Dopamine Transporter Structure and Function

多巴胺转运蛋白的结构和功能

• 批准号: 7636438
• 负责人: CHRISTOPHER K SURRATT
• 金额: $ 31.27万
• 依托单位: DUQUESNE UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2009
• 资助国家: 美国
• 起止时间: 2009-07-01 至 2011-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7636438
• 关键词: 3-Dimensional; Address; Affinity; Alanine; Algorithms; Alkylation; Amino Acid Substitution; Amino Acids; Amphetamines; Antidepressive Agents; Anxiety Disorders; Applications Grants; Attention; Attention deficit hyperactivity disorder; Behavioral; Benztropine; Binding; Binding Sites; Biological Assay; Brain; Carrier Proteins; Cell Line; Cell membrane; Cell surface; Cells; Central Nervous System Diseases; Cocaine; Complementary DNA; Complex; Computer Simulation; Covalent Interaction; Crystallization; Cysteine; Data; Databases; Development; Discrimination; Docking; Dopamine; Drug Binding Site; Goals; Grant; Immunoblotting; Isothiocyanates; Lead; Leucine; Libraries; Ligand Binding; Ligands; Mammalian Cell; Maps; Methylphenidate; Modeling; Molecular; Molecular Conformation; Molecular Models; Monitor; Mutagenesis; Mutate; Mutation; Narcolepsy; Nature; Neurotransmitters; Nucleic Acids; Parkinson Disease; Pharmaceutical Preparations; Pharmacology; Process; Property; Proteins; Proteolysis; Publishing; Radiolabeled; Research; Resolution; Rewards; Screening procedure; Series; Site; Site-Directed Mutagenesis; Structure; Substance abuse problem; Techniques; Testing; Therapeutic; Transmembrane Domain; Tropanes; Western Blotting; Work; addiction; analog; base; chronic pain; combat; comparative; computer generated; crosslink; depression; design; dopamine transporter; inhibitor/antagonist; insight; molecular modeling; molecular transporter; mutant; novel; preference; psychostimulant; public health relevance; pyrovalerone; radiotracer; receptor; research study; small molecule; stimulant abuse; uptake; virtual

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.

描述（由申请人提供）：几十年来一直在进行进行精神刺激滥用和成瘾的科学搜索，以打击精神刺激性滥用和成瘾，迄今为止尚无临床可用的代理。新型抗抑郁药，注意力缺陷治疗和其他CNS作用剂的理性，脑受体蛋白引导的设计也是多巴胺转运蛋白（DAT）研究领域的未实现目标。主要的障碍是有关质膜DAT蛋白的结构和功能的有限信息。药理学和行为研究表明，DAT是主要负责可卡因和苯丙胺的奖励/增强特性的大脑受体。该蛋白质进一步与许多中枢神经系统疾病有关。该应用程序的长期目标是了解心理刺激剂和其他CNS活性抑制剂如何与DAT相互作用，DAT需要在氨基酸残基的水平上绘制DAT配体结合口袋。现在，这种分辨率级别可能是一种可能性，因为同源Leut Transporter的最近发表的晶体结构可以用作创建3-D DAT计算机模型的模板。该提案的具体目的是创建基于Leut的3-D DAT模型，并使用该模型来识别和表征通过DAT位置定向的诱变/药理学和新型DAT配体的交联，DAT残基的配体识别的关键。这些发现将在寻找新型DAT配体时完善结构库筛选的DAT分子模型。 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通过免疫印迹和半胱氨酸烷基化实验进行蛋白质，以及一系列新型DAT配体的共同附着后，DAT蛋白溶质物的免疫化学分析。提出的实验的结果应揭示有关可卡因如何通过DAT发挥其影响的具体见解，以及有关识别滥用潜力较小的药物（如苯甲胺和甲基苯甲酸酯）的Dat识别的信息。在氨基酸残留水平上阐明滥用滥用和非滥用底物和抑制剂的机制可能会为合理设计的药物设计提供蓝图，而这些药物的可卡因作用钝化而又不带来滥用和成瘾的潜力。此外，这些研究可能会导致其他与DAT相关疾病的治疗方法，包括抑郁症，焦虑症，注意力缺陷多动障碍，发作性疾病，慢性疼痛和帕金森氏病。公共卫生相关性：拟议的研究试图提供3维图，以说明多巴胺转运蛋白（DAT）如何与其CNS活性抑制剂相互作用；此类地图将用于筛选在DAT起作用的新型化合物的结构数据库。在此过程中，MAP细节包括如何阐明可卡因（高滥用潜力）和哌醋甲酯（低滥用潜力），包括特定的DAT蛋白成分如何促进歧视。这种性质的结果为与DAT相关的疾病的理性设计打开了大门，包括药物滥用和成瘾，抑郁症，焦虑症，注意力缺陷多动障碍，发毒症，慢性疼痛，慢性疼痛和帕金森氏病。