A β-Lactamase-Dependent Gal4-Estrogen Receptor β Transactivation Assay for the Ultra-High Throughput Screening of Estrogen Receptor β Agonists in a 3,456-Well Format

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Abstract
Estrogen action is mediated via two estrogen receptor (ER) subtypes, ERα and ERβ. Selective ER modulators with balanced high affinity for ERα and ERβ have been developed as therapeutics for the treatment of a variety of diseases, including hormone-responsive breast cancer and osteoporosis. Recent data based primarily on the evaluation of ER-knockout mice have revealed that ERα and ERβ may regulate separate and distinct biological processes. The identification of ERβ specific ligands could further enhance our understanding of ERβ biology. In addition, compounds targeting ERβ may prove useful as therapeutic agents with activity profiles distinguishable from that of estradiol. To discover novel selective ligands for ERβ, we developed and characterized a cell-based Gal4-ERβ β-lactamase reporter gene assay (GERTA) in CHO cells for the ligand-induced activation of the human ERβ. The sensitivity and selectivity of this assay were found to be comparable to those of an ER ligand-binding assay. The assay was optimized for screening in an ultra high throughput 3,456-well nanoplate format and was successfully used to screen a large compound collection for ERβ agonists. Compounds identified in a primary screen were tested in an in vitro ligand-binding assay to characterize further the selectivity and potency for ERβ.