Identifying CAR Modulators Utilizing a Reporter Gene Assay.

Area of Science:

• Pharmacology
• Molecular Biology
• Biochemistry

Background:

• The constitutive androstane receptor (CAR, NR1I3) is a nuclear receptor regulating hepatic drug metabolism and transport.
• CAR activation occurs via direct ligand binding or ligand-independent pathways, with nuclear translocation being essential for both.
• Constitutive nuclear localization and high basal activity of CAR in immortalized cell lines pose challenges for in vitro assays.

Purpose of the Study:

• To detail a quantitative high-throughput screening method for identifying human CAR modulators.
• To introduce a double stable cell line designed to overcome the challenges of CAR's high basal activity in vitro.
• To enable the identification of both activators and deactivators of the CAR nuclear receptor.

Main Methods:

• Development and utilization of a double stable cell line for CAR modulation screening.
• Implementation of quantitative high-throughput screening (HTS) assays.
• Assays designed to measure CAR activity, accounting for its constitutive nuclear localization.

Main Results:

• Successful establishment of a screening system using a double stable cell line.
• Demonstration of the system's capability to identify compounds modulating CAR activity.
• The developed method allows for the detection of both CAR activators and deactivators.

Conclusions:

• The described quantitative high-throughput screening method employing a double stable cell line is effective for identifying human CAR modulators.
• This approach provides a robust solution for studying the challenging nuclear receptor CAR in vitro.
• The developed cell line and screening strategy facilitate the discovery of novel therapeutic agents targeting CAR.