RESUMEN
The vitamin D receptor (VDR) plays a critical role in the regulation of mineral and bone homeostasis. Upon binding of 1α,25-dihydroxyvitamin D3 to the VDR, the activation function 2 (AF2) domain repositions and recruits coactivators for the assembly of the transcriptional machinery required for gene transcription. In contrast to coactivator-induced transcriptional activation, the functional effects of coactivator-independent VDR signaling remain unclear. In humans, mutations in the AF2 domain are associated with hereditary vitamin D-resistant rickets, a genetic disorder characterized by impaired bone mineralization and growth. In the present study, we used mice with a systemic or conditional deletion of the VDR-AF2 domain (VdrΔAF2) to study coactivator-independent VDR signaling. We confirm that ligand-induced transcriptional activation was disabled because the mutant VDRΔAF2 protein was unable to interact with coactivators. Systemic VdrΔAF2 mice developed short, undermineralized bones with dysmorphic growth plates, a bone phenotype that was more pronounced than that of systemic Vdr knockout (Vdr-/-) mice. Interestingly, a rescue diet that is high in calcium, phosphate, and lactose, normalized this phenotype in Vdr-/-, but not in VdrΔAF2 mice. However, osteoblast- and osteoclast-specific VdrΔAF2 mice did not recapitulate this bone phenotype indicating coactivator-independent VDR effects are more important in other organs. In addition, RNA-sequencing analysis of duodenum and kidney revealed a decreased expression of VDR target genes in systemic VdrΔAF2 mice, which was not observed in Vdr-/- mice. These genes could provide new insights in the compensatory (re)absorption of minerals that are crucial for bone homeostasis. In summary, coactivator-independent VDR effects contribute to mineral and bone homeostasis.
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Calcio , Lactosa , Fosfatos , Receptores de Calcitriol , Raquitismo , Transducción de Señal , Animales , Receptores de Calcitriol/metabolismo , Receptores de Calcitriol/genética , Ratones , Raquitismo/metabolismo , Raquitismo/genética , Raquitismo/patología , Raquitismo/prevención & control , Fosfatos/metabolismo , Calcio/metabolismo , Lactosa/metabolismo , Ratones Noqueados , Dieta , Ratones Endogámicos C57BLRESUMEN
Exogenous glucocorticoids are frequently used to treat inflammatory disorders and as adjuncts for the treatment of solid cancers. However, their use is associated with severe side effects and therapy resistance. Novel glucocorticoid receptor (GR) ligands with a patient-validated reduced side effect profile have not yet reached the clinic. GR is a member of the nuclear receptor family of transcription factors and heavily relies on interactions with coregulator proteins for its transcriptional activity. To elucidate the role of the GR interactome in the differential transcriptional activity of GR following treatment with the selective GR agonist and modulator dagrocorat compared to classic (ant)agonists, we generated comprehensive interactome maps by high-confidence proximity proteomics in lung epithelial carcinoma cells. We found that dagrocorat and the antagonist RU486 both reduced GR interaction with CREB-binding protein/p300 and the mediator complex compared to the full GR agonist dexamethasone. Chromatin immunoprecipitation assays revealed that these changes in GR interactome were accompanied by reduced GR chromatin occupancy with dagrocorat and RU486. Our data offer new insights into the role of differential coregulator recruitment in shaping ligand-specific GR-mediated transcriptional responses.
