Novel human Constitutive Androstane Receptor Activators and their Effects on Hepatic Energy Homeostasis

Abstract

The constitutive androstane receptor (CAR, NR1I3) serves as a ligand-activated transcription factor transforming chemical stimuli into cellular responses by regulating the expression of its target genes. CAR is well-recognized as a key mediator affecting drug metabolism and transport in response to a broad range of xenobiotics. It is predominantly expressed in the liver and intestine; where it regulates the inductive expression of phase I and II drug metabolizing enzyme target genes such as cytochrome P450 (CYP) 2B6, CYP3A4, sulfotransferases, and glutathione s-transferases, and drug transporters such as multidrug resistance protein 1 and organic anion-transporting polypeptides. Recent animal studies revealed that CAR also influences gluconeogenesis, lipogenesis, and fatty acid synthesis, and potentially ameliorates diabetes and obesity. Thus, discovery of compounds activating or deactivating CAR offers a promising avenue for the prediction of induction-mediated drug-drug interactions and a potential therapeutic strategy for metabolic diseases. Strategies incorporating computational based virtual screening with biological approaches were employed to identify and validate novel human (h) CAR modulators. Luciferase reporter assays were performed by transfecting various hCAR expression and CYP2B6 reporter plasmids into HepG2 cells. A stable cell line expressing both CYP2B6 luciferase and hCAR vectors was generated to identify novel hCAR activators and deactivators in a high-throughput manner. Human primary hepatocytes were utilized to explore the effects of CAR activation on target gene expression and hepatic energy homeostasis. Overall, this project revealed that a combined computational and biological approach is effective for identifying hCAR activators. The novel double stable cell line generated here offers a unique cell model in identification of both hCAR agonists and antagonists at high-throughput levels. The most distinctive finding obtained through this research however, was the species difference between hCAR and mouse (m) CAR energy metabolism. Activation of mCAR represses the expression of genes associated with gluconeogenesis, lipogenesis, and fatty acid synthesis, while activation of hCAR selectively inhibits gluconeogenesis without suppressing fatty acid synthesis. These findings warrant caution with respect to the interpretation and extrapolation of results obtained from animal models to humans.