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Screening Method for the Identification of Compounds That Activate Pregnane X Receptor
Caitlin Lynch1, Srilatha Sakamuru1, Menghang Xia1
1National Center for Advancing Translational Sciences, National Institutes of Health, Rockville, Maryland, USA.
Current Protocols
|December 5, 2022
Summary
A new high-throughput screening method efficiently identifies compounds that activate the pregnane X receptor (PXR). This approach accelerates the discovery of potential therapeutics and toxicity modulators by testing many compounds rapidly.
Area of Science:
- Pharmacology and Molecular Biology
- Drug Discovery and Development
- Nuclear Receptor Signaling
Background:
- The pregnane X receptor (PXR) is a key regulator of drug metabolism and has roles in homeostasis, immunity, and cancer.
- Identifying PXR modulators is crucial for understanding drug toxicity and therapeutic potential.
- Traditional methods of testing compounds for PXR activity are time-consuming and inefficient for large libraries.
Purpose of the Study:
- To develop and describe a high-throughput screening (HTS) method for identifying human PXR (hPXR) activators.
- To enable rapid screening of large compound libraries for compounds that modulate PXR activity.
- To provide a framework for subsequent confirmation of identified hPXR activators.
Main Methods:
- Utilized HepG2 cells stably transfected with hPXR and a CYP3A4-promoter-driven luciferase reporter.
- Developed a 1536-well plate-based assay for plating, treating, and reading luciferase reporter gene activity.
- Incorporated data analysis, pharmacological characterization, and quantitative polymerase chain reaction (qPCR) for result confirmation.
Main Results:
- Successfully established a robust HTS method for identifying hPXR activators.
- Demonstrated the ability to screen compound libraries efficiently using the developed assay.
- Outlined protocols for confirming identified activators through secondary assays.
Conclusions:
- The described HTS method significantly enhances the efficiency of identifying hPXR activators.
- This approach facilitates the discovery of compounds with therapeutic potential or those causing downstream toxicity.
- The protocol provides a comprehensive workflow from initial screening to confirmation of hPXR modulators.
