RESUMO
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.
Assuntos
Receptor alfa de Estrogênio , Estrogênios , Simulação de Dinâmica Molecular , Multimerização Proteica , Receptor alfa de Estrogênio/metabolismo , Receptor alfa de Estrogênio/química , Regulação Alostérica , Humanos , Ligantes , Estrogênios/metabolismo , Estrogênios/química , Sítios de Ligação , Ligação Proteica , Conformação ProteicaRESUMO
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.
Assuntos
Antineoplásicos Hormonais/farmacologia , Neoplasias da Mama/tratamento farmacológico , Citocinas/metabolismo , Resistencia a Medicamentos Antineoplásicos , Receptor alfa de Estrogênio/efeitos dos fármacos , Mediadores da Inflamação/metabolismo , Neoplasias Hormônio-Dependentes/tratamento farmacológico , Moduladores Seletivos de Receptor Estrogênico/farmacologia , Tamoxifeno/análogos & derivados , Neoplasias da Mama/genética , Neoplasias da Mama/metabolismo , Neoplasias da Mama/patologia , Relação Dose-Resposta a Droga , Resistencia a Medicamentos Antineoplásicos/genética , Receptor alfa de Estrogênio/química , Receptor alfa de Estrogênio/genética , Receptor alfa de Estrogênio/metabolismo , Feminino , Regulação Neoplásica da Expressão Gênica , Células HeLa , Células Hep G2 , Humanos , Quinase I-kappa B/genética , Quinase I-kappa B/metabolismo , Interleucina-1beta/metabolismo , Células MCF-7 , Simulação de Dinâmica Molecular , Neoplasias Hormônio-Dependentes/genética , Neoplasias Hormônio-Dependentes/metabolismo , Neoplasias Hormônio-Dependentes/patologia , Fosforilação , Conformação Proteica , Interferência de RNA , Transdução de Sinais/efeitos dos fármacos , Relação Estrutura-Atividade , Tamoxifeno/farmacologia , Transcrição Gênica , Transfecção , Microambiente Tumoral , Fator de Necrose Tumoral alfa/metabolismoRESUMO
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.
Assuntos
Neoplasias da Mama/tratamento farmacológico , Antagonistas de Estrogênios/química , Antagonistas de Estrogênios/farmacologia , Receptor alfa de Estrogênio/antagonistas & inibidores , Neoplasias da Mama/metabolismo , Neoplasias da Mama/patologia , Cristalografia por Raios X , Feminino , Humanos , Ligação Proteica , Conformação Proteica , Relação Estrutura-Atividade , Células Tumorais CultivadasRESUMO
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.
Assuntos
Anti-Inflamatórios/farmacologia , Transportador de Glucose Tipo 4/genética , Atrofia Muscular/tratamento farmacológico , Receptores de Glucocorticoides/química , Transdução de Sinais/efeitos dos fármacos , Células A549 , Regulação Alostérica , Animais , Anti-Inflamatórios/síntese química , Linhagem Celular Transformada , Regulação da Expressão Gênica , Glucose/metabolismo , Transportador de Glucose Tipo 4/metabolismo , Humanos , Lipopolissacarídeos/administração & dosagem , Masculino , Potencial da Membrana Mitocondrial/efeitos dos fármacos , Camundongos , Camundongos Endogâmicos C57BL , Mitocôndrias/efeitos dos fármacos , Mitocôndrias/metabolismo , Fibras Musculares Esqueléticas/efeitos dos fármacos , Fibras Musculares Esqueléticas/metabolismo , Fibras Musculares Esqueléticas/patologia , Atrofia Muscular/induzido quimicamente , Atrofia Muscular/genética , Atrofia Muscular/metabolismo , Mioblastos/efeitos dos fármacos , Mioblastos/metabolismo , Ratos , Receptores de Glucocorticoides/genética , Receptores de Glucocorticoides/metabolismo , Relação Estrutura-AtividadeRESUMO
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.
Assuntos
Proteína de Ligação ao Elemento de Resposta ao AMP Cíclico/fisiologia , Metabolismo Energético , Jejum , Resistência à Insulina , Músculo Esquelético/fisiologia , Fatores de Transcrição/fisiologia , Redução de Peso/fisiologia , Animais , Masculino , Camundongos , Camundongos TransgênicosRESUMO
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.
