High-throughput screening assays for estrogen receptor by using coumestrol, a natural fluorescence compound

Estrogen receptor (ER) is a ligand-inducible transcriptional factor involving in cell growth, differentiation, and diseases, so detection and identification of compounds having estrogenic effects are of great importance in the drug discovery industry. We have developed and validated a rapid, simple, and homogeneous method that can detect estrogenic compounds. This human ERα/β binding assay uses fluorescence polarization (FP) by applying an autofluorescent phytoestrogen, coumestrol (CS). A nonspecific adsorption assay shows that no obvious nonspecific adsorption is detected between CS and ERs. In the Scatchard plot analysis, the convex curve exhibits a positive cooperative binding, indicating that the binding of CS induces a conformational change of the ER to form a dimer and increases the affinity for the additional CS. In the Hill plot analysis, CS shows moderate binding affinity with both ERα and ERβ, and the measured Kd of CS is 32.66 µM and 36.14 µM, respectively, indicating that CS is applicable to the ER binding assay for determination of potent ligands of moderate binding affinity. Four typical ligands are selected to verify the ER binding assays, and the results are consistent with the reported data. All of above make the FP method based on CS suitable for high-throughput screening.