Multiple mechanisms of ligand interaction with the human organic cation transporter, OCT2

Abstract

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.

Lack of Influence of Substrate on Ligand Interaction with the Human Multidrug and Toxin Extruder, MATE1

“…In both studies the test inhibitors exerted a significantly greater inhibition of metformin transport than of MPP transport. These data were cited as being consistent with the view expressed by others (Zhang et al, 2005;Koepsell, 2011;Egenberger et al, 2012;Harper and Wright, 2013) that ligand interaction with OCT transporters may involve interaction at a binding surface that can support binding of two or more ligands at once. The observation here of inhibitor interactions with MATE1 that consistently displayed the same apparent inhibitor constants, regardless of substrate identity, suggests that substrates and inhibitory ligands typically interact at a kinetically common binding site at the external face of MATE1.…”

Organic Anion Transporter 1 Is Inhibited by Multiple Mechanisms and Shows a Transport Mode Independent of Exchange

“…From these data we speculated that for inhibitors where IC 50 values shifted when a-ketoglutarate was present, they act in a competitive manner to reduce transport activity, whereas for inhibitors where a shift was not observed, they are noncompetitive inhibitors. Given our previous results, and recent data showing that a related transporter, organic cation transporter 2, is inhibited by multiple mechanisms (Harper and Wright, 2013), we hypothesized that OAT1 can be inhibited by multiple mechanisms as well.…”

Substrate-Dependent Ligand Inhibition of the Human Organic Cation Transporter OCT2

“…We routinely use CHO cells as an expression system to study OCT2 transport (e.g., Suhre et al, 2005;Pelis et al, 2007Pelis et al, , 2012Harper and Wright, 2013). However, it is more common to use HEK293 cells as an expression for studying OCT-mediated transport (Nies et al, 2011).…”

“…It should be noted that whereas the metformin substrate concentration used in the determination of inhibitory ligand IC 50 values by Zolk et al (2009) was , , than the apparent K t for OCT2-mediated metformin transport, that was not the case for MPP; as noted earlier, the MPP substrate concentration used in those experiments was 10 mM, which is sufficiently close to the apparent K t for MPP transport these authors measured (20 mM) that it is likely that at least some of the IC 50 values they reported represented modest (∼50%) overestimates of the "true" K i values for any of the ligands studied that interacted competitively with MPP. However, to the extent that was the case (and we have shown that for at least some of these ligands, the interactions are noncompetitive or mixed-type rather than competitive) (Harper and Wright, 2013), the MPP IC 50 values still exceed by more than 7-fold those for metformin. We build on this idea in an upcoming section, but here we show that this observation by Zolk et al is one we could reproduce.…”