A novel microplate reader-based high-throughput assay for estrogen receptor binding

Coumestrol is a well-known ligand for the estrogen receptor (ER). The compound itself is fluorescent, and its fluorescence intensity at 408 nm increases upon binding to the ER. Here we describe a novel binding assay in 96-well plate format for estrogenic compounds, based on the competition between fluorescent coumestrol and estrogenic compounds for binding to the ligand binding domain (LBD) of the ER-alpha. Displacement of coumestrol was measured as a decrease in fluorescence intensity using a Victor2 1420 multilabel reader. Competitive binding curves for the well-known estrogenic compounds, 17β-estradiol (E2), ethinylestradiol, 4-nonylphenol, 4-octylphenol, genistein, bisphenol A, tamoxifen and diethylstilbestrol were constructed by using 7–10 different concentrations of the compounds and a fixed concentration of ER-α-LBD (14 nmol) and coumestrol (100 nmol). IC50 values and relative potencies (compared to E2) of the estrogenic compounds were determined. The assay was validated by comparing the relative potencies to those from standard radioligand binding assays in the literature. Within day and between day variations were determined and the performance of the assay was assessed by determining the coefficients of variation and Z′ values. The present fluorescent binding assay has proven to be fast and easy, and allows accurately quantifying the binding affinity of estrogenic ligands. The method is also suitable as a high-throughput screening assay for ER ligands.

[1]  A. Kortenkamp,et al.  Something from "nothing"--eight weak estrogenic chemicals combined at concentrations below NOECs produce significant mixture effects. , 2002, Environmental science & technology.

[2]  J. Westendorf,et al.  Comparison of an array of in vitro assays for the assessment of the estrogenic potential of natural and synthetic estrogens, phytoestrogens and xenoestrogens. , 2001, Toxicology.

[3]  G. Barrows,et al.  Cytofluorometric analysis for estrogen receptors using fluorescent estrogen probes. , 1983, Cytometry.

[4]  F. Young.  Biochemistry , 1955, The Indian Medical Gazette.

[5]  E. Hulme,et al.  Receptor-ligand interactions : a practical approach , 1992 .

[6]  T. Wiese,et al.  Rapid screening of environmental chemicals for estrogen receptor binding capacity. , 1998, Environmental health perspectives.

[7]  D. Seigneurin.  [Cytometry]. , 2020, Annales de Pathologie.

[8]  D. Moras,et al.  Overexpression, purification, and crystal structure of native ER alpha LBD. , 2001, Protein expression and purification.

[9]  J. van der Greef,et al.  Reversed-phase liquid chromatography coupled on-line to receptor affinity detection based on the human estrogen receptor. , 1996, Analytical chemistry.

[10]  Makoto Usami,et al.  Estrogen receptor binding assay of chemicals with a surface plasmon resonance biosensor , 2002, The Journal of Steroid Biochemistry and Molecular Biology.

[11]  G. G. Stokes.  "J." , 1890, The New Yale Book of Quotations.

[12]  H. Irth,et al.  High Resolution Screening of Plant Natural Product Extracts for Estrogen Receptor a and f3 Binding Activity Using an Online HPLC-MS Biochemical Detection System , 2001, Journal of biomolecular screening.

[13]  R. Flanigan,et al.  Estrogenicity of coumestrol in the mouse: fluorescence detection of interaction with estrogen receptors. , 1984, Biochemistry.

[14]  S. Kharb.  Toxicology , 1936 .

[15]  J. Schwartz,et al.  Ligand-mediated modulation of estrogen receptor conformation by estradiol analogs. , 1993, Biochemistry.

[16]  H. Harris,et al.  The ligand binding profiles of estrogen receptors α and β are species dependent , 2002, Steroids.

[17]  J. Meerman,et al.  Detection of estrogenic potency in wastewater and surface water with three in vitro bioassays , 2002, Environmental toxicology and chemistry.

[18]  Dianne Dumanoski,et al.  Our Stolen Future , 1996 .

[19]  Clay W Scott,et al.  A medium-throughput functional assay of KCNQ2 potassium channels using rubidium efflux and atomic absorption spectrometry. , 2003, Analytical biochemistry.

[20]  J H Zhang,et al.  Confirmation of primary active substances from high throughput screening of chemical and biological populations: a statistical approach and practical considerations. , 2000, Journal of combinatorial chemistry.

[21]  Gregory J. Parker,et al.  Development of High Throughput Screening Assays Using Fluorescence Polarization: Nuclear Receptor-Ligand-Binding and Kinase/Phosphatase Assays , 2000, Journal of biomolecular screening.