Substrate-dependent ligand inhibition of the human organic cation transporter OCT2

Abstract

Organic cation transporter 2 (OCT2) mediates the initial step in renal secretion of organic cations: uptake from the blood, across the basolateral membrane, and into the renal proximal tubule cells. Because of its potential as a target for unwanted drug-drug interactions (DDIs), considerable attention has been directed toward understanding the basis of OCT2 selectivity. These studies typically assess selectivity based on ligand inhibition profiles for OCT2-mediated transport of a probe substrate. However, little attention has been given to the potential influence of the substrate on the profile of ligand inhibition. Here we compared the IC50 values obtained for a set of structurally distinct inhibitors against OCT2-mediated transport of three structurally distinct substrates: 1-methyl-4-phenylpyridinium (MPP); metformin; and a novel fluorescent substrate, N,N,N-trimethyl-2-[methyl(7-nitrobenzo[c][l,2,5]oxadiazol-4-yl)amino]ethanaminium iodide (NBD-MTMA). The median IC50 value for inhibition of MPP transport was 9-fold higher than that for inhibition of metformin transport. Similarly, the median IC50 value for inhibition of MPP transport was 5-fold higher than that for NBD-MTMA transport. However, this was not a systematic difference in inhibitory efficacy; the ratio of IC50 values, MPP versus NBD-MTMA, ranged from 88-fold (ipratropium) to 0.3-fold (metformin). These data show that 1) the choice of OCT2 substrate significantly influences both quantitative and qualitative inhibitory interactions with cationic drugs; and 2) ligand interactions with OCT2 are not restricted to competition for a common ligand binding site, consistent with a binding surface characterized by multiple, possibly overlapping interaction sites. Development of predictive models of DDIs with OCT2 must take into account the substrate dependence of ligand interaction with this protein.

Fig. 1.

(A) Effect of increasing concentration of unlabeled MPP on the rate of [³H]MPP uptake into HEK293 cells or CHO cells that stably expressed OCT2. Each point is the mean (± S.E.) of 1-minute uptakes of 33 nM [³H]MPP (determined in triplicate) measured in three separate experiments with each cell line. Inset: the same data presented as total MPP uptake (labeled plus unlabeled MPP) as a function of increasing MPP concentration. (B) The effect on increasing metformin concentration on the rate of [³H]MPP uptake into HEK293 or CHO cells that stably expressed OCT2. Each point is the mean (± S.E.) of 1-minute uptakes of 12 nM [³H]MPP (determined in triplicate) measured in five (HEK293) or three (CHO) separate experiments. Uptakes were normalized to that measured in the absence of metformin. For the purpose of comparing absolute rates of transport, clearance of tracer in the control condition averaged (± S.E.) 2.8 ± 0.3 and 2.5 ± 0.5 μl cm⁻² min⁻¹ for HEK293 and CHO cells, respectively. (C) Relationship between the log of K_t values for OCT2-mediated substrate transport (open circles) or IC₅₀ values for inhibition of OCT2-mediated MPP transport measured in stably transfected CHO cells (x-axis) or HEK293 cells (y-axis). The dashed line indicates the line of identity (i.e., equal kinetic values in the two cell types). The heavy solid line reflects linear regression (r = 0.99) of the log of the IC₅₀ values (with the 95% confidence interval).

Fig. 2.

Relationship between IC₅₀ values for inhibition of OCT2-mediated MPP transport determined in the present study (x-axis; Table 1) and those reported in Table 1 of the study by Zolk et al. (2009) (y-axis). The dashed line indicates the line of identity (equal IC₅₀ values). The heavy solid line reflects linear regression (r = 0.96) of the log of all the IC₅₀ values (light curved lines indicate 95% confidence interval), except chloroquine (●; see Results).

Fig. 3.

