Title:Transactivation Assays that Identify Indirect and Direct Activators of Human Pregnane X Receptor (PXR, NR1I2) and Constitutive Androstane Receptor (CAR, NR1I3)
VOLUME: 11 ISSUE: 2
Author(s):Marija Pinne*, Elsa Ponce and Judy L. Raucy
Affiliation:Puracyp, Inc., Carlsbad, CA 92010, Puracyp, Inc., Carlsbad, CA 92010, Puracyp, Inc., Carlsbad, CA 92010
Keywords:Ligand binding assays, full length nuclear receptor, PXR, CAR, indirect activators, phenobarbital, phosphorylation,
DPX2 cells, nuclear receptors, drug-drug interactions.
Abstract:Background: Nuclear Receptors (NRs), including PXR and CAR, are presumed to be
ligand-dependent transcription factors, but ligand binding is not an absolute requirement for activation.
Indeed, many compounds activate PXR and CAR by indirect mechanisms. Detecting these indirect activators
of specific nuclear receptors in vitro has been difficult. As NR activation of either or both PXR
and CAR can lead to drug-drug interactions and adverse drug effects, false negatives obtained with
screening tools incapable of detecting indirect activators could present liabilities.
Objective: The aim of this study was to establish assays that identify indirect activators of human PXR
and CAR.
Methods: Commercially available human PXR and CAR transactivation assays were used for analyses.
Results: We show that transactivation assays containing full-length nuclear receptors with native promoters
can identify indirect activators of human CAR and PXRwhen compared to those of commercially
available assays containing only the LBD of PXR and CAR. Of these two assay systems, only
human PXR and CAR1 assays with full-length receptors and native promoters are capable of detecting
indirect and ligand activators. With this capability, several kinase inhibitors were identified that activate
PXR and CAR by indirect mechanisms. Furthermore by using both the LBD and full-length receptors,
phenobarbital and midostaurin were found to be direct and indirect activators of PXR while
human CAR activation by phenobarbital occurs by indirect mechanisms only.
Conclusion: Cell based transactivation assays employing the full-length receptors and native promoters
identify both direct and indirect activators of either or both human PXR and CAR.
