Monoamine Transporter Structure-Function Studies

单胺转运蛋白结构功能研究

基本信息

批准号：
7305399
负责人：
CHRISTOPHER K SURRATT
金额：
$ 17.9万
依托单位：
DUQUESNE UNIVERSITY
依托单位国家：
美国
项目类别：
财政年份：
2003
资助国家：
美国
起止时间：
2003-08-01 至 2010-07-31
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/7305399
关键词：
3-Dimensional Address Affinity Amino Acid Substitution Amino Acids Amphetamines Antidepressive Agents Anxiety Disorders Applications Grants Attention deficit hyperactivity disorder Behavioral Benztropine Binding Binding Sites Biological Assay Brain Carrier Proteins Cell Line Cell membrane Cell surface Cells Chromosome Pairing Cocaine Complementary DNA Computer Assisted Computer Simulation Condition Crystallization Data Development Discrimination Disruption Dopamine Goals Grant Immunoblotting Lead Leucine Libraries Ligand Binding Ligands Mammalian Cell Maps Mental Depression Methamphetamine Methylphenidate Migraine Modeling Monitor Moods Mutagenesis Narcolepsy Neurons Neurotransmitters Parents Parkinson Disease Pharmaceutical Preparations Pharmacology Play Process Property Protein Binding Proteins Publishing Purpose Radioactive Radiolabeled Resolution Rewards Roentgen Rays Role Screening procedure Serotonin Site-Directed Mutagenesis Structure Synapses Techniques Testing Therapeutic Transmembrane Domain Tryptamines Work addiction base computer generated design dopamine transporter ecstasy inhibitor/antagonist insight molecular modeling monoamine mutant neurotransmission novel psychostimulant radioligand radiotracer receptor research study serotonin transporter small molecule stimulant abuse tool tryptamine uptake

