Discovery of Novel Human Constitutive Androstane Receptor Agonists with the Imidazo[1,2-a]pyridine Structure

Abstract

The nuclear constitutive androstane receptor (CAR, NR1I3) plays significant roles in many hepatic functions, such as fatty acid oxidation, biotransformation, liver regeneration, as well as clearance of steroid hormones, cholesterol, and bilirubin. CAR has been proposed as a hypothetical target receptor for metabolic or liver disease therapy. Currently known prototype high-affinity human CAR agonists such as CITCO (6-(4-chlorophenyl)imidazo[2,1-b][1,3]thiazole-5-carbaldehyde-O-(3,4-dichlorobenzyl)oxime) have limited selectivity, activating the pregnane X receptor (PXR) receptor, a related receptor of the NR1I subfamily. We have discovered several derivatives of 3-(1H-1,2,3-triazol-4-yl)imidazo[1,2-a]pyridine that directly activate human CAR in nanomolar concentrations. While compound 39 regulates CAR target genes in humanized CAR mice as well as human hepatocytes, it does not activate other nuclear receptors and is nontoxic in cellular and genotoxic assays as well as in rodent toxicity studies. Our findings concerning potent human CAR agonists with in vivo activity reinforce the role of CAR as a possible therapeutic target.

Filling the Blank Space: Branched 4-Nonylphenol Isomers Are Responsible for Robust Constitutive Androstane Receptor (CAR) Activation by Nonylphenol

“…HepG2 cells were purchased from the European Collection of Authenticated Cell Cultures (Salisbury, UK) and cultured at 37 °C in a 5% CO 2 humidified atmosphere in antibiotic-free DMEM (Thermo Fischer Scientific, Waltham, MA, United States) supplemented with 10% fetal bovine serum (FBS, Merck, Darmstadt, Germany) and sodium pyruvate (Merck, Darmstadt, Germany). Reporter gene assays with CAR1, CAR2, CAR3 variant and with murine Car expression vectors have been performed as we described before (Supporting Information − extended methods 2.5.1 16 ).…”

“…32 Relative activation of the luciferase construct was expressed as a percentage of the vehicle (0.1% DMSO)-treated samples (100%). HepaRG cells cultivation and subsequent RT-qPCR to analyze CYP3A4, CYP2C9, and CYP2B6 mRNA expression has been performed as we decided before (Supporting Information − extended methods 2.5.2, 16 ). In all experiments, results are presented as means and SD from at least three independent experiments (n = 3) performed in triplicates.…”

“…15 Many environ-mental toxicants or industrial chemicals as well as some drugs have been identified as CAR activators. 16 CAR has a typical nuclear receptor structure consisting of the DNA-binding domain (DBD) connected to the ligandbinding domain (LBD, Figure 1A,B) via a hinge region. CAR interacts with the promoter response elements of target genes with its N-terminal DBD.…”

“…29 Nowadays it is clear that CAR1 and CAR3 can be activated by different ligands. 16,30 Due to the low number of available CAR1 structures and, to date, no crystal structures of CAR3, the discussion on this chemically diverse set of ligands remains a challenge. Meanwhile, docking approaches are limited as they are unable to capture CAR's characteristic LBD flexibility.…”

“…Therefore, molecular dynamics (MD) simulations were successfully employed to study the binding of novel human CAR ligands. 16,19 In this study, we focus on elucidating the binding mechanism of four 4-NP isomers into CAR1 and CAR3 LBDs using extended MD simulations. By analyzing the conformational dynamics, we compare these changes to the well-known CAR agonist CITCO.…”