Estrogen Receptor-α Ligand-binding Domain Research Articles

Dual-mechanism estrogen receptor inhibitors

Efforts to improve estrogen receptor-α (ER)-targeted therapies in breast cancer have relied upon a single mechanism, with ligands having a single side chain on the ligand core that extends outward to determine antagonism of breast cancer growth. Here, we describe inhibitors with two ER-targeting moieties, one of which uses an alternate structural mechanism to generate full antagonism, freeing the side chain to independently determine other critical properties of the ligands. By combining two molecular targeting approaches into a single ER ligand, we have generated antiestrogens that function through new mechanisms and structural paradigms to achieve antagonism. These dual-mechanism ER inhibitors (DMERIs) cause alternate, noncanonical structural perturbations of the receptor ligand-binding domain (LBD) to antagonize proliferation in ER-positive breast cancer cells and in allele-specific resistance models. Our structural analyses with DMERIs highlight marked differences from current standard-of-care, single-mechanism antiestrogens. These findings uncover an enhanced flexibility of the ER LBD through which it can access nonconsensus conformational modes in response to DMERI binding, broadly and effectively suppressing ER activity.

Multi-Residue Method for the Analysis of Stilbene Estrogens in Milk.

The rapid analysis of stilbene estrogens is crucially important in the environment, food and health sectors, but quantitation of lower detection limit for stilbene estrogens persists as a severe challenge. We herein described a homologous and sensitive fluorescence polarization (FP) assay based on estrogen receptor α ligand binding domain (ER-LBD) to monitor stilbene estrogens in milk. Under optimal conditions, the half maximal inhibitory concentrations (IC50) of the FP assay were 9.27 nM, 12.94 nM, and 22.38 nM for hexestrol, dienestrol and diethylstilbestrol, respectively. And the corresponding limits of detection (LOD) values were 2.94 nM, 2.89 nM, and 3.12 nM. Finally, the assay was applied to determine the stilbenes in milk samples where the mean recoveries ranged from 95.76% to 112.78% and the coefficients of variation (CV) below 12.00%. Furtherly, we have focused our study on high cross-reactivity phenomena by using two in silico approaches, including molecular docking analysis and topology analysis. Overall, docking results show that several residues in the hydrophobic pocket produce hydrophobic interactions with the tested drug molecules, which contribute to the stability of their binding. In this paper, we conclude that the FP method is suitable for the rapid detection of stilbenes in milk samples, requiring no expensive analytical equipment or time-consuming sample preparation. This work offers a practical approach that applies bioscience technology in food safety testing and improves analytical speed and laboratory efficiency.

Synthesis of benzoylbenzamide derivatives of 17α-E-vinyl estradiol and evaluation as ligands for the estrogen receptor-α ligand binding domain

A series consisting of substituted benzoylbenzamide derivatives of 17α-E-vinyl estradiol 6a-i and 7a-d was prepared in good overall yields from the corresponding novel iodinated benzoylbenzamide precursors using Pd(0)-catalyzed Stille coupling. Biological evaluation using competitive binding assays indicated that all compounds were effective ligands for the ERα- and ERβ-LBD (RBA = 0.5–10.0% of estradiol). Most of the compounds expressed lower stimulatory (agonist) potency (RSA <0.2–0.5%) compared to their binding affinity, however, the meta-substituted isomer 6h demonstrated a level of efficacy (RSA = 5.7%) comparable to its affinity (RBA = 9.5%). Docking studies of 6b, 6h, and 6i with the 2YAT crystal structure suggested that higher affinity and efficacy of 6h are due to an effective set of interactions with exposed receptor sidechains not observed with the ortho- and para- isomers. In this binding model, the terminal ring of the ligand is exposed to the solvent space, which would explain both the small variation in RBA values and the narrow SAR for the diverse structural features.

