A Short-Incubation Reporter-Gene Assay for High-Throughput Screening of Estrogen Receptor-α Antagonists

Abstract

Estrogen receptor (ER) α and β are ligand-activated nuclear transcription factors that mediate the effects of the steroid hormone 17β-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 ERα transactivation assay for the screening of ERα-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.