Molecular Organization of the Organic Cation-Proton Exchanger, MATE1

性状（由申请方提供）：肝脏和肾脏从体内分泌大量具有生理学、药理学和毒理学意义的带正电荷的有机分子。载体介导的这些“有机阳离子”（OC）的分泌，特别是肾脏分泌，对这些化合物的药代动力学具有深远的影响，重要的是，OC分泌是许多临床上显著的药物-药物相互作用的位点。OC分泌的活性和限速步骤涉及载体介导的累积OC穿过肾和肝上皮细胞的管腔膜的排出。该过程的分子鉴定涉及新鉴定的多药和毒素挤出蛋白MATE 1和MATE 2-K。虽然现在清楚地了解到OC分泌中发挥重要作用，但实际上对底物与这些转运蛋白相互作用的分子决定因素一无所知。在这个修改后的建议，我们利用最近的解决方案的X-射线结构的原型成员的MATE家族的运输蛋白（NorM）。我们已经使用了NorM结构来开发人MATE 1的同源模型，并且在该提议中，我们概述了两组实验，其设计用于开发药物与MATE转运蛋白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开发MATE 1和MATE 2-K与底物/抑制剂相互作用的3D-QSAR/药效团模型。这些数据将在肾分泌上皮模型中这些转运蛋白综合活性的平行研究背景下进行解释（反过来，将在这些转运蛋白在人肾脏中差异分布的研究背景下进行解释）。目标2将采用基于靶点的方法，使用定点研究来探测MATE 1的拓扑结构和表面可及性，从而测试我们的同源模型所产生的预测，并建立一个旨在探测蛋白质功能结构的数据库，该蛋白质的功能结构是在并行努力中确定的，以解决人MATE 1的X射线结构。目的2还将在应用（i）蛋白质组学方法鉴定与OC/H+交换器的光活化探针特异性相互作用的肽和氨基酸残基;和（ii）应用计算方法（导向分子动力学）鉴定影响底物易位的氨基酸残基的研究中研究MATE 1的底物易位途径。这些研究将在建立模型中发挥关键作用，这些模型可以准确预测，理想情况下，可以预先阻止阳离子药物在肾脏和肝脏中的不必要的相互作用。

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.