RT Journal Article
SR Electronic
T1 Coproporphyrin-I: A fluorescent, endogenous optimal probe substrate for ABCC2 (MRP2) that is suitable for vesicle based MRP2 inhibition assay
JF Drug Metabolism and Disposition
JO Drug Metab Dispos
FD American Society for Pharmacology and Experimental Therapeutics
SP dmd.116.074740
DO 10.1124/dmd.116.074740
A1 Ravindranath Reddy Gilibili
A1 Sagnik Chatterjee
A1 Pravin Bagul
A1 Kthleen W. Mosure
A1 Bokka Venkata Murali
A1 T. Thanga Mariappan
A1 Sandhya Mandlekar
A1 Yurong Lai
YR 2017
UL http://dmd.aspetjournals.org/content/early/2017/03/21/dmd.116.074740.abstract
AB 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.