Asymmetric allostery in estrogen receptor-α homodimers drives responses to the ensemble of estrogens in the hormonal milieu.

The estrogen receptor-α (ER) is thought to function only as a homodimer but responds to a variety of environmental, metazoan, and therapeutic estrogens at subsaturating doses, supporting binding mixtures of ligands as well as dimers that are only partially occupied. Here, we present a series of flexible ER ligands that bind to receptor dimers with individual ligand poses favoring distinct receptor conformations-receptor conformational heterodimers-mimicking the binding of two different ligands. Molecular dynamics simulations showed that the pairs of different ligand poses changed the correlated motion across the dimer interface to generate asymmetric communication between the dimer interface, the ligands, and the surface binding sites for epigenetic regulatory proteins. By examining the binding of the same ligand in crystal structures of ER in the agonist vs. antagonist conformers, we also showed that these allosteric signals are bidirectional. The receptor conformer can drive different ligand binding modes to support agonist vs. antagonist activity profiles, a revision of ligand binding theory that has focused on unidirectional signaling from the ligand to the coregulator binding site. We also observed differences in the allosteric signals between ligand and coregulator binding sites in the monomeric vs. dimeric receptor, and when bound by two different ligands, states that are physiologically relevant. Thus, ER conformational heterodimers integrate two different ligand-regulated activity profiles, representing different modes for ligand-dependent regulation of ER activity.

Synthesis and estrogenic activity of BODIPY-labeled estradiol conjugates.

Novel BODIPY-estradiol conjugates have been synthesized by selecting position C-3-O for labeling. The conjugation strategy was based on Cu(I)-catalyzed azide-alkyne cycloaddition (CuAAC) or etherification. Estradiol derivatives used as azide partners bearing an ω-azidoalkyl function through C4-C8-long linkers have been prepared. CuAAC reactions of estradiol azides with BODIPY alkyne furnished fluorescent 3-O-labeled conjugates bearing the triazole ring as a coupling moiety. Williamson etherifications of 3-O-(ω-bromoalkyl)-17ß-estradiol derivatives with BODIPY-OH resulted in labeled conjugates connected with an ether moiety. Interactions of the conjugates with estrogen receptor (ER) were investigated using molecular docking calculations in comparison with estradiol. The conjugates occupied both the classical and alternative binding sites on human ERα, with slightly lower binding affinity to references estradiol and diethystilbestrol. All compounds have displayed reasonable estrogenic activity. They increased the proliferation of ER-positive breast cancer cell line MCF7 contrary to ER-negative SKBR-3 cell line. The most potent compound 13a induced the transcriptional activity of ER in dose-dependent manner in dual luciferase recombinant reporter model and increased progesterone receptor's expression, proving the retained estrogenic activity. The fluorescence of candidate compound 13a co-localised with the ERα. The newly synthesized labeled compounds might serve as good starting point for further development of fluorescent probes for modern biological applications. In addition to studying steroid uptake and transport in cells, e.g. in the processes of biodegradation of estrogen-hormones micropollutants, they could also be utilized in examination of estrogen-binding proteins.

Rational evolution for altering the ligand preference of estrogen receptor alpha.

Estrogen receptor α is commonly used in synthetic biology to control the activity of genome editing tools. The activating ligands, estrogens, however, interfere with various cellular processes, thereby limiting the applicability of this receptor. Altering its ligand preference to chemicals of choice solves this hurdle but requires adaptation of unspecified ligand-interacting residues. Here, we provide a solution by combining rational protein design with multi-site-directed mutagenesis and directed evolution of stably integrated variants in Saccharomyces cerevisiae. This method yielded an estrogen receptor variant, named TERRA, that lost its estrogen responsiveness and became activated by tamoxifen, an anti-estrogenic drug used for breast cancer treatment. This tamoxifen preference of TERRA was maintained in mammalian cells and mice, even when fused to Cre recombinase, expanding the mammalian synthetic biology toolbox. Not only is our platform transferable to engineer ligand preference of any steroid receptor, it can also profile drug-resistance landscapes for steroid receptor-targeted therapies.