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Benzamidas , Cromatina , Fenantrenos , Receptores de Glucocorticoides , Humanos , Receptores de Glucocorticoides/genética , Mifepristona/farmacología , Complejo Mediador/metabolismo , Glucocorticoides/farmacología , Glucocorticoides/metabolismo , Dexametasona/farmacologíaRESUMEN
Glucocorticoid stress hormones are produced in response to hypothalamic-pituitary-adrenal (HPA) axis activation. Glucocorticoids are essential for physiology and exert numerous actions via binding to the glucocorticoid receptor (GR). Relacorilant is a highly selective GR antagonist currently undergoing a phase 3 clinical evaluation for the treatment of endogenous Cushing's syndrome. It was found that increases in serum adrenocorticotropic hormone (ACTH) and cortisol concentrations after relacorilant treatment were substantially less than the increases typically observed with mifepristone, but it is unclear what underlies these differences. In this study, we set out to further preclinically characterize relacorilant in comparison to the classical but non-selective GR antagonist mifepristone. In human HEK-293 cells, relacorilant potently antagonized dexamethasone- and cortisol-induced GR signaling, and in human peripheral blood mononuclear cells, relacorilant largely prevented the anti-inflammatory effects of dexamethasone. In mice, relacorilant treatment prevented hyperinsulinemia and immunosuppression caused by increased corticosterone exposure. Relacorilant treatment reduced the expression of classical GR target genes in peripheral tissues but not in the brain. In mice, relacorilant induced a modest disinhibition of the HPA axis as compared to mifepristone. In line with this, in mouse pituitary cells, relacorilant was generally less potent than mifepristone in regulating Pomc mRNA and ACTH release. This contrast between relacorilant and mifepristone is possibly due to the distinct transcriptional coregulator recruitment by the GR. In conclusion, relacorilant is thus an efficacious peripheral GR antagonist in mice with only modest disinhibition of the HPA axis, and the distinct properties of relacorilant endorse the potential of selective GR antagonist treatment for endogenous Cushing's syndrome.
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Síndrome de Cushing , Mifepristona , Humanos , Ratones , Animales , Mifepristona/farmacología , Hidrocortisona/metabolismo , Receptores de Glucocorticoides/metabolismo , Sistema Hipotálamo-Hipofisario/metabolismo , Leucocitos Mononucleares , Células HEK293 , Sistema Hipófiso-Suprarrenal/metabolismo , Glucocorticoides/farmacología , Glucocorticoides/metabolismo , Hormona Adrenocorticotrópica/metabolismo , Dexametasona/farmacologíaRESUMEN
Phospholipids are ligands for nuclear hormone receptors (NRs) that regulate transcriptional programs relevant to normal physiology and disease. Here, we demonstrate that mimicking phospholipid-NR interactions is a robust strategy to improve agonists of liver receptor homolog-1 (LRH-1), a therapeutic target for colitis. Conventional LRH-1 modulators only partially occupy the binding pocket, leaving vacant a region important for phospholipid binding and allostery. Therefore, we constructed a set of molecules with elements of natural phospholipids appended to a synthetic LRH-1 agonist. We show that the phospholipid-mimicking groups interact with the targeted residues in crystal structures and improve binding affinity, LRH-1 transcriptional activity, and conformational changes at a key allosteric site. The best phospholipid mimetic markedly improves colonic histopathology and disease-related weight loss in a murine T cell transfer model of colitis. This evidence of in vivo efficacy for an LRH-1 modulator in colitis represents a leap forward in agonist development.
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Colitis , Fosfolípidos , Receptores Citoplasmáticos y Nucleares , Animales , Colitis/tratamiento farmacológico , Ligandos , Ratones , Fosfolípidos/uso terapéutico , Receptores Citoplasmáticos y Nucleares/agonistasRESUMEN
INTRODUCTION: Estrogens used in women's healthcare have been associated with increased risks of venous thromboembolism (VTE) and breast cancer. Estetrol (E4), an estrogen produced by the human fetal liver, has recently been approved for the first time as a new estrogenic component of a novel combined oral contraceptive (E4/drospirenone [DRSP]) for over a decade. In phase 3 studies, E4/DRSP showed good contraceptive efficacy, a predictable bleeding pattern, and a favorable safety and tolerability profile. AREAS COVERED: This narrative review discusses E4's pharmacological characteristics, mode of action, and the results of preclinical and clinical studies for contraception, as well as for menopause and oncology. EXPERT OPINION: Extensive studies have elucidated the properties of E4 that underlie its favorable safety profile. While classical estrogens (such as estradiol) exert their actions via both activation of nuclear and membrane estrogen receptor α (ERα), E4 presents a specific profile of ERα activation: E4 binds and activates nuclear ERα but does not induce the activation of membrane ERα signaling pathways in specific tissues. E4 has a small effect on normal breast tissue proliferation and minimally affects hepatic parameters. This distinct profile of ERα activation, uncoupling nuclear and membrane activation, is unique.
