Abstract
Therapeutic block of estrogen action is typically achieved with conventional antagonists (CAs), compounds that displace estradiol from the estrogen receptor (ER) and induce formation of an ER conformation that cannot bind to coactivator proteins, such as the steroid receptor coactivators (SRCs). As an alternative mode for blocking estrogen action, the authors seek small molecules that act as coactivator binding inhibitors (CBIs)-that is, they compete directly with SRC3 for interaction with estradiol-bound ER. CBIs would be interesting mechanistic probes of estrogen action and might also provide an alternative, more durable endocrine therapy for hormone-responsive breast cancer, where cellular adaptations lead to resistance to CAs. The authors have designed and optimized a set of time-resolved fluorescence resonance energy transfer (TR-FRET) assays to monitor the interaction of ER with SRC3 and ligands, and they have used them in high-throughput screens to discover small-molecule CBIs that are able to disrupt this interaction. These assays also distinguish CBIs from CAs. These robust and sensitive "mix-and-measure" assays use low concentrations of ER labeled with a europium chelate as FRET donor and a Cy5-labeled SRC as acceptor. This multiplexed protocol produces excellent signal-to-noise ratios (>100) and Z' values (>0.8).