Identification and characterisation of a novel interaction between oestrogen receptor alpha and FOXP2.

Forkhead box P2 (FOXP2) regulates expression of various genes and is associated with language, speech and neural development as well as cancer. Since there may be a putative link between sex and language and because transcription factors rarely function in isolation, this study aims to investigate whether FOXP2 directly associates with oestrogen receptor α (ER1), a nuclear receptor responsible for sexual differentiation that is also associated with cancer. Isothermal titration calorimetry and fluorescence anisotropy were used to investigate the interaction between the DNA-binding forkhead domain (FHD) of FOXP2, the N-terminal region (NT) of FOXP2, and the ligand-binding domain (LBD) of ER1. ER1 LBD does not interact with FOXP2 NT but associates with apo-FOXP2 FHD in an enthalpically favourable manner. The affinity of this interaction is inversely correlated to the salt concentration. Additionally, FOXP2 FHD that is bound to ER1 LBD, has reduced ability to interact with its cognate DNA. This research identifies a novel interaction between ER1 LBD and FOXP2 FHD and shows that the interaction is regulated by salt. Moreover, FOXP2 FHD cannot bind to both ER1 LBD and DNA simultaneously, suggesting that this interaction could be involved in regulating the transcriptional pathway of FOXP2 should the interaction be found in vivo. This study could serve as a foundation for uncovering the basis of sexual dimorphism in speech and language development and related disorders and potentially offers an alternate for targeted cancer therapies.

Nontargeted Analysis Reveals a Broad Range of Bioactive Pollutants in Drinking Water by Estrogen Receptor Affinity-Mass Spectrometry.

Exposure to environmental endocrine-disrupting chemicals (EDCs) can cause extensive health issues. However, specific EDCs remain elusive. This work aimed at performing nontargeted identification of estrogen receptor α (ERα)-active compounds using an ERα protein affinity assay combined with high-resolution mass spectrometry in the source and drinking water sampled from major rivers in China. Fifty-one potential ERα-active compounds across 13 categories were identified. For the first time, diisodecyl phenyl phosphate was found to have antiestrogenic activity, and three chemicals (galaxolidone, bensulfuron methyl, and UV234) were plausible ERα ligands. Among the 51 identified compounds, 12 were detected in the aquatic environment for the first time, and the concentration of N-phenyl-2-naphthylamine, a widely used antioxidant in rubber products, was up to 1469 and 1190 ng/L in source and drinking water, respectively. This study demonstrated the widespread presence of known and unknown ERα estrogenic and antiestrogenic pollutants in the major rivers that serve as key sources of drinking water in China and the low removal efficiency of these chemicals in drinking water treatment plants.

Dynamacophore model for breast cancer estrogen receptor alpha as an effective lead generation screening technique.

Regardless to overwhelming quantum of cancer research worldwide, there are few drugs on the market to treat disease conditions. This is owing to multiple process inferences of drug targets in integrated pathways for invasion, growth, and metastasis. Over the past years, the death rate due to breast cancer has been increasing, that set the stage for improved better treatment. Therefore, there is a persistent and vital demand for innovative development of drugs to treat breast cancer. Many studies have reported that more than 60% of breast cancers are Estrogen receptor-α (ERα)-positive tumours and a key transcription factor, Estrogen receptor-α (ERα) was believed to promote proliferation of breast cancer cells. In this study, 150 ns of molecular dynamics was performed for protein-ligand complex to retrieve the potential stable conformations. The most populated dynamics cluster of 4-Hydroxytamoxifen intact with active site amino acid was selected to generate dynamacophore model (dynamic pharmacophore). Further, internal model validation with AU-ROC values ∼0.93 indicate the best model to screen library. The refined hits are funnelled in pharmacokinetics/dynamics, CDOCKER molecular docking, MM-GBSA and density functional theory to identify the promising ERα ligand candidates.Communicated by Ramaswamy H. Sarma.