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Estetrol , Anticoncepción , Anticonceptivos Orales Combinados , Estetrol/efectos adversos , Estrógenos/efectos adversos , Femenino , Humanos , MenopausiaRESUMEN
Angelman Syndrome (AS) is a severe neurodevelopmental disorder, caused by the neuronal absence of the ubiquitin protein ligase E3A (UBE3A). UBE3A promotes ubiquitin-mediated protein degradation and functions as a transcriptional coregulator of nuclear hormone receptors, including the glucocorticoid receptor (GR). Previous studies showed anxiety-like behavior and hippocampal-dependent memory disturbances in AS mouse models. Hippocampal GR is an important regulator of the stress response and memory formation, and we therefore investigated whether the absence of UBE3A in AS mice disrupted GR signaling in the hippocampus. We first established a strong cortisol-dependent interaction between the GR ligand binding domain and a UBE3A nuclear receptor box in a high-throughput interaction screen. In vivo, we found that UBE3A-deficient AS mice displayed significantly more variation in circulating corticosterone levels throughout the day compared to wildtypes (WT), with low to undetectable levels of corticosterone at the trough of the circadian cycle. Additionally, we observed an enhanced transcriptomic response in the AS hippocampus following acute corticosterone treatment. Surprisingly, chronic corticosterone treatment showed less contrast between AS and WT mice in the hippocampus and liver transcriptomic responses. This suggests that UBE3A limits the acute stimulation of GR signaling, likely as a member of the GR transcriptional complex. Altogether, these data indicate that AS mice are more sensitive to acute glucocorticoid exposure in the brain compared to WT mice. This suggests that stress responsiveness is altered in AS which could lead to anxiety symptoms.
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Síndrome de Angelman , Ratones , Animales , Síndrome de Angelman/genética , Síndrome de Angelman/metabolismo , Corticosterona/metabolismo , Hipocampo/metabolismo , Encéfalo/metabolismo , Neuronas/metabolismo , Receptores de Glucocorticoides/genética , Receptores de Glucocorticoides/metabolismo , Ubiquitina-Proteína Ligasas/metabolismo , Modelos Animales de EnfermedadRESUMEN
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.
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Neoplasias de la Mama/tratamiento farmacológico , Antagonistas de Estrógenos/química , Antagonistas de Estrógenos/farmacología , Receptor alfa de Estrógeno/antagonistas & inhibidores , Neoplasias de la Mama/metabolismo , Neoplasias de la Mama/patología , Cristalografía por Rayos X , Femenino , Humanos , Unión Proteica , Conformación Proteica , Relación Estructura-Actividad , Células Tumorales CultivadasRESUMEN
Given the unequivocal benefits of menopause hormone therapies (MHT) and combined oral contraceptives (COC), there is a clinical need for new formulations devoid of any risk of breast cancer promotion. Accumulating data from preclinical and clinical studies support that estetrol (E4) is a promising natural estrogen for MHT and COC. Nevertheless, we report here that E4 remains active on the endometrium, even under a dose that is neutral on breast cancer growth and lung metastasis dissemination. This implies that a progestogen should be combined with E4 to protect the endometrium of non-hysterectomized women from hyperplasia and cancer. Through in vivo observations and transcriptomic analyses, our work provides evidence that combining a progestogen to E4 is neutral on breast cancer growth and dissemination, with very limited transcriptional impact. The assessment of breast cancer risk in patients during the development of new MHT or COC is not possible given the requirement of long-term studies in large populations. This translational preclinical research provides new evidence that a therapeutic dose of E4 for MHT or COC, combined with progesterone or drospirenone, may provide a better benefit/risk profile towards breast cancer risk compared to hormonal treatments currently available for patients.
