RESUMEN
Insufficient telomerase activity, stemming from low telomerase reverse transcriptase (TERT) gene transcription, contributes to telomere dysfunction and aging pathologies. Besides its traditional function in telomere synthesis, TERT acts as a transcriptional co-regulator of genes pivotal in aging and age-associated diseases. Here, we report the identification of a TERT activator compound (TAC) that upregulates TERT transcription via the MEK/ERK/AP-1 cascade. In primary human cells and naturally aged mice, TAC-induced elevation of TERT levels promotes telomere synthesis, blunts tissue aging hallmarks with reduced cellular senescence and inflammatory cytokines, and silences p16INK4a expression via upregulation of DNMT3B-mediated promoter hypermethylation. In the brain, TAC alleviates neuroinflammation, increases neurotrophic factors, stimulates adult neurogenesis, and preserves cognitive function without evident toxicity, including cancer risk. Together, these findings underscore TERT's critical role in aging processes and provide preclinical proof of concept for physiological TERT activation as a strategy to mitigate multiple aging hallmarks and associated pathologies.
Asunto(s)
Envejecimiento , Metilación de ADN , Telomerasa , Telomerasa/metabolismo , Telomerasa/genética , Humanos , Animales , Ratones , ADN (Citosina-5-)-Metiltransferasas/metabolismo , ADN (Citosina-5-)-Metiltransferasas/genética , Senescencia Celular , Regiones Promotoras Genéticas , ADN Metiltransferasa 3B , Encéfalo/metabolismo , Telómero/metabolismo , Ratones Endogámicos C57BL , Masculino , Factor de Transcripción AP-1/metabolismo , Inhibidor p16 de la Quinasa Dependiente de Ciclina/metabolismo , Inhibidor p16 de la Quinasa Dependiente de Ciclina/genética , NeurogénesisRESUMEN
KEAP1 (Kelch-like ECH-associated protein), a cytoplasmic repressor of the oxidative stress responsive transcription factor Nuclear factor erythroid 2-related factor 2 (NRF2), senses the presence of electrophilic agents by modification of its sensor cysteine residues. In addition to xenobiotics, several reactive metabolites have been shown to covalently modify key cysteines on KEAP1, although the full repertoire of these molecules and their respective modifications remain undefined. Here, we report the discovery of sAKZ692, a small molecule identified by high-throughput screening that stimulates NRF2 transcriptional activity in cells by inhibiting the glycolytic enzyme pyruvate kinase. sAKZ692 treatment promotes the buildup of glyceraldehyde 3-phosphate, a metabolite which leads to S-lactate modification of cysteine sensor residues of KEAP1, resulting in NRF2-dependent transcription. This work identifies a posttranslational modification of cysteine derived from a reactive central carbon metabolite and helps further define the complex relationship between metabolism and the oxidative stress-sensing machinery of the cell.
Asunto(s)
Cisteína , Factor 2 Relacionado con NF-E2 , Proteína 1 Asociada A ECH Tipo Kelch/química , Factor 2 Relacionado con NF-E2/metabolismo , Cisteína/metabolismo , Transducción de Señal , Estrés OxidativoRESUMEN
The transcriptional coactivator Yes-associated protein 1 (YAP) orchestrates a proproliferative transcriptional program that controls the fate of somatic stem cells and the regenerative responses of certain tissues. As such, agents that activate YAP may hold therapeutic potential in disease states exacerbated by insufficient proliferative repair. Here we report the discovery of a small molecule, termed PY-60, which robustly activates YAP transcriptional activity in vitro and promotes YAP-dependent expansion of epidermal keratinocytes in mouse following topical drug administration. Chemical proteomics revealed the relevant target of PY-60 to be annexin A2 (ANXA2), a protein that directly associates with YAP at the cell membrane in response to increased cell density. PY-60 treatment liberates ANXA2 from the membrane, ultimately promoting a phosphatase-bound, nonphosphorylated and transcriptionally active form of YAP. This work reveals ANXA2 as a previously undescribed, druggable component of the Hippo pathway and