Received for publication February 25, 2008.
Revised May 20, 2008.
Accepted for publication May 20, 2008.

The Peripheral Benzodiazepine Receptor Ligand PK11195 is a Novel Antagonist of Human Constitutive Androstane Receptor

Abstract

As a promiscuous xenobiotic sensor, the constitutive androstane receptor (CAR; NR1I3) regulates the expression of multiple drug metabolizing enzymes and transporters in liver. The constitutively activated nature of CAR in the cell-based transfection assays has hindered its utilization as a predictor of metabolism-based drug-drug interactions. Here we have identified PK11195 [1-(2-Chlorophenyl-methylpropyl)-3-isoquinoline-carboxamide], a typical peripheral benzodiazepine receptor (PBR) ligand, as a selective and potent inhibitor of human (h) CAR. In cell-based transfection assays, PK11195 inhibited the constitutive activity of hCAR more than 80% at the concentration of 10 µM, and the PK11195-inhibited activity was efficiently reactivated by the direct CAR activator, CITCO but not by the indirect hCAR activator, phenobarbital. Mammalian two hybrid, and GST pull-down assays showed that PK11195 repressed the interactions of hCAR with the co-activators SRC1 and GRIP1 to inhibit hCAR activity. The inhibition by PK11195 specifically occurred to the hCAR: PK1195 strongly activated human PXR, while did not alter the activity of the mouse CAR and mouse PXR. In addition, PBR played no role in the PK11195 inhibition of hCAR since the inhibition fully occurred in the HeLa cells in which the PBR was knocked down by siRNA. In the Car^-/- mouse liver, PK11195 translocated EYFP-hCAR into the nucleus. These results are consistent with the conclusion that PK11195 is a novel hCAR specific antagonist that represses the CAR-co-activator interactions to inhibit the receptor activity inside the nucleus. Thus, PK11195 can be used as a chemical tool for studying the molecular basis of CAR function.

Key words: Transcriptional coactivators, Regulation of gene expression, Cytochrome P450, Regulation - xenobiotic, Cholesterol metabolism/lipoproteins