RESUMEN
OBJECTIVE: Carbonyl reductase 1 (Cbr1), a recently discovered contributor to tissue glucocorticoid metabolism converting corticosterone to 20ß-dihydrocorticosterone (20ß-DHB), is upregulated in adipose tissue of obese humans and mice and may contribute to cardiometabolic complications of obesity. This study tested the hypothesis that Cbr1-mediated glucocorticoid metabolism influences glucocorticoid and mineralocorticoid receptor activation in adipose tissue and impacts glucose homeostasis in lean and obese states. METHODS: The actions of 20ß-DHB on corticosteroid receptors in adipose tissue were investigated first using a combination of in silico, in vitro, and transcriptomic techniques and then in vivo administration in combination with receptor antagonists. Mice lacking one Cbr1 allele and mice overexpressing Cbr1 in their adipose tissue underwent metabolic phenotyping before and after induction of obesity with high-fat feeding. RESULTS: 20ß-DHB activated both the glucocorticoid and mineralocorticoid receptor in adipose tissue and systemic administration to wild-type mice induced glucose intolerance, an effect that was ameliorated by both glucocorticoid and mineralocorticoid receptor antagonism. Cbr1 haploinsufficient lean male mice had lower fasting glucose and improved glucose tolerance compared with littermate controls, a difference that was abolished by administration of 20ß-DHB and absent in female mice with higher baseline adipose 20ß-DHB concentrations than male mice. Conversely, overexpression of Cbr1 in adipose tissue resulted in worsened glucose tolerance and higher fasting glucose in lean male and female mice. However, neither Cbr1 haploinsfficiency nor adipose overexpression affected glucose dyshomeostasis induced by high-fat feeding. CONCLUSIONS: Carbonyl reductase 1 is a novel regulator of glucocorticoid and mineralocorticoid receptor activation in adipose tissue that influences glucose homeostasis in lean mice.
Asunto(s)
Tejido Adiposo/metabolismo , Oxidorreductasas de Alcohol/metabolismo , Glucocorticoides/metabolismo , Intolerancia a la Glucosa/metabolismo , Obesidad/metabolismo , Receptores de Glucocorticoides/metabolismo , Transducción de Señal/genética , Oxidorreductasas de Alcohol/genética , Animales , Corticosterona/análogos & derivados , Corticosterona/sangre , Corticosterona/farmacología , Dieta Alta en Grasa/efectos adversos , Modelos Animales de Enfermedad , Femenino , Técnicas de Silenciamiento del Gen , Glucosa/metabolismo , Intolerancia a la Glucosa/genética , Células HEK293 , Homeostasis/genética , Humanos , Masculino , Ratones , Ratones Endogámicos C57BL , Ratones Transgénicos , Obesidad/genética , Receptores de Mineralocorticoides/metabolismo , Transducción de Señal/efectos de los fármacosRESUMEN
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.
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Antiinflamatorios/farmacología , Transportador de Glucosa de Tipo 4/genética , Atrofia Muscular/tratamiento farmacológico , Receptores de Glucocorticoides/química , Transducción de Señal/efectos de los fármacos , Células A549 , Regulación Alostérica , Animales , Antiinflamatorios/síntesis química , Línea Celular Transformada , Regulación de la Expresión Génica , Glucosa/metabolismo , Transportador de Glucosa de Tipo 4/metabolismo , Humanos , Lipopolisacáridos/administración & dosificación , Masculino , Potencial de la Membrana Mitocondrial/efectos de los fármacos , Ratones , Ratones Endogámicos C57BL , Mitocondrias/efectos de los fármacos , Mitocondrias/metabolismo , Fibras Musculares Esqueléticas/efectos de los fármacos , Fibras Musculares Esqueléticas/metabolismo , Fibras Musculares Esqueléticas/patología , Atrofia Muscular/inducido químicamente , Atrofia Muscular/genética , Atrofia Muscular/metabolismo , Mioblastos/efectos de los fármacos , Mioblastos/metabolismo , Ratas , Receptores de Glucocorticoides/genética , Receptores de Glucocorticoides/metabolismo , Relación Estructura-ActividadRESUMEN
Identification of chemicals that affect hormone-regulated systems will help to predict endocrine disruption. In our previous study, a 46 gene biomarker was found to be an accurate predictor of estrogen receptor (ER) α modulation in chemically treated MCF-7 cells. Here, potential ERα modulators were identified using the biomarker by screening a microarray compendium consisting of â¼1600 gene expression comparisons representing exposure to â¼1200 chemicals. A total of â¼170 chemicals were identified as potential ERα modulators. In the Connectivity Map 2.0 collection, 75 and 39 chemicals were predicted to activate or suppress ERα, and they included 12 and six known ERα agonists and antagonists/selective ERα modulators, respectively. Nineteen and eight of the total number were also identified as active in an ERα transactivation assay carried out in an MCF-7-derived cell line used to screen the Tox21 10K chemical library in agonist or antagonist modes, respectively. Chemicals predicted to modulate ERα in MCF-7 cells were examined further using global and targeted gene expression in wild-type and ERα-null cells, transactivation assays, and cell-free ERα coregulator interaction assays. Environmental chemicals classified as weak and very weak agonists were confirmed to activate ERα including apigenin, kaempferol, and oxybenzone. Novel activators included digoxin, nabumetone, ivermectin, and six progestins. Novel suppressors included emetine, mifepristone, niclosamide, and proscillaridin. Our strategy will be useful to identify environmentally relevant ERα modulators in future high-throughput transcriptomic screens.