Assuntos
Antineoplásicos/farmacologia , Neoplasias da Mama/tratamento farmacológico , Compostos Bicíclicos Heterocíclicos com Pontes/farmacologia , Receptores de Estrogênio/antagonistas & inibidores , Antineoplásicos/química , Neoplasias da Mama/metabolismo , Neoplasias da Mama/patologia , Compostos Bicíclicos Heterocíclicos com Pontes/química , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Relação Dose-Resposta a Droga , Feminino , Humanos , Ligantes , Modelos Moleculares , Estrutura Molecular , Receptores de Estrogênio/metabolismo , Relação Estrutura-AtividadeRESUMO
Despite tremendous progress made in the understanding of the ERα signaling pathway and the approval of many therapeutic agents, ER+â¯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.
Assuntos
Antineoplásicos/farmacologia , Benzopiranos/farmacologia , Receptor alfa de Estrogênio/química , Antineoplásicos/química , Benzopiranos/química , Cristalização , Humanos , Células MCF-7 , Modelos Moleculares , Estrutura Molecular , Conformação Proteica , Transdução de Sinais , Relação Estrutura-AtividadeRESUMO
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.
Assuntos
Regulação da Expressão Gênica , Inflamação/genética , Receptores de Glucocorticoides/genética , Elementos de Resposta/genética , Fator de Transcrição AP-1/genética , Sequência de Bases , Sítios de Ligação/genética , Linhagem Celular Tumoral , Cristalografia por Raios X , DNA/química , DNA/genética , DNA/metabolismo , Células HEK293 , Humanos , Modelos Moleculares , Mutação , Conformação de Ácido Nucleico , Ligação Proteica , Estrutura Terciária de Proteína , Receptores de Glucocorticoides/química , Receptores de Glucocorticoides/metabolismo , Fator de Transcrição AP-1/química , Fator de Transcrição AP-1/metabolismoRESUMO
During the stress response to intense exercise, the sympathetic nervous system (SNS) induces rapid catabolism of energy reserves through the release of catecholamines and subsequent activation of protein kinase A (PKA). Paradoxically, chronic administration of sympathomimetic drugs (ß-agonists) leads to anabolic adaptations in skeletal muscle, suggesting that sympathetic outflow also regulates myofiber remodeling. Here, we show that ß-agonists or catecholamines released during intense exercise induce Creb-mediated transcriptional programs through activation of its obligate coactivators Crtc2 and Crtc3. In contrast to the catabolic activity normally associated with SNS function, activation of the Crtc/Creb transcriptional complex by conditional overexpression of Crtc2 in the skeletal muscle of transgenic mice fostered an anabolic state of energy and protein balance. Crtc2-overexpressing mice have increased myofiber cross-sectional area, greater intramuscular triglycerides and glycogen content. Moreover, maximal exercise capacity was enhanced after induction of Crtc2 expression in transgenic mice. Collectively these findings demonstrate that the SNS-adrenergic signaling cascade coordinates a transient catabolic stress response during high-intensity exercise, which is followed by transcriptional reprogramming that directs anabolic changes for recovery and that augments subsequent exercise performance.
Assuntos
Tolerância ao Exercício/genética , Músculo Esquelético/metabolismo , Sistema Nervoso Simpático/metabolismo , Fatores de Transcrição/fisiologia , Animais , Animais Recém-Nascidos , Catecolaminas/metabolismo , Catecolaminas/farmacologia , Células Cultivadas , Tolerância ao Exercício/efeitos dos fármacos , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Condicionamento Físico Animal/fisiologiaRESUMO
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.
Assuntos
Receptor alfa de Estrogênio/química , Receptor alfa de Estrogênio/metabolismo , Transdução de Sinais/efeitos dos fármacos , Sítios de Ligação , Proliferação de Células/efeitos dos fármacos , Receptor alfa de Estrogênio/genética , Feminino , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Humanos , Ligantes , Células MCF-7 , Modelos Moleculares , Estrutura Molecular , Biblioteca de Peptídeos , Ligação ProteicaRESUMO
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.
