Affinity Labeling the Dopamine Transporter Active Site

描述(申请人提供)：可卡因和苯丙胺在大脑中的主要作用部位之一是多巴胺转运体(DAT)，这是一种神经元蛋白，负责在突触传递后迅速重新摄取多巴胺。可卡因和其他精神刺激剂通过抑制多巴胺再摄取来发挥作用，这会导致突触多巴胺水平增加，并增强下游神经活动。一些治疗药物如哌醋甲酯也作用于DAT，DAT活性障碍可能与多巴胺能障碍有关，如帕金森-S病、抑郁症、注意力缺陷多动障碍和精神分裂症。目前尚不清楚可卡因和其他DAT抑制剂是否与DAT上的相同或不同部位结合，或者它们如何阻止DAT的转运。对这些化合物作用的分子基础缺乏基本的了解，部分原因是对DAT的结构和活性部位知之甚少，这是开发DAT特异性药物的主要障碍，这些药物可能有助于治疗精神刺激剂滥用或其他多巴胺能障碍。这项研究的目的是用可卡因和其他摄取阻滞剂的不可逆类似物标记DAT，并使用蛋白分解、表位特异性免疫沉淀和质谱学来鉴定与配体相互作用的蛋白质结构域。有待开发的配体将是先前分析的结合部位已知的不可逆配体的修饰，新配体的掺入模式与先前的发现相结合，将有助于更好地理解DAT的三维结构以及有助于拮抗和运输活性部位的结构域。具有不同结构的新配体也将被研究。这项研究的长期目标是确定DAT结构域是如何在空间上排列来产生抑制剂的结合位点的，并利用这些信息来阐明运输和运输抑制的分子机制。这些知识将有助于开发针对药物滥用和其他多巴胺能系统障碍的改进试剂和治疗药物，并可能有助于产生用于DAT成像的新试剂。DAT与去甲肾上腺素和5-羟色胺转运体的相似性表明，这些研究有可能适用于其他转运体。

DESCRIPTION (provided by applicant): One of the major sites of action for cocaine and amphetamine in the brain is the dopamine transporter (DAT), a neuronal protein responsible for rapid re-uptake of dopamine after synaptic transmission. Cocaine and other psychostimulants act by inhibiting dopamine reuptake, which results in increased levels of synaptic dopamine and enhanced downstream neural activity. Some therapeutic agents such as methylphenidate also act at DAT, and dysfunctions in DAT activity may be related to dopaminergic disorders such as Parkinson s disease, depression, Attention Deficity Hyperactivity Disorder, and schizophrenia. It is currentlynot known if cocaine and other DAT inhibitors bind to the same or different sites on DAT or how they prevent transport. This basic lack of understanding of the molecular basis of the action of these compounds arises in part because so little is known about DAT structure and active sites, and is a major obstacle in the development of DAT-specific drugs that could be useful for treating psychostimulant abuse or other dopaminergic disorders. The goal of this study is to label DAT with irreversible analogs of cocaine and other uptake blockers and use proteolysis, epitope-specific immunoprecipitation, and mass spectrometry to identify the domains of the protein that interact with the ligands. The ligands to be developed will be modifications of previously-analyzed irreversible ligands whose sites of binding are known, and the patterns of incorporation of the new ligands, in conjunction with the previous findings, will lead to better understanding of the three-dimensional structure of DAT and the domains that contribute to antagonist and transport active sites. New ligands with distinct structures will also be examined. The long term objective of this research is to determine how DAT domains are spatially arranged to generate the binding sites for inhibitors, and to use this information to elucidate the molecular mechanisms underlying transport and transport inhibition. This knowledge will aid in the development of improved agents and therapeutic medications for drug abuse and other dopaminergic system disorders, and may be useful for generation of new agents for DAT imaging. The similarity of DAT to the norepinephrine and serotonin transporters presents the potential for these studies to be applicable to these other transporters.