LIGAND BINDING SITES ON THE DOPAMINE TRANSPORTER

描述(改编自申请人的摘要)： 开发治疗可卡因滥用的药物是#年的一个重要目标 解决全国性的药物滥用问题。为了开发新药 阻止可卡因与其靶标多巴胺转运体结合， 了解可卡因和多巴胺在体内的结合部位是非常有用的。 转运蛋白。一种定位结合位点的方法 可卡因一直在探索可卡因类似物的结构特征 影响对转运蛋白的结合。此方法的一个扩展是 开发不可逆转地与转运蛋白结合的抑制剂。这一类人 化合物与蛋白质反应，在抑制剂之间形成共价键 和蛋白质。了解这种键是在哪里形成的，也就是说，哪种氨基 序列中的酸参与，使结合部位开始 本地化。这项拟议的工作使用质谱仪来确定 人的多巴胺转运蛋白上的不可逆配体的连接。 一系列不可逆转的抑制剂，包括不可逆转的可卡因 类似地，可以构建结合位点的地图。此外，类似于 多巴胺，转运蛋白的底物，不可逆转地与 转运体将用于识别底物结合区。因此， 将确定在抑制和运输方面都很重要的区域。这个 综合结果将有助于构建配体结合区的图像 对人类多巴胺转运蛋白的功能和抑制至关重要。 如果没有足够的结构信息，就不可能理解 膜结合蛋白在正常和非正常工作状态下的作用机制 对急性或慢性药物滥用的反应。确定所涉及的域 在底物和抑制剂结合是理解的重要第一步 这种转运蛋白和相关转运蛋白的机制。

DESCRIPTION(Adapted from applicant's abstract): Development of medications to treat cocaine abuse is an important goal in addressing the national problem of drug abuse. In order to develop new drugs which prevent the binding of cocaine to its target, the dopamine transporter, it would be very useful to know where both cocaine and dopamine bind on the transporter protein. One approach to localization of the binding site for cocaine has been to explore the structural features of cocaine analogs which affect binding to the transporter. An extension of this approach has been to develop inhibitors which bind irreversibly to the transporter. This class of compounds reacts with the protein to form a covalent bond between the inhibitor and the protein. Knowledge of where this bond is formed, that is, which amino acid in the sequence is involved, allows the binding site to begin to be localized. The proposed work uses mass spectrometry to identify the sites of attachment of the irreversible ligands at the human dopamine transporter. With a range of irreversible inhibitors, including irreversible cocaine analogs, a map of the binding site can be constructed. In addition, analogs of dopamine, the substrate for the transporter, that irreversibly react with the transporter will be used to identify the substrate binding region. Thus, regions important in both inhibition and transport will be identified. The combined results will help to construct a picture of the ligand binding regions critical for human dopamine transporter function and inhibition. Without adequate structural information, it is not possible to understand the mechanism of this membrane bound protein when functioning normally or in response to acute or chronic drugs of abuse. Identification of domains involved in substrate and inhibitor binding is an important first step in understanding the mechanism of this and related transporters.