High-Throughput Screening Assays for Estrogen Receptor by Using Coumestrol, a Natural Fluorescence Compound

Abstract

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 Kdof 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.

Amyloid Fibril Formation of α‐Synuclein Is Modulated via the Estrogen Receptor Ligand Binding Domain of Estrogen Receptor α Bound with Tamoxifen‐based Small Molecules

“…Upon binding of CS to ERLBD, fluorescence polarization increases as a result of producing less freely rotating complexes. The IC 50 of CS for ERLBD protein was measured as 56 nM in our hands, which exhibits a 3‐fold tighter binding compared to the literature value (170 nM) . This observation suggests that the purified ERLBD was properly folded, resulting in functional binding with a cognate ligand.…”

“…In order to confirm that the purified ERLBD protein was properly folded and thereby functional, we tried the coumestrol (CS) binding‐based fluorescence polarization assay with a minor modification of the protocol described elsewhere . Purified ERLBD protein at 10 μM was mixed with a different concentration of CS and mP value was measured by fluorescence polarization using microplate reader (Figure (a)).…”

“…Binding of coumestrol (CS) for ERLBD protein was monitored by fluorescence polarization . Purified ERLBD protein at 10 μM was mixed with different concentrations of CS in 100 mM phosphate buffer (pH 7.4) containing 200 μg/mL bovine gamma globulin (BGG) in 386‐well plates for a total volume of 40 μL.…”

Selenophenes: Introducing a New Element into the Core of Non‐Steroidal Estrogen Receptor Ligands

“…After being ultrasonicated in an icy bath, the supernatant was applied to a column of GSH‐resin. The collection was dialyzed in ice buffer for 4 h. After being checked by a combination of sodium dodecyl sulfate‐polyacrylamide gel electrophoresis and Western blotting, the protein was prepared as a 10‐m m stock in potassium phosphate and stored at −80 °C …”

“…The collection was dialyzed in ice buffer for 4h.A fter being checked by ac ombination of sodium dodecyl sulfate-polyacrylamide gel electrophoresis and Western blotting, the protein was prepared as a1 0-mm stock in potassium phosphate and stored at À80 8C. [49] Estrogen receptor binding affinity.R elative binding affinities were determined by ac ompetitive fluorimetric binding assay as previously described. Briefly,4 0nm fluorescence tracer (coumestrol, Sigma-Aldrich, St. Louis, MO, USA) and 0.8 mm purified human ERa or ERb ligand binding domain (LBD) were diluted in 100 mm potassium phosphate buffer (pH 7.4), containing 100 mgmL À1 bovine gamma globulin (Sigma-Aldrich).…”