TY - JOUR T1 - Coproporphyrin-I: A Fluorescent, Endogenous Optimal Probe Substrate for ABCC2 (MRP2) Suitable for Vesicle-Based MRP2 Inhibition Assay JF - Drug Metabolism and Disposition JO - Drug Metab Dispos SP - 604 LP - 611 DO - 10.1124/dmd.116.074740 VL - 45 IS - 6 AU - Ravindranath Reddy Gilibili AU - Sagnik Chatterjee AU - Pravin Bagul AU - Kathleen W. Mosure AU - Bokka Venkata Murali AU - T. Thanga Mariappan AU - Sandhya Mandlekar AU - Yurong Lai Y1 - 2017/06/01 UR - http://dmd.aspetjournals.org/content/45/6/604.abstract N2 - 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-17β-glucuronide (E17βG) remains the probe substrate that is frequently used in MRP2 inhibition assays. Here we recapitulated the sigmoidal kinetics of MRP2-mediated transport of E17βG, with apparent Michaelis-Menten constant (Km) and Vmax values of 170 ±17 µM and 1447 ± 137 pmol/mg protein/min, respectively. The 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. 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 Km and Vmax values were 7.7 ± 0.7 µM 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 E17βG for appreciating MRP2 interactions while minimizing potential false-negative results for MRP2 inhibition due to stimulatory effects. ER -