Coproporphyrin-I: A Fluorescent, Endogenous Optimal Probe Substrate for ABCC2 (MRP2) Suitable for Vesicle-Based MRP2 Inhibition Assay.
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) andVmaxvalues of 170 ±17µM 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 (apparentKmandVmaxvalues were 7.7 ± 0.7µM 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.
Pharmaceutical Candidate Optimization, Biocon Bristol-Myers Squibb R&D Center (BBRC), Syngene International Ltd., Bangalore, India (R.R.G., S.C., P.B., B.V.M., T.T.M.); Pharmaceutical Candidate Optimization, Bristol-Myers Squibb Company, Princeton, New Jersey (K.W.M., Y.L.); and Pharmaceutical Candidate Optimization, Biocon Bristol-Myers Squibb R&D Center (BBRC), Bangalore, India (S.M.).
Ravindranath Reddy
RR
Gilibili
Pharmaceutical Candidate Optimization, Biocon Bristol-Myers Squibb R&D Center (BBRC), Syngene International Ltd., Bangalore, India (R.R.G., S.C., P.B., B.V.M., T.T.M.); Pharmaceutical Candidate Optimization, Bristol-Myers Squibb Company, Princeton, New Jersey (K.W.M., Y.L.); and Pharmaceutical Candidate Optimization, Biocon Bristol-Myers Squibb R&D Center (BBRC), Bangalore, India (S.M.) sagnik.chatterjee@syngeneintl.com.
Sagnik
S
Chatterjee
Pharmaceutical Candidate Optimization, Biocon Bristol-Myers Squibb R&D Center (BBRC), Syngene International Ltd., Bangalore, India (R.R.G., S.C., P.B., B.V.M., T.T.M.); Pharmaceutical Candidate Optimization, Bristol-Myers Squibb Company, Princeton, New Jersey (K.W.M., Y.L.); and Pharmaceutical Candidate Optimization, Biocon Bristol-Myers Squibb R&D Center (BBRC), Bangalore, India (S.M.).
Pravin
P
Bagul
Pharmaceutical Candidate Optimization, Biocon Bristol-Myers Squibb R&D Center (BBRC), Syngene International Ltd., Bangalore, India (R.R.G., S.C., P.B., B.V.M., T.T.M.); Pharmaceutical Candidate Optimization, Bristol-Myers Squibb Company, Princeton, New Jersey (K.W.M., Y.L.); and Pharmaceutical Candidate Optimization, Biocon Bristol-Myers Squibb R&D Center (BBRC), Bangalore, India (S.M.).
Kathleen W
KW
Mosure
Pharmaceutical Candidate Optimization, Biocon Bristol-Myers Squibb R&D Center (BBRC), Syngene International Ltd., Bangalore, India (R.R.G., S.C., P.B., B.V.M., T.T.M.); Pharmaceutical Candidate Optimization, Bristol-Myers Squibb Company, Princeton, New Jersey (K.W.M., Y.L.); and Pharmaceutical Candidate Optimization, Biocon Bristol-Myers Squibb R&D Center (BBRC), Bangalore, India (S.M.).
Bokka Venkata
BV
Murali
Pharmaceutical Candidate Optimization, Biocon Bristol-Myers Squibb R&D Center (BBRC), Syngene International Ltd., Bangalore, India (R.R.G., S.C., P.B., B.V.M., T.T.M.); Pharmaceutical Candidate Optimization, Bristol-Myers Squibb Company, Princeton, New Jersey (K.W.M., Y.L.); and Pharmaceutical Candidate Optimization, Biocon Bristol-Myers Squibb R&D Center (BBRC), Bangalore, India (S.M.).
T Thanga
TT
Mariappan
Pharmaceutical Candidate Optimization, Biocon Bristol-Myers Squibb R&D Center (BBRC), Syngene International Ltd., Bangalore, India (R.R.G., S.C., P.B., B.V.M., T.T.M.); Pharmaceutical Candidate Optimization, Bristol-Myers Squibb Company, Princeton, New Jersey (K.W.M., Y.L.); and Pharmaceutical Candidate Optimization, Biocon Bristol-Myers Squibb R&D Center (BBRC), Bangalore, India (S.M.).
Sandhya
S
Mandlekar
Pharmaceutical Candidate Optimization, Biocon Bristol-Myers Squibb R&D Center (BBRC), Syngene International Ltd., Bangalore, India (R.R.G., S.C., P.B., B.V.M., T.T.M.); Pharmaceutical Candidate Optimization, Bristol-Myers Squibb Company, Princeton, New Jersey (K.W.M., Y.L.); and Pharmaceutical Candidate Optimization, Biocon Bristol-Myers Squibb R&D Center (BBRC), Bangalore, India (S.M.).
Yurong
Y
Lai
D001665:Binding Sites / Q000187:drug effects; D001692:Biological Transport / Q000187:drug effects; D003306:Coproporphyrins / Q000494:pharmacology*; D004958:Estradiol / Q000031:analogs & derivatives / Q000378:metabolism; D005456:Fluorescent Dyes / Q000494:pharmacology*; D006801:Humans; D007700:Kinetics; D026901:Membrane Transport Proteins / Q000378:metabolism; D000089762:Multidrug Resistance-Associated Protein 2; D027425:ATP-Binding Cassette, Sub-Family C Proteins / Q000032:analysis*
D003306:Coproporphyrins; D004958:Estradiol; D005456:Fluorescent Dyes; D026901:Membrane Transport Proteins; D000089762:Multidrug Resistance-Associated Protein 2; D027425:ATP-Binding Cassette, Sub-Family C Proteins; C000715087:ABCC2 protein, human; C025483:estradiol-17 beta-glucuronide; C031467:coproporphyrin I