Amphetamine-related photoaffinity probes

安非他明相关光亲和探针

• 批准号: 7300457
• 负责人: NICHOLAS V COZZI
• 金额: $ 9.59万
• 依托单位: UNIVERSITY OF WISCONSIN-MADISON
• 依托单位国家: 美国
• 财政年份: 2006
• 资助国家: 美国
• 起止时间: 2006-09-01 至 2007-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7300457
• 关键词: X ray crystallography; affinity labeling; amphetamines; binding sites; biotechnology; chemical synthesis; drug design /synthesis /production; ketones; laboratory rat; matrix assisted laser desorption ionization; molecular probes; neurotransmitter transport; nuclear magnetic resonance spectroscopy; pharmacokinetics; protein purification; protein sequence; proteolysis; radiotracer; stereoisomer; synaptosomes; thin layer chromatography; transport inhibitor; two dimensional gel electrophoresis

DESCRIPTION (provided by applicant): This is an R21 exploratory/developmental proposal. The goal is to investigate the feasibility of employing novel light sensitive amphetamine-like molecules as photoaffinity labels to identify and characterize amphetamine-related binding sites. This goal will be achieved through chemical synthesis and biochemical analysis. The emphasis will be to synthesize compounds known as aminoalkylphenones, The new compounds will be evaluated for their functional similarities to amphetamine in affecting monoamine neurotransmitter uptake and release and will be used to identify amphetamine-related binding sites using photoaffinity labeling techniques and MALDI-TOF analysis. The information generated will provide insight into the structural requirements for binding and efficacy of amphetamine-like molecules at the monoamine uptake transporters and may identify other amphetamine binding proteins that would not be discovered using other approaches. Ultimately, this research will aid in the rational design of therapeutic agents with greater specificity for the treatment of drug abuse, depression, and other psychiatric diseases associated with perturbed monoamine homeostasis and may also identify new biological targets for pharmacological intervention. The specific aims are: 1) To design, synthesize, and physically characterize novel amphetamine-related drugs belonging to a chemical class known as aminoalkylphenones. Compounds with various substituents on the phenyl ring and on the alkylamine side-chain will be synthesized for pharmacological evaluation. When chiral molecules are possible, stereoisomers will either be individually synthesized using enantioselective syntheses or racemates will be resolved using fractional crystallization or chiral chromatography. Radiolabeled variants will be synthesized as potential photoaffinity labels or radiotracers. All new compounds will be characterized by physical and chemical methods. 2) To generate pharmacological profiles of the newly synthesized compounds in vitro. Because inhibition of monoamine uptake and stimulation of monoamine release are major mechanisms of action for amphetamine-like drugs, the new compounds will be tested for their abilities to inhibit neurotransmitter uptake into cells stably expressing the cloned human monoamine transporters and they will be evaluated for their abilities to evoke the release of stored monoamine neurotransmitters under superfusion conditions. 3) To identify and biochemically characterize binding sites for new amphetamine-related photoaffinity labels. The photoaffinity labels synthesized under Specific Aim 1 will be used to identify binding domains and contact residues for amphetamine-like molecules within monoamine transporters or other protein binding sites. After photoaffinity labeling of cultured cells or native tissues, 2-D PAGE, MALDI-TOF analysis, limited proteolysis, N-terminal sequencing, and radiosequencing of the purified, protectably labeled proteins will identify domains and individual amino acids that form the drug acceptor sites.

描述（由申请人提供）：这是一项 R21 探索性/开发性提案。目标是研究采用新型光敏苯丙胺类分子作为光亲和标签来识别和表征苯丙胺相关结合位点的可行性。这一目标将通过化学合成和生化分析来实现。重点是合成氨基烷基苯酮化合物，将评估新化合物在影响单胺神经递质摄取和释放方面与安非他明的功能相似性，并将用于使用光亲和标记技术和 MALDI-TOF 分析来识别安非他明相关的结合位点。生成的信息将深入了解苯丙胺类分子在单胺摄取转运蛋白上的结合和功效的结构要求，并可能识别使用其他方法无法发现的其他苯丙胺结合蛋白。最终，这项研究将有助于合理设计具有更大特异性的治疗药物，用于治疗药物滥用、抑郁症和其他与单胺稳态紊乱相关的精神疾病，并且还可能确定药物干预的新生物靶点。具体目标是： 1) 设计、合成和物理表征属于氨基烷基苯酮化学类别的新型苯丙胺相关药物。将合成苯环和烷基胺侧链上具有各种取代基的化合物用于药理学评价。当手性分子可能时，立体异构体将使用对映选择性合成单独合成，或使用分级结晶或手性色谱法拆分外消旋体。放射性标记的变体将被合成为潜在的光亲和标记或放射性示踪剂。所有新化合物都将通过物理和化学方法进行表征。 2) 生成新合成化合物的体外药理学特征。由于抑制单胺摄取和刺激单胺释放是苯丙胺类药物的主要作用机制，因此将测试新化合物抑制稳定表达克隆人单胺转运蛋白的细胞摄取神经递质的能力，并评估它们的作用在灌注条件下激发储存的单胺神经递质释放的能力。 3) 识别新的苯丙胺相关光亲和标记的结合位点并对其进行生化表征。具体目标 1 下合成的光亲和标记将用于识别单胺转运蛋白或其他蛋白质结合位点内苯丙胺样分子的结合域和接触残基。对培养细胞或天然组织进行光亲和标记后，对纯化的、受保护标记的蛋白质进行 2-D PAGE、MALDI-TOF 分析、有限蛋白水解、N 末端测序和放射测序，将识别形成药物受体位点的结构域和单个氨基酸。