Kinetic validation of the use of carboxydichlorofluorescein as a drug surrogate for MRP5-mediated transport.
Multidrug resistance protein-5 (MRP5, ABCC5) is a member of the ATP-binding cassette transporter superfamily that effluxes a broad range of natural and xenobiotic compounds such as cyclic GMP, antiviral compounds, and cancer chemotherapeutic agents including nucleoside-based drugs, antifolate agents and platinum compounds. In cellular assays, MRP5 transfectants are less fluorescent after incubation with 5-chloromethylfluorescein diacetate (CMFDA). The present study examines the uptake of a close fluorescent analog, carboxydichlorofluorescein (CDCF), and drug substrates into inside-out membrane vesicles prepared from MRP transfected cells. MRP5-mediated uptake of CDCF was ATP-dependent and GSH-independent and possessed a Km of 12 microM and a Vmax of 56 pmol/min/mg prot. Comparison of kinetic parameters with drug substrates such as methotrexate (MTX), pemetrexed (Alimta), and the metabolite of 5-fluorouracil, 5-fluorodeoxyuridine monophosphate (5-FdUMP) (Km values of 0.3-1.3 mM) indicated that MRP5 has a 25-100-fold higher affinity for CDCF than for these drugs and that they share a common transport binding site. In addition, the potency of MRP5 inhibitors such as probenecid, MK571, and the phosphodiesterase 5 inhibitors correlated well between the uptake of CDCF and MTX. A survey of CDCF uptake by other MRPs revealed that MRP2 (ABCC2) also demonstrated ATP-dependent uptake with a Km of 19 microM and Vmax of 95.5 pmol/min/mg prot, while MRP1 (ABCC1) and MRP4 (ABCC4) had little to no uptake. Taken together, these data indicate that CDCF is a useful fluorescent drug surrogate with which to measure ATP-dependent MRP5-mediated transport.
Pratt
Susan
S
Lilly Research Laboratories, Lilly Corporate Center, Indianapolis IN 46285, USA. pratt_susan@lilly.com
Chen
Victor
V
Perry
William I
WI
Starling
James J
JJ
Dantzig
Anne H
AH
D015106:3',5'-Cyclic-GMP Phosphodiesterases; D000964:Antimetabolites, Antineoplastic / Q000378:metabolism; D054706:Cyclic Nucleotide Phosphodiesterases, Type 5; D005452:Fluoresceins / Q000378:metabolism*; D005456:Fluorescent Dyes / Q000378:metabolism*; D005971:Glutamates / Q000378:metabolism; D006147:Guanine / Q000031:analogs & derivatives / Q000378:metabolism; D006367:HeLa Cells; D006801:Humans; D007700:Kinetics; D026901:Membrane Transport Proteins / Q000235:genetics / Q000378:metabolism; D008727:Methotrexate / Q000378:metabolism; D000089762:Multidrug Resistance-Associated Protein 2; D027425:ATP-Binding Cassette, Sub-Family C Proteins / Q000037:antagonists & inhibitors / Q000235:genetics / Q000378:metabolism*; D000068437:Pemetrexed; D010726:Phosphodiesterase Inhibitors / Q000494:pharmacology; D010727:Phosphoric Diester Hydrolases / Q000378:metabolism; D011339:Probenecid / Q000494:pharmacology; D011422:Propionates / Q000494:pharmacology; D011804:Quinolines / Q000494:pharmacology; D015203:Reproducibility of Results; D013194:Staining and Labeling / Q000379:methods; D014162:Transfection
D015106:3',5'-Cyclic-GMP Phosphodiesterases; D000964:Antimetabolites, Antineoplastic; D054706:Cyclic Nucleotide Phosphodiesterases, Type 5; D005452:Fluoresceins; D005456:Fluorescent Dyes; D005971:Glutamates; D006147:Guanine; D026901:Membrane Transport Proteins; D008727:Methotrexate; D000089762:Multidrug Resistance-Associated Protein 2; D027425:ATP-Binding Cassette, Sub-Family C Proteins; D000068437:Pemetrexed; D010726:Phosphodiesterase Inhibitors; D010727:Phosphoric Diester Hydrolases; D011339:Probenecid; D011422:Propionates; D011804:Quinolines; C000715087:ABCC2 protein, human; C120809:ABCC5 protein, human; C053897:5(6)-carboxy-2',7'-dichlorofluorescein; C069306:5-chloromethylfluorescein; C059141:verlukast; C517381:PDE5A protein, human