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Automation of a Multiplexed Cell-based Assay to Measure Simultaneously Inhibition and Induction of the Cytochrome P450 Isoform 3A4 by Small Molecule CompoundsDownload
January 26, 2010
Authors: Brad Larson, Peter Banks, BioTek Instruments, Inc., P.O. Box 998, Highland Park,Winooski, VT 05404, USA; James Cali, Mary Sobol, Promega Corporation, 2800 Woods Hollow Road, Madison, WI 53711, USA
The role that CYP3A4 plays in the metabolism of drugs has been well documented. Therefore, it is essential to understand how this P450 isoform is affected by xenobiotics, to avoid possible drug-drug interactions. Compounds have been shown to either inhibit the activity of CYP3A4 or induce its expression. What is less understood is how a drug can induce gene expression, and inhibit activity of the same isoform. Here we demonstrate the automation of a cell-based multiplexed assay which provides this information. Inhibition and induction were measured using a luminogenic, cell-membrane permeable 3A4-specific substrate and a luciferin based reporter gene assay, respectively. Normalization to cell number was also performed using a fluorescent cell viability assay. DPX-2 cells were dosed for 48 hours with variable concentrations of 54 individual compounds. The combined information from the triplex assay provides a comprehensive picture of a drug’s effects on CYP3A4.