Switching from Aromatase Inhibitors to Dual Targeting Flavonoid-Based Compounds for Breast Cancer Treatment.

Despite the significant outcomes attained by scientific research, breast cancer (BC) still represents the second leading cause of death in women. Estrogen receptor-positive (ER+) BC accounts for the majority of diagnosed BCs, highlighting the disruption of estrogenic signalling as target for first-line treatment. This goal is presently pursued by inhibiting aromatase (AR) enzyme or by modulating Estrogen Receptor (ER) α. An appealing strategy for fighting BC and reducing side effects and resistance issues may lie in the design of multifunctional compounds able to simultaneously target AR and ER. In this paper, previously reported flavonoid-related potent AR inhibitors were suitably modified with the aim of also targeting ERα. As a result, homoisoflavone derivatives 3b and 4a emerged as well-balanced submicromolar dual acting compounds. An extensive computational study was then performed to gain insights into the interactions the best compounds established with the two targets. This study highlighted the feasibility of switching from single-target compounds to balanced dual-acting agents, confirming that a multi-target approach may represent a valid therapeutic option to counteract ER+ BC. The homoisoflavone core emerged as a valuable natural-inspired scaffold for the design of multifunctional compounds.

Replacing animal-derived components in in vitro test guidelines OECD 455 and 487.

The evaluation of single substances or environmental samples for their genotoxic or estrogenic potential is highly relevant for human- and environment-related risk assessment. To examine the effects on a mechanism-specific level, standardized cell-based in vitro methods are widely applied. However, these methods include animal-derived components like fetal bovine serum (FBS) or rat-derived liver homogenate fractions (S9-mixes), which are a source of variability, reduced assay reproducibility and ethical concerns. In our study, we evaluated the adaptation of the cell-based in vitro OECD test guidelines TG 487 (assessment of genotoxicity) and TG 455 (detection of estrogenic activity) to an animal-component-free methodology. Firstly, the human cell lines A549 (for OECD TG 487), ERα-CALUX® and GeneBLAzer™ ERα-UAS-bla GripTite™ (for OECD TG 455) were investigated for growth in a chemically defined medium without the addition of FBS. Secondly, the biotechnological S9-mix ewoS9R was implemented in comparison to the induced rat liver S9 to simulate in vivo metabolism capacities in both OECD test guidelines. As a model compound, Benzo[a]pyrene was used due to its increased genotoxicity and endocrine activity after metabolization. The metabolization of Benzo[a]Pyrene by S9-mixes was examined via chemical analysis. All cell lines (A549, ERα-CALUX® and GeneBLAzer™ Erα-UAS-bla GripTite™) were successfully cultivated in chemically defined media without FBS. The micronucleus assay could not be conducted in chemically defined medium due to formation of cell clusters. The methods for endocrine activity assessment could be conducted in chemically defined media or reduced FBS content, but with decreased assay sensitivity. The biotechnological ewoS9R showed potential to replace rat liver S9 in the micronucleus in FBS-medium with A549 cells and in the ERα-CALUX® assay in FBS- and chemically defined medium. Our study showed promising steps towards an animal-component free toxicity testing. After further improvements, the new methodology could lead to more reproducible and reliable results for risk assessment.

A computational study of potent series of selective estrogen receptor degraders for breast cancer therapy.

