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 clinic, estradiol-17β-glucuronide (E17βG) remains the probe substrate frequently used in MRP2 inhibition assays. Here we re-capitulated the sigmoidal kinetics of MRP2 mediated uptake of E17βG, with apparent Km and Vmax values 170 ±17 μM and 1447 ± 137 pmol/mg protein/min, respectively. Hill coefficient (2.05 ± 0.1), suggests multiple substrate binding sites for E17βG transport with cooperative interactions. Using E17βG as a probe substrate, 51 of 97 compounds tested (53%) showed up to 6-fold stimulatory effects. Alternatively, we demonstrated that coproporphyrin-I (CP-I) is a MRP2 substrate in membrane vesicles, for the first time. The uptake of CP-I followed a hyperbolic relationship, adequately described by the standard Michaelis-Menten equation (apparent Km and Vmax values were 7.7 ± 0.7 μM and 48 ± 11 pmol/mg protein/min, respectively), suggesting the involvement of single binding site. Of 47 compounds tested, thirty compounds were inhibitors of human MRP2 and eight compounds (17%) stimulated MRP2-medaited CP-I transport. The stimulators were found to share basic backbone structure of the physiological 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 E17βG for appreciating MRP2 interactions while minimizing potential false-negatives for MRP2 inhibition due to stimulatory effects.
- drug-drug interactions
- efflux transporters (P-gp, BCRP, MRP, MATE, BSEP, etc)
- membrane-protein interactions
- Transporter-mediated drug/metabolite disposition
- The American Society for Pharmacology and Experimental Therapeutics