RESUMEN
A scaffold hopping strategy, including intellectual property availability assessment, was successfully applied for the discovery of novel PI3K inhibitors. Compounds were designed based on the chemical structure of the lead compound ETP-46321, a potent PI3K inhibitor, previously reported by our group. The new generated compounds showed good in vitro potency and selectivity, proved to inhibit potently the phosphorylation of AKTSer473 in cells and demonstrated to be orally bioavailable, thus becoming potential back-up candidates for ETP-46321.
Asunto(s)
Inhibidores de las Quinasa Fosfoinosítidos-3 , Inhibidores de Proteínas Quinasas/metabolismo , Administración Oral , Animales , Química Farmacéutica , Evaluación Preclínica de Medicamentos , Semivida , Imidazoles/química , Imidazoles/metabolismo , Concentración 50 Inhibidora , Ratones , Ratones Endogámicos BALB C , Fosfatidilinositol 3-Quinasas/metabolismo , Fosforilación , Isoformas de Proteínas/antagonistas & inhibidores , Isoformas de Proteínas/metabolismo , Inhibidores de Proteínas Quinasas/química , Inhibidores de Proteínas Quinasas/farmacocinética , Proteínas Proto-Oncogénicas c-akt/metabolismo , Pirazinas/química , Pirazinas/metabolismo , Serina-Treonina Quinasas TOR/antagonistas & inhibidores , Serina-Treonina Quinasas TOR/metabolismoRESUMEN
BACKGROUND: The CCAAT/enhancer-binding protein ß (C/EBPß) is a transcription factor implicated in the control of proliferation, differentiation, and inflammatory processes mainly in adipose tissue and liver; although more recent results have revealed an important role for this transcription factor in the brain. Previous studies from our laboratory indicated that CCAAT/enhancer-binding protein ß is implicated in inflammatory process and brain injury, since mice lacking this gene were less susceptible to kainic acid-induced injury. More recently, we have shown that the complement component 3 gene (C3) is a downstream target of CCAAT/enhancer-binding protein ß and it could be a mediator of the proinflammatory effects of this transcription factor in neural cells. METHODS: Adult male Wistar rats (8-12 weeks old) were used throughout the study. C/EBPß+/+ and C/EBPß-/- mice were generated from heterozygous breeding pairs. Animals were injected or not with kainic acid, brains removed, and brain slices containing the hippocampus analyzed for the expression of both CCAAT/enhancer-binding protein ß and C3. RESULTS: In the present work, we have further extended these studies and show that CCAAT/enhancer-binding protein ß and C3 co-express in the CA1 and CA3 regions of the hippocampus after an excitotoxic injury. Studies using CCAAT/enhancer-binding protein ß knockout mice demonstrate a marked reduction in C3 expression after kainic acid injection in these animals, suggesting that indeed this protein is regulated by C/EBPß in the hippocampus in vivo. CONCLUSIONS: Altogether these results suggest that CCAAT/enhancer-binding protein ß could regulate brain disorders, in which excitotoxic and inflammatory processes are involved, at least in part through the direct regulation of C3.
Asunto(s)
Proteína beta Potenciadora de Unión a CCAAT/metabolismo , Complemento C3/genética , Agonistas de Aminoácidos Excitadores/toxicidad , Regulación de la Expresión Génica/efectos de los fármacos , Hipocampo , Ácido Kaínico/toxicidad , Degeneración Nerviosa/inducido químicamente , Animales , Proteína beta Potenciadora de Unión a CCAAT/genética , Antígeno CD11b/metabolismo , Complemento C3/metabolismo , Modelos Animales de Enfermedad , Fluoresceínas/metabolismo , Regulación de la Expresión Génica/genética , Proteína Ácida Fibrilar de la Glía/metabolismo , Hipocampo/efectos de los fármacos , Hipocampo/metabolismo , Hipocampo/patología , Interleucina-1beta/metabolismo , Masculino , Ratones , Ratones Transgénicos , Degeneración Nerviosa/patología , Neuroglía/metabolismo , Neuroglía/patología , ARN Mensajero/metabolismo , Ratas , Ratas WistarRESUMEN