A detailed multistep framework combining quantitative structure-activity relationship, global reactivity, absorption, distribution, metabolism and elimination properties, molecular docking and molecular dynamics simulation (MD) on a series of Selective Estrogen Receptor Down-Regulators (SERDs) interacting with Estrogen Receptor α (ERα) has been performed. The partial least squares regression method derived an empirical model with better predictive capability. The results of global reactivity descriptors revealed that all the compounds are considered strong electrophiles, allowing them to participate in polar reactions more easily. The Brain Or IntestinaL EstimateD permeation diagram revealed that compounds 49 and 31 were predicted to be well absorbed by the human gastrointestinal tract and would not enter the brain. The elucidation of the binding mode between the most active compounds that comply with Lipinski's and Veber's rules from the dataset and ERα targets was explored by molecular docking. The MD simulations were performed for 100 ns on the best compounds, which indicated their stability state under dynamics simulations. These findings are expected to help predict the anticancer activities of the studied SERD compounds and better understand their binding mechanism with ERα targets.Communicated by Ramaswamy H. Sarma.

Study of potential inhibition of the estrogen receptor α by cannabinoids using an in silico approach: Agonist vs antagonist mechanism.

Breast cancer is the main cancer type with more than 2.2 million cases in 2020, and is the principal cause of death in women; with 685000 deaths in 2020 worldwide. The estrogen receptor is involved at least in 70% of breast cancer diagnoses, and the agonist and antagonist properties of the drug in this receptor play a pivotal role in the control of this illness. This work evaluated the agonist and antagonist mechanisms of 30 cannabinoids by employing molecular docking and dynamic simulations. Compounds with docking scores < -8 kcal/mol were analyzed by molecular dynamic simulation at 300 ns, and relevant insights are given about the protein's structural changes, centered on the helicity in alpha-helices H3, H8, H11, and H12. Cannabicitran was the cannabinoid that presented the best relative binding-free energy (-34.96 kcal/mol), and based on rational modification, we found a new natural-based compound with relative binding-free energy (-44.83 kcal/mol) better than the controls hydroxytamoxifen and acolbifen. Structure modifications that could increase biological activity are suggested.

The Clinical Utility of ESR1 Mutations in Hormone Receptor-Positive, HER2-Negative Advanced Breast Cancer.

The estrogen receptor is a key driver of estrogen receptor-positive breast cancers. Accumulating evidence indicates that the ESR1 ligand-binding domain mutations have an important role in acquired endocrine resistance, mainly to treatment with aromatase inhibitors. The identification, monitoring, and targeting of ESR1 mutations is an evolving field of major interest given the potential of improved outcomes in metastatic hormone receptor-positive breast cancers. Herein, the authors review the current evidence and rationale for exploiting the ESR1 mutations as a potential biomarker and therapeutic target. The authors discuss the role of ESR1 testing and current therapeutic efforts to target these mutations.

An Extended DNA Binding Domain of the Estrogen Receptor Alpha Directly Interacts with RNAs in Vitro.

Estrogen receptor alpha (ERα) is a ligand-responsive transcription factor critical for sex determination and development. Recent reports challenge the canonical view of ERα function by suggesting an activity beyond binding dsDNA at estrogen-responsive promotor elements: association with RNAs in vivo. Whether these interactions are direct or indirect remains unknown, which limits the ability to understand the extent, specificity, and biological role of ERα-RNA binding. Here we demonstrate that an extended DNA-binding domain of ERα directly binds a wide range of RNAs in vitro with structural specificity. ERα binds RNAs that adopt a range of hairpin-derived structures independent of sequence, while interacting poorly with single- and double-stranded RNA. RNA affinities are only 4-fold weaker than consensus dsDNA and significantly tighter than nonconsensus dsDNA sequences. Moreover, RNA binding is competitive with DNA binding. Together, these data show that ERα utilizes an extended DNA-binding domain to achieve a high-affinity/low-specificity mode for interacting with RNA.

Labeling of a mutant estrogen receptor with an Affimer in a breast cancer cell line.

