Monoamine Transporter Structure-Function Studies

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。实现这一目标的一个主要障碍是关于质膜单胺转运蛋白的结构和功能的信息有限。药理学和行为学研究表明，多巴胺转运蛋白(DAT)是大脑受体，主要负责可卡因和苯丙胺的奖赏/增强特性。5-羟色胺转运蛋白(SERT)在这些药物改变情绪的效果中也起着关键作用，特别是在安非他明(如MDMA(摇头丸))的情况下。DAT和SERT负责通过从神经元突触摄取单胺类神经递质来终止神经传递。这项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水平上绘制这些药物的DAT和SERT结合区段。这种水平的分辨率现在可能是可能的，因为最近发表的LeuTAa转运蛋白的X射线晶体结构在序列上与DAT和SERT同源，可以作为创建三维DAT和SERT计算机模型的模板。这项建议的两个具体目标是创建基于LeuTAa的3-D DAT和SERT模型，并使用这些模型通过定点突变和药理学识别在底物(例如多巴胺、5-羟色胺、各种苯丙胺)和抑制剂(例如可卡因、哌酸甲酯)识别中起关键作用的DAT和SERT残基。由此产生的药理学数据将被用来改进分子模型，使其可用于电子筛选包含数十万种“小分子”化合物的结构文库，以寻找新的DAT和SERT配体。这些具体目标将通过DAT和SERT计算机辅助分子建模来实现，以确定合理的突变靶点，编码DAT和SERT蛋白的cDNAs的定点突变，用野生型或突变型DAT或SERT cDNA转染哺乳动物细胞系，检测转基因细胞结合和摄取放射性标记的DAT或SERT配体，抑制放射性配体与竞争的DAT或SERT配体的结合或摄取，以及通过免疫印迹技术评估转运蛋白的细胞表面表达。拟议实验的结果应该揭示可卡因和苯丙胺如何通过这些单胺转运体发挥作用的具体见解，以及关于苯妥拉平和哌醋甲酯等滥用潜力较小的药物的DAT识别和抗抑郁药物的SERT识别的信息。在氨基酸残基水平上阐明区分滥用和未滥用底物和抑制剂的机制应为合理设计药物提供一个蓝图，这些药物可以阻止可卡因和苯丙胺的作用，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或SERT相关的疾病，包括抑郁症、焦虑症、注意力缺陷多动障碍、偏头痛、发作性睡病和帕金森氏症。拟议的多巴胺转运体(DAT)和5-羟色胺转运体(SERT)研究了低滥用潜力(苯妥拉平和哌酸甲酯)和高滥用潜力(可卡因和甲基苯丙胺)的精神刺激剂的结合要求，甚至应该允许区分可卡因、甲基苯丙胺和亚甲二氧基安非他明(“摇头丸”)的单胺转运体识别机制。探索特定的DAT和SERT蛋白成分如何有助于识别滥用和未滥用的药物，应该为设计阻止可卡因和苯丙胺的作用而不反过来具有滥用和成瘾的可能性的药物提供一个蓝图。此外，这些研究可能导致治疗其他与DAT或SERT相关的疾病，包括抑郁症、焦虑症、注意力缺陷多动障碍、偏头痛、发作性睡病和帕金森氏症。

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. 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.