Assuntos
Receptores de Glucocorticoides/agonistas , Receptores de Glucocorticoides/antagonistas & inibidores , Esteroides/farmacologia , Animais , Relação Dose-Resposta a Droga , Células Hep G2 , Humanos , Interleucina-6/antagonistas & inibidores , Interleucina-6/metabolismo , Lipopolissacarídeos/antagonistas & inibidores , Lipopolissacarídeos/farmacologia , Camundongos , Estrutura Molecular , Fibras Musculares Esqueléticas/efeitos dos fármacos , Fibras Musculares Esqueléticas/metabolismo , Receptores de Glucocorticoides/metabolismo , Esteroides/síntese química , Esteroides/química , Relação Estrutura-AtividadeRESUMO
Ligand-binding dynamics control allosteric signaling through the estrogen receptor-α (ERα), but the biological consequences of such dynamic binding orientations are unknown. Here, we compare a set of ER ligands having dynamic binding orientation (dynamic ligands) with a control set of isomers that are constrained to bind in a single orientation (constrained ligands). Proliferation of breast cancer cells directed by constrained ligands is associated with DNA binding, coactivator recruitment and activation of the estrogen-induced gene GREB1, reflecting a highly interconnected signaling network. In contrast, proliferation driven by dynamic ligands is associated with induction of ERα-mediated transcription in a DNA-binding domain (DBD)-dependent manner. Further, dynamic ligands showed enhanced anti-inflammatory activity. The DBD-dependent profile was predictive of these signaling patterns in a larger diverse set of natural and synthetic ligands. Thus, ligand dynamics directs unique signaling pathways and reveals a new role of the DBD in allosteric control of ERα-mediated signaling.
Assuntos
Receptor alfa de Estrogênio/metabolismo , Transdução de Sinais , Regulação Alostérica , Proliferação de Células , Células HEK293 , Humanos , Ligantes , Células MCF-7 , Modelos Moleculares , Simulação de Dinâmica Molecular , Estrutura MolecularRESUMO
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.
RESUMO
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⺠and/or ERß are co-expressed in various cancer cell types, including breast and prostate cancer cells. ER⺠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. ERâº-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.
Assuntos
Anidrases Carbônicas , Neoplasias da Próstata , Humanos , Masculino , Anidrases Carbônicas/metabolismo , Estrutura Molecular , Relação Estrutura-Atividade , Receptores de Estrogênio , Ligantes , Anidrase Carbônica IX/metabolismo , Antígenos de Neoplasias/metabolismo , Inibidores da Anidrase Carbônica/química , Microambiente TumoralRESUMO
Compounds that block estrogen action through the estrogen receptor (ER) or downregulate ER levels are useful for the treatment of breast cancer and endocrine disorders. In our search for structurally novel estrogens having three-dimensional core scaffolds, we found some compounds with a 7-oxabicyclo[2.2.1]heptene core that bound well to the ERs. The best of these compounds, a phenyl sulfonate ester (termed OBHS for oxabicycloheptene sulfonate), was a partial antagonist on both ERα and ERß. Although OBHS bears no structural resemblance to other estrogen antagonists, it appears to achieve its partial antagonist character by stabilizing a novel conformation of the ER that involves a significant distortion of helix-11. To enhance the antagonist properties of these oxabicyclo[2.2.1]heptane core ligands, we expanded the functional diversity of OBHS by replacing the sulfonate with secondary or tertiary sulfonamides (-SO(2)NR-), isoelectronic and potentially isostructural molecular replacements. An array of 16 OBHS sulfonamide analogues were prepared through a Diels-Alder reaction of a 3,4-diarylfuran using various N-aryl vinyl sulfonamide dienophiles. While the more polar secondary sulphonamides were weak ligands, certain of the tertiary sulfonamides had very good ER binding affinity. In HepG2 cell reporter gene assays, the sulphonamides had moderate potency, but they showed lower intrinsic transcriptional activity on ERα than the selective estrogen receptor modulator (SERM) hydroxytamoxifen or OBHS, and they were inverse agonists on ERß. Thus, the behaviour of these OBH-sulfonamides more closely mirrors the activity of full antagonists like the drug fulvestrant (ICI 182 780), and their greater antagonist biocharacter appears to arise from an accentuated distortion of helix-11.
Assuntos
Compostos Bicíclicos com Pontes/síntese química , Compostos Bicíclicos com Pontes/farmacologia , Estrogênios/farmacologia , Receptores de Estrogênio/antagonistas & inibidores , Sulfonamidas/química , Sulfonamidas/farmacologia , Compostos Bicíclicos com Pontes/química , Estrogênios/síntese química , Estrogênios/química , Humanos , Ligantes , Modelos Moleculares , Estrutura Molecular , Relação Estrutura-Atividade , Sulfonamidas/síntese químicaRESUMO
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.
Assuntos
COVID-19 , Glicoproteína da Espícula de Coronavírus , Animais , Cricetinae , Humanos , Receptores de Estrogênio , Receptor alfa de Estrogênio , SARS-CoV-2RESUMO
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.