Luminogenic cytochrome P450 assays

Abstract

Luminogenic cytochrome P450 (CYP) assays couple CYP enzyme activity to firefly luciferase luminescence in a technology called P450-Glo(TM) (Promega). Luminogenic substrates are used in assays of human CYP1A1, -1A2, -1B1, -2C8, -2C9, -2C19, -2D6, -2J2, -3A4, -3A7, -4A11, -4F3B, -4F12 and -19. The assays detect dose-dependent CYP inhibition by test compounds against recombinant CYP enzymes or liver microsomes. Induction or inhibition of CYP activities in cultured hepatocytes is measured in a nonlytic approach that leaves cells intact for additional analysis. Luminogenic CYP assays offer advantages of speed and safety over HPLC and radiochemical-based methods. Compared with fluorogenic methods the approach offers advantages of improved sensitivity and decreased interference between optical properties of test compound and CYP substrate. These homogenous assays are sensitive and robust tools for high-throughput CYP screening in early drug discovery.

Changes in expression of hepatic cytochrome P450 subfamily enzymes during development of adjuvant-induced arthritis in rats

“…Hepatic CYP3A activity was determined using a P450-Glo TM CYP3A4 Assay Kit (Promega, Madison, MI, USA) according to the manufacturer's instructions. In this system, luciferin-6'-pentafluorobenzyl ether (Luciferin-PFBE) was used as a substrate for CYP3A subfamily enzymes (Cali et al, 2006). Briefly, a 25-μl reaction mixture of 0.1 M sodium phosphate buffer (pH 7.4) containing hepatic microsomes (20 μg) and Luciferin-PFBE (100 μM) was placed in each well of a 96-well plate (Corning Inc.) and preincubated at room temperature for 10 min under light shielding.…”

“…2 Therefore, we examined the changes in the activities and expression levels of hepatic CYP3A and CYP2B subfamily enzymes and in the amounts of hepatic inflammatory cytokines, namely TNF-α, IL-1α, IL-1β and IL-6, at 1, 12, and 25 days after MT adjuvant injection. The hepatic activities of CYP3A and CYP2B subfamily enzymes were assessed using the substrates, Luciferin-PFBE for CYP3A activities (Cali et al, 2006) and pentoxyresorufin for CYP2B activities (Lubet et al, 1985;Burke et al, 1994). All of the enzyme activities examined were drastically decreased, even at 1 day after MT adjuvant injection, and the decreases persisted up to day 25.…”

In Vitro Evaluation of Reversible and Irreversible Cytochrome P450 Inhibition: Current Status on Methodologies and their Utility for Predicting Drug–Drug Interactions

“…Here, CYP activity results in the generation of a luciferin analogue which can be made to luminesce by addition of a development reagent (5). The advantage of luminescence technology is the greatly improved signal to noise ratio which can be achieved, although there are the drawbacks of needing an extra "development" step in the assay process and not being able to read the signal generation in real time.…”

Human monocyte-derived cells with individual hepatocyte characteristics: a novel tool for personalized in vitro studies

“…Briefly, cells were induced for various periods with known CYP-inducers in media without additives. 28 Luminescence values were normalized for protein content.…”

“…28 MH cells exhibit activities of the CYP450 enzymes 3A4, 2C9 and 1A2. CYP3A4 shows highest luminescence values, followed by 1A2 and 2C9 (Figure 1a).…”