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.
The kidney and liver actively secrete many drugs from the body, and unwanted drug-drug interactions at the sites of secretion in these organs are a source of substantial morbidity and mortality. The rate-limiting step in the secretion of cationic drugs by the human liver and kidney involves the mediated exchange of organic cations (OCs) for hydrogen ion (H+), a transport process that, until recently, was undescribed at the molecular level. Recent work identified two transporters, MATE1 and MATE2-K, as these "OC/H+ exchangers." Despite their biological importance, and their clinical significance in humans, virtually nothing is known about the relationship of structure and function for these transporters. In this proposal, we outline experiments that will establish the 3D structure of human MATE transporters, identify specific sites within these proteins that influence drug binding, and develop predictive models of substrate/inhibitor interaction with MATE transporters. The results of these studies will help predict and, ideally, preempt unwanted drug-drug interactions in both the kidney and liver, and can be expected to assist in development of programs of structure-based rational drug design.
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