Decision tree–based predictions of potential DDIs with multidrug transporters are complicated when the quantitative profile of inhibition of transport by a potential perpetrator is influenced by the choice of substrate used to assess transport activity (e.g., Hacker et al., 2015). , 2002; Roth et al., 2011; Belzer et al., 2013; Hacker et al., 2015), the extent to which ligand interaction with MATE1 displays a similar substrate dependence is not clear. The two screens of inhibitor interaction with MATE1 reported to date focused on profiles generated against transport of single substrates, i.e., MPP (Astorga et al., 2012) or ASP (Wittwer et al., 2013). However, we did recently report that two structurally distinct ionic liquids (1-methyl-3-butylimidazolium and N-butyl-N-methylpyrrolidinium) had ICfifty values for inhibition of MATE1-mediated transport of [ 3 H]MPP that were about 4-fold lower than the values observed for inhibition of transport of [ 3 H]triethylmonomethylammonium, consistent with the concept of substrate-dependent ligand interaction with MATE transporters (). However, the current results suggest that substrate identity exerts comparatively little influence on ligand interaction with MATE1.
This conclusion was based on the assessment of transport of four structurally diverse MATE1 substrates, two drugs in common clinical use (metformin and cimetidine) and two probe OCs (MPP and NBD-MTMA) (Fig. 1). When tested as inhibitors of each other’s transport, there were no significant differences between each substrate’s Ktapp value and the IC50 values they displayed against transport of the others (Figs. 3 and 4). This was followed by a low-resolution screen of 400 compounds from the NCC that provided a broadly based assessment of the influence of structural diversity on ligand interaction with MATE1. Although the rank order of inhibitory effectiveness varied slightly for the four test substrates (Fig. 5), no systematic differences were noted. In other words, the results of the low-resolution screen revealed no indication that transport of one of the test substrates was more efficiently reduced by exposure to inhibitory ligands than any of the other substrates (Fig. 6; Supplemental Fig. 1). Finally, substrate-to-substrate pairwise comparisons of IC50 values determined for the structurally diverse subset of the NCC also revealed no differences for the inhibitory interaction of the test compounds against transport of the test substrates (Fig. 8). These data are consistent with the four test substrates and the set of test inhibitors competing for interaction at a common binding site (or a set of mutually exclusive sites) at the external face of the transporter.
The qualifier, the external face of the transporter, is important. The present observations, indeed those from virtually all studies on MATE transport to date (Wright, 2014), focused on the kinetic characteristics of the transporter operating in an uptake mode. However, in its normal physiologic role as the second step in OC secretion, MATE1 mediates efflux of its organic substrates. The emphasis on influx largely reflects the technical challenges associated with accurate assessment of rates of efflux. Cytoplasmic substrate activity is difficult to quantify, and because cells are small the cytoplasmic substrate concentration during efflux changes very rapidly; the combination of these issues typically confounds efforts to measure the kinetics of efflux. It should be acknowledged that although there are thermodynamic constraints on the kinetic properties of influx versus efflux, they need not be symmetrical (Stein, 1986); in other words, under so-called zero-trans conditions the apparent affinity for substrate (or inhibitor) of the cytoplasmic face of MATE1 need not be the same as that of the extracellular face. Thus, whereas the rank ordering of ligand affinity may be expected to be qualitatively similar at the two faces of the membrane (e.g., both membrane faces of monium and corticosterone than for TEA and choline) (Volk et al., 2003), the few studies that have made such measurements suggest that the absolute Kt or IC50 value can differ by 10-fold (or more) (Stein, 1986).
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