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.

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 sub-saturating 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 binding of the same ligand in crystal structures of ER in the agonist versus antagonist conformers, we also showed that these allosteric signals are bidirectional. The receptor conformer can drive different ligand binding modes to support agonist versus antagonist activity profiles, a revision of ligand binding theory that has focused on unidirectional signaling from ligand to the coregulator binding site. We also observed differences in the allosteric signals between ligand and coregulator binding sites in the monomeric versus 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 new modes for ligand-dependent regulation of ER activity. Significance: The estrogen receptor-α (ER) regulates transcription in response to a hormonal milieu that includes low levels of estradiol, a variety of environmental estrogens, as well as ER antagonists such as breast cancer anti-hormonal therapies. While ER has been studied as a homodimer, the variety of ligand and receptor concentrations in different tissues means that the receptor can be occupied with two different ligands, with only one ligand in the dimer, or as a monomer. Here, we use X-ray crystallography and molecular dynamics simulations to reveal a new mode for ligand regulation of ER activity whereby sequence-identical homodimers can act as functional or conformational heterodimers having unique signaling characteristics, with ligand-selective allostery operating across the dimer interface integrating two different signaling outcomes.

Design, synthesis, biological evaluation and crystal structure determination of dual modulators of carbonic anhydrases and estrogen receptors.

Multi-target compounds have become increasingly important for the development of safer and more effective drug candidates. In this work, we devised a combined ligand-based and structure-based multi-target repurposing strategy and applied it to a series of hexahydrocyclopenta[c]quinoline compounds synthesized previously. The in silico analyses identified human Carbonic Anhydrases (hCA) and Estrogen Receptors (ER) as top scoring candidates for dual modulation. hCA isoforms IX and XII, and ER subtypes ER⍺ and/or ERß are co-expressed in various cancer cell types, including breast and prostate cancer cells. ER⍺ is the primary target of anti-estrogen therapy in breast cancer, and the hCA IX isoform is a therapeutic target in triple-negative breast cancer. ER⍺-mediated transcriptional programs and hCA activity in cancer cells promote favorable microenvironments for cell proliferation. Interestingly, several lines of evidence indicate that the combined modulation of these two targets may provide significant therapeutic benefits. Moving from these first results, two additional hexahydrocyclopenta[c]quinoline derivatives bearing a sulfonamide zinc binding group (hCA) and a phenolic hydroxyl (ER) pharmacophoric group placed at the appropriate locations were designed and synthesized. Interestingly, these compounds were able to directly modulate the activities of both hCA and ER targets. In cell-based assays, they inhibited proliferation of breast and prostate cancer cells with micromolar potency and cell type-selective efficacy. The compounds inhibited hCA activity with nanomolar potency and isoform-selectivity. In transactivation assays, they reduced estrogen-driven ER activity with micro-molar potency. Finally, crystal structures of the synthesized ligands in complex with the two targets revealed that the compounds bind directly to the hCA active site, as well as to the ER ligand-binding domain, providing structural explanation to the observed activity and a rationale for optimization of their dual activity. To the best of our knowledge, this work describes the design, synthesis and biological characterization of the first dual modulators of hCA and ER, laying the ground for the structure-based optimization of their multi-target activity.

The SARS-CoV-2 spike protein binds and modulates estrogen receptors.

The severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) spike (S) protein binds angiotensin-converting enzyme 2 as its primary infection mechanism. Interactions between S and endogenous proteins occur after infection but are not well understood. We profiled binding of S against >9000 human proteins and found an interaction between S and human estrogen receptor α (ERα). Using bioinformatics, supercomputing, and experimental assays, we identified a highly conserved and functional nuclear receptor coregulator (NRC) LXD-like motif on the S2 subunit. In cultured cells, S DNA transfection increased ERα cytoplasmic accumulation, and S treatment induced ER-dependent biological effects. Non-invasive imaging in SARS-CoV-2-infected hamsters localized lung pathology with increased ERα lung levels. Postmortem lung experiments from infected hamsters and humans confirmed an increase in cytoplasmic ERα and its colocalization with S in alveolar macrophages. These findings describe the discovery of a S-ERα interaction, imply a role for S as an NRC, and advance knowledge of SARS-CoV-2 biology and coronavirus disease 2019 pathology.

The SARS-CoV-2 spike protein binds and modulates estrogen receptors.

The severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) spike (S) protein binds angiotensin-converting enzyme 2 (ACE2) at the cell surface, which constitutes the primary mechanism driving SARS-CoV-2 infection. Molecular interactions between the transduced S and endogenous proteins likely occur post-infection, but such interactions are not well understood. We used an unbiased primary screen to profile the binding of full-length S against >9,000 human proteins and found significant S-host protein interactions, including one between S and human estrogen receptor alpha (ERα). After confirming this interaction in a secondary assay, we used bioinformatics, supercomputing, and experimental assays to identify a highly conserved and functional nuclear receptor coregulator (NRC) LXD-like motif on the S2 subunit and an S-ERα binding mode. In cultured cells, S DNA transfection increased ERα cytoplasmic accumulation, and S treatment induced ER-dependent biological effects and ACE2 expression. Noninvasive multimodal PET/CT imaging in SARS-CoV-2-infected hamsters using [ 18 F]fluoroestradiol (FES) localized lung pathology with increased ERα lung levels. Postmortem experiments in lung tissues from SARS-CoV-2-infected hamsters and humans confirmed an increase in cytoplasmic ERα expression and its colocalization with S protein in alveolar macrophages. These findings describe the discovery and characterization of a novel S-ERα interaction, imply a role for S as an NRC, and are poised to advance knowledge of SARS-CoV-2 biology, COVID-19 pathology, and mechanisms of sex differences in the pathology of infectious disease.

Activation of Crtc2/Creb1 in skeletal muscle enhances weight loss during intermittent fasting.

The Creb-Regulated Transcriptional Coactivator (Crtc) family of transcriptional coregulators drive Creb1-mediated transcription effects on metabolism in many tissues, but the in vivo effects of Crtc2/Creb1 transcription on skeletal muscle metabolism are not known. Skeletal muscle-specific overexpression of Crtc2 (Crtc2 mice) induced greater mitochondrial activity, metabolic flux capacity for both carbohydrates and fats, improved glucose tolerance and insulin sensitivity, and increased oxidative capacity, supported by upregulation of key metabolic genes. Crtc2 overexpression led to greater weight loss during alternate day fasting (ADF), selective loss of fat rather than lean mass, maintenance of higher energy expenditure during the fast and reduced binge-eating during the feeding period. ADF downregulated most of the mitochondrial electron transport genes, and other regulators of mitochondrial function, that were substantially reversed by Crtc2-driven transcription. Glucocorticoids acted with AMPK to drive atrophy and mitophagy, which was reversed by Crtc2/Creb1 signaling. Crtc2/Creb1-mediated signaling coordinates metabolic adaptations in skeletal muscle that explain how Crtc2/Creb1 regulates metabolism and weight loss.

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.

Chemical systems biology reveals mechanisms of glucocorticoid receptor signaling.

Glucocorticoids display remarkable anti-inflammatory activity, but their use is limited by on-target adverse effects including insulin resistance and skeletal muscle atrophy. We used a chemical systems biology approach, ligand class analysis, to examine ligands designed to modulate glucocorticoid receptor activity through distinct structural mechanisms. These ligands displayed diverse activity profiles, providing the variance required to identify target genes and coregulator interactions that were highly predictive of their effects on myocyte glucose disposal and protein balance. Their anti-inflammatory effects were linked to glucose disposal but not muscle atrophy. This approach also predicted selective modulation in vivo, identifying compounds that were muscle-sparing or anabolic for protein balance and mitochondrial potential. Ligand class analysis defined the mechanistic links between the ligand-receptor interface and ligand-driven physiological outcomes, a general approach that can be applied to any ligand-regulated allosteric signaling system.

Unexpected equivalent potency of a constrained chromene enantiomeric pair rationalized by co-crystal structures in complex with estrogen receptor alpha.

Despite tremendous progress made in the understanding of the ERα signaling pathway and the approval of many therapeutic agents, ER+ breast cancer continues to be a leading cause of cancer death in women. We set out to discover compounds with a dual mechanism of action in which they not only compete with estradiol for binding with ERα, but also can induce the degradation of the ERα protein itself. We were attracted to the constrained chromenes containing a tetracyclic benzopyranobenzoxepine scaffold, which were reported as potent selective estrogen receptor modulators (SERMs). Incorporation of a fluoromethyl azetidine side chain yielded highly potent and efficacious selective estrogen receptor degraders (SERDs), such as 16aa and surprisingly, also its enantiomeric pair 16ab. Co-crystal structures of the enantiomeric pair 16aa and 16ab in complex with ERα revealed default (mimics the A-D rings of endogenous ligand estradiol) and core-flipped binding modes, rationalizing the equivalent potency observed for these enantiomers in the ERα degradation and MCF-7 anti-proliferation assays.

