Molecular Organization of the Organic Cation-Proton Exchanger, MATE1

描述(申请人提供)：肝脏和肾脏从体内排泄出一系列具有生理、药理和毒理学意义的正电荷有机分子。载体介导的这些“有机阳离子”(OCs)的分泌，特别是通过肾脏，对这些化合物的药代动力学有深远的影响，重要的是，OC的分泌是许多临床上重要的药物相互作用的场所。OC分泌的活跃和限速步骤涉及载体介导的累积的OCs穿过肾和肝上皮的管腔膜退出。这一过程的分子鉴定涉及新发现的多药物和毒素排出蛋白MATE1和Mate2-K。虽然现在清楚地知道在OC的分泌中起着重要作用，但对于底物与这些转运蛋白相互作用的分子决定因素几乎一无所知。在这个修订的提案中，我们利用了最近对运输蛋白Mate家族(NORM)原型成员的X射线结构的解决方案。我们使用范数结构来开发一个人类MATE1的同源模型，在这个建议中，我们概述了两组实验，旨在开发一个药物与Mate转运体相互作用的预测模型。在目标1中，我们采用基于配体的方法建立了底物/抑制剂与MATE1和Mate2-K相互作用的3D-QSAR/药效团模型。这些数据将在关于这些转运蛋白在肾脏分泌的上皮模型中的整合活性的平行研究的背景下被解释(这将在关于这些转运体在人类肾脏中的不同分布的研究的背景下被解释)。AIM 2将使用基于目标的方法，使用定点研究来探索MATE1的拓扑和表面可及性，从而测试来自我们的同源模型的预测，并建立一个数据库，旨在探索蛋白质的功能结构，该结构是在并行努力中确定的，以解决人类MATE1的X射线结构。目的2还将在以下研究中研究MATE1的底物转位途径：(I)应用蛋白质组学方法识别与OC/H+交换器的光活化探针特异性相互作用的多肽和氨基酸残基；以及(Ii)应用计算方法(导向分子动力学)识别影响底物转位的氨基酸残基。这些研究将在建立准确预测阳离子药物在肾脏和肝脏中不必要的相互作用的模型方面发挥关键作用，最好是先发制人。

DESCRIPTION (provided by applicant): The liver and kidney excrete from the body a wide array of positively charged organic molecules of physiological, pharmacological and toxicological significance. Carrier-mediated secretion of these "organic cations" (OCs), particularly by the kidney, has a profound influence on the pharmacokinetics of these compounds and, importantly, OC secretion is the site of many clinically significant drug-drug interactions. The active and rate-limiting step in OC secretion involves the carrier-mediated exit of accumulated OCs across the luminal membrane of renal and hepatic epithelia. The molecular identify of this process involves the newly identified Multidrug And Toxin Extrusion proteins, MATE1 and MATE2-K. Although now clearly understood to play a significant role in OC secretion, virtually nothing is known about the molecular determinants of substrate interaction with these transporters. In this revised proposal, we take advantage of the recent solution of the x- ray structure of a prototypic member of the MATE family of transport protein (NorM). We have used the NorM structure to develop a homology model of human MATE1 and, in this proposal, we outline two sets experiments designed to develop a predictive model of drug interaction with MATE transporters. In Aim 1, we take a ligand-based approach to develop 3D-QSAR/pharmacophore models of substrate/inhibitor interaction with MATE1 and MATE2-K. These data will be interpreted in the context of parallel studies on the integrated activity of these transporters in epithelial models of renal secretion (which, in turn, will be interpreted in the context of studies on the differential distribution of these transporters in human kidney). Aim 2 will employ a target-based approach, using site-directed studies to probe the topology and surface accessibility of MATE1, thereby testing predictions arising from our homology model, and establishing a database designed to probe the functional structure of the protein as determined in a parallel effort to solve the x-ray structure of human MATE1. Aim 2 will also study the substrate translocation pathway of MATE1 in studies that apply (i) proteomic methods to identify peptides and amino acid residues that specifically interact with a photoactivatable probe of the OC/H+ exchanger; and (ii) apply computational methods (steered molecular dynamics) to identify amino acid residues that influence substrate translocation. These studies will play a critical role in establishing models that accurately predict and, ideally, preempt unwanted interactions of cationic drugs in both the kidney and liver.