Computational and Biochemical Docking of Dopamine Transporter Antagonists

描述(由申请人提供)：这个项目将调查药物化合物如何与多巴胺转运体(DAT)相互作用，使用结合生化和计算方法的综合方法。生化分析将使用新型的光亲和抑制剂，这种抑制剂不可逆转地附着在转运蛋白上。这些研究将确定药物和DAT之间的接触点以及结合位置上配体的分子取向。同时，我们将进行计算研究，以建立DAT的比较模型，该模型基于与相关细菌转运蛋白Leut的竞争对手结合的晶体结构的同源性。比较模型将与生化结果相结合，以计算方式将光亲和配体对接到DAT中。最近在计算蛋白质折叠方面的进展已经使其在整体膜蛋白建模中的使用合法化。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精炼。从生化和计算分析获得的结果将导致假说，这些假说将通过使用定点突变、半胱氨酸扫描可及性和DAT拮抗剂类似物库来评估分子预测而进行实验测试。综合这些方法的发现，将提供与DAT活性部位的结构和拮抗剂如何在运输中发挥作用有关的重要信息。 公共卫生相关性：多巴胺转运体是几种药物滥用的主要目标，与成瘾和寻药行为有关，因此是一种具有高度临床相关性的蛋白质。然而，关于DAT中药物识别的基础，或者各种DAT药物如何导致特定的行为结果，我们仍然不了解许多细节。了解这些特性的分子基础可以在DAT的临床靶向以及相关的去甲肾上腺素和5-羟色胺转运体方面取得重要进展，所有这些都在我们的神经和心理健康中发挥关键作用。

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.