The SERM/SERD bazedoxifene disrupts ESR1 helix 12 to overcome acquired hormone resistance in breast cancer cells.

Acquired resistance to endocrine therapy remains a significant clinical burden for breast cancer patients. Somatic mutations in the ESR1 (estrogen receptor alpha (ERα)) gene ligand-binding domain (LBD) represent a recognized mechanism of acquired resistance. Antiestrogens with improved efficacy versus tamoxifen might overcome the resistant phenotype in ER +breast cancers. Bazedoxifene (BZA) is a potent antiestrogen that is clinically approved for use in hormone replacement therapies. We found that BZA possesses improved inhibitory potency against the Y537S and D538G ERα mutants compared to tamoxifen and has additional inhibitory activity in combination with the CDK4/6 inhibitor palbociclib. In addition, comprehensive biophysical and structural biology studies show BZA's selective estrogen receptor degrading (SERD) properties that override the stabilizing effects of the Y537S and D538G ERα mutations.

Cryptic glucocorticoid receptor-binding sites pervade genomic NF-κB response elements.

Glucocorticoids (GCs) are potent repressors of NF-κB activity, making them a preferred choice for treatment of inflammation-driven conditions. Despite the widespread use of GCs in the clinic, current models are inadequate to explain the role of the glucocorticoid receptor (GR) within this critical signaling pathway. GR binding directly to NF-κB itself-tethering in a DNA binding-independent manner-represents the standing model of how GCs inhibit NF-κB-driven transcription. We demonstrate that direct binding of GR to genomic NF-κB response elements (κBREs) mediates GR-driven repression of inflammatory gene expression. We report five crystal structures and solution NMR data of GR DBD-κBRE complexes, which reveal that GR recognizes a cryptic response element between the binding footprints of NF-κB subunits within κBREs. These cryptic sequences exhibit high sequence and functional conservation, suggesting that GR binding to κBREs is an evolutionarily conserved mechanism of controlling the inflammatory response.

Tethering not required: the glucocorticoid receptor binds directly to activator protein-1 recognition motifs to repress inflammatory genes.

The glucocorticoid receptor (GR) is a ligand-regulated transcription factor that controls the expression of extensive gene networks, driving both up- and down-regulation. GR utilizes multiple DNA-binding-dependent and -independent mechanisms to achieve context-specific transcriptional outcomes. The DNA-binding-independent mechanism involves tethering of GR to the pro-inflammatory transcription factor activator protein-1 (AP-1) through protein-protein interactions. This mechanism has served as the predominant model of GR-mediated transrepression of inflammatory genes. However, ChIP-seq data have consistently shown GR to occupy AP-1 response elements (TREs), even in the absence of AP-1. Therefore, the current model is insufficient to explain GR action at these sites. Here, we show that GR regulates a subset of inflammatory genes in a DNA-binding-dependent manner. Using structural biology and biochemical approaches, we show that GR binds directly to TREs via sequence-specific contacts to a GR-binding sequence (GBS) half-site found embedded within the TRE motif. Furthermore, we show that GR-mediated transrepression observed at TRE sites to be DNA-binding-dependent. This represents a paradigm shift in the field, showing that GR uses multiple mechanisms to suppress inflammatory gene expression. This work further expands our understanding of this complex multifaceted transcription factor.

Structural and Molecular Mechanisms of Cytokine-Mediated Endocrine Resistance in Human Breast Cancer Cells.

Human breast cancers that exhibit high proportions of immune cells and elevated levels of pro-inflammatory cytokines predict poor prognosis. Here, we demonstrate that treatment of human MCF-7 breast cancer cells with pro-inflammatory cytokines results in ERα-dependent activation of gene expression and proliferation, in the absence of ligand or presence of 4OH-tamoxifen (TOT). Cytokine activation of ERα and endocrine resistance is dependent on phosphorylation of ERα at S305 in the hinge domain. Phosphorylation of S305 by IKKß establishes an ERα cistrome that substantially overlaps with the estradiol (E2)-dependent ERα cistrome. Structural analyses suggest that S305-P forms a charge-linked bridge with the C-terminal F domain of ERα that enables inter-domain communication and constitutive activity from the N-terminal coactivator-binding site, revealing the structural basis of endocrine resistance. ERα therefore functions as a transcriptional effector of cytokine-induced IKKß signaling, suggesting a mechanism through which the tumor microenvironment controls tumor progression and endocrine resistance.

