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 drug-metabolizing 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 drug-drug 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 microM, 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(-/-) 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.

Figure 1. PK11195 deactivates constitutive activity of hCAR in HepG2 cells

(A) Structure of PK11195. (B) HepG2 cells were transfected with CYP2B6-2.2 kb reporter, and hCAR expression vectors. Transfected cells were treated with PK11195, CITCO, or PB at indicated concentrations for 24 hrs. Luciferase activities were determined and expressed relative to vehicle control (CT). Data represent the mean ± S.D. (n=3) (**, P<0.01 denotes comparison with DMSO group; #, P<0.05, and ##, P<0.01 denote comparison with 10 µM PK11195 group).

Figure 2. PK11195 selectively inhibited the constitutive activity of hCAR

HepG2 cells were transfected with CYP2B6-1.8kb (A), (NR1)₅pGL3-tk (B), or CYP3A4-PXRE/XREM (C) reporter construct in the presence of mCAR, hCAR, mPXR, or hPXR expression vectors. Transfected cells were then treated with PK11195 (10 µM) or 0.1% DMSO as vehicle control (CT). Dual luciferase activities were determined and expressed relative to CT. All data are presented as mean ± S.D. (n=3) (**, p<0.01).

Figure 3. Induction of CYP2B6 and CYP3A4 expression in human primary hepatocyte cultures

Human hepatocytes (UM-HL-001 and UM-HL-002) cultured in WME were treated for 24 hrs with PK11195 10 µM, SFN 25 µM, Picrotoxin 10 µM, Ro5-4868 10 µM, FGIN-1-29 10 µM and CMZ 30 µM, as well as co-treatment of SFN with PK11195, RIF, and CITCO, respectively. Total RNA was collected, reverse transcribed, and subjected to TaqMan real-time PCR. CYP2B6 and CYP3A4 expression levels were normalized against β-actin. Induction of CYP2B6 and CYP3A4 relative to control was calculated as described under “Materials and Methods”. All data are presented as mean ± S.D. (n=3), (*, p<0.05; **, p<0.01).

Figure 4. The effect of PK11195 on hCAR or hPXR induced expression of CYP3A4

HepG2 and Huh7 cells were transfected with hCAR (A), or hPXR (B) expression vectors, and treated with PK11195 (10 µM), CITCO (1 µM), or RIF (10 µM) for 24 hrs. Total RNA was collected, reverse transcribed, and subjected to TaqMan real-time PCR as described in “Materials and Methods”. All data are expressed as mean ± S.D. (n=3), (*, p<0.05; **, p<0.01).

Figure 5. The effects of PBR Ligands on the activity of hCAR and hPXR

Four structurally different PBR ligands (A) were selected in this reporter assay. HepG2 cells were transfected with hCAR (B), hPXR (C), or hCAR3 (D) expression vectors in the presence of CYP2B6-2.2kb reporter construct. Transfected cells were then treated with PBR ligands (PK11195 10 µM, Picrotoxin 10 µM, Ro5-4868 10 µM, FGIN-1-29 10 µM, or CMZ 30 µM) for 24 hrs. CITCO 1 µM and RIF 10 µM were used as positive control for hCAR and hPXR activation, respectively. Luciferase activities were determined and expressed relative to CT. Data represent the mean ± S.D. (n=3) (*, p<0.05; **, p<0.01).

Figure 6. PK11195 deactivation of hCAR is independent of PBR

HeLa cells were transfected with siRNA-NT or siRNA-PBR. Forty-eight hour after transfection, downregulation of PBR gene was detected by real-time PCR (A). In parallel experiment, HeLa cells were first subjected to the specific siRNA-PBR for 24 hrs, the cells were subsequently transfected with hCAR expression vector and CYP2B6-2.2 kb reporter vector (B). The transfected cells were then treated with PK11195 (10 µM) or CITCO (1 µM). Luciferase activities were determined and expressed relative to CT. Data represent the mean ± S.D. (n=3) (**, p<0.01).

Figure 7. PK11195 translocates hCAR in CAR−/− mouse liver

Car^−/− mice were injected with 10 µg of expression plasmid encoding EYFP-tagged hCAR or EYFP-mCAR as described under “Materials and Methods” and treated 3 hrs later with vehicle (DMSO, CTL), 100 mg/kg PB, or 10 mg/kg PK11195. Mice livers were harvested 8 hrs after tail vein delivery and slides of frozen liver sections were prepared for confocal microscopy. Approximately 100 EYFP expressed cells were classified according to hCAR (A) or mCAR (B) localization status. Representative images depict examples of hCAR localization as EYFP-hCAR in yellow and nuclear staining in blue (C and D).

Figure 8. PK11195 disrupts co-activators binding to hCAR

Mammalian two-hybrid, assays were performed in COS1 cells transiently transfected with expression plasmids encoding VP16-AD/hCAR fusion proteins and GAL4-DBD/coregulators fusion proteins, as indicated, together with the reporter gene plasmid pG5luc. Cells were treated with DMSO (0.1% v/v), CITCO (1 µM), or PK11195 (10 µM) for an additional 24 hrs before determination of luciferase activities (A, B, C, and D). Data represent mean±S.D. of three independent transfections (**, p<0.01). GST pull-down assay (E). In vitro-translated 35S-labeled SRC-1 and GRIP1 were incubated with bacterially expressed GST-hCAR fusion protein in the presence of 0.1% DMSO, 100 µM PK11195, or 1 µM CITCO as indicated. GST was used as a negative control for binding, in vitro incubation, gel electrophoresis and autoradiography were done as described in “Material and Methods”.