Understanding how steroid hormones regulate physiological functions has been significantly advanced by structural biology approaches. However, progress has been hampered by significant misfolding of the ligand binding domains in heterologous expression systems and by conformational flexibility that interferes with crystallization. Here, we show that protein folding problems common to steroid hormone receptors are circumvented by a mutations that stabilize well-characterized conformations of the receptor. We use this approach to present the first structure of an apo steroid receptor, revealing a ligand-accessible channel, allowing soaking of preformed crystals. Furthermore, crystallization of different pharmacological classes of compounds allowed us to define the structural basis of NFκB selective signaling through ER, revealing a unique conformation of the receptor that allows selective suppression of inflammatory gene expression. The ability to crystallize many receptor-ligand complexes with distinct pharmacophores allows one to define the structural features of signaling specificity that would not be apparent in a single structure.
A new series of ligands for the estrogen receptor (ER) based on a three-dimensional structural motif consisting of a bridged oxabicyclic core (7-oxabicyclo[2.2.1]heptene or heptadiene) were synthesized and examined for their receptor binding activity and as regulators of transcription through the two ER subtypes, ER alpha and ER beta. The prototypical ligands also contain a 1,2-diarylethylene motif, common to many nonsteroidal estrogens, as an embellishment on the oxabicyclic core. Thus, these ligands bear peripheral groups typically found in ER ligands, built here upon an overall three-dimensional core topology that is unusual for these targets. Most of these compounds were conveniently synthesized by a Diels-Alder reaction of various 3,4-diarylfurans with a variety of dienophiles, neat and under mild conditions in the absence of catalysts. Some of the synthesized compounds display good binding affinity for the ER, and in transcription assays, the highest affinity compounds are antagonists on both ERs. Molecular modeling studies suggest a structural basis for the antagonist activity of these compounds. These compounds, based on the bicyclo[2.2.1]core system, expand the structural diversity of ligands that can be antagonists for the estrogen receptors.
A strategy to develop chemotherapeutic agents by combining several active groups into a single molecule as a conjugate that can modulate multiple cellular pathways may produce compounds having higher efficacy compared to that of single-target drugs. In this article, we describe the synthesis and evaluation of an array of dual-acting ER and histone deacetylase inhibitors. These novel hybrid compounds combine an indirect antagonism structure motif of ER (OBHS, oxabicycloheptene sulfonate) with the HDAC inhibitor suberoylanilide hydroxamic acid (SAHA). These OBHS-HDACi conjugates exhibited good ER binding affinity and excellent ERα antagonistic activity, and they also exhibited potent inhibitory activities against HDACs. Compared with the approved drug tamoxifen, these conjugates exhibited higher antitumor potency in ERα-positive breast cancer cells (MCF-7). Moreover, these conjugates not only showed selective anticancer activity that was more potent against MCF-7 cells than DU 145 (prostate cancer), but they had no toxicity toward normal cells.
To increase the chemical diversity of bioactive molecules by incorporating unusual elements, we have examined the replacement of a C=C double bond with the isoelectronic, isostructural B-N bond in the context of nonsteroidal estrogen receptor (ER) ligands. While the B-N bond was hydrolytically labile in the unhindered cyclofenil system, the more hindered anilino dimesitylboranes, analogs of triarylethylene estrogens, were easily prepared, hydrolytically stable, and demonstrated substantial affinity for ERs. X-ray analysis of one ERalpha-ligand complex revealed steric clashes with the para methyl groups distorting the receptor; removal of these groups resulted in an increase in affinity, potency, and transcriptional efficacy. These studies define the structural determinants of stability and cellular bioactivity of a B-N for C=C substitution in nonsteroidal estrogens and provide a framework for further exploration of "elemental isomerism" for diversification of drug-like molecules.
Some estrogen receptor‐α (ERα)‐targeted breast cancer therapies such as tamoxifen have tissue‐selective or cell‐specific activities, while others have similar activities in different cell types. To identify biophysical determinants of cell‐specific signaling and breast cancer cell proliferation, we synthesized 241 ERα ligands based on 19 chemical scaffolds, and compared ligand response using quantitative bioassays for canonical ERα activities and X‐ray crystallography. Ligands that regulate the dynamics and stability of the coactivator‐binding site in the C‐terminal ligand‐binding domain, called activation function‐2 (AF‐2), showed similar activity profiles in different cell types. Such ligands induced breast cancer cell proliferation in a manner that was predicted by the canonical recruitment of the coactivators NCOA1/2/3 and induction of the GREB1 proliferative gene. For some ligand series, a single inter‐atomic distance in the ligand‐binding domain predicted their proliferative effects. In contrast, the N‐terminal coactivator‐binding site, activation function‐1 (AF‐1), determined cell‐specific signaling induced by ligands that used alternate mechanisms to control cell proliferation. Thus, incorporating systems structural analyses with quantitative chemical biology reveals how ligands can achieve distinct allosteric signaling outcomes through ERα.
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