BACKGROUND: The CCAAT/enhancer-binding protein ß (C/EBPß) is a transcription factor, which was first identified as a regulator of differentiation and inflammatory processes mainly in adipose tissue and liver; however, its function in the brain was largely unknown for many years. Previous studies from our laboratory indicated that C/EBPß is implicated in inflammatory process and brain injury, since mice lacking this gene were less susceptible to kainic acid-induced injury. METHODS: We first performed cDNA microarrays analysis using hippocampal RNA isolated from C/EBPß (+/+) and C/EBPß (-/-) mice. Immunocytochemical and immunohistochemical studies were done to evaluate C/EBPß and C3 levels. Transient transfection experiments were made to analyze transcriptional regulation of C3 by C/EBPß. To knockdown C/EBPß and C3 expression, mouse astrocytes were infected with lentiviral particles expressing an shRNA specific for C/EBPß or an siRNA specific for C3. RESULTS: Among the genes displaying significant changes in expression was complement component 3 (C3), which showed a dramatic decrease in mRNA content in the hippocampus of C/EBPß (-/-) mice. C3 is the central component of the complement and is implicated in different brain disorders. In this work we have found that C/EBPß regulates C3 levels in rodents glial in vitro and in the rat Substantia nigra pars compacta (SNpc) in vivo following an inflammatory insult. Analysis of the mouse C3 promoter showed that it is directly regulated by C/EBPß through a C/EBPß consensus site located at position -616/-599 of the gene. In addition, we show that depletion of C/EBPß by a specific shRNA results in a significant decrease in the levels of C3 together with a reduction in the increased levels of pro-inflammatory agents elicited by lipopolysaccharide treatment. CONCLUSIONS: Altogether, these results indicate that C3 is a downstream target of C/EBPß, and it could be a mediator of the pro-inflammatory effects of this transcription factor in neural cells.
Asunto(s)
Proteína beta Potenciadora de Unión a CCAAT/metabolismo , Complemento C3/genética , Regulación de la Expresión Génica/genética , Neuronas/metabolismo , Animales , Animales Recién Nacidos , Proteína beta Potenciadora de Unión a CCAAT/genética , Línea Celular Tumoral , Células Cultivadas , Complemento C3/metabolismo , Ciclooxigenasa 2/metabolismo , Regulación de la Expresión Génica/efectos de los fármacos , Hipocampo/citología , Interleucina-1beta/metabolismo , Masculino , Ratones , Ratones Transgénicos , Análisis por Micromatrices , Neuroblastoma , Neuroglía/efectos de los fármacos , Neuroglía/metabolismo , Fragmentos de Péptidos/metabolismo , Polisacáridos/farmacología , Ratas , Ratas WistarRESUMEN
We report a medium-throughput drug-screening platform (METPlatform) based on organotypic cultures that allows to evaluate inhibitors against metastases growing in situ. By applying this approach to the unmet clinical need of brain metastasis, we identified several vulnerabilities. Among them, a blood-brain barrier permeable HSP90 inhibitor showed high potency against mouse and human brain metastases at clinically relevant stages of the disease, including a novel model of local relapse after neurosurgery. Furthermore, in situ proteomic analysis applied to metastases treated with the chaperone inhibitor uncovered a novel molecular program in brain metastasis, which includes biomarkers of poor prognosis and actionable mechanisms of resistance. Our work validates METPlatform as a potent resource for metastasis research integrating drug-screening and unbiased omic approaches that is compatible with human samples. Thus, this clinically relevant strategy is aimed to personalize the management of metastatic disease in the brain and elsewhere.
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Antineoplásicos , Neoplasias Encefálicas , Animales , Antineoplásicos/farmacología , Antineoplásicos/uso terapéutico , Barrera Hematoencefálica , Neoplasias Encefálicas/tratamiento farmacológico , Ratones , Recurrencia Local de Neoplasia , ProteómicaRESUMEN
Telomerase deficiency leads to age-related diseases and shorter lifespans. Inhibition of the mechanistic target of rapamycin (mTOR) delays aging and age-related pathologies. Here, we show that telomerase deficient mice with short telomeres (G2-Terc-/-) have an hyper-activated mTOR pathway with increased levels of phosphorylated ribosomal S6 protein in liver, skeletal muscle and heart, a target of mTORC1. Transcriptional profiling confirms mTOR activation in G2-Terc-/- livers. Treatment of G2-Terc-/- mice with rapamycin, an inhibitor of mTORC1, decreases survival, in contrast to lifespan extension in wild-type controls. Deletion of mTORC1 downstream S6 kinase 1 in G3-Terc-/- mice also decreases longevity, in contrast to lifespan extension in single S6K1-/- female mice. These findings demonstrate that mTOR is important for survival in the context of short telomeres, and that its inhibition is deleterious in this setting. These results are of clinical interest in the case of human syndromes characterized by critically short telomeres.