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Biomarcadores de Tumor/genética , Moduladores de los Receptores de Estrógeno/análisis , Receptor alfa de Estrógeno/genética , Moduladores de los Receptores de Estrógeno/farmacología , Receptor alfa de Estrógeno/agonistas , Receptor alfa de Estrógeno/antagonistas & inhibidores , Femenino , Perfilación de la Expresión Génica , Humanos , Células MCF-7 , Células Tumorales CultivadasRESUMEN
A homozygous missense mutation in the gene encoding the estrogen receptor α (ERα) was previously identified in a female patient with estrogen insensitivity syndrome. We investigated the molecular features underlying the impaired transcriptional response of this mutant (ERα-Q375H) and four other missense mutations at this position designed to query alternative mechanisms. The identity of residue 375 greatly affected the sensitivity of the receptor to agonists without changing the ligand binding affinity. Instead, the mutations caused changes in the affinity of coactivator binding and alterations in the balance of coactivator and corepressor recruitment. Comparisons among the transcriptional regulatory responses of these six ERα genotypes to a set of ER agonists showed that both steric and electrostatic factors contributed to the functional deficits in gene regulatory activity of the mutant ERα proteins. ERα-coregulator peptide binding in vitro and RIME (rapid immunoprecipitation mass spectrometry of endogenous) analysis in cells showed that the degree of functional impairment paralleled changes in receptor-coregulator binding interactions. These findings uncover coupling between ligand binding and coregulator recruitment that affects the potency rather than the efficacy of the receptor response without substantially altering ligand binding affinity. This highlights a molecular mechanism for estrogen insensitivity syndrome involving mutations that perturb a bidirectional allosteric coupling between ligand binding and coregulator binding that determines receptor transcriptional output.
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Receptor alfa de Estrógeno/genética , Estrógenos/metabolismo , Mutación Missense , Coactivador 1 de Receptor Nuclear/genética , Coactivador 3 de Receptor Nuclear/genética , Sitios de Unión/genética , Resistencia a Medicamentos/genética , Receptor alfa de Estrógeno/química , Receptor alfa de Estrógeno/metabolismo , Estrógenos/farmacología , Regulación de la Expresión Génica , Células HEK293 , Células Hep G2 , Humanos , Cinética , Ligandos , Simulación de Dinámica Molecular , Coactivador 1 de Receptor Nuclear/metabolismo , Coactivador 3 de Receptor Nuclear/metabolismo , Unión Proteica , Dominios ProteicosRESUMEN
LRH-1 is a nuclear receptor that regulates lipid metabolism and homeostasis, making it an attractive target for the treatment of diabetes and non-alcoholic fatty liver disease. Building on recent structural information about ligand binding from our labs, we have designed a series of new LRH-1 agonists that further engage LRH-1 through added polar interactions. While the current synthetic approach to this scaffold has, in large part, allowed for decoration of the agonist core, significant variation of the bridgehead substituent is mechanistically precluded. We have developed a new synthetic approach to overcome this limitation, identified that bridgehead substitution is necessary for LRH-1 activation, and described an alternative class of bridgehead substituents for effective LRH-1 agonist development. We determined the crystal structure of LRH-1 bound to a bridgehead-modified compound, revealing a promising opportunity to target novel regions of the ligand binding pocket to alter LRH-1 target gene expression.