Homology models of mouse and rat estrogen receptor-α ligand-binding domain created by in silico mutagenesis of a human template: Molecular docking with 17β-estradiol, diethylstilbestrol, and paraben analogs

Crystal structures exist for human, but not rodent, estrogen receptor-α ligand-binding domain (ERα-LBD). Consequently, rodent studies involving binding of compounds to ERα-LBD are limited in their molecular-level interpretation and extrapolation to humans. Because the sequences of rodent and human ERα-LBDs are >95% identical, we expected their 3D structures and ligand binding to be highly similar. To test this hypothesis, we used the human ERα-LBD structure (PDB 3UUD) as a template to produce rat and mouse homology models. Employing the rodent models and human structure, we generated docking poses of 23 Group A ligands (17β-estradiol, diethylstilbestrol, and 21 paraben analogs) in AutoDock Vina for interspecies comparisons. Ligand RMSDs (Å) (median, 95% CI) were 0.49 (0.21–1.82) (human-mouse) and 1.19 (0.22–1.82) (human-rat), well below the 2.0–2.5 Å range for equivalent docking poses. Numbers of interspecies ligand-receptor residue contacts were highly similar, with Sorensen Sc (%) = 96.8 (90.0–100) (human-mouse) and 97.7 (89.5–100) (human-rat). Likewise, numbers of interspecies ligand-receptor residue contacts were highly correlated: Pearson r = 0.913 (human-mouse) and 0.925 (human-rat). Numbers of interspecies ligand-receptor atom contacts were even more tightly correlated: r = 0.979 (human-mouse) and 0.986 (human-rat). Pyramid plots of numbers of ligand-receptor atom contacts by residue exhibited high interspecies symmetry and had Spearman rs = 0.977 (human-mouse) and 0.966 (human-rat). Group B ligands included 15 ring-substituted parabens recently shown experimentally to exhibit decreased binding to human ERα and to exert increased antimicrobial activity. Ligand efficiencies calculated from docking ligands into human ERα-LBD were well correlated with those derived from published experimental data (Pearson partial rp = 0.894 and 0.918; Groups A and B, respectively). Overall, the results indicate that our constructed rodent ERα-LBDs interact with ligands in like manner to the human receptor, thus providing a high level of confidence in extrapolations of rodent to human ligand-receptor interactions.

Abstract GS2-02: Direct regulation of estrogen receptor-α (ER) transcriptional activity by NF1

Abstract Inactivating germline mutations in the NF1 gene (encoding neurofibromin) cause neurofibromatosis type 1. In addition to peripheral nervous system tumors, NF1 patients are at higher risk for other cancers, including breast cancer. Tumor exome-sequencing studies demonstrate that approximately 20% of all human cancers have somatic NF1 mutations. NF1 has been best known for its ability to inactivate Ras as a GAP (GTPase Activating Protein). However, this function is served by a small GAP domain in a very large protein. Recurrent missense mutations inactivating the GAP activity are infrequent. In contrast, it is common to detect frameshift (FS) and nonsense (NS) NF1 mutations, which can create an NF1-null state deleting not only GAP, but also, potentially, undefined NF1 functions whose loss could also drive tumorigenesis. As we reported at SABCS previously, in 600+ patients treated by tamoxifen adjuvant monotherapy, we found that FS/NS NF1 mutations independently correlate with relapse risk (HR=2.6, p=0.03). To explore this finding, we silenced NF1 in preclinical models of ER+ breast cancer, which markedly enhanced ER transcriptional activities, causing estradiol (E2) hypersensitivity and converted tamoxifen into an agonist (in vitro and in vivo). Most important, these activities depend on ER, but not on NF1's GAP activity. These findings readily explain the poor patient outcomes associated with NS/FS NF1 mutations, and reveal a previously unrecognized function for NF1 in ER regulation. In the presence of an agonist, liganded ER repels co-repressors and recruits co-activators, while the reverse is true with an antagonist such as tamoxifen. Many co-regulators contain leucine/isoleucine rich motifs, which bind directly to the ligand-binding domain (LBD) in ER. NF1 has several of these motifs that are much more highly conserved in species with a functional ER pathway, and some of these are mutated in cancers (e.g., in our patient cohort). Furthermore, we found that NF1 canbind directly to ER, and that this binding is mediated between the ER LBD and the NF1 leucine-rich regions. Like a classic co-repressor, wildtype NF1 (but not mutants lacking GAP activity or the Leu-rich motif) binds to ER, and is recruited by ER to the ERE in the presence of tamoxifen, but not E2. Further preclinical treatment studies indicate that while NF1-deficient ER+ breast cancer should not be treated by tamoxifen or AIs, fulvestrant remains effective. Furthermore, when fulvestrant is combined with dabrafinib and trametinib to inhibit Ras effectors Raf and MEK, apoptosis is induced in vitro, and tumor regression is observed in vivo. In conclusion, we have demonstrated that NF1 is a dual negative regulator at the intersection of two potent oncogenic signaling pathways, Ras and ER, and that NF1-deficient ER+ breast cancer patients may be more effectively treated by co-targeting the Ras and ER signaling. These patients, up to 10% of those with advanced ER+ breast cancer, can be readily identified for treatment by ctDNA analysis. A clinical trial is under development. Citation Format: Chang EC, Zheng Z, Philip L, Burcu C, Lei J, Singh P, Anurag M, Chan D, Li JD, Du XP, Shafaee MN, Banks K, Sacker S, Song W, Nguyen T, Cao J, Chen X, Haricharan S, Kavuri M, Kim B-J, Zhang B, Gutmann DH, Lanman RB, Foulds C, Ellis M. Direct regulation of estrogen receptor-α (ER) transcriptional activity by NF1 [abstract]. In: Proceedings of the 2017 San Antonio Breast Cancer Symposium; 2017 Dec 5-9; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2018;78(4 Suppl):Abstract nr GS2-02.