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Envejecimiento/genética , ARN/genética , Serina-Treonina Quinasas TOR/metabolismo , Telomerasa/genética , Telómero/genética , Envejecimiento/efectos de los fármacos , Animales , Daño del ADN/efectos de los fármacos , Femenino , Longevidad/efectos de los fármacos , Longevidad/genética , Masculino , Ratones Endogámicos C57BL , Ratones Noqueados , Neoplasias/genética , Fosforilación , Proteínas Quinasas S6 Ribosómicas 90-kDa/genética , Sirolimus/farmacología , Tasa de Supervivencia , Serina-Treonina Quinasas TOR/genética , Telómero/efectos de los fármacos , Telómero/metabolismoRESUMEN
Despite significant efforts to improve pancreatic ductal adenocarcinoma (PDAC) clinical outcomes, overall survival remains dismal. The poor response to current therapies is partly due to the existence of pancreatic cancer stem cells (PaCSCs), which are efficient drivers of PDAC tumorigenesis, metastasis and relapse. To find new therapeutic agents that could efficiently kill PaCSCs, we screened a chemical library of 680 compounds for candidate small molecules with anti-CSC activity, and identified two compounds of a specific chemical series with potent activity in vitro and in vivo against patient-derived xenograft (PDX) cultures. The anti-CSC mechanism of action of this specific chemical series was found to rely on induction of lysosomal membrane permeabilization (LMP), which is likely associated with the increased lysosomal mass observed in PaCSCs. Using the well characterized LMP-inducer siramesine as a tool molecule, we show elimination of the PaCSC population in mice implanted with tumors from two PDX models. Collectively, our approach identified lysosomal disruption as a promising anti-CSC therapeutic strategy for PDAC.
RESUMEN
Palbociclib is a selective inhibitor of cyclin-dependent kinases 4 and 6 (CDK4/6) approved for the treatment of some cancers. The main mechanism of action of palbociclib is to induce cell cycle arrest and senescence on responsive cells. Here, we report that palbociclib concentrates in intracellular acidic vesicles, where it can be readily observed due to its intrinsic fluorescence, and it is released from these vesicles upon dilution or washing out of the extracellular medium. This reversible storage of drugs into acidic vesicles is generally known as lysosomal trapping and, based on this, we uncover novel properties of palbociclib. In particular, a short exposure of cells to palbociclib is sufficient to produce a stable cell-cycle arrest and long-term senescence. Moreover, after washing out the drug, palbociclib-treated cells release the drug to the medium and this conditioned medium is active on susceptible cells. Interestingly, cancer cells resistant to palbociclib also accumulate and release the drug producing paracrine senescence on susceptible cells. Finally, other lysosomotropic drugs, such as chloroquine, interfere with the accumulation of palbociclib into lysosomes, thereby reducing the minimal dose of palbociclib required for cell-cycle arrest and senescence. In summary, lysosomal trapping explains the prolonged temporal activity of palbociclib, the paracrine activity of exposed cells, and the cooperation with lysosomotropic drugs. These are important features that may help to improve the therapeutic dosing and efficacy of palbociclib. Finally, two other clinically approved CDK4/6 inhibitors, ribociclib and abemaciclib, present a similar behavior as palbociclib, suggesting that lysosomal trapping is a property common to all three clinically-approved CDK4/6 inhibitors.
Asunto(s)
Antineoplásicos/farmacocinética , Lisosomas/efectos de los fármacos , Lisosomas/metabolismo , Piperazinas/farmacocinética , Piridinas/farmacocinética , Naranja de Acridina/química , Aminopiridinas/farmacocinética , Bencimidazoles/farmacocinética , Línea Celular Tumoral , Senescencia Celular/efectos de los fármacos , Cloroquina/farmacología , Quinasa 4 Dependiente de la Ciclina/antagonistas & inhibidores , Citocinas/metabolismo , Colorantes Fluorescentes/química , Humanos , Proteínas de Membrana de los Lisosomas/metabolismo , Inhibidores de Proteínas Quinasas/farmacocinética , Purinas/farmacocinéticaRESUMEN
In the version of this article originally published, the names of three authors were incorrect. The authors were listed as "Coral Fustero-Torres", "Elena Pineiro" and "Melchor Sánchez-Martínez". Their respective names are "Coral Fustero-Torre", "Elena Piñeiro-Yáñez" and "Melchor Sanchez-Martinez". The errors have been corrected in the print, HTML and PDF versions of this article.
