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Using TR-FRET to Investigate Protein-Protein Interactions: A Case Study of PXR-Coregulator Interaction

Wenwei Lin1, Taosheng Chen1

  • 1St. Jude Children's Research Hospital, Memphis, TN, United States.

Advances in Protein Chemistry and Structural Biology
|February 8, 2018
PubMed

Insights

A novel pregnane X receptor (PXR) assay using time-resolved fluorescence resonance energy transfer (TR-FRET) effectively characterizes ligand binding and distinguishes agonists from antagonists. This robust assay shows promise for high-throughput screening of PXR modulators.

Area of Science:

  • Biochemistry
  • Molecular Biology
  • Pharmacology

Background:

  • Time-resolved fluorescence resonance energy transfer (TR-FRET) assays are crucial for studying nuclear receptor-ligand interactions.
  • Limited progress has been made in applying TR-FRET assays to the pregnane X receptor (PXR).

Purpose of the Study:

  • To develop and validate a novel TR-FRET coactivator interaction assay for PXR.
  • To characterize PXR ligand binding affinities and differentiate between agonists and antagonists.

Main Methods:

  • Development of a PXR TR-FRET coactivator interaction assay based on cocrystal structural data.
  • Assay validation for robustness, stability, DMSO tolerance, and signal-to-noise ratio.

Main Results:

  • The novel PXR TR-FRET assay successfully characterizes ligand binding affinities.
  • The assay can effectively differentiate between PXR agonists and antagonists.
  • The assay demonstrates robustness with stable signals over long incubation times (up to 300 min), high DMSO tolerance (up to 5%), and a high signal-to-noise ratio (six).

Conclusions:

  • The developed PXR TR-FRET coactivator interaction assay is a robust tool for PXR research.
  • This assay has significant potential for high-throughput screening to identify novel PXR modulators.
Keywords:
AgonistAntagonistCoactivatorCoregulatorCorepressorNuclear receptorPregnane X receptorTR-FRET

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