Profiling of engineering hotspots identifies an allosteric CRISPR-Cas9 switch.

The CRISPR-associated protein Cas9 from Streptococcus pyogenes is an RNA-guided DNA endonuclease with widespread utility for genome modification. However, the structural constraints limiting the engineering of Cas9 have not been determined. Here we experimentally profile Cas9 using randomized insertional mutagenesis and delineate hotspots in the structure capable of tolerating insertions of a PDZ domain without disrupting the enzyme’s binding and cleavage functions. Orthogonal domains or combinations of domains can be inserted into the identified sites with minimal functional consequence. To illustrate the utility of the identified sites, we construct an allosterically regulated Cas9 by insertion of the Estrogen Receptor α Ligand Binding Domain. This protein displayed robust, ligand-dependent activation in prokaryotic and eukaryotic cells, establishing a versatile one-component system for inducible and reversible Cas9 activation. Thus, domain insertion profiling facilitates the rapid generation of new Cas9 functionalities and provides useful data for future engineering of Cas9.

Unusual Nitrogenous Phenalenone Derivatives from the Marine-Derived Fungus Coniothyrium cereale.

The new phenalenone metabolites 1, 2, 4, and 6 were isolated from the marine-derived endophytic fungus Coniothyrium cereale, in addition to the ergostane-type sterol (3) and entatrovenetinone (5). Compounds 1 and 2 represent two unusual nitrogen-containing compounds, which are composed of a sterol portion condensed via two bonds to phenalenone derivatives. Compound 6, which contains unprecedented imine functionality between two carbonyl groups to form a oxepane -imine-dione ring, exhibited a moderate cytotoxicity against K562, U266, and SKM1 cancer cell lines. Moreover, molecular docking studies were done on estrogen receptor α-ligand binding domain (ERα-LBD) to compounds 1 and 2 to correlate with binding energies and affinities calculated from molecular docking to the anti-proliferative activity.

Intestinal-specific activatable Myb initiates colon tumorigenesis in mice.

