A new small molecule inhibitor of estrogen receptor α binding to estrogen response elements blocks estrogen-dependent growth of cancer cells

Estrogen receptor alpha(ER alpha) plays an important role in several human cancers. Most current ER alpha 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 ER alpha binding to consensus estrogen response element (cERE) DNA. We identified small molecule inhibitors of ER alpha 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 ER alpha binding to the flcERE (IC50 similar to 3 mu M) and inhibited ER alpha-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 ER alpha, TPBM inhibited 17 beta-estradiol (E-2)-ER alpha (IC50 9 mu M) and 4-hydroxytamoxifen ER alpha-mediated gene expression. Chromatin immunoprecipitation showed TPBM reduced E-2.ER alpha recruitment to an endogenous estrogen-responsive gene. TPBM inhibited E-2-dependent growth of ER alpha-positive cancer cells (IC50 of 5 mu M). 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 ER alpha inhibitors. Using a simple high throughput screen for inhibitors of ER alpha binding to the cERE, a small molecule inhibitor has been identified that selectively inhibits ER alpha-mediated gene expression and estrogen-dependent growth of cancer cells.