(A) Relationship between IC₅₀ values for the inhibition of OCT2-mediated transport of metformin (x-axis) and MPP (y-axis), as shown in Fig. 6c in Zolk et al. (2009). The dashed line indicates the line of identity [equal IC₅₀ values; light curved lines indicate 95% confidence interval (CI)]. The heavy solid line reflects linear regression of the log of the IC₅₀ values. (B) Relationship between IC₅₀ values for inhibition of OCT2-mediated transport of metformin (x-axis) and MPP (y-axis) determined in the present report. The dashed line indicates the line of identity (equal IC₅₀ values. The heavy solid line reflects linear regression (r = 0.92 or 0.97) of the log of the IC₅₀ values; light curved lines indicate 95% CI). In both figures, the points represent mean values (± S.E.) determined in separate experiments (2–6 in the present study; reported as 3 in Zolk et al., 2009). Gray symbols indicate ligands tested by both Zolk et al. (2009) and the present study; circular symbols indicate ligands tested in the present study only; square symbols by Zolk et al. (2009) only (refer to Table 3). Different ligands indicated by different shapes: octagon, imipramine; triangle, clonidine; inverted triangle, verapamil; diamond, quinidine; open square, carvedilol; half-filled square up, mexiletine; half-filled square down, flecanide; open circle, cimetidine; half-filled circle left, chloroquine (refer to Table 3).

Fig. 4.

Two-dimensional structures of the three OCT2 substrates used in this study: MPP, metformin, and NBD-MTMA. The Tanimoto similarities were calculated using ChemMine Web Tools (http://chemmine.ucr.edu/).

Fig. 5.

The effect of increasing NBD-MTMA concentration on uptake of [³H]MPP (●) or [¹⁴C]metformin (○) into CHO cells that stably expressed OCT2. Each point is the mean (± S.E.) of 1-minute uptakes (expressed as the percentage of uptake measured in the absence of NBD-MTMA) of 16 nM [³H]MPP or 12 μM [¹⁴C]metformin (determined in triplicate) measured in three separate experiments.

Fig. 6.

(A) Time course of uptake of 10 μM NBD-MTMA, expressed as raw accumulated fluorescence (arbitrary units), into wild-type (WT) CHO cells or cells that expressed OCT2. Each point is the mean (± S.E.) uptake measured in six wells of a 96-well plate in a single representative experiment. Lines were fit by linear regression (with 95% confidence limits). (B) Effect of increasing concentration (1, 10, or 100 μM) of NBD-MTMA on the rate of OCT2-specific accumulation of fluorescence. Uptakes determined as in (A), in a single, representative experiment, with the WT-CHO signal subtracted from the CHO-OCT2 signal. Lines were fit by linear regression. arb, arbitrary. (C) Time course of uptake of 6 nM [³H]NBD-MTMA in OCT2-expressing (filled symbols) and WT (open symbols) CHO cells, measured in the absence (circles) or presence (squares) of 1 mM unlabeled MPP. Each point is the mean (± S.E.) of triplicate determinations of accumulation of radiolabeled substrate determined in a single representative experiment; the lines were fit to the data using second-order polynomial expansion. (D) Kinetics of NBD-MTMA transport into OCT2-expressing CHO cells determined from simultaneous accumulation of fluorescence (filled circles) or radioactively labeled substrate (open circles). Each point is the mean (± S.E.) of 5-minute uptakes (6 wells), measured in three separate experiments.

Fig. 7.

The effect of increasing inhibitor concentration on the rate of NBD-MTMA uptake into CHO cells that stably expressed OCT2. Each point is the mean (± S.E.) of five minute uptakes (expressed as the percentage of uptake measured in the absence of inhibitor) of 10 nM [³H]MPP (determined in triplicate) measured in three to four separate experiments.

Fig. 8.

(A) Relationship between IC₅₀ values for inhibition of OCT2-mediated transport of NBD-MTMA (x-axis; Table 1 in the present report) and MPP (y-axis; as reported in Table 1 of Zolk et al., 2009). The dashed line indicates the line of identity (equal IC₅₀ values; light curved lines indicate 95% confidence interval). The heavy solid line reflects linear regression (r = 0.84) of the log of the IC₅₀ values. The points represent mean values (± S.E.) determined in separate experiments (two to six in the present study, Table 1; reported as three in Zolk et al., 2009). (B) Ratio of IC₅₀ value for inhibition of OCT2-mediated MPP transport to the value for inhibition of NBD-MTMA transport. The dashed line indicates the median value (5.1) for this ratio.

Fig. 9.

Relationship between IC₅₀ values for inhibition of OCT2-mediated transport of NBD-MTMA (x-axis; Table 2 in the present report) and metformin (y-axis; open symbols indicate values measured by us; solid symbols show the values reported by Zolk et al., 2009) (refer to Table 3). The dashed line indicates the line of identity (equal IC₅₀ values; light curved lines indicate 95% confidence interval). The heavy solid line reflects linear regression (r = 0.89) of the log of the IC₅₀ values.