Abstract
The constitutive androstane receptor (CAR) is a critical nuclear receptor in the gene regulation of xenobiotic and endobiotic metabolism. The LanthaScreenTM TR-FRET CAR coactivator assay provides a simple and reliable method to analyze the affinity of a ligand to the human CAR ligand-binding domain (LBD) with no need to use cellular models. This in silico assay thus enables the study of direct CAR ligands and the ability to distinguish them from the indirect CAR activators that affect the receptor via the cell signaling-dependent phosphorylation of CAR in cells. For the current paper we characterized the pharmacodynamic interactions of three known CAR inverse agonists/antagonists—PK11195, clotrimazole and androstenol—with the prototype agonist CITCO (6-(4-chlorophenyl)imidazo[2,1-b][1,3]thiazole-5-carbaldehyde-O-(3,4-dichlorobenzyl)oxime) using the TR-FRET LanthaScreenTM assay. We have confirmed that all three compounds are inverse agonists of human CAR, with IC50 0.51, 0.005, and 0.35 μM, respectively. All the compounds also antagonize the CITCO-mediated activation of CAR, but only clotrimazole was capable to completely reverse the effect of CITCO in the tested concentrations. Thus this method allows identifying not only agonists, but also antagonists and inverse agonists for human CAR as well as to investigate the nature of the pharmacodynamic interactions of CAR ligands.
Highlights
The LanthaScreenTM Nuclear Receptor Assay is commercially provided by Invitrogen for several nuclear receptors, including Farnesoid X receptor (FXR), LXRα, PPARα and PXR
In contrast to other nuclear receptor LanthaScreenTM assays, which are frequently reported in literature, to our knowledge no paper has been published on the characterization, validation or use of the time-resolved Förster resonance energy transfer (TR-FRET) Constitutive Androstane Receptor (CAR) Coactivator Assay until quite recently, when we published the first article demonstrating the value of the assay in the characterization of indirect CAR ligands [1]
One of the modifications was to add the ligand (CITCO), a fluorescein-labeled PGC1α coactivator and terbium-labeled anti GSH antibody and the CAR-ligand-binding domain (LBD) solution. Another modification consisted of adding CAR-LBD and fluorescein-/terbium-labeled reagents solutions before the agonist
Summary
The LanthaScreenTM Nuclear Receptor Assay is commercially provided by Invitrogen ( a division of Thermo) for several nuclear receptors, including FXR, LXRα, PPARα and PXR. Constitutive Androstane Receptor Coactivator Assay is a TR-FRET based assay with terbium and fluorescein fluorophores to detect ligands of the Constitutive Androstane Receptor (CAR) nuclear receptor. In contrast to other nuclear receptor LanthaScreenTM assays, which are frequently reported in literature, to our knowledge no paper has been published on the characterization, validation or use of the TR-FRET CAR Coactivator Assay until quite recently, when we published the first article demonstrating the value of the assay in the characterization of indirect CAR ligands [1]. The constitutive androstane receptor (CAR, NR1I3) is a ligand-activated transcription factor with high constitutive activity belonging to the nuclear receptor group NR1I. CAR has been demonstrated to regulate the gene expression of major phase I and phase II xenobiotic-metabolizing enzymes (DMEs) and drug transporters such as cytochrome P450 CYP3A4 and CYP2B6 enzymes, the ABC efflux transporter MRP2 and the conjugation enzyme UGT1A1 [2].
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