Amphetamine-related photoaffinity probes

安非他明相关光亲和探针

• 批准号: 6952234
• 负责人: NICHOLAS V COZZI
• 金额: $ 4.11万
• 依托单位: PHYSIOGENIX, INC.
• 依托单位国家: 美国
• 财政年份: 2004
• 资助国家: 美国
• 起止时间: 2004-09-30 至 2006-07-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6952234
• 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R21探索/发展建议。目的是研究采用新型光敏感的苯丙胺样分子作为光亲和力标签的可行性，以识别和表征苯丙胺相关的结合位点。该目标将通过化学合成和生化分析来实现。 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.所产生的信息将为单胺摄取转运蛋白的结合和有效性提供结合和功效的结构要求，并可能鉴定出其他方法不会使用其他方法发现的其他苯丙胺结合蛋白。最终，这项研究将有助于对治疗药物滥用，抑郁症和其他与扰动的单胺稳态相关的药物滥用，抑郁症和其他精神疾病的特异性的理性设计，并且还可能确定药理学干预的新生物学靶标。具体目的是：1）设计，合成和物理表征属于氨基烷基球酮的化学类别的新型苯丙胺相关药物。将合成苯环和烷基侧链上各种取代基的化合物进行药理评估。当可能的手性分子可以使用对映选择性合成的单独合成，或者使用分数结晶或手性色谱分析立体异构体。放射标记的变体将合成为潜在的光性标签或放射性示例。所有新化合物将以物理和化学方法的特征。 2）在体外生成新合成化合物的药理谱。由于抑制单胺摄取和单胺释放的刺激是苯丙胺样药物的主要作用机制，因此将测试新化合物的能力，以抑制神经递质摄取神经递质的能力，以稳定地表达稳固的人类单胺转运蛋白，以使其在释放的释放中，以释放出来的人，以使其释放出来。 状况。 3）识别和生化表征新的苯丙胺相关光性标签的结合位点。在特定目标1下合成的光亲和力标记将用于鉴定单胺转运蛋白转运蛋白或其他蛋白质结合位点内苯丙胺样分子的结合结构域和接触残基。经过培养细胞或天然组织的光性标记后，2-D页，MALDI-TOF分析，有限的蛋白水解，N末端测序以及纯化的，可保护的标记蛋白的放射测序将识别形成药物受体位点的域和单个氨基酸。