Exploration of Dimensions of Estrogen Potency

Abstract

The estrogen receptors, ER␣ and ER␤, are ligand-regulated transcription factors that control gene expression programs in target tissues. The molecular events underlying estrogen action involve minimally two steps, hormone binding to the ER ligandbinding domain followed by coactivator recruitment to the ER⅐ligand complex; this ligand⅐receptor⅐coactivator triple complex then alters gene expression. Conceptually, the potency of an estrogen in activating a cellular response should reflect the affinities that characterize both steps involved in the assembly of the active ligand⅐receptor⅐coactivator complex. Thus, to better understand the molecular basis of estrogen potency, we developed a completely in vitro system (using radiometric and timeresolved FRET assays) to quantify independently three parameters: (a) the affinity of ligand binding to ER, (b) the affinity of coactivator binding to the ER⅐ligand complex, and (c) the potency of ligand recruitment of coactivator. We used this system to characterize the binding and potency of 12 estrogens with both ER␣ and ER␤. Some ligands showed good correlations between ligand binding affinity, coactivator binding affinity, and coactivator recruitment potency with both ERs, whereas others showed correlations with only one ER subtype or displayed discordant coactivator recruitment potencies. When ligands with low receptor binding affinity but high coactivator recruitment potencies to ER␤ were evaluated in cell-based assays, elevation of cellular coactivator levels significantly and selectively improved their potency. Collectively, our results indicate that some low affinity estrogens may elicit greater cellular responses in those target cells that express higher levels of specific coactivators capable of binding to their ER complexes with high affinity.Estrogens of diverse structure are used for many clinical needs and various health benefits. Ethynylestradiol is used for fertility regulation (1, 2), and the drug Premarin (3, 4), which contains a mixture of 10 structurally different equine estrogens, is widely prescribed for menopausal hormone replacement therapy. Non-steroidal estrogens, such as diethylstilbestrol, are used to suppress androgen production in the treatment of prostate cancer (5, 6), and soy isoflavone extracts that contain the phytoestrogen genistein are consumed by older women as estrogen supplement for its possible beneficial effect in relieving some of the post-menopausal symptoms (7,8). In addition, estrogens selective for one of the estrogen receptor (ER) 2 subtypes, ER␤, are under active investigation for the management of breast, prostate, and colon cancers and specific cases of cardiovascular and central nervous system disorders (9, 10). Little is known, however, about what structural features of a particular estrogen and what molecular interactions it undergoes during its course of action contribute to its potency and to its selectivity of action through the two ER subtypes, ER␣ and ER␤.ER␣ and ER␤ are members of the nuclear receptor (NR) family of li...

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“…71 A lack of correlation between binding affinities (RBA), and both potency for coactivator recruitment, and cellular ERE-reporter activation was also apparent from a study of ERα ligands commonly used as chemical probes. 66 This is compatible with the thermodynamics of ligand binding to ERα in a multi-protein complex being influenced by other binding partners in that complex.…”

“…Neither RBA ral nor RBA for isolated ERα correlated with potency in cell cultures. Various studies have made similar observations on the discordance between biochemical affinity and potency in cell cultures, 66, 71-73 and alternate methodologies have been proposed. 74 Poor correlation was observed between RBA and growth inhibition in MCF-7 cells for a series of raloxifene analogues with similar RBAs, but up to 100-fold differences in potency in cell cultures.…”

“…66 In the SRC3 titration assay, the 3X concentrations of Fluorescein-labeled SRC3 (5 μl) prepared in buffer A was added to a 96-well black microplate containing premixed streptavidin-terbium (SA-Tb) and biotinylated ERα LBD (5 μL). This was followed by the addition of 3X concentration of ligands 1 , 23 and 30a (5 μL) prepared in buffer B.…”

“…63, 64 Time-resolved fluorescence (or Förster) resonance energy transfer (TR-FRET) assay of the interaction of ERα-LBD with an LxxLL-containing recognition sequence of SRC3, in the presence of E 2 , has been used to identify small molecules that directly inhibit protein-protein binding. 65, 66 Ligand stabilization of the ERα/SRC3 complex is an initial and essential step in transmitting a functional, transcriptional signal and, unlike RBA measurements, will define agonist and antagonist activity. A full agonist is predicted to stabilize the ERα/SRC3 complex in a similar conformation to the E 2 -bound complex, yielding a similar TR-FRET signal; whereas, an antagonist in the presence of E 2 will disrupt the ERα/SRC3 complex destroying the FRET signal.…”

An exposure:activity profiling method for interpreting high-throughput screening data for estrogenic activity—Proof of concept

“…In fact, squelching is apparent in the responses to GEN in measurements of cellular growth kinetics and responses at high doses had to be removed from the dose-response fit (Rotroff et al, 2013b). Coregulatory proteins can modulate both potency and efficacy in estrogen-induced gene expression (Jeyakumar et al, 2008(Jeyakumar et al, , 2011Simons, 2008;Simons and Kumar, 2013).…”