Determining the Kinetic Mechanism of Metformin and NBD Interaction at the Multidrug Transporter OCT2: a Mathematical and Experimental Approach
Authors: Sandoval Philip J , Secomb Timothy W , Wright Stephen H 
Published: November 2023

Approximately 40 percent of commonly prescribed drugs are positively charged at physiological pH and, consequently, referred to as ‘organic cations’ (OCs). This structurally diverse array of compounds is primarily cleared from the blood by renal secretion. The first step in this process, entry from the blood across the basolateral membrane of proximal tubule cells, is mediated by the Organic Cation Transporter, OCT2. The necessary ‘multispecificity’ of OCT2 sets the stage for adverse drug‐drug interactions when two or more OCs compete for the transporter. Current predictive models of ligand interaction with OCT2 are based on inhibition of the transport of a model substrate produced by a group of structurally diverse ligands. While these models provide insight into the molecular determinants of inhibitor interaction with OCT2, they ignore the kinetic basis of ligand binding to a protein that appears to have a complex binding surface rather than a simple, common binding ‘site.’ Complicating the assessment of such interactions are several confounding issues associated with the measurement of transport in cell systems that overexpress introduced transport proteins, including unstirred water layers (UWLs; Shibayama et al, Pharmaceut Res, 32:293, 2015) and efflux of accumulated substrate, both of which influence estimates of the kinetics of substrate influx. We have developed a MATLAB‐based simulation of the influence these factors have on the apparent kinetics of metformin's inhibition of OCT2‐mediated transport of the probe OC substrate, NBD‐MTMA. The presence of an UWL of 1000 μm was predicted to result in a ~2.5‐fold overestimate of the true Kt for NBD‐MTMA transport (from 4 μM to 10 μM); and the influence of OCT2‐mediated efflux of accumulated substrate leads to a ~33% underestimate of the Jmax of transport (30 vs. 45 pmol cm−2 min−1). However, the simulation also revealed that these factors should not influence the kinetic profile of ligand inhibition of OCT2; a competitive inhibitor should still increase the apparent Kt of substrate transport while having no effect on the apparent Jmax of the process (even though the quantitative values of each parameter differ from those based on true unidirectional influx). We tested this by measuring the influence metformin on the kinetics of NBD‐MTMA transport. The presence of 1 mM metformin increased the apparent Kt of OCT2‐mediated NBD‐MTMA transport from 10.0 to 32.5 μM (P<0.0001), but had no effect on Jmax (29.8 vs. 32.3 pmol cm−2 min−1; P=0.7). These values are similar to our simulation‐derived values that predicted that 1mM metformin would increase the apparent Kt of OCT2‐mediated NBD transport from 10 μM to 27.8 μM with no effect on the apparent Jmax (33.2 vs 33.6 pmol cm−2 min−1). This simulation should permit assessment of the kinetic basis of OCT2 inhibition produced by an array of structurally diverse ligands fully informed of the experimental complexity of such measurements.

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supported by NIH Award 5R01DK058251