Measurement of CYP3A4 enzyme activity

CYP3A4 enzyme activity was measured using the luminogenic CYP3A4 substrate Luciferin-IPA from Promega (Madison, Wisconsin, USA) (catalog number: V840A). The substrate was incubated with insect control supersomes (Corning, New York, USA) or human CYP3A4 supersomes from Corning (catalog number: 456202) (Corning, New York, USA). The latter convert the substrate to d-luciferin. Detection was performed with the luciferin detection reaction (catalog number: V859A) dissolved in reconstitution buffer with esterase (catalog number: V144A) from Promega (Madison, Wisconsin, USA). Experiments were performed in white 96-well plates in triplicates. For one well 7.35 µL water, 5 µL sodium phosphate buffer (1 M, pH 7.4), 0.05 µL Luciferin-IPA (3 mM), and 0.1 µL CYP3A4 supersomes were mixed. Experiments were conducted by adding 12.5 µL of test compound (4 × concentrated to compensate for the dilution effect in the assay), or a CYP3A4 inhibition control (ketoconazole, final concentration 1 µM) or water as negative control to each well. After this, 12.5 µL of the mix with control supersomes as negative control or CYP3A4 supersomes was added, mixed on a plate shaker and pre-incubated for 10 min at 37 °C. The reaction was started by adding 25 µL of cofactor solution, containing 33 mM potassium chloride, 8 mM magnesium chloride, 1 mM NADP and 5 mM glucose-6-phosphate. Plate was again shaken and incubated for 30 min at 37 °C. Finally, 50 µL of luciferin detection reagent was added to each well and the plate was incubated for 20 min at room temperature. After this, luminescence was measured with 1 s integration time.