Affinity Labeling the Dopamine Transporter Active Site

多巴胺转运蛋白活性位点的亲和标记

• 批准号: 6868946
• 负责人: ROXANNE A VAUGHAN
• 金额: $ 26.92万
• 依托单位: UNIVERSITY OF NORTH DAKOTA
• 依托单位国家: 美国
• 财政年份: 2003
• 资助国家: 美国
• 起止时间: 2003-04-01 至 2007-03-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6868946
• 关键词: active sites; affinity labeling; analog; binding sites; chemical synthesis; cocaine; dopamine transporter; epitope mapping; immunoprecipitation; laboratory rat; ligands; mass spectrometry; matrix assisted laser desorption ionization; neurotransmitter antagonist; neurotransmitter transport; piperidine; protein binding; protein structure; protein structure function; proteolysis; tissue /cell culture; tropanes

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.

描述（由申请方提供）：可卡因和安非他明在大脑中的主要作用部位之一是多巴胺转运蛋白（DAT），这是一种负责突触传递后多巴胺快速再摄取的神经元蛋白。可卡因和其他精神兴奋剂通过抑制多巴胺再摄取起作用，这导致突触多巴胺水平增加和下游神经活动增强。一些治疗剂如哌醋甲酯也作用于DAT，DAT活性的功能障碍可能与多巴胺能疾病如帕金森病、抑郁症、注意缺陷多动障碍和精神分裂症有关。目前尚不清楚可卡因和其他DAT抑制剂是否与DAT上相同或不同的位点结合，以及它们如何阻止转运。对这些化合物作用的分子基础缺乏了解，部分原因是对DAT结构和活性位点知之甚少，这是开发可用于治疗精神兴奋剂滥用或其他多巴胺能疾病的DAT特异性药物的主要障碍。本研究的目的是标记DAT与可卡因和其他摄取阻滞剂的不可逆类似物，并使用蛋白水解，表位特异性免疫沉淀和质谱法来确定与配体相互作用的蛋白质的结构域。要开发的配体将修改以前分析的不可逆配体的结合位点是已知的，并结合新的配体的模式，与以前的研究结果，将导致更好地了解DAT的三维结构和有助于拮抗剂和运输活性位点的域。还将研究具有不同结构的新配体。本研究的长期目标是确定DAT结构域如何在空间上排列以产生抑制剂的结合位点，并利用这些信息来阐明转运和转运抑制的分子机制。这些知识将有助于开发用于药物滥用和其他多巴胺能系统疾病的改进药物和治疗药物，并可能有助于生成用于DAT成像的新药物。 DAT与去甲肾上腺素和5-羟色胺转运蛋白的相似性表明这些研究有可能适用于这些其他转运蛋白。

项目成果

A novel photoaffinity ligand for the dopamine transporter based on pyrovalerone.

基于吡咯戊酮的多巴胺转运蛋白的新型光亲和配体。

• DOI: 10.1016/j.bmc.2009.04.057
• 发表时间: 2009
• 期刊: Bioorganic & medicinal chemistry
• 影响因子: 3.5
• 作者: Lapinsky,DavidJ;Aggarwal,Shaili;Huang,Yurong;Surratt,ChristopherK;Lever,JohnR;Foster,JamesD;Vaughan,RoxanneA
• 通讯作者: Vaughan,RoxanneA

Evolution of a Compact Photoprobe for the Dopamine Transporter Based on (±)-threo-Methylphenidate.

基于(±)-苏型哌甲酯的多巴胺转运蛋白紧凑型光探针的演变。

• DOI: 10.1021/ml3000098
• 发表时间: 2012
• 期刊: ACS medicinal chemistry letters
• 影响因子: 4.2
• 作者: Lapinsky,DavidJ;Yarravarapu,Nageswari;Nolan,TammyL;Surratt,ChristopherK;Lever,JohnR;Tomlinson,Michael;Vaughan,RoxanneA;Deutsch,HowardM
• 通讯作者: Deutsch,HowardM

Localization of cocaine analog [125I]RTI 82 irreversible binding to transmembrane domain 6 of the dopamine transporter.

可卡因类似物 [125I]RTI 82 与多巴胺转运蛋白跨膜结构域 6 不可逆结合的定位。

• DOI: 10.1074/jbc.m610633200
• 发表时间: 2007
• 期刊: The Journal of biological chemistry
• 作者: Vaughan,RoxanneA;Sakrikar,DhananjayS;Parnas,MLaura;Adkins,Steven;Foster,JamesD;Duval,RomainA;Lever,JohnR;Kulkarni,SantoshS;Hauck-Newman,Amy
• 通讯作者: Hauck-Newman,Amy

Azido-iodo-N-benzyl derivatives of threo-methylphenidate (Ritalin, Concerta): Rational design, synthesis, pharmacological evaluation, and dopamine transporter photoaffinity labeling.

苏型哌甲酯的叠氮基碘-N-苄基衍生物（利他林，Concerta）：合理设计、合成、药理学评价和多巴胺转运蛋白光亲和标记。

• DOI: 10.1016/j.bmc.2010.11.002
• 发表时间: 2011
• 期刊: Bioorganic & medicinal chemistry
• 影响因子: 3.5
• 作者: Lapinsky,DavidJ;Velagaleti,Ranganadh;Yarravarapu,Nageswari;Liu,Yi;Huang,Yurong;Surratt,ChristopherK;Lever,JohnR;Foster,JamesD;Acharya,Rejwi;Vaughan,RoxanneA;Deutsch,HowardM
• 通讯作者: Deutsch,HowardM