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Compuestos de Anilina/farmacología , Desarrollo de Medicamentos , Receptores Citoplasmáticos y Nucleares/agonistas , Compuestos de Anilina/síntesis química , Compuestos de Anilina/química , Relación Dosis-Respuesta a Droga , Humanos , Simulación del Acoplamiento Molecular , Estructura Molecular , Oxidación-Reducción , Procesos Fotoquímicos , Receptores Citoplasmáticos y Nucleares/genética , Relación Estructura-ActividadRESUMEN
Glucocorticoids mediate numerous essential processes in the human body via binding to the glucocorticoid receptor (GR). Excessive GR signaling can cause disease, and GR antagonists can be used to treat many symptoms of glucocorticoid-induced pathology. The purpose of this study was to characterize the tissue-specific properties of the selective GR antagonist CORT125281. We evaluated the antagonistic effects of CORT125281 upon acute and subchronic corticosterone exposure in mice. In the acute corticosterone setting, hypothalamus-pituitary-adrenal-axis activity was investigated by measurement of basal- and stress-induced corticosterone levels, adrenocorticotropic hormone levels and pituitary proopiomelanocortin expression. GR signaling was evaluated by RT-PCR analysis of GR-responsive transcripts in liver, muscle, brown adipose tissue (BAT), white adipose tissue (WAT) and hippocampus. Pretreatment with a high dose of CORT125281 antagonized GR activity in a tissue-dependent manner. We observed complete inhibition of GR-induced target gene expression in the liver, partial blockade in muscle and BAT and no antagonism in WAT and hippocampus. Tissue distribution only partially explained the lack of effective antagonism. CORT125281 treatment did not disinhibit the hypothalamus-pituitary-adrenal neuroendocrine axis. In the subchronic corticosterone setting, CORT125281 partially prevented corticosterone-induced hyperinsulinemia, but not hyperlipidemia and immune suppression. In conclusion, CORT125281 antagonizes GR transcriptional activity in a tissue-dependent manner and improves corticosterone-induced hyperinsulinemia. Tailored dosing of CORT125281 may allow tissue-specific inhibition of GR transcriptional activity.
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Sistema Hipófiso-Suprarrenal/efectos de los fármacos , Sistema Hipófiso-Suprarrenal/metabolismo , Receptores de Glucocorticoides/antagonistas & inhibidores , Receptores de Glucocorticoides/metabolismo , Tejido Adiposo Pardo/efectos de los fármacos , Tejido Adiposo Pardo/metabolismo , Tejido Adiposo Blanco/efectos de los fármacos , Tejido Adiposo Blanco/metabolismo , Animales , Corticosterona/farmacología , Hipocampo/efectos de los fármacos , Hipocampo/metabolismo , Hiperinsulinismo/inducido químicamente , Hiperinsulinismo/prevención & control , Hiperlipidemias/metabolismo , Hiperlipidemias/prevención & control , Sistema Hipotálamo-Hipofisario/efectos de los fármacos , Sistema Hipotálamo-Hipofisario/metabolismo , Hígado/efectos de los fármacos , Hígado/metabolismo , Ratones , Mifepristona/farmacología , Músculos/efectos de los fármacos , Músculos/metabolismoRESUMEN
Liver X Receptors (LXRs) are ligand dependent transcription factors activated by oxidized cholesterol metabolites (oxysterols) that play fundamental roles in the transcriptional control of lipid metabolism, cholesterol transport and modulation of inflammatory responses. In the last decade, LXRs have become attractive pharmacological targets for intervention in human metabolic diseases and thus, several efforts have concentrated on the development of synthetic analogues able to modulate LXR transcriptional response. In this sense, we have previously found that cholestenoic acid analogues with a modified side chain behave as LXR inverse agonists. To further investigate the structure-activity relationships and to explore how cholestenoic acid derivatives interact with the LXRs, we evaluated the LXR biological activity of new analogues containing a C24-C25 double bond. Furthermore, a microarray assay was performed to evaluate the recruitment of coregulators to recombinant LXR LBD upon ligand binding. Also, conventional and accelerated molecular dynamics simulations were applied to gain insight on the molecular determinants involved in the inverse agonism. As LXR inverse agonists emerge as very promising candidates to control LXR activity, the cholestenoic acid analogues here depicted constitute a new relevant steroidal scaffold to inhibit LXR action.
