Compound profiling for ABCC2 (MRP2) using a fluorescent microplate assay system

Purpose: To establish a fluorescent dye (Glutathione methylfluorescein, GSMF) based assay to rapidly screen compounds for drug efflux interactions with the ABC-protein ABCC2 (MRP2). Methods: MDCK-cells overexpressing ABCC2 were cultured until confluency in 96-well plates. Cells were incubated with chloromethylfluorescein-diacetate (CMFDA) in the absence and presence of increasing concentrations of potential substrates and inhibitors of ABCC2. After formation of GSMF the extent of intracellular fluorescence was monitored with a fluorescence plate reader in a time- and a concentration-dependent manner. Results: MDCK cells showed stable expression of ABCC2 and, as a consequence, GSMF was extruded by the cells across the apical membrane in an energy-dependent manner. The incubation conditions (optimum CMFDA concentration; glutathione dependency, membrane toxicity) were elaborated. Determination of intracellular glutathione concentration indicated that under the chosen conditions glutathione is not rate limiting for the assay performance. Known inhibitors of ABCB1 (P-GP) and ABCG2 (BCRP) did not influence intracellular fluorescence intensity, but a significant increase of intracellular fluorescence was observed in the presence of MRP2-substrates and inhibitors accompanied with a concomitant decrease of GSMF efflux. Conclusions: The GSMF-assay based on fluorescence accumulation in MRP2-overexpressing MDCK cells can be used as a rapid microplate screening system for interactions of drugs with MRP2 and therefore represents a useful tool in drug profiling. (C) 2007 Elsevier B.V. All rights reserved.