A New Small Molecule Inhibitor of Estrogen Receptor α Binding to Estrogen Response Elements Blocks Estrogen-dependent Growth of Cancer Cells*

Estrogen receptor α (ERα) plays an important role in several human cancers. Most current ERα antagonists bind in the receptor ligand binding pocket and compete for binding with estrogenic ligands. Instead of the traditional approach of targeting estrogen binding to ER, we describe a strategy using a high throughput fluorescence anisotropy microplate assay to identify small molecule inhibitors of ERα binding to consensus estrogen response element (cERE) DNA. We identified small molecule inhibitors of ERα binding to the fluorescein-labeled (fl)cERE and evaluated their specificity, potency, and efficacy. One small molecule, theophylline, 8-[(benzylthio)methyl]-(7CI,8CI) (TPBM), inhibited ERα binding to the flcERE (IC50 ∼ 3 μm) and inhibited ERα-mediated transcription of a stably transfected ERE-containing reporter gene. Inhibition by TPBM was ER-specific, because progesterone and glucocorticoid receptor transcriptional activity were not significantly inhibited. In tamoxifen-resistant breast cancer cells that overexpress ERα, TPBM inhibited 17β-estradiol (E2)-ERα (IC50 9 μm) and 4-hydroxytamoxifen-ERα-mediated gene expression. Chromatin immunoprecipitation showed TPBM reduced E2·ERα recruitment to an endogenous estrogen-responsive gene. TPBM inhibited E2-dependent growth of ERα-positive cancer cells (IC50 of 5 μm). TPBM is not toxic to cells and does not affect estrogen-independent cell growth. TPBM acts outside of the ER ligand binding pocket, does not act by chelating the zinc in ER zinc fingers, and differs from known ERα inhibitors. Using a simple high throughput screen for inhibitors of ERα binding to the cERE, a small molecule inhibitor has been identified that selectively inhibits ERα-mediated gene expression and estrogen-dependent growth of cancer cells.