Mutations of the intracellular estrogen receptor alpha (ERα) is implicated in 70% of breast cancers. Therefore, it is of considerable interest to image various mutants (L536S, Y537S, D538G) in living cancer cell lines, particularly as a function of various anticancer drugs. We therefore developed a small (13 kDa) Affimer, which, after fluorescent labeling, is able to efficiently label ERα by traveling through temporary pores in the cell membrane, created by the toxin streptolysin O. The Affimer, selected by a phage display, predominantly labels the Y537S mutant and can tell the difference between L536S and D538G mutants. The vast majority of Affimer-ERαY537S is in the nucleus and is capable of an efficient, unrestricted navigation to its target DNA sequence, as visualized by single-molecule fluorescence. The Affimer can also differentiate the effect of selective estrogen receptor modulators. More generally, this is an example of a small binding reagent-an Affimer protein-that can be inserted into living cells with minimal perturbation and high efficiency, to image an endogenous protein.

Diversity-Oriented Synthesis of ERα Modulators via Mitsunobu Macrocyclization.

The diversity of cyclic peptides was expanded by elaborating Mitsunobu macrocyclization, tethering various hydroxy acid building blocks with different Nε-amine substituents. This new strategy was then applied in synthesizing peptidomimetic estrogen receptor modulator (PERM) analogs on the solid support. The PERM analogs exhibited increased serum peptidase stability, cell penetration, and estrogen receptor α binding affinity. Studying diversity-oriented methods for preparing azacyclopeptides provides a new tool for macrocycle construction and further structural information for optimizing ERα modulators for ER positive breast cancers.

Segregation of nuclear and membrane-initiated actions of estrogen receptor using genetically modified animals and pharmacological tools.

Estrogen receptor alpha (ERα) and beta (ERß) are members of the nuclear receptor superfamily, playing widespread functions in reproductive and non-reproductive tissues. Beside the canonical function of ERs as nuclear receptors, in this review, we summarize our current understanding of extra-nuclear, membrane-initiated functions of ERs with a specific focus on ERα. Over the last decade, in vivo evidence has accumulated to demonstrate the physiological relevance of this ERα membrane-initiated-signaling from mouse models to selective pharmacological tools. Finally, we discuss the perspectives and future challenges opened by the integration of extra-nuclear ERα signaling in physiology and pathology of estrogens.

ERα is an RNA-binding protein sustaining tumor cell survival and drug resistance.

Estrogen receptor α (ERα) is a hormone receptor and key driver for over 70% of breast cancers that has been studied for decades as a transcription factor. Unexpectedly, we discover that ERα is a potent non-canonical RNA-binding protein. We show that ERα RNA binding function is uncoupled from its activity to bind DNA and critical for breast cancer progression. Employing genome-wide cross-linking immunoprecipitation (CLIP) sequencing and a functional CRISPRi screen, we find that ERα-associated mRNAs sustain cancer cell fitness and elicit cellular responses to stress. Mechanistically, ERα controls different steps of RNA metabolism. In particular, we demonstrate that ERα RNA binding mediates alternative splicing of XBP1 and translation of the eIF4G2 and MCL1 mRNAs, which facilitates survival upon stress conditions and sustains tamoxifen resistance of cancer cells. ERα is therefore a multifaceted RNA-binding protein, and this activity transforms our knowledge of post-transcriptional regulation underlying cancer development and drug response.

Elucidation of Agonist and Antagonist Dynamic Binding Patterns in ER-α by Integration of Molecular Docking, Molecular Dynamics Simulations and Quantum Mechanical Calculations.

