OCT2 is the entry step for organic cation (OC) secretion by renal proximal tubules. Although many drugs inhibit OCT2 activity, neither the mechanistic basis of their inhibition nor their transport status is generally known. Using representatives of several structural classes of OCT2-inhibitory ligands described recently (Kido Y, Matsson P, Giacomini KM. J Med Chem 54: 4548-4558, 2011), we determined the kinetic basis of their inhibition of 1-methyl-4-phenylpyridinium (MPP) transport into Chinese hamster ovary cells that stably expressed hOCT2. The "cluster II" inhibitors (which contain known OCT2 substrates) metformin and cimetidine interacted competitively with MPP. However, other cluster II compounds, including tetraethylammonium (TEA), diphenidol and phenyltoloxamine, were mixed-type inhibitors of MPP transport (i.e., decreasing J(max) and increasing K(t)). A cluster III (neutral steroid) representative, adrenosterone, and a cluster I (large, flexible cation) representative, carvedilol, displayed noncompetitive inhibitory profiles. Competitive counterflow (CCF) was used to determine whether the inhibitory ligands served as substrates of hOCT2. Carvedilol (cluster I) and adrenosterone (cluster III) did not support CCF, consistent with the prediction that members of these structural classes are likely to be nontransported inhibitors of OCT2. The cluster II representatives MPP, metformin, cimetidine, and TEA all supported CCF, consistent with independent assessments of their OCT2-mediated transport. However, the other cluster II representatives, diphenidol and phenyltoloxamine, failed to support CCF, suggesting that neither compound is transported by OCT2. An independent assessment of diphenidol transport (using liquid chromatography with tandem mass spectroscopy) confirmed this observation. The results underscore the caution required for development of predictive models of ligand interaction with multidrug transporters.

The organic cation (OC) transporter 2 (OCT2; SLC22A2) is the entry step of the OC secretory pathway of human renal proximal tubules. We used Competitive Exchange Diffusion (CED) to indirectly assess whether inhibitors of OCT2‐mediated transport also serve as substrates for transport. In CED, CHO cells that stably expressed hOCT2 were preloaded with [3H]MPP (‘M*’) to steady‐state (30 min). The solution was then replaced with one containing either (i) the pre‐loading solution (PLS), or (ii) PLS plus unlabeled test ligand. Whereas PLS alone supported no change in M* content over 5 min, presence of a transported ligand supported CED and a decrease in M* content. When the ligand was 100 uM unlabeled MPP (20× its Kt), M* decreased by 40% within 1 min. Saturating concentrations of the other OCT2 substrates (20× their Kt values), TEA, metformin and cimetidine supported M* efflux comparable to that supported by MPP (10 mM glucose, sucrose, or mannitol supported no CED). We then tested two compounds recently identified as strong inhibitors of OCT2 (Kido et al, J Med Chem, 54(13):4548–58, 2011): the sterol, adrenosterone (AS; IC50 of 6 uM) and the strong base, phenyltoloxamine (PT; IC50 of 6 uM). Interestingly, saturating AS or PT did not support CED‐induced efflux of M*. AS and PT both proved to be non‐competitive inhibitors of OCT2‐mediated MPP transport (decreasing Jmax with little or no change in Kt). NIH grant 5R01DK58251.