Affinity purification using recombinant PXR as a tool to characterize environmental ligands

Area of Science:

• Environmental Chemistry
• Molecular Toxicology
• Biochemistry

Background:

• Environmental endocrine-disrupting compounds (EDCs) frequently interact with nuclear receptors.
• The human pregnane X receptor (hPXR) is activated by diverse environmental chemicals, including pesticides and PCBs.
• Previous studies identified hPXR ligands in environmental samples but could not identify specific compounds.

Purpose of the Study:

• To develop and validate a PXR-affinity column for purifying environmental hPXR ligands.
• To assess the column's ability to capture ligands based on their binding affinity.
• To analyze an environmental sample for PXR-active compounds and their potential cross-reactivity with other receptors.

Main Methods:

• Immobilization of recombinant human pregnane X receptor (hPXR) onto a solid support to create a PXR-affinity column.
• Testing the column's ligand-binding capacity using known PXR ligands with varying affinities (EC50 values).
• Application of the PXR-affinity column to analyze a complex environmental sample for PXR activity and shared ligand interactions.

Main Results:

• The PXR-affinity column preferentially captured PXR ligands with medium to high binding affinities (EC50 < 1 μM).
• Approximately 50% of the PXR activity in the environmental sample was attributed to high-affinity PXR ligands.
• Significant ligand overlap was observed between PXR and other nuclear receptors, including ERα, AR, and AhR.

Conclusions:

• The developed PXR-affinity column is a valuable tool for purifying and characterizing environmental PXR-active compounds.
• This method facilitates the identification of specific EDCs and their binding affinities.
• The findings highlight the promiscuity of nuclear receptor ligand binding and the potential for cross-talk between receptors.