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.