A short-incubation reporter-gene assay for high-throughput screening of estrogen receptor-alpha antagonists.
Estrogen receptor (ER) alpha and beta are ligand-activated nuclear transcription factors that mediate the effects of the steroid hormone 17beta-estradiol. Tissue-selective ER modulators have been developed for the treatment of a variety of diseases, including osteoporosis and hormone-dependent breast cancer. Second- and third-generation selective ER modulators are in development, with the goal of reducing toxicity and improving tissue-selective efficacy. Novel tissue-selective and ERsubtype specific ligands may have the potential of providing a new paradigm for maintaining the health of women. The traditional cell-based screening assays for nuclear receptors require 16-18 h of incubation, which limits the assay miniaturization for ultra-high-throughput screening. We have developed a new cell-based ERalpha transactivation assay for the screening of ERalpha-specific antagonists with only 4 h of incubation time. The assay was optimized and used for a fully automated ultrahigh-throughput screen in 3,456-well nanoplate format. The screening throughput was 250,000-300,000 compounds per day, and a number of valuable leads were identified.
Shi
Xiaoqing
X
Department of Automated Biotechnology, Merck Research Laboratories, North Wales, PA, USA.
Zheng
Wei
W
Schneeweis
Jonathan E
JE
Fischer
Paul A
PA
Strulovici
Berta
B
Peekhaus
Norbert T
NT
D000818:Animals; D016466:CHO Cells; D006224:Cricetinae; D003412:Cricetulus; D004305:Dose-Response Relationship, Drug; D004353:Drug Evaluation, Preclinical / Q000379:methods; D004958:Estradiol / Q000031:analogs & derivatives / Q000494:pharmacology; D004965:Estrogen Antagonists / Q000032:analysis / Q000494:pharmacology*; D047628:Estrogen Receptor alpha / Q000037:antagonists & inhibitors* / Q000235:genetics / Q000378:metabolism; D031541:Fluorescence Resonance Energy Transfer / Q000379:methods; D000077267:Fulvestrant; D005822:Genetic Vectors; D020849:Raloxifene Hydrochloride / Q000494:pharmacology; D011993:Recombinant Fusion Proteins / Q000378:metabolism; D020845:Selective Estrogen Receptor Modulators / Q000494:pharmacology; D013629:Tamoxifen / Q000494:pharmacology; D014157:Transcription Factors / Q000235:genetics / Q000378:metabolism; D015533:Transcriptional Activation; D014162:Transfection; D001618:beta-Lactamases / Q000235:genetics / Q000378:metabolism
D004965:Estrogen Antagonists; D047628:Estrogen Receptor alpha; D011993:Recombinant Fusion Proteins; D020845:Selective Estrogen Receptor Modulators; D014157:Transcription Factors; D013629:Tamoxifen; D000077267:Fulvestrant; D020849:Raloxifene Hydrochloride; D004958:Estradiol; D001618:beta-Lactamases