LIGAND BINDING SITES ON THE DOPAMINE TRANSPORTER

描述（改编自申请人摘要）： 开发治疗可卡因滥用的药物是一个重要的目标， 解决全国性的药物滥用问题。为了开发新药 阻止可卡因与其靶点多巴胺转运蛋白结合， 如果能知道可卡因和多巴胺在大脑中的结合位置， 转运蛋白一种定位结合位点的方法， 研究可卡因的目的是探索可卡因类似物的结构特征， 影响与转运蛋白的结合。这种方法的一个延伸是， 产生与转运蛋白不可逆结合的抑制剂。这类 化合物与蛋白质反应，在抑制剂与蛋白质之间形成共价键， 和蛋白质。知道这个键是在哪里形成的，也就是说，哪个氨基 序列中的一个酸参与，允许结合位点开始被 本地化。拟议的工作使用质谱法来确定 不可逆配体在人多巴胺转运蛋白上的附着。 一系列不可逆抑制剂，包括不可逆可卡因 类似物，可以构建结合位点的图谱。此外， 多巴胺是转运蛋白的底物，它与多巴胺发生不可逆的反应。 转运蛋白将用于鉴定底物结合区。因此，在本发明中， 将鉴定在抑制和转运中均重要的区域。的 综合结果将有助于构建配体结合区域的图像 对人类多巴胺转运蛋白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 这种膜结合蛋白的机制，当功能正常或 对急性或慢性药物滥用的反应。确定涉及的领域 在底物和抑制剂结合中是理解 其机制及相关转运蛋白。

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.