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

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 drug‐drug interactions (DDIs). It is estimated that 1–5% 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 OCT2‐mediated 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 OCT2‐mediated transport of the fluorescent OC, NBD‐MTMA produced by four known OCT2 substrates (MPP, TEA, cimetidine, and metformin). In each case presence of inhibitor increased the apparent K t for NBD‐MTMA transport without affecting J max , 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 NBD‐MTMA), 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, ‘substrate‐dependent ligand interaction’ was noted between several other substrate pairs. In particular, MPP is the least sensitive substrate to these inhibitors, with estimated IC 50 's 2–5 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 over‐ or underestimates of the inhibitor's effectiveness. Support or Funding Information supported by NIH 5R01DK058251