-The basal transcriptional activity of unliganded human constitutive androstane receptor (hCAR) was shown to be repressed by the potent liver X receptor (LXR) agonist, T0901317, in a concentration-dependent manner using a reporter assay in cultured cells. T0901317 also repressed the basal transcriptional activity of both mouse and rat CAR. The certified hCAR agonist, CITCO, partially reversed this repressive effect of T0901317 on hCAR basal activity. Unlike hCAR, a three alanine insertion mutant and the splice variant 2 of hCAR require agonists, such as CITCO, to become transcriptionally active and the CITCO-induced reporter activity was repressed by T0901317. As has been previously shown for the typical hCAR inverse agonist, PK11195, T0901317 blocked the interaction of hCAR with steroid receptor co-activator 1 (SRC1). In contrast, the interaction between hCAR and nuclear receptor corepressor 1 (NCoR1) was promoted by PK11195 and T0901317. Furthermore, the hCAR-mediated basal induction of endogenous cytochrome P450 2B6 (CYP2B6) mRNA was adversely affected by co-treatment with T0901317.
The constitutive androstane receptor (CAR, NR1I3) is very important for drug development and for understanding pharmacokinetic drug–drug interactions. We screened by mammalian one hybrid assay among natural compounds to discover novel ligands of human constitutive androstane receptor (hCAR). hCAR transcriptional activity was measured by luciferase assay and mRNA levels of CYP2B6 and CYP3A4 in HepTR-hCAR cells and human primary hepatocytes were measured by real-time RT-PCR. Nigramide J (NJ) whose efficacy is comparable to those of hitherto known inverse agonists such as clotrimazole, PK11195, and ethinylestradiol. NJ is a naturally occurring cyclohexane-type amide alkaloid that was isolated from the roots of Piper nigrum. The suppressive effect of NJ on the CAR-dependent transcriptional activity was found to be species specific, in the descending order of hCAR, rat CAR, and mouse CAR. The unliganded hCAR-dependent transactivation of reporter and endogenous genes was suppressed by NJ at concentrations higher than 5 μmol/L. The ligand-binding cavity of hCAR was shared by NJ and CITCO, because they were competitive in the binding to hCAR. NJ interfered with the interaction of hCAR with coactivator SRC-1, but not with its interaction with the corepressor NCoR1. Furthermore, NJ is agonist of human pregnane X receptor (hPXR). NJ is a dual ligand of hCAR and hPXR, being an agonist of hPXR and an inverse agonist of hCAR.
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