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J. Biol. Chem., Vol. 283, Issue 19, 12819-12830, May 9, 2008

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

Chengjian Mao, Nicole M. Patterson, Milu T. Cherian, Irene O. Aninye¹, Chen Zhang, Jamie Bonéy Montoya, Jingwei Cheng, Karson S. Putt^¶, Paul J. Hergenrother^¶, Elizabeth M. Wilson^||, Ann M. Nardulli, Steven K. Nordeen^**, and David J. Shapiro²

From the Departments of Biochemistry, Molecular and Integrative Physiology, and ^¶Chemistry, University of Illinois, Urbana, Illinois 61810-3602, the ^||Laboratories for Reproductive Biology and Lineberger Comprehensive Cancer Center, University of North Carolina, Chapel Hill, North Carolina 27599-7500, and the ^**Department of Pathology, University of Colorado and Health Sciences Center, Denver, Colorado 80045

Estrogen receptor (ER) plays an important role in several human cancers. Most current ER 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 binding to consensus estrogen response element (cERE) DNA. We identified small molecule inhibitors of ER 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 binding to the flcERE (IC₅₀ 3 µM) and inhibited ER-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, TPBM inhibited 17β-estradiol (E₂)-ER (IC₅₀ 9 µM) and 4-hydroxytamoxifen-ER-mediated gene expression. Chromatin immunoprecipitation showed TPBM reduced E₂·ER recruitment to an endogenous estrogen-responsive gene. TPBM inhibited E₂-dependent growth of ER-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 ER inhibitors. Using a simple high throughput screen for inhibitors of ER binding to the cERE, a small molecule inhibitor has been identified that selectively inhibits ER-mediated gene expression and estrogen-dependent growth of cancer cells.

Received for publication, December 5, 2007 , and in revised form, March 12, 2008.

^* This work was supported, in whole or in part, by National Institutes of Health Grants RO1 DK-071909 (to D. J. S.), RO1 DK 53884 (to A. N. M.) and by NICHD, NIH Grant HD16910 (to E. M. W.). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

¹ An NIH predoctoral trainee in cell and molecular biology.

² To whom correspondence should be addressed: Dept. of Biochemistry, University of Illinois, 600 S. Mathews Ave. Urbana, IL 61801. Tel.: 217-333-1788; Fax: 217-244-5858; E-mail: djshapir{at}uiuc.edu.