A High Throughput Screening Assay to Screen for CYP2E1 Metabolism and Inhibition Using a Fluorogenic Vivid^® P450 Substrate

Abstract

Large-scale screening of multiple compound libraries and combinatorial libraries for pharmacological activity is one of the novel approaches of the modern drug discovery process. The application of isozyme-specific high-throughput screening (HTS) assays for characterizing the interactions of potential drug candidates with major human drug-metabolizing cytochrome p450 enzymes (p450s) is newly becoming an essential part of this process. Fluorescence-based HTS assays have been successfully employed for in vitro assessment of drug-drug interactions and enzyme inhibition with several p450 isoforms, including CYP3A4, CYP2D6, CYP2C9, and CYP2C19. Here we describe a fluorescence-based HTS assay for detecting drug metabolism and inhibition with human CYP2E1. CYP2E1 plays an important role in the metabolism of several drugs, many solvents, and toxins and therefore has been repeatedly linked to numerous pathologies, including cancer, liver and kidney toxicity, diabetes, and alcoholism. The assay is based on the ability of a drug to compete with the fluorogenic Vivid CYP2E1 Blue Substrate for CYP2E1 metabolism and thus enables rapid screening of lead molecules for their inhibitory potential. We have used this assay to screen a panel of drugs and compounds for their effects on CYP2E1 metabolism and inhibition. Our results demonstrate the assay's usefulness in identifying CYP2E1 substrates and inhibitors and in enabling in-depth characterization of their interactions with the CYP2E1 isozyme. We also present detailed characteristics of the assay, including its dynamic range and Z'-factor values, which indicate that this robust assay is well suited for kinetic and inhibition studies in HTS formats.

Pungent ginger components modulates human cytochrome P450 enzymes in vitro

“…Although microsomal assays are commonly used to evaluate the activity of P450 enzymes, fluorescent methods for determining P450 activity allow for high-throughput screening and more reproducible results [21][22][23][24][25] . Bell et al found that nearly 75% of marketed drugs yielded acceptable correlations between fluorogenic and HLM+LC-MS/MS assays for CYPs 3A4, 2D6, and 2C9, which supports the use of fluorogenic assays for rapid early risk binning based on IC 50 values [26] .…”

A simultaneous assessment of CYP3A4 metabolism and induction in the DPX-2 cell line

“…Although the induction potency for compounds examined here varied significantly, most were consistent with previously reported data allowing discrimination between potent, moderate, and weak CYP3A4 inducers. For example, maximal levels of induction (up to 35-fold) were obtained for rifampicin, a previously reported prototypical CYP3A4 inducer of high potency, 14 whereas incubation in the presence of paclitaxel, a previously reported weak CYP3A inducer, 15 resulted in only a 2-fold induction of luciferase reporter gene activity.…”

In Vitro Drug Interactions of Cytochrome P450: An Evaluation of Fluorogenic to Conventional Substrates

“…Burke and Mayer (1974) first used the P450-catalyzed O-dealkylation of alkoxyresorufins to examine the induction of hepatic P450 activity by fluorescence. Since then, various O-alkyl derivatives of resorufin, fluorescein, 7-hydroxycoumarins, and 6-hydroxyquinolines have been examined as substrates, and some have been commercialized for use in P450-mediated drug interaction studies (Clarke, 1999;Stresser et al, 2000;Bapiro et al, 2001;Crespi and Stresser, 2001;Miller et al, 2001;Marks et al, 2002). The complex kinetic patterns known for P450 enzymes with several probe substrates and inhibitors (Tang and Stearns, 2001;Hutzler, 2002) suggested that similar complexity is to be expected with fluorogenic substrates.…”