James J. Cali, Dongping Ma, Mary Sobol and Poncho Meisenheimer
Promega Corporation, 2800 Woods Hollow Rd, Madison, WI 53711
CYP3A4 enzyme induction and inhibition by drugs and other xenobiotics is a significant cause of adverse drug-drug interactions. To predict the potential for these outcomes early in drug discovery, compounds are tested for their capacity to induce or inhibit conversion of a probe substrate to a specific product by the CYP3A4 enzyme. A new, highly selective and sensitive CYP3A4 probe substrate, luciferin isopropyl acetal (Luciferin-IPA), was synthesized for use in rapid bioluminescent cell-based and cell-free CYP3A4 assays. Bioluminescent enzyme substrates are proluciferins that are converted by the enzymes of interest to a luciferin that is detected in a bioluminescent reaction with luciferase. This enables assays that correlate light output with target enzyme activity.
Out of 21 human CYP enzymes luciferin-IPA only showed activity with the CYP3A subfamily, having 14 and 161 fold selectivity for CYP3A4 over CYP3A5 and CYP3A7, respectively. Cell free luciferin-IPA enzyme assays were sensitive to inhibition by known CYP3A4 inhibitors and were readily configured for IC50 determinations using either recombinant CYP3A4 or human liver microsomes. CYP3A4 induction was measured in rapid, 96-well, human hepatocyte assays where activity was increased by known CYP3A4 inducers. The luciferin-IPA/CYP3A4 hepatocyte assay was easily configured in a multiplex application with a cell viability assay that provided a means for normalizing CYP enzyme activity to viable cell number. While luciferin-IPA/CYP3A4 induction and inhibition data was virtually identical to data from conventional CYP3A4 testosterone 6-b hydroxylation assays, the luminescent assays were simpler, more sensitive and substantially quicker. Luciferin-IPA assays enable early ADME screening with high throughput and rapid turn around times.