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Inside-out-oriented membrane vesicles are useful tools to investigate whether a compound can be an inhibitor of efflux transporters such as multidrug resistance-associated protein 2 (MRP2). However, because of technical limitations of substrate diffusion and low dynamic uptake windows for interacting drugs used in the clinic, estradiol-17 β -glucuronide (E17 β G) remains the probe substrate that is frequently used in MRP2 inhibition assays. Here we recapitulated the sigmoidal kinetics of MRP2-mediated transport of E17 β G, with apparent Michaelis-Menten constant ( K  m ) and V  max values of 170 ±17 µ M and 1447 ± 137 pmol/mg protein/min, respectively. The Hill coefficient (2.05 ± 0.1) suggests multiple substrate binding sites for E17 β G transport with cooperative interactions. Using E17 β G as a probe substrate, 51 of 97 compounds tested (53%) showed up to 6-fold stimulatory effects. Here, we demonstrate for the first time that coproporphyrin-I (CP-I) is a MRP2 substrate in membrane vesicles. The uptake of CP-I followed a hyperbolic relationship, adequately described by the standard Michaelis-Menten equation (apparent K  m and V  max values were 7.7 ± 0.7 µ M and 48 ± 11 pmol/mg protein/min, respectively), suggesting the involvement of a single binding site. Of the 47 compounds tested, 30 compounds were inhibitors of human MRP2 and 8 compounds (17%) stimulated MRP2-mediated CP-I transport. The stimulators were found to share the basic backbone structure of the physiologic steroids, which suggests a potential in vivo relevance of in vitro stimulation of MRP2 transport. We concluded that CP-I could be an alternative in vitro probe substrate replacing E17 β G for appreciating MRP2 interactions while minimizing potential false-negative results for MRP2 inhibition due to stimulatory effects.

Coproporphyrin-I: A Fluorescent, Endogenous Optimal Probe Substrate for ABCC2 (MRP2) Suitable for Vesicle-Based MRP2 Inhibition Assay