，可卡因（高滥用可能性）和哌醋甲酯（低滥用可能性）可能被阐明。这种性质的发现为DAT相关疾病的治疗方法的合理设计打开了大门，这些疾病包括物质滥用和成瘾，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.