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Abstract for Novel Bioluminescent Substrates to Measure CYP3A4
Activity in Cell-Based and Microsome Assays
Mary Sobol, Dongping Ma, Thomas Yeager, Dan Simpson, Troy Good, David Liu,
James J. Cali
Promega Corp., Madison, WI, USA
We have developed three distinct luminogenic substrates for measuring the
enzyme activity of CYP3A4, the principal drug-metabolizing cytochrome
P450 (CYP) in the liver and small intestine. The substrates are
luciferin derivatives used with the luciferase based P450-Glo™
technology. Factors that influence the choice of substrate include CYP
enzyme selectivity, DMSO sensitivity and source of CYP3A4 (microsomes
vs. cells). The selectivity of each substrate for CYP enzymes is
demonstrated in assays with recombinant CYPs. These microsome assays can
utilize any of the three substrates for detecting inhibition and
measuring IC50s. Assays of CYP3A4 from cultured cells use the substrates
in a non-lytic approach that detects basal and induced CYP3A enzyme
activity in freshly isolated and cryopreserved hepatocytes and DPX-2
cells (a stable cell-line over-expressing the PXR nuclear receptor). The
cell-based assays can be multiplexed with a cell viability assay to
measure cytotoxicity of test compounds and for normalization of CYP
activity measurements to cell number. These cell- and microsome-based
assays are homogeneous and easily configured in high throughput
multiwell formats to measure CYP gene induction or enzyme inhibition by
new chemical entities. |
