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.