Computational and Biochemical Docking of Dopamine Transporter Antagonists

描述（由申请人提供）：该项目将利用结合生化和计算方法的综合方法研究药物化合物如何与多巴胺转运体（DAT）相互作用。生化分析将使用新的光亲和抑制剂，不可逆地附着在转运体上。这些研究将确定药物与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，我们将开展计算研究，基于与相关细菌转运体LeuT的竞争结合晶体结构的同源性，建立DAT的比较模型。比较模型将与生化结果结合使用，以计算将光亲和配体停靠到DAT中。计算蛋白质折叠的最新进展使其在整体膜蛋白建模中的应用合法化。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改进。从生化和计算分析中获得的结果将导致假设，这些假设将使用位点定向诱变、半胱氨酸扫描可及性和DAT拮抗剂类似物库进行实验测试，以评估分子预测。整合这些方法的发现将提供与DAT活性位点结构和拮抗剂如何对运输产生影响有关的重要信息。

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. PUBLIC HEALTH RELEVANCE: The dopamine transporter is a major target of several drugs of abuse and has been linked to addiction and drug seeking behaviors, and as such is a highly clinically relevant protein. However, we still do not understand many of the details regarding the basis of drug recognition at DAT, or how various DAT drugs induce particular behavioral outcomes. Understanding the molecular basis of these properties could lead to important advances in clinical targeting of DAT as well as the related norepinephrine and serotonin transporters, all which play critical roles in our neurological and psychological health.