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

Organic cation transporter (OCT) 2 mediates the entry step for organic cation secretion by renal proximal tubule cells and is a site of unwanted drug-drug interactions (DDIs). But reliance on decision tree-based predictions of DDIs at OCT2 that depend on IC50 values can be suspect because they can be influenced by choice of transported substrate; for example, IC50 values for the inhibition of metformin versus MPP transport can vary by 5- to 10-fold. However, it is not clear whether the substrate dependence of a ligand interaction is common among OCT2 substrates. To address this question, we screened the inhibitory effectiveness of 20 mu M concentrations of several hundred compounds against OCT2-mediated uptake of six structurally distinct substrates: MPP, metformin, N,N,N-trimethyl-2-[methyl(7-nitrobenzo[c][1,2,5]-oxadiazol-4-yl)amino]ethanaminium (NBD-MTMA), TEA, cimetidine, and 4-4-dimethylaminostyryl-N-methylpyridinium (ASP). Of these, MPP transport was least sensitive to inhibition. IC50 values for 20 structurally diverse compounds confirmed this profile, with IC50 values for MPP averaging 6-fold larger than those for the other substrates. Bayesian machine-learning models of ligand-induced inhibition displayed generally good statistics after cross-validation and external testing. Applying our ASP model to a previously published large-scale screening study for inhibition of OCT2-mediated ASP transport resulted in comparable statistics, with approximately 75% of active inhibitors predicted correctly. The differential sensitivity of MPP transport to inhibition suggests that multiple ligands can interact simultaneously with OCT2 and supports the recommendation that MPP not be used as a test substrate for OCT2 screening. Instead, metformin appears to be a comparatively representative OCT2 substrate for both in vitro and in vivo (clinical) use.