A new small molecule inhibitor of estrogen receptor alpha binding to estrogen response elements blocks estrogen-dependent growth of cancer cells.
Estrogen receptor alpha (ERalpha) plays an important role in several human cancers. Most current ERalpha 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 ERalpha binding to consensus estrogen response element (cERE) DNA. We identified small molecule inhibitors of ERalpha 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 ERalpha binding to the flcERE (IC(50) approximately 3 microm) and inhibited ERalpha-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 ERalpha, TPBM inhibited 17beta-estradiol (E(2))-ERalpha (IC(50) 9 microm) and 4-hydroxytamoxifen-ERalpha-mediated gene expression. Chromatin immunoprecipitation showed TPBM reduced E(2).ERalpha recruitment to an endogenous estrogen-responsive gene. TPBM inhibited E(2)-dependent growth of ERalpha-positive cancer cells (IC(50) of 5 microm). 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 ERalpha inhibitors. Using a simple high throughput screen for inhibitors of ERalpha binding to the cERE, a small molecule inhibitor has been identified that selectively inhibits ERalpha-mediated gene expression and estrogen-dependent growth of cancer cells.
Mao
Chengjian
C
Department of Biochemistry, and Chemistry, University of Illinois, Urbana, Illinois 61810-3602, USA.
Patterson
Nicole M
NM
Cherian
Milu T
MT
Aninye
Irene O
IO
Zhang
Chen
C
Montoya
Jamie Bonéy
JB
Cheng
Jingwei
J
Putt
Karson S
KS
Hergenrother
Paul J
PJ
Wilson
Elizabeth M
EM
Nardulli
Ann M
AM
Nordeen
Steven K
SK
Shapiro
David J
DJ
D045744:Cell Line, Tumor; D049109:Cell Proliferation / Q000187:drug effects; D002614:Chelating Agents / Q000494:pharmacology; D047369:Chromatin Immunoprecipitation; D004354:Drug Screening Assays, Antitumor; D047628:Estrogen Receptor alpha / Q000037:antagonists & inhibitors* / Q000235:genetics / Q000378:metabolism*; D004967:Estrogens / Q000494:pharmacology*; D015972:Gene Expression Regulation, Neoplastic / Q000187:drug effects; D006801:Humans; D008024:Ligands; D015394:Molecular Structure; D009369:Neoplasms / Q000235:genetics / Q000378:metabolism* / Q000473:pathology*; D011485:Protein Binding; D020218:Response Elements; D012680:Sensitivity and Specificity; D014158:Transcription, Genetic / Q000187:drug effects / Q000235:genetics; D015032:Zinc / Q000378:metabolism
D002614:Chelating Agents; D047628:Estrogen Receptor alpha; D004967:Estrogens; D008024:Ligands; D015032:Zinc