Identification of farnesoid X receptor modulators by a fluorescence polarization-based interaction assay
Ki-Cheol Han1, Jung Hwan Kim, Kook-Han Kim
1Life Sciences Research Division, Korea Institute of Science and Technology, Seoul 130-650, Republic of Korea.
Abstract:
Farnesoid X receptor (FXR) serves as a receptor for chenodeoxycholic acid (CDCA) and other bile acids, and it coordinates cholesterol and lipid metabolism. Because targeting the FXR-CDCA interaction might provide a way to regulate lipid homeostasis, we developed an FXR binding assay based on fluorescence polarization. Employing a fluorescently labeled CDCA (CDCA-F), we showed that CDCA-F selectively bound to the ligand binding domain of FXR (FXR-LBD) among nuclear receptors. The assay was then used for screening inhibitors against the FXR-CDCA interaction, thereby discovering four relatively potent inhibitors. The selected inhibitors were further studied for changes in intrinsic tryptophan fluorescence of FXR-LBD to gain structural insights into the interaction. Furthermore, transactivation effects of the inhibitors on the human bile salt excretory pump (BSEP) promoter were examined to reveal their cellular activities in the FXR-mediated pathway. Therefore, we demonstrated that the developed assay would offer an efficient primary screening tool for identifying FXR modulators.
Insights
Researchers developed a fluorescence polarization assay to screen for Farnesoid X receptor (FXR) modulators. This efficient method identified four potent inhibitors of the FXR-chenodeoxycholic acid interaction, aiding lipid homeostasis research.
Area of Science:
- Biochemistry
- Molecular Biology
- Pharmacology
Background:
- Farnesoid X receptor (FXR) regulates cholesterol and lipid metabolism.
- FXR is activated by bile acids like chenodeoxycholic acid (CDCA).
- Targeting the FXR-CDCA interaction offers a potential strategy for managing lipid homeostasis.
Purpose of the Study:
- To develop a fluorescence polarization-based assay for screening FXR modulators.
- To identify novel inhibitors of the FXR-CDCA interaction.
- To evaluate the cellular activity and structural insights of identified inhibitors.
Main Methods:
- Development of a fluorescence polarization binding assay using fluorescently labeled CDCA (CDCA-F).
- Screening of potential inhibitors against the FXR-CDCA interaction.
- Analysis of inhibitor binding using intrinsic tryptophan fluorescence of FXR ligand-binding domain (FXR-LBD).
- Assessment of transactivation effects on the bile salt excretory pump (BSEP) promoter.
Main Results:
- CDCA-F selectively bound to FXR-LBD.
- The assay successfully identified four potent inhibitors of the FXR-CDCA interaction.
- Structural insights into inhibitor binding were obtained.
- Inhibitors demonstrated cellular activity within the FXR-mediated pathway.
Conclusions:
- A fluorescence polarization assay was successfully developed for efficient primary screening of FXR modulators.
- The identified inhibitors provide a basis for further drug development targeting FXR.
- This assay facilitates the discovery of compounds that modulate lipid homeostasis via FXR.
