Philip Sandoval's Abstracts

      Philip Sandoval
      Ph.D. Student
      Physiological Sciences GIDP

      Conference Summary
      Experimental Biology
      Chicago, Illinois

Abstract

Assessment of Substrate Dependent Ligand Interactions at the Organic Cation Transporter OCT2
Using Six Model Substrates

Philip Joseph Sandoval, Stephen H Wright. Physiology, University of Arizona, Tucson, AZ

The term “organic cation” describes a family of structurally diverse organic compounds that are positively charged at physiological pH. Approximately 40% of prescribed drugs are considered to be OCs. In spite of their structural diversity most OCs are substrates for (or inhibitors of) the Organic Cation Transporter, OCT2, which is expressed in the basolateral membrane of proximal tubule cells and is the initial (‘entry’) step in renal OC secretion. While the multispecificity of OCT2 allows it be extremely versatile in removing a wide array of compounds from the body, it also sets the stage for unwanted drugdrug interactions (DDIs). It is estimated that 15% of emergency room visits can be attributed to the adverse effects that result from DDIs; and for pharmaceutical companies the prediction and prevention of potential DDIs carries a significant cost in both time and money. The primary approach for characterizing the multispecificity of OCT2 has been to screen the inhibitory effectiveness of structurally diverse compounds against the OCT2mediated transport activity of a model substrate in cultured cells, with the profile of inhibition then used to develop pharmacophore models that highlight the molecular determinants of transporter selectivity. However, these models do not take into account the mechanism of substrate and inhibitor interaction at the transporter, nor do they consider the potential influence of substrate structure on inhibitor efficacy; both of these issues are critical for understanding if ligands interact at a single binding site or at multiple sites. We used two approaches to assess the mechanism of ligand interaction with OCT2. First, we determined the kinetic basis of inhibition of OCT2mediated transport of the fluorescent OC, NBDMTMA produced by four known OCT2 substrates (MPP, TEA, cimetidine, and metformin). In each case presence of inhibitor increased the apparent Kt for NBDMTMA transport without affecting Jmax, consistent with a competitive interaction of all these substrates at a common binding site. The second approach involved the conventional screening of inhibition of OCT2 activity produced by a 20 μM concentration of each of 320 test compounds, extended to cover transport of six structurally diverse substrates (radiolabeled MPP, TEA, metformin, cimetidine; and the fluorescent compounds ASP and NBDMTMA), thereby testing the hypothesis that interaction of all compounds with a common binding site will result in each test ligand producing the same degree of inhibition of each test substrate. Although pairwise comparisons of inhibition profiles for inhibition of three of the substrates (metformin, cimetidine and TEA) revealed no systematic influence of substrate structure on ligand interaction with the transporter, ‘substratedependent ligand interaction’ was noted between several other substrate pairs. In particular, MPP is the least sensitive substrate to these inhibitors, with estimated IC50’s 25 fold greater than the other substrates tested. This substrate dependence should be taken into consideration when testing inhibitors against a single substrate because it may result in significant overor underestimates of the inhibitor’s effectiveness.

Lay Abstract

One of the principal functions of the kidney is to remove toxic compounds from the body using membrane drug transporters such as OCT2 (Organic Cation Transporter 2). One of the unique features of OCT2 is that it is “mutlispecific” or capable of transporting a variety of substrates with different structures. OCT2 is found primarily in the proximal tubule cells of the kidney and is a key transporter involved in the removal of a family of compounds designated as “organic cations” from the blood and into the urine. It is estimated that nearly 40% of prescribed drugs come in the form of organic cations and as a result OCT2 could potentially be involved in the removal of nearly half of prescribed drugs. While OCT2 multispecificity enables it to be very versatile in removing toxins from the body it also sets the stage for potential drug-drug interactions. Drug-drug interactions occur when two or more substrates compete for transport by OCT2 which could prevent their removal and result in toxicity. Adverse drug-drug interactions carry a significant healthcare cost with an estimated 1-5% of emergency room visits being attributed to adverse drug reactions. Additionally, pharmaceutical companies invest years and millions of dollars in order to assess potential adverse reactions their novel compounds may cause. It is the goal of our lab and others to understand OCT2 multispecificity in order to prevent future drug-drug interactions and to accelerate pharmaceutical research. The primary approach in characterizing OCT2 specificity is to screen the inhibitory activity of a set of structurally diverse inhibitors against the transport activity of a model substrate in order to develop a pharmacophore model that highlights the molecular features that make a good inhibitor of OCT2. However, these models do not include the mechanism of ligand interaction at the transporter which is critical for understanding whether there is a single or multiple binding sites for ligands to interact at OCT2. My research is, to date, the most extensive study of the mechanism of ligand interaction at OCT2 in order to further characterize ligand interaction at the transporter in order to further develop more accurate models of OCT2 selectivity. In order to do this we utilized two approaches. First, we determined the effect of model substrates (cimetidine, TEA, MPP, and Metformin) on the kinetics of OCT2 mediated uptake of the fluorescent organic cation NBD-MTMA. In each case the presence of a model substrate increased the apparent Kt of NBD-MTMA uptake with no change in Jmax. This is the classic profile for a competitive interaction at the transporter suggesting that NBD-MTMA interacts at a single binding site with these model substrates. The second approach that we used was to screen the inhibitory effectiveness of 20uM concentrations of 400 compounds from the national clinical collection against OCT2 mediated transport activity of the six model substrates MPP, Metformin, NBD-MTMA, TEA, ASP and Cimetidine. If each of these substrates bind to a single common binding site at OCT2 then the inhibitors from the national clinical collection should inhibit the uptake of each substrate to the same extent. Pairwise comparisons of inhibition profiles showed that this was the case for four of our tested substrates (NBD-MTMA, Cimetidine, MPP, and TEA) while MPP was the least sensitive to inhibition and ASP was the most sensitive. Our results indicate that ligands and substrates interact at a single binding site with some exceptions that must be taken into consideration when using MPP or ASP as a substrate.