Transcription factor Myb is overexpressed in most colorectal cancers (CRC). Patients with CRC expressing the highest Myb are more likely to relapse. We previously showed that mono-allelic loss of Myb in an Adenomatous polyposis coli (APC)-driven CRC mouse model (ApcMin/+) significantly improves survival. Here we directly investigated the association of Myb with poor prognosis and how Myb co-operates with tumor suppressor genes (TSGs) (Apc) and cell cycle regulator, p27. Here we generated the first intestinal-specific, inducible transgenic model; a MybER transgene encoding a tamoxifen-inducible fusion protein between Myb and the estrogen receptor-α ligand-binding domain driven by the intestinal-specific promoter, Gpa33. This was to mimic human CRC with constitutive Myb activity in a highly tractable mouse model. We confirmed that the transgene was faithfully expressed and inducible in intestinal stem cells (ISCs) before embarking on carcinogenesis studies. Activation of the MybER did not change colon homeostasis unless one p27 allele was lost. We then established that MybER activation during CRC initiation using a pro-carcinogen treatment, azoxymethane (AOM), augmented most measured aspects of ISC gene expression and function and accelerated tumorigenesis in mice. CRC-associated symptoms of patients including intestinal bleeding and anaemia were faithfully mimicked in AOM-treated MybER transgenic mice and implicated hypoxia and vessel leakage identifying an additional pathogenic role for Myb. Collectively, the results suggest that Myb expands the ISC pool within which CRC is initiated while co-operating with TSG loss. Myb further exacerbates CRC pathology partly explaining why high MYB is a predictor of worse patient outcome.

Selective antitumour activity and ERα molecular docking studies of newly synthesizedd-homo fused steroidal tetrazoles

Here we report a convenient “click” synthesis for D-homo fused steroidal tetrazoles 11–14 from androstane and estratriene 16,17-seco-16-nitrile-17-mesyloxy derivatives 5–8, via intramolecular 1,3-dipolar cycloaddition from in situ generated 16,17-seco-17-azido-16-nitriles 5a–8a. Products were validated by 1H/13C-NMR, IR, HRMS, and structurally characterized by X-ray crystallography and computational methods. Compounds were evaluated as potential anti-proliferative agents against a panel of human cancer cell lines. D-Homo fused steroidal tetrazoles 13 and 14 appear to have specific, selective anti-proliferative effects against estrogen receptor positive (ER+) breast adenocarcinoma cells, which correlate with binding energies calculated from molecular docking to the estrogen receptor α-ligand binding domain (ERα-LBD). Moreover, molecular docking suggests that D-ring fused steroidal tetrazoles 13 and 14 could bind to ERα-LBD in a manner similar to known anti-estrogenic compounds. Addition of a D-homo fused tetrazole group appears to structurally mimic the hydrogen bonding potential of β-estradiol, suggesting their general utility in designing novel steroidal tetrazole derivatives as anti-estrogens or inhibitors of steroidogenic enzymes.

Synthesis and evaluation of 4-(substituted styryl/alkenyl)-3,5-bis(4-hydroxyphenyl)-isoxazoles as ligands for the estrogen receptor

A series of 3,5-bis (4-hydroxyphenyl) isoxazoles bearing a styryl/alkyl vinyl group at the 4-position were prepared and evaluated as ligands for the estrogen receptor-alpha (ERα). The target compounds were prepared using the Suzuki reaction to couple an iodo-isoxazole intermediate with a series of styryl/alkenyl boronic acids, followed by O-demethylation. The products were evaluated for their estrogen receptor-α ligand binding domain (ERα-LBD) binding affinity using a competitive binding assay. The 4-(4-hydroxystyryl) derivative 4h displays binding properties similar to those of the previously described pyrazole class of ER ligands, indicating that the ERα-LBD tolerates the presence of the added vinyl group at the 4-position of the isoxazole ring.

Synthesis and evaluation of 17α-(dimethylphenyl)vinyl estradiols as probes of the estrogen receptor-α ligand binding domain

As part of our program to explore the influence of small structural modifications on the biological response of the estrogen receptor-α (ERα), we prepared and evaluated a series of mono-and di-substituted phenyl vinyl estradiols. The target compounds were prepared in 45–80% yields using the Stille coupling reaction and evaluated using competitive binding analysis with the ERα-ligand binding domain (hERα-LBD) and estrogenic activity (induction of alkaline phosphatase in Ishikawa cells). Results indicated that the 2,4- and 2,5-dimethyl derivatives, 5b and 5c, had the highest relative binding affinity (RBA=20.5 and 37.3%) and relative stimulatory activity (RSA=101.0% and 12.3%) of the di-methyl series.

