New in Vitro Tools to Study Human Constitutive Androstane Receptor (CAR) Biology: Discovery and Comparison of Human CAR Inverse Agonists

Abstract

The human constitutive androstane receptor (CAR, NR1I3) is one of the key regulators of xenobiotic and endobiotic metabolism. The unique properties of human CAR, such as the high constitutive activity and the complexity of signaling, as well as the lack of functional and predictive cell-based assays to study the properties of the receptor, have hindered the discovery of selective human CAR ligands. Here we report a novel human CAR inverse agonist, 1-[(2-methylbenzofuran-3-yl)methyl]-3-(thiophen-2-ylmethyl) urea (S07662), which suppresses human CAR activity, recruits the corepressor NCoR in cell-based assays, and attenuates the phenytoin- and 6-(4-chlorophenyl)imidazo[2,1-b][1,3]thiazole-5-carbaldehyde O-(3,4-dichlorobenzyl)oxime (CITCO)-induced expression of CYP2B6 mRNA in human primary hepatocytes. The properties of S07662 are also compared with those of known human CAR inverse agonists by using an array of different in vitro and in silico assays. The identified compound S07662 can be used as a chemical tool to study the biological functions of human CAR and also as a starting point for the development of new drugs for various conditions involving the receptor.

Application of Micropatterned Cocultured Hepatocytes to Evaluate the Inductive Potential and Degradation Rate of Major Xenobiotic Metabolizing Enzymes

“…Phenobarbital showed the highest inductive response for CYP2C9 expression and activity in the HepatoPac model, perhaps owing to its dual activation of PXR and CAR. Phenytoin, a preferential hCAR transactivator (Küblbeck et al, 2011) failed to significantly increase CYP2C9 mRNA or activity in either culture model. A previous report by Sahi et al (2009) has also shown that phenytoin does not induce CYP2C9.…”

Small-molecule modulators of the constitutive androstane receptor

“…Interestingly, this compound was reported to be an mCAR agonist instead. However, other studies did not validate meclizine as being an hCAR inverse agonist [29, 30]. …”

An update on the constitutive androstane receptor (CAR)

“…The mammalian two-hybrid assay measures the ligand-dependent interaction of CAR with a selected co-regulator peptide. This assay appears to be more sensitive in identifying weak or partial agonists that may elicit both co-activator and corepressor recruitment and very useful in dissecting the co-regulator profile of human CAR [ 57 ] and to gain support for human CAR/ligand interactions [ 35 , 63 ].…”