Nigramide J is a novel potent inverse agonist of the human constitutive androstane receptor

Abstract

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.

Nigramide C Is a Natural Agonist of Human Pregnane X Receptor

“…NigC at concentrations of up to 10 mM had no effect on the basal transactivity of hCAR (Supplemental Fig. 1 and our previous report, Kanno et al, 2014). Furthermore, we investigated whether NigC could reverse inverse agonist-mediated suppression.…”

“…These observations indicate that NigC is not a ligand of hCAR. It is notable that compound 3 (NigJ), which differs in its structure from 1 by only C-6 aliphatic moiety, has been identified as a potent inverse agonist of hCAR (Kanno et al, 2014).…”

Differences in Gene Regulation by Dual Ligands of Nuclear Receptors Constitutive Androstane Receptor (CAR) and Pregnane X Receptor (PXR) in HepG2 Cells Stably Expressing CAR/PXR

“…and PBREM-Luc reporter assays. However, our previous report showed that CITCO can reverse inverse agonistmediated CAR repression (Kanno et al, 2014). These observations suggest that CITCO has high affinity, but weak inverse agonistic activity.…”

“…pIRES-puro/human PXR (hPXR) was constructed by inserting an hPXR fragment, which was amplified from cDNA of HepG2 cells by polymerase chain reaction, into the pIRES-puro vector (Clontech Laboratories, Inc., Palo Alto, CA). The construction of expression plasmids for GAL4/DNAbinding domain (DBD)-fused human CAR (hCAR)/ligand binding domain (LBD) (GAL4/DBD-hCAR/LBD), GAL4/DBD-hCAR/LBD + three amino acid (+3 a.a.), and GAL4/DBD-hPXR/LBD has been reported previously (Kanno and Inouye, 2010;Kanno et al, 2014). Preparation of the hCAR (pcDNA5-hCAR) expression plasmid and penobarbital-responsive enhancer module (PBREM)-driven luciferase reporter plasmid (PBREM-Luc) has also been described previously (Kanno et al, 2012).…”

“…Androstenol (Forman et al, 1998), clotrimazole (Moore et al, 2000), PK11195 (Li et al, 2008), S07662 (Kublbeck et al, 2011, nigramide J (Kanno et al, 2014), TO901317 (Kanno et al, 2013)…”