Estrogen receptor alpha (ERα) is a ligand-dependent transcriptional factor in the nuclear receptor superfamily. Many structures of ERα bound with agonists and antagonists have been determined. However, the dynamic binding patterns of agonists and antagonists in the binding site of ERα remains unclear. Therefore, we performed molecular docking, molecular dynamics (MD) simulations, and quantum mechanical calculations to elucidate agonist and antagonist dynamic binding patterns in ERα. 17ß-estradiol (E2) and 4-hydroxytamoxifen (OHT) were docked in the ligand binding pockets of the agonist and antagonist bound ERα. The best complex conformations from molecular docking were subjected to 100 nanosecond MD simulations. Hierarchical clustering was conducted to group the structures in the trajectory from MD simulations. The representative structure from each cluster was selected to calculate the binding interaction energy value for elucidation of the dynamic binding patterns of agonists and antagonists in the binding site of ERα. The binding interaction energy analysis revealed that OHT binds ERα more tightly in the antagonist conformer, while E2 prefers the agonist conformer. The results may help identify ERα antagonists as drug candidates and facilitate risk assessment of chemicals through ER-mediated responses.

In silico identification of novel inhibitors targeting the DNA-binding domain of the human estrogen receptor alpha.

The human estrogen receptor alpha (ERα) is an important regulator in breast cancer development and progression. The frequent ERα mutations in the ligand-binding domain (LBD) can increase the resistance of antiestrogen drugs, highlighting the need to develop new drugs to target ERα-positive breast cancer. In this study, we combined molecular docking, molecular dynamics simulations and binding free energy calculations to develop a structure-based virtual screening workflow to identify hit compounds capable of interfering with the recognition of ERα by the specific response element of DNA. A druggable pocket on the DNA binding domain (DBD) of ERα was identified as the potential binding site. The hits binding modes were further analyzed to reveal the structural characteristics of the DBD-inhibitor complexes. The core structure of the lead molecules was synthesized and was found to inhibit the E2-induced cell proliferation in MCF-7 cell lines. These findings provide an insight into the structural basis of ligand-ERα for alternate sites beyond the LBD-based pocket. The core structure proposed in this study could potentially be used as the lead molecule for further rational optimization of the antiestrogen drug structure with stronger binding of DBD and higher activity.

Approaches to Introduce Helical Structure in Cysteine-Containing Peptides with a Bimane Group.

An i-i+4 or i-i+3 bimane-containing linker was introduced into a peptide known to target Estrogen Receptor alpha (ERα), in order to stabilise an α-helical geometry. These macrocycles were studied by CD and NMR to reveal the i-i+4 constrained peptide adopts a 310 -helical structure in solution, and an α-helical conformation on interaction with the ERα coactivator recruitment surface in silico. An acyclic bimane-modified peptide is also helical, when it includes a tryptophan or tyrosine residue; but is significantly less helical with a phenylalanine or alanine residue, which indicates such a bimane modification influences peptide structure in a sequence dependent manner. The fluorescence intensity of the bimane appears influenced by peptide conformation, where helical peptides displayed a fluorescence increase when TFE was added to phosphate buffer, compared to a decrease for less helical peptides. This study presents the bimane as a useful modification to influence peptide structure as an acyclic peptide modification, or as a side-chain constraint to give a macrocycle.

Dynamics-Based Discovery of Novel, Potent Benzoic Acid Derivatives as Orally Bioavailable Selective Estrogen Receptor Degraders for ERα+ Breast Cancer.

The estrogen receptor α (ERα) is identified as an effective target for the treatment of ERα+ breast cancer; thus, discovery of novel selective estrogen receptor degraders (SERDs) are developed as an effective method to overcome the resistance of breast cancer. Herein, the hot-spot residues for protein-ligand interaction between SERDs and ERα are analyzed by molecular dynamic simulation technology, focusing on the hot-spot residues for four series of designed and synthesized SERDs. SAR studies revealed that while the acrylic acid moiety of AZD9496 is scaffold hopping into benzoic acid, compound D24 exhibits potent binding affinity with ERα, good degradation efficacy of ERα, and inhibitory effect against the MCF-7 breast cancer cell line. Besides, D24 also displays good antitumor efficacy in the MCF-7 human breast cancer xenograft model in vivo, favorable pharmacokinetic properties, excellent druggability, and good safety property, making D24 as a promising drug candidate of SERD for further evaluation.