Computational and Biochemical Docking of Dopamine Transporter Antagonists

描述（由申请人提供）：本项目将研究药物化合物如何利用结合生物化学和计算方法的综合方法与多巴胺转运蛋白（DAT）相互作用。生化分析将使用新型的光亲和性抑制剂，不可逆地连接到转运蛋白。这些研究将确定药物和DAT之间的接触点以及结合位点中配体的分子取向。与此同时，我们将进行计算研究，以建立DAT的比较模型的基础上同源性的竞争对手结合的晶体结构，从相关的细菌转运蛋白，LeuT。比较模型将被用于与生物化学的结果计算对接到DAT的光亲和配体。最近的进展，计算蛋白质折叠合法化它的使用在建模的完整的膜蛋白。与当前和正在进行的生化数据一致的对接结构将使用分子动力学进一步完善。从生物化学和计算分析获得的结果将导致的假设，将实验测试使用定点诱变，半胱氨酸扫描的可访问性，和一个数据库的DAT拮抗剂类似物，以评估分子预测。这些方法的结果的整合将提供有关DAT活性位点的结构以及拮抗剂如何对转运产生影响的重要信息。

DESCRIPTION (provided by applicant): This project will investigate how drug compounds interact with dopamine transporter (DAT) utilizing an integrated approach combining biochemical and computational methodologies. Biochemical analyses will use novel photoaffinity inhibitors that irreversibly attach to the transporter. These studies will determine points of contact between the drug and DAT and the molecular orientation of the ligand in the binding site. In parallel, we will carry out computational studies to build comparative models of DAT based on homology to a competitor-bound crystal structure from a related bacterial transporter, LeuT. The comparative models will be used in conjunction with the biochemical results to computationally dock the photoaffinity ligands into DAT. Recent advancements in computational protein folding have legitimized its use in modeling integral membrane proteins. Docked structures consistent with current and ongoing biochemical data will be further refined using molecular dynamics. The results obtained from biochemical and computational analyses will lead to hypotheses that will be experimentally tested using site-directed mutagenesis, cysteine-scanning accessibility, and a library of DAT antagonist analogs to evaluate the molecular predictions. The integration of findings from these approaches will provide significant information related to the structure of the DAT active site and how antagonists exert effects on transport.