RESUMEN
The brain microenvironment imposes a particularly intense selective pressure on metastasis-initiating cells, but successful metastases bypass this control through mechanisms that are poorly understood. Reactive astrocytes are key components of this microenvironment that confine brain metastasis without infiltrating the lesion. Here, we describe that brain metastatic cells induce and maintain the co-option of a pro-metastatic program driven by signal transducer and activator of transcription 3 (STAT3) in a subpopulation of reactive astrocytes surrounding metastatic lesions. These reactive astrocytes benefit metastatic cells by their modulatory effect on the innate and acquired immune system. In patients, active STAT3 in reactive astrocytes correlates with reduced survival from diagnosis of intracranial metastases. Blocking STAT3 signaling in reactive astrocytes reduces experimental brain metastasis from different primary tumor sources, even at advanced stages of colonization. We also show that a safe and orally bioavailable treatment that inhibits STAT3 exhibits significant antitumor effects in patients with advanced systemic disease that included brain metastasis. Responses to this therapy were notable in the central nervous system, where several complete responses were achieved. Given that brain metastasis causes substantial morbidity and mortality, our results identify a novel treatment for increasing survival in patients with secondary brain tumors.
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Astrocitos/patología , Neoplasias Encefálicas/secundario , Factor de Transcripción STAT3/metabolismo , Animales , Encéfalo/patología , Neoplasias Encefálicas/patología , Supervivencia Celular , Marcación de Gen , Proteína Ácida Fibrilar de la Glía/metabolismo , Humanos , Inmunidad Innata , Ratones , Fosforilación , Microambiente TumoralRESUMEN
The CCAAT/Enhancer binding protein ß (C/EBPß) is a transcription factor involved in numerous physiological as well as pathological conditions in the brain. However, little is known regarding its possible role in neurodegenerative disorders. We have previously shown that C/EBPß regulates the expression of genes involved in inflammatory processes and brain injury. Here, we have analyzed the effects of C/EBPß interference in dopaminergic cell death and glial activation in the 6-hydroxydopamine model of Parkinson's disease. Our results showed that lentivirus-mediated C/EBPß deprivation conferred marked in vitro and in vivo neuroprotection of dopaminergic cells concomitant with a significant attenuation of the level of the inflammatory response and glial activation. Additionally, C/EBPß interference diminished the induction of α-synuclein in the substantia nigra pars compacta of animals injected with 6-hydroxydopamine. Taking together, these results reveal an essential function for C/EBPß in the pathways leading to inflammatory-mediated brain damage and suggest novel roles for C/EBPß in neurodegenerative diseases, specifically in Parkinson's disease, opening the door for new therapeutic interventions.
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Proteína beta Potenciadora de Unión a CCAAT/metabolismo , Enfermedad de Parkinson/patología , Animales , Apoptosis/efectos de los fármacos , Proteína beta Potenciadora de Unión a CCAAT/antagonistas & inhibidores , Proteína beta Potenciadora de Unión a CCAAT/genética , Células Cultivadas , Modelos Animales de Enfermedad , Neuronas Dopaminérgicas/citología , Neuronas Dopaminérgicas/efectos de los fármacos , Neuronas Dopaminérgicas/metabolismo , Humanos , Masculino , Mesencéfalo/citología , Mesencéfalo/efectos de los fármacos , Mesencéfalo/metabolismo , Oxidopamina/farmacología , Enfermedad de Parkinson/metabolismo , Porción Compacta de la Sustancia Negra/efectos de los fármacos , Porción Compacta de la Sustancia Negra/metabolismo , Interferencia de ARN , ARN Interferente Pequeño/metabolismo , Ratas , Ratas Wistar , alfa-Sinucleína/metabolismoRESUMEN
Different studies have suggested that the nucleotide cyclic adenosine 3', 5'-monophosphate can actively play an important role as a neuroprotective and anti-inflammatory agent after a brain injury. The phosphodiesterase 7 (PDE7) enzyme is one of the enzymes responsible for controlling specifically the intracellular levels of cyclic adenosine 3', 5'-monophosphate in the immune and central nervous systems. Therefore, this enzyme could play an important role in brain inflammation and neurodegeneration. In this regard, using different chemical inhibitors of PDE7 we have demonstrated their neuroprotective and anti-inflammatory activity in different models of neurodegenerative disorders, including Parkinson's disease (PD). In the present study, we have used the toxin 6-hydroxydopamine and lipopolysaccharide to model PD and explore the protective effects of PDE7B deficiency in dopaminergic neurons cell death. Lentivirus-mediated PDE7B deprivation conferred marked in vitro and in vivo neuroprotection against 6-hydroxydopamine and lipopolysaccharide toxicity in dopaminergic neurons and preserved motor function involving the dopamine system in mouse. Our results substantiate previous data and provide a validation of PDE7B enzyme as a valuable new target for therapeutic development in the treatment of PD.