Correlation between in Vitro Peptide Binding Profiles and Cellular Activities for Estrogen Receptor-Modulating Compounds

Numerous biochemical and structural studies have shown that the conformation of the estrogen receptor α (ERα) can be influenced by ligand binding. In turn, the conformational state of ERα affects the ability of the receptor to interact with a wide variety of protein accessory factors. To globally investigate ligand-based cofactor recruitment activities of ERα, we have applied a flow cytometric multiplexed binding assay to determine the simultaneous binding of ERα to over 50 different peptides derived from both known cofactor proteins and random peptide phage display. Using over 400 ERα-binding compounds, we have observed that the multiplexed in vitro peptide-binding profiles are distinct for a number of compounds and that these profiles can predict the effect that ERα ligands have on various cellular activities. These cell-based activities include transcriptional regulation at an estrogen response element, MCF-7 cell proliferation, and Ishikawa endometrial cell stimulation. The majority of the compound-induced diversity in the peptide profiling assay is provided by the unique phage display peptides. Importantly, some of these peptides show a sequence relationship with the corepressor motif, suggesting that peptides identified via phage display might represent natural binding partners of ERα. These in vitro:cellular correlations may in part explain tissue-specific activities of ERα-modulating compounds.