Identification of farnesoid X receptor modulators by a fluorescence polarization-based interaction assay.
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.
Han
Ki-Cheol
KC
Life Sciences Research Division, Korea Institute of Science and Technology, Seoul 130-650, Republic of Korea.
Kim
Jung Hwan
JH
Kim
Kook-Han
KH
Kim
Eunice EunKyeong
EE
Seo
Jin-Ho
JH
Yang
Eun Gyeong
EG
D000818:Animals; D002635:Chenodeoxycholic Acid / Q000378:metabolism; D004353:Drug Evaluation, Preclinical / Q000379:methods*; D005454:Fluorescence Polarization; D005456:Fluorescent Dyes / Q000378:metabolism; D017930:Genes, Reporter; D006801:Humans; D008024:Ligands; D051379:Mice; D008958:Models, Molecular; D011485:Protein Binding; D017434:Protein Structure, Tertiary; D018160:Receptors, Cytoplasmic and Nuclear / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D013050:Spectrometry, Fluorescence; D013379:Substrate Specificity; D015533:Transcriptional Activation; D000099150:Receptor, Farnesoid X-Activated
D002635:Chenodeoxycholic Acid; D005456:Fluorescent Dyes; D008024:Ligands; D018160:Receptors, Cytoplasmic and Nuclear; D000099150:Receptor, Farnesoid X-Activated