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High-throughput Functional Screening using a Homemade Dual-glow Luciferase Assay

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Quantitative High-Throughput Luciferase Screening in Identifying CAR Modulators.

Caitlin Lynch¹, Jinghua Zhao¹, Hongbing Wang²

¹National Center for Advancing Translational Sciences, National Institutes of Health, Building C, MSC: 3375, 9800 Medical Center Drive, Bethesda, MD, 20892, USA.

Methods in Molecular Biology (Clifton, N.J.)

|August 13, 2016

Summary

This study presents a novel method using a double stable cell line to screen for modulators of the constitutive androstane receptor (CAR). This approach overcomes challenges with basal CAR activity in cell models, enabling identification of activators and deactivators.

Keywords:

Constitutive Androstane Receptor (CAR)Cytochrome P450 2B6 (CYP2B6)Luciferase Quantitative high-throughput screening (qHTS)

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Area of Science:

Pharmacology
Molecular Biology
Biochemistry

Background:

The constitutive androstane receptor (CAR, NR1I3) regulates drug-metabolizing enzymes and transporters.
CAR activation occurs via direct ligand binding or ligand-independent pathways, involving cytoplasmic-to-nuclear translocation.
Constitutive CAR activity in immortalized cells poses challenges for in vitro assay development due to high basal receptor activity.

Purpose of the Study:

To detail a quantitative high-throughput screening (HTS) method for identifying modulators of human CAR (hCAR1).
To develop a reliable in vitro assay model that circumvents issues with high basal CAR activity in immortalized cells.
To enable the identification of both activators and deactivators of the nuclear receptor CAR.

Main Methods:

Development and utilization of a double stable cell line engineered for CAR modulation studies.
Implementation of quantitative high-throughput screening (HTS) assays.
Assay validation for identifying compounds that modulate CAR activity, including activators and deactivators.

Main Results:

Successful establishment of a double stable cell line suitable for CAR activity screening.
Demonstration of the cell line's capability to identify modulators of CAR.
Overcoming the challenge of high basal CAR activity in standard immortalized cell lines.

Conclusions:

The developed double stable cell line provides a robust platform for quantitative HTS of CAR modulators.
This method effectively addresses the challenge of constitutive CAR activity, facilitating drug discovery and mechanistic studies.
The approach allows for the identification of novel activators and deactivators of the important nuclear receptor CAR.