FOXO1 Regulates the Expression of 4E-BP1 and Inhibits mTOR Signaling in Mammalian Skeletal Muscle

The mammalian target of rapamycin (mTOR) is regulated by growth factors to promote protein synthesis. In mammalian skeletal muscle, the Forkhead-O1 transcription factor (FOXO1) promotes catabolism by activating ubiquitin-protein ligases. Using C2C12 mouse myoblasts that stably express inducible FOXO1-ER fusion proteins and transgenic mice that specifically overexpress constitutively active FOXO1 in skeletal muscle (FOXO(++/+)), we show that FOXO1 inhibits mTOR signaling and protein synthesis. Activation of constitutively active FOXO1 induced the expression of eukaryotic initiation factor 4E-binding protein 1 (4E-BP1) mRNA via binding to the promoter. This resulted in an increased total 4E-BP1 abundance and a reduced 4E-BP1 (Thr-37/46) phosphorylation. The reduction in 4E-BP1 phosphorylation was associated with a reduction in the abundance of Raptor and mTOR proteins, Raptor-associated mTOR, reduced phosphorylation of the downstream protein p70S6 kinase, and attenuated incorporation of [(14)C]phenylalanine into protein. The FOXO(++/+) mice, characterized by severe skeletal muscle atrophy, displayed similar patterns of mRNA expression and protein abundance to those observed in the constitutively active FOXO1 C2C12 myotubes. These data suggest that FOXO1 may be an important therapeutic target for human diseases where anabolism is impaired.

Combination of receptor-binding assays and designed mutant receptors for discerning agonists and antagonists

Competitive receptor-binding assays are convenient for analyzing interactions between receptors and their ligands and for screening pharmaceutical drugs and potential endocrine-disrupting chemicals. Although these assays can be used for high-throughput screening, they cannot discern antagonists and agonists. Based on three-dimensional structures of complexes between ligand-binding domain of human estrogen receptor-α and its ligands, we designed mutant receptors with modified mode of ligand-binding. In the current studies we examined the binding of endogenous ligands, artificial ligands, and potent endocrine-disrupting chemicals to wild-type and Asp351 mutants of the human estrogen receptor-α ligand-binding domain. The new combination assay showed the decrease of relative biding affinity (RBA) values for antagonists. For example, RBA for tamoxifen was changed from 4.8 (using the Asp351 receptor) to less than 1.5 (using the Glu351 receptor). On the other hand, the agonists showed increase of RBA values. For example, RBA for bisphenol A was changed from 0.011 (using the Asp351 receptor) to less than 0.030 (using the Glu351 receptor). The variation of RBA was dependant on the type of mutant receptors. The change of RBA from wild-type to mutant-type can be an index for discerning agonists and antagonists. Comparison of RBA values obtained by assays using wild-type and mutant receptors is a simple way of discerning agonists and antagonists, and this approach could be extended to other types of receptors, if information of the receptors was enough to construct a designed mutant receptor.

Potent estrogen agonists based on carborane as a hydrophobic skeletal structure: A new medicinal application of boron clusters

Background: Carboranes (dicarba- closo-dodecaboranes) are a class of carbon-containing polyhedral boron-cluster compounds having remarkable thermal stability and exceptional hydrophobicity. Applications of the unique structural and chemical properties offered by icosahedral carboranes in boron neutron capture therapy have received increasing attention over the past 30 years. However, these features of carboranes may allow another application as a hydrophobic pharmacophore in biologically active molecules that interact hydrophobically with receptors. Results: We have designed candidate estrogen-receptor-binding compounds having carborane as a hydrophobic skeletal structure and synthesized them. The most potent compound bearing a carborane cage exhibited activity at least 10-fold greater than that of 17β-estradiol in the luciferase reporter gene assay. Estrogen receptor-α-binding data for the compound were consistent with the results of the luciferase reporter gene assay. The compound also showed potent in vivo effects on the recovery of uterine weight and bone loss in ovariectomized mice. Conclusion: Further development of the potent carborane-containing estrogenic agonists described here, having a new skeletal structure and unique characteristics, should yield novel therapeutic agents, especially selective estrogen receptor modulators. Furthermore, the suitability of the spherical carborane cage for binding to the cavity of the estrogen receptor-α ligand-binding domain should provide a basis for a similar approach to developing novel ligands for other steroid receptors.