项目摘要

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. A major hindrance toward this goal has been the limited information on the structure and function of the plasma membrane monoamine transporter proteins. Pharmacological and behavioral studies indicate that the dopamine transporter protein (DAT) is the brain receptor chiefly responsible for the reward/reinforcing properties of cocaine and the amphetamines. The serotonin transporter protein (SERT) also plays a key role in the mood-altering effects of these drugs, especially in the case of amphetamines such as MDMA ("ecstasy"). The DAT and SERT are responsible for termination of neurotransmission by uptake of the monoamine neurotransmitter from the neuronal synapse. The long-term objective of this application is to understand how cocaine and the amphetamines interact with the DAT and SERT, which entails mapping the DAT and SERT binding pockets of these drugs at the level of the amino acid residue. This level of resolution may now be a possibility, as the recently published X-ray crystal structure of the LeuTAa transporter protein, homologous in sequence to the DAT and SERT, can serve as a template for creation of 3-dimensional DAT and SERT computer models. The two specific aims of this proposal are to create LeuTAa-based 3-D DAT and SERT models, and use the models to identify and characterize via site-directed mutagenesis and pharmacology the DAT and SERT residues key in substrate (e.g., dopamine, serotonin, various amphetamines) and inhibitor (e.g., cocaine, methylphenidate) recognition. The resultant pharmacologic data will be used to refine the molecular models to the point that they can be employed in in silico screening of structural libraries containing hundreds of thousands of "small molecule" compounds in the search for novel DAT and SERT ligands. These specific aims will be addressed by DAT and SERT computer-aided molecular modeling to identify logical mutagenesis targets, site-directed mutagenesis of the cDNAs encoding the DAT and SERT proteins, transfecting mammalian cell lines with wildtype or mutant DAT or SERT cDNAs, assaying transfected cells for binding and uptake of radiolabeled DAT or SERT ligands, inhibition of radioligand binding or uptake with non-radioactive competing DAT or SERT ligands, and assessment of cell surface expression of the transporter proteins via immunoblotting techniques. Results from the proposed experiments should reveal specific insights as to how cocaine and the amphetamines exert their effects via these monoamine transporters, as well as information on DAT recognition of drugs with less abuse potential such as benztropine and methylphenidate and SERT recognition of antidepressant drugs. Elucidating at the amino acid residue level the mechanisms of discriminating abused and non-abused substrates and inhibitors should provide a blueprint for rational design of medications that block cocaine and amphetamine actions without in turn carrying the potential for abuse and addiction. Additionally, these studies may lead to therapeutics for other DAT- or SERT-related conditions including depression, anxiety disorders, attention deficit hyperactivity disorder, migraine, narcolepsy and Parkinson's disease. The proposed dopamine transporter (DAT) and serotonin transporter (SERT) studies tease apart binding requirements for psychostimulants of low abuse potential (benztropine and methylphenidate) and high abuse potential (cocaine and methamphetamine), and should even allow discrimination of monoamine transporter recognition mechanisms for cocaine, methamphetamine and MDMA ("ecstasy"). Exploring exactly how specific DAT and SERT protein components contribute to recognition of abused and non-abused drugs should provide a blueprint for designing medications that block actions of cocaine and the amphetamines without in turn carrying the potential for abuse and addiction. Additionally, these studies may lead to therapeutics for other DAT- or SERT-related conditions including depression, anxiety disorders, attention deficit hyperactivity disorder, migraine, narcolepsy 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。针对该目标的主要障碍是有关质膜单胺转运蛋白的结构和功能的有限信息。药理学和行为研究表明，多巴胺转运蛋白（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。 5-羟色胺转运蛋白（SERT）在这些药物的改变情绪效应中也起着关键作用，尤其是在诸如MDMA（“狂喜”）等苯丙胺的情况下。 DAT和SERT负责通过从神经元突触中摄取单胺神经递质来终止神经传递。该应用的长期目标是了解可卡因和苯丙胺如何与DAT和SERT相互作用，这需要在氨基酸残基的水平上映射这些药物的DAT和SERT结合口袋。现在，这种分辨率现在可能是一种可能性，因为与DAT和SERT序列同源的Leutaa转运蛋白的最近发表的X射线晶体结构可以用作创建3维DAT和SERT计算机模型的模板。该提案的两个具体目的是创建基于Leutaa的3-D DAT和SERT模型，并使用这些模型通过位置定向的诱变和药理学来识别和表征底物中的DAT和SERT残基关键（例如多巴胺，血清素，羟色胺，各种氨基胺）和抑制剂（例如，甲基甲基甲基化的）。所得的药理学数据将用于完善分子模型，以至于它们可以用于用于在寻找新颖的DAT和Sert配体的结构文库中筛选结构文库中。 These specific aims will be addressed by DAT and SERT computer-aided molecular modeling to identify logical mutagenesis targets, site-directed mutagenesis of the cDNAs encoding the DAT and SERT proteins, transfecting mammalian cell lines with wildtype or mutant DAT or SERT cDNAs, assaying transfected cells for binding and uptake of radiolabeled DAT or SERT ligands, inhibition of使用非放射性竞争性DAT或SERT配体的放射性配体结合或摄取，以及通过免疫印迹技术评估转运蛋白蛋白的细胞表面表达。提出的实验的结果应揭示有关可卡因和苯丙胺如何通过这些单胺转运蛋白发挥其作用的特定见解，以及有关识别具有较小滥用潜力的药物的信息，例如苯甲胺和甲基苯甲酸甲酯和SERT识别抗抑郁药。在氨基酸残基水平上阐明滥用滥用和未施加的底物和抑制剂的机制应提供蓝图，用于对可卡因和苯丙胺作用的合理设计，而无需遭受滥用和成瘾的潜力。此外，这些研究可能会导致针对其他与DAT或SERT相关的疾病的治疗方法，包括抑郁症，焦虑症，注意力缺陷多动障碍，偏头痛，睡病和帕金森氏病。拟议的多巴胺转运蛋白（DAT）和5-羟色胺转运蛋白转运蛋白（SERT）研究分开了对低滥用潜力的精神刺激因素（苯二甲状腺素和甲基苯甲酸酯）和高滥用潜力（可卡因和甲基苯丙胺）的结合要求，甚至应该允许甲基甲基（Methampane）的单胺胺识别机械学（Mothamine Transporter）识别机械学（Methampasy and Methampane）。准确探索特定的DAT和SERT蛋白成分如何有助于识别滥用和未虐待药物的药物应为设计可卡因和苯丙胺作用的药物提供蓝图，而不会反过来却带来滥用和成瘾的潜力。此外，这些研究可能会导致针对其他与DAT或SERT相关的疾病的治疗方法，包括抑郁症，焦虑症，注意力缺陷多动障碍，偏头痛，睡病和帕金森氏病。