The Peripheral Benzodiazepine Receptor Ligand 1-(2-Chlorophenyl-methylpropyl)-3-isoquinoline-carboxamide 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 drugmetabolizing enzymes and transporters in liver. The constitutively activated nature of CAR in the cell-based transfection assays has hindered its use as a predictor of metabolism-based drugdrug interactions. Here, we have identified 1-(2-chlorophenylmethylpropyl)-3-isoquinoline-carboxamide (PK11195), 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 M, and the PK11195-inhibited activity was efficiently reactivated by the direct CAR activator, 6-(4-chlorophenyl)imidazo[2,1-b][1,3]thiazole-5-carbaldehyde-O-(3,4-dichlorobenzyl) oxime, 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 coactivators steroid receptor coactivator-1 and glucocorticoid receptor-interacting protein 1 to inhibit hCAR activity. The inhibition by PK11195 specifically occurred to the hCAR: PK1195 strongly activated human pregnane X receptor (PXR), whereas it did not alter the activity of the mouse CAR and mouse PXR. In addition, PBR played no role in the PK11195 inhibition of hCAR because the inhibition fully occurred in the HeLa cells in which the PBR was knocked down by small interfering RNA. In the Car Ϫ/Ϫ mouse liver, PK11195 translocated enhanced yellow fluorescent protein-hCAR into the nucleus. These results are consistent with the conclusion that PK11195 is a novel hCAR-specific antagonist that represses the CAR-coactivator 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.The constitutive androstane receptor (CAR, NR1I3) is an important transcription factor that transfers endogenous and exogenous stimuli into cellular responses by regulating the expression of its target genes that code for cytochrome P450 (P450) and transport proteins Tzameli et al., 2000;Ueda et al., 2002;Huang et al., 2004;Maglich et al., 2004). Thus, CAR organizes a cellular defense system against xenobiotic insults by increasing hepatic capacity for metabolism and excretion. Besides its role in drug metabolism, CAR is also reported to have the ability to regulate

p38 MAP Kinase Links CAR Activation and Inactivation in the Nucleus via Phosphorylation at Threonine 38

“…dmd.aspetjournals.org looping, possibly enabling the promoter to recruit RNA polymerase II for effective promoter activation Negishi, 2008, 2009). In addition to early growth response 1, various other transcription factors were shown to interact with and activate the CYP2B promoters together with CAR, such as peroxisome proliferator-activated receptor -g coactivator-1 alpha (PGC-1a), steroid receptor coactivator-1 (SRC-1), HNF-4a, and CCAAT/enhancer-binding protein alpha (Shiraki et al, 2003;Li et al, 2008;Benet et al, 2010). Since p38 MAPK can activate these factors (Lim et al, 1998;Knutti et al, 2001;Guo et al, 2007;Qiao et al, 2006;Fernandez-Marcos and Auwerx, 2011;Antoon et al, 2012), it may enable CAR to bind to PBREM and to loop the promoter by phosphorylating one or more of them.…”

Methadone Induces the Expression of Hepatic Drug-Metabolizing Enzymes through the Activation of Pregnane X Receptor and Constitutive Androstane Receptor

“…Total RNA was isolated using the RNeasy Mini Kit (QIAGEN, Valencia, CA) and reverse-transcribed using the High-Capacity cDNA Archive Kit (Applied Biosystems) per manufacturers' instructions. Primers and probes for CYP2B6, CYP3A4, UGT1A1, and MDR1 genes (Table 1) were designed using Primer Express version 2.0 (Applied Biosystems) and entered into the National Center for Biotechnology Information BLAST to ensure specificity as described previously (Maglich et al, 2002;Smith et al, 2005;Faucette et al, 2007;Li et al, 2008). The mRNA expression of CYP2B6, CYP3A4, UGT1A1, and MDR1 was normalized against that of human ␤-actin, which was detected using a predeveloped primer/probe mixture (Applied Biosystems).…”

A consecutive three alanine residue insertion mutant of human CAR: a novel CAR ligand screening system in HepG2 cells

“…Secondly, the effects of Andro, CLT, artemisinin (Simonsson et al, 2006), and PK11195 (Li et al, 2008), all ligands of CAR, on the transactivation potential of GAL4/DBD-hCAR/LBD(+3a.a.) were evaluated.…”