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Colestenos/farmacología , Colesterol/metabolismo , Receptores X del Hígado/química , Oxiesteroles/metabolismo , Colestenos/química , Colesterol/genética , Regulación de la Expresión Génica/efectos de los fármacos , Humanos , Ligandos , Metabolismo de los Lípidos , Receptores X del Hígado/genética , Receptores X del Hígado/ultraestructura , Análisis por Micromatrices , Conformación Molecular , Simulación de Dinámica Molecular , Resonancia Magnética Nuclear Biomolecular , Oxiesteroles/química , Unión Proteica/efectos de los fármacos , Conformación Proteica , Transducción de Señal/efectos de los fármacos , Relación Estructura-ActividadRESUMEN
PURPOSE: Androgen receptor (AR)-targeting prostate cancer drugs, which are predominantly competitive ligand-binding domain (LBD)-binding antagonists, are inactivated by common resistance mechanisms. It is important to develop next-generation mechanistically distinct drugs to treat castration- and drug-resistant prostate cancers. EXPERIMENTAL DESIGN: Second-generation AR pan antagonist UT-34 was selected from a library of compounds and tested in competitive AR binding and transactivation assays. UT-34 was tested using biophysical methods for binding to the AR activation function-1 (AF-1) domain. Western blot, gene expression, and proliferation assays were performed in various AR-positive enzalutamide-sensitive and -resistant prostate cancer cell lines. Pharmacokinetic and xenograft studies were performed in immunocompromised rats and mice. RESULTS: UT-34 inhibits the wild-type and LBD-mutant ARs comparably and inhibits the in vitro proliferation and in vivo growth of enzalutamide-sensitive and -resistant prostate cancer xenografts. In preclinical models, UT-34 induced the regression of enzalutamide-resistant tumors at doses when the AR is degraded; but, at lower doses, when the AR is just antagonized, it inhibits, without shrinking, the tumors. This indicates that degradation might be a prerequisite for tumor regression. Mechanistically, UT-34 promotes a conformation that is distinct from the LBD-binding competitive antagonist enzalutamide and degrades the AR through the ubiquitin proteasome mechanism. UT-34 has a broad safety margin and exhibits no cross-reactivity with G-protein-coupled receptor kinase and nuclear receptor family members. CONCLUSIONS: Collectively, UT-34 exhibits the properties necessary for a next-generation prostate cancer drug.
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Antagonistas de Receptores Androgénicos/farmacología , Antineoplásicos/farmacología , Resistencia a Antineoplásicos , Feniltiohidantoína/análogos & derivados , Neoplasias de la Próstata/metabolismo , Administración Oral , Antagonistas de Receptores Androgénicos/administración & dosificación , Antagonistas de Receptores Androgénicos/efectos adversos , Antagonistas de Receptores Androgénicos/farmacocinética , Animales , Antineoplásicos/administración & dosificación , Antineoplásicos/efectos adversos , Antineoplásicos/farmacocinética , Benzamidas , Biomarcadores de Tumor , Línea Celular Tumoral , Modelos Animales de Enfermedad , Relación Dosis-Respuesta a Droga , Resistencia a Antineoplásicos/genética , Expresión Génica , Humanos , Masculino , Ratones , Mutación , Nitrilos , Feniltiohidantoína/administración & dosificación , Feniltiohidantoína/efectos adversos , Feniltiohidantoína/farmacología , Neoplasias de la Próstata/tratamiento farmacológico , Neoplasias de la Próstata/patología , Complejo de la Endopetidasa Proteasomal/metabolismo , Unión Proteica , Proteolisis , Ratas , Receptores Androgénicos/genética , Receptores Androgénicos/metabolismo , Transducción de Señal/efectos de los fármacos , Ubiquitina/metabolismo , Ensayos Antitumor por Modelo de XenoinjertoRESUMEN
Estrogen receptor-positive (ER+) breast cancers frequently remain dependent on ER signaling even after acquiring resistance to endocrine agents, prompting the development of optimized ER antagonists. Fulvestrant is unique among approved ER therapeutics due to its capacity for full ER antagonism, thought to be achieved through ER degradation. The clinical potential of fulvestrant is limited by poor physicochemical features, spurring attempts to generate ER degraders with improved drug-like properties. We show that optimization of ER degradation does not guarantee full ER antagonism in breast cancer cells; ER "degraders" exhibit a spectrum of transcriptional activities and anti-proliferative potential. Mechanistically, we find that fulvestrant-like antagonists suppress ER transcriptional activity not by ER elimination, but by markedly slowing the intra-nuclear mobility of ER. Increased ER turnover occurs as a consequence of ER immobilization. These findings provide proof-of-concept that small molecule perturbation of transcription factor mobility may enable therapeutic targeting of this challenging target class.
