Development of a strategy to identify and evaluate direct and indirect activators of constitutive androstane receptor...

Area of Science:

• Pharmacology
• Molecular Biology
• Toxicology

Background:

• The constitutive androstane receptor (CAR) is a nuclear receptor crucial for drug metabolism and disposition.
• CAR activation influences drug efficacy and toxicity, and its dysregulation is linked to liver tumor development in rodents.
• CAR can be activated by direct ligands or indirect signaling pathways, presenting a challenge for comprehensive assessment.

Purpose of the Study:

• To identify direct and indirect activators of rat CAR (rCAR).
• To develop a robust method for evaluating chemical compounds' effects on rCAR activity.
• To differentiate between direct ligand binding and indirect activation mechanisms of CAR.

Main Methods:

• Utilized reporter assays with a modified rat CAR (rCAR-3A-G354Q) mutant exhibiting high ligand responsiveness.
• Assessed the induction of Cyp2b1 mRNA levels in primary rat hepatocytes to identify potential rCAR activators.
• Determined CYP2B6 mRNA levels in human HepaRG cells to identify compounds acting as indirect rCAR activators, leveraging minimal species differences.

Main Results:

• Identified several compounds that directly activated the rCAR-3A-G354Q mutant, suggesting they are rCAR ligands.
• Discovered six compounds that increased Cyp2b1 mRNA in rat hepatocytes but did not activate the rCAR mutant, indicating potential indirect rCAR activation.
• Demonstrated that combined analysis of rat Cyp2b1 expression and rCAR-3A-G354Q reporter assays effectively evaluates rCAR activation.

Conclusions:

• The study provides a refined approach for characterizing rCAR activators.
• Distinguishing between direct and indirect CAR activation is critical for understanding its role in drug metabolism and toxicology.
• The developed assay system offers a valuable tool for screening chemicals and predicting their impact on CAR signaling.