Systems Structural Biology Analysis of Ligand Effects on ERα Predicts Cellular Response to Environmental Estrogens and Anti-hormone Therapies.

Environmental estrogens and anti-hormone therapies for breast cancer have diverse tissue- and signaling-pathway-selective outcomes, but how estrogen receptor alpha (ERα) mediates this phenotypic diversity is poorly understood. We implemented a statistical approach to allow unbiased, parallel analyses of multiple crystal structures, and identified subtle perturbations of ERα structure by different synthetic and environmental estrogens. Many of these perturbations were in the sub-Å range, within the noise of the individual structures, but contributed significantly to the activities of synthetic and environmental estrogens. Combining structural perturbation data from many structures with quantitative cellular activity profiles of the ligands enabled identification of structural rules for ligand-specific allosteric signaling-predicting activity from structure. This approach provides a framework for understanding the diverse effects of environmental estrogens and for guiding iterative medicinal chemistry efforts to generate improved breast cancer therapies, an approach that can be applied to understanding other ligand-regulated allosteric signaling pathways.

Exploring the Structural Compliancy versus Specificity of the Estrogen Receptor Using Isomeric Three-Dimensional Ligands.

The estrogen receptors (ERs) bind with high affinity to many structurally diverse ligands by significantly distorting the contours of their ligand-binding pockets. This raises a question: To what degree is ER able to distinguish between structurally related regioisomers and enantiomers? We have explored the structural compliance and specificity of ERα with a set of ligands having a 7-oxa-bicyclo[2.2.1]hept-5-ene sulfonate core and basic side chains typical of selective ER modulators (SERMs). These ligands have two regioisomers, each of which is a racemate of enantiomers. Using orthogonal protecting groups and chiral HPLC, we isolated all 4 isomers and assigned their absolute stereochemistry by X-ray analysis. The 1S,2R,4S isomer has a 80-170-fold higher affinity for ERα than the others, and it profiles as a partial agonist/antagonist in cellular reporter gene assays and in suppressing proliferation of MCF-7 breast cancer cells with subnanomolar potency, far exceeding that of the other isomers. It is the only isomer found bound to ERα by X-ray analysis after crystallization with four-isomer mixtures of closely related analogs. Thus, despite the general compliance of this receptor for binding a large variety of ligand structures, ER demonstrates marked structural specificity and stereospecificity by selecting a single component from a mixture of structurally related isomers to drive ER-regulated cellular activity. Our findings lay the necessary groundwork for seeking unique ER-mediated pharmacological profiles by rational structural perturbations of two different types of side chains in this unprecedented class of ER ligands, which may prove useful in developing more effective endocrine therapies for breast cancer.

Synthesis of novel steroidal agonists, partial agonists, and antagonists for the glucocorticoid receptor.

Adverse effects of glucocorticoids could be limited by developing new compounds that selectively modulate anti-inflammatory activity of the glucocorticoid receptor (GR). We have synthesized a novel series of steroidal GR ligands, including potent agonists, partial agonists and antagonists with a wide range of effects on inhibiting secretion of interleukin-6. Some of these new ligands were designed to directly impact conformational stability of helix-12, in the GR ligand-binding domain (LBD). These compounds modulated GR activity and glucocorticoid-induced gene expression in a manner that was inversely correlated to the degree of inflammatory response. In contrast, compounds designed to directly modulate LBD epitopes outside helix-12, led to dissociated levels of GR-mediated gene expression and inflammatory response. Therefore, these new series of compounds and their derivatives will be useful to dissect the ligand-dependent features of GR signaling specificity.

Full antagonism of the estrogen receptor without a prototypical ligand side chain.

Resistance to endocrine therapies remains a major clinical problem for the treatment of estrogen receptor-α (ERα)-positive breast cancer. On-target side effects limit therapeutic compliance and use for chemoprevention, highlighting an unmet need for new therapies. Here we present a full-antagonist ligand series lacking the prototypical ligand side chain that has been universally used to engender antagonism of ERα through poorly understood structural mechanisms. A series of crystal structures and phenotypic assays reveal a structure-based design strategy with separate design elements for antagonism and degradation of the receptor, and access to a structurally distinct space for further improvements in ligand design. Understanding structural rules that guide ligands to produce diverse ERα-mediated phenotypes has broad implications for the treatment of breast cancer and other estrogen-sensitive aspects of human health including bone homeostasis, energy metabolism, and autoimmunity.

Predictive features of ligand-specific signaling through the estrogen receptor.

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.