Asunto(s)
Neoplasias de la Mama/metabolismo , Antagonistas del Receptor de Estrógeno/farmacología , Fulvestrant/farmacología , Receptores de Estrógenos/antagonistas & inhibidores , Receptores de Estrógenos/metabolismo , Animales , Neoplasias de la Mama/tratamiento farmacológico , Neoplasias de la Mama/patología , Proliferación Celular/efectos de los fármacos , Cinamatos/farmacología , Resistencia a Antineoplásicos , Antagonistas del Receptor de Estrógeno/uso terapéutico , Femenino , Fulvestrant/uso terapéutico , Células HEK293 , Xenoinjertos , Humanos , Indazoles/farmacología , Ligandos , Células MCF-7 , Ratones , Ratones Endogámicos NOD , Ratones Desnudos , Ratones SCID , Polimorfismo de Nucleótido Simple , Proteolisis/efectos de los fármacos , Transducción de Señal/efectos de los fármacos , Transcripción Genética/efectos de los fármacosRESUMEN
Regulatory factors that control gene transcription in multicellular organisms are assembled in multicomponent complexes by combinatorial interactions. In this context, nuclear receptors provide well-characterized and physiologically relevant systems to study ligand-induced transcription resulting from the integration of cellular and genomic information in a cell- and gene-specific manner. Here, we developed a mathematical model describing the interactions between the glucocorticoid receptor (GR) and other components of a multifactorial regulatory complex controlling the transcription of GR-target genes, such as coregulator peptides. We support the validity of the model in relation to gene-specific GR transactivation with gene transcription data from A549 cells and in vitro real time quantification of coregulator-GR interactions. The model accurately describes and helps to interpret ligand-specific and gene-specific transcriptional regulation by the GR. The comprehensive character of the model allows future insight into the function and relative contribution of the molecular species proposed in ligand- and gene-specific transcriptional regulation.
RESUMEN
Glucocorticoid hormones have important effects on brain function in the context of acute and chronic stress. Many of these are mediated by the glucocorticoid receptor (GR). GR has transcriptional activity which is highly context-specific and differs between tissues and even between cell types. The outcome of GR-mediated transcription depends on the interactome of associated coregulators. Selective GR modulators (SGRMs) are a class of GR ligands that can be used to activate only a subset of GR-coregulator interactions, thereby giving the possibility to induce a unique combination of agonistic and antagonistic GR properties. We describe SGRM action in animal models of brain function and pathology, and argue for their utility as molecular filters, to characterize context-specific GR interactome and transcriptional activity that are responsible for particular glucocorticoid-driven effects in cognitive processes such as memory consolidation. The ultimate objective of this approach is to identify molecular processes that are responsible for adaptive and maladaptive effects of glucocorticoids in the brain.
Asunto(s)
Encéfalo/metabolismo , Glucocorticoides/metabolismo , Receptores de Glucocorticoides/metabolismo , Animales , HumanosRESUMEN
Androgen deprivation therapy for prostate cancer (PCa) benefits patients with early disease, but becomes ineffective as PCa progresses to a castration-resistant state (CRPC). Initially CRPC remains dependent on androgen receptor (AR) signaling, often through increased expression of full-length AR (ARfl) or expression of dominantly active splice variants such as ARv7. We show in ARv7-dependent CRPC models that ARv7 binds together with ARfl to repress transcription of a set of growth-suppressive genes. Expression of the ARv7-repressed targets and ARv7 protein expression are negatively correlated and predicts for outcome in PCa patients. Our results provide insights into the role of ARv7 in CRPC and define a set of potential biomarkers for tumors dependent on ARv7.