April 1, 2010 (Vol. 30, No. 7)

Demonstration of the Use of a Selective Luciferin-IPA CYP3A4 Assay

The cytochromes P450 (CYPs) are the main enzyme family for metabolism of therapeutic drugs. CYP3A4 is the most active drug metabolizing CYP, and it plays a prominent role in adverse drug-drug interactions (DDIs). A drug that inhibits CYP3A4 enzyme activity or induces its expression respectively either slows or increases clearance of a co-administered CYP3A4-metabolized drug, leading in the first case to toxicity and in the second to reduced efficacy.

To predict the potential for these outcomes early in drug discovery, compounds are tested in vitro for their capacity to induce or inhibit the conversion of a probe substrate by CYP3A4 to a specific product. CYP3A4 inhibition is typically measured with a cell-free enzyme assay.

Inductions that are initiated at the transcription level require a cell-based assay, but the induction can be monitored at a post-transcriptional level by measuring changes in CYP3A4 enzyme activity.

There are various CYP assay methods including mass spectrometry, absorbance, radioactivity, fluorescence, and bioluminescence. Promega’s P450-Glo™ bioluminescent CYP assays give highly predictive results and typically rank highest for sensitivity and ease of use.

The P450-Glo assay technology is a rapid high-throughput approach that monitors the conversion by CYPs of inactive D-luciferin derivatives to an active form that makes light when firefly luciferase is added to the reaction mixture. Light intensity is proportional to CYP activity.

Figure 1. Selectivity of Luciferin-IPA for human CYP enzymes


Four distinct P450-Glo CYP3A4 substrates are now available.

The original CYP3A4 substrate, Luciferin-BE, was followed by Luciferin PPXE and Luciferin-PFBE; the latest substrate is the luciferin isopropyl acetal or Luciferin-IPA. With the development of each new substrate a general trend was followed toward increased sensitivity, improved CYP3A4 selectivity, and better cell-based assay performance.

An increased capacity to detect a wider range of CYP3A4 inhibitors was also achieved, along with decreased sensitivity to inhibition by the common solvent DMSO. A detailed comparison of Luciferin-BE, Luciferin-PPXE, and Luciferin-PFBE was published previously, so we will focus here on the newest bioluminescent CYP3A4 substrate, Luciferin-IPA.

Recombinant CYP enzymes were used initially to characterize Luciferin-IPA as a CYP substrate. The activity profile of Luciferin-IPA shows high selectivity for CYP3A4 with minimal cross-reactivity with the closely related enzymes CYP3A5 and 3A7 (Figure 1).

The Luciferin-IPA reaction with recombinant CYP3A4 was linear with increasing enzyme concentrations up to at least 0.25 pmol CYP3A4/50 µL reaction (5 nM CYP3A4; r2 = 0.99) (data not shown). The enzyme concentration curve demonstrates that Luciferin-IPA is a highly sensitive probe for detecting CYP3A4 activity with greater sensitivity than earlier generation luminogenic substrate and much greater sensitivity than fluorescent substrates.

A large signal-to-noise ratio (mean value=2,580) was observed at 0.16 pmol CYP3A4 per 50 µL reaction (0.3 nM CYP3A4), the lowest enzyme concentration tested. This indicates that the CYP3A4/Luciferin-IPA reaction can be employed using substantially less than the 20–30 nM CYP3A4 enzyme concentrations that are often used in CYP3A4 assays.

Figure 2. Using Luciferin-IPA to detect CYP3A4 inhibition

Detecting CYP3A4 Inhibitors

The CYP3A4/Luciferin-IPA proved to be a sensitive probe reaction for detecting CYP3A4 inhibitors. Dose-dependent inhibition by midazolam, testosterone, and nifedipine was observed (Figure 2). These three compounds are widely used as CYP3A4 probe substrates, so they are acting here as competitive inhibitors of the CYP3A4/Luciferin-IPA reaction. Because the CYP3A4/Luciferin-IPA reaction is inhibited by these three compounds it is expected in turn to detect any inhibitor also detected by each of these compounds when they are used as CYP3A4 probe substrates.

Since the subsets of CYP3A4 inhibitors detected by midazolam, testosterone, and nifedipine reactions do not show complete overlap, it can be expected that Luciferin-IPA will prove to be a broader spectrum probe substrate than any one of these three.

Luciferin-IPA was also used to detect CYP3A4 gene induction by the well-known CYP3A4 inducer rifampicin in a cell-based assay with fresh human hepatocytes (Figure 3).

Because Luciferin-IPA is cell permeant, as is the luciferin product of the CYP3A4 reaction, it is possible to configure a nonlytic assay that detects the CYP reaction product in the cell culture medium. This provides the possibility of performing an assay such as a cell viability assay in multiplex.

A substantial basal signal representing CYP3A4 enzyme activity endogenous to hepatocytes was detected from the vehicle-treated wells showing a signal-to-background ratio of 366. The basal signal was increased 18.5-fold by rifampicin, reflecting an increase in CYP3A4 enzyme activity due to CYP3A4 gene induction.

Luciferin-IPA, a bioluminescent CYP3A4 substrate, is a highly sensitive and selective probe for detecting CYP3A4 activity. This sensitivity means that the CYP3A4 enzyme can be used at substantially lower concentrations than are typically used in CYP3A4 assays. Because Luciferin-IPA is cell permeant and CYP3A4-selective, it can be used to detect CYP3A4 gene induction and be multiplexed with a cell viability assay.

The CYP3A4/Luciferin-IPA assay is also a sensitive means for detecting CYP3A4 inhibitors in a dose-dependent  manner. This new-generation bioluminescent substrate proves to be sensitive, selective and versatile for all bioluminescent CYP3A4 assay applications. 

Figure 3. Detection of CYP3A4 gene induction in a cell-based assay

James J. Cali ([email protected]) is senior R&D manager, Mary Sobol is senior research scientist, and Dongping Ma is R&D scientist at Promega. H. Tetsuo Uyeda and Poncho Meisenheimer are R&D scientists at Promega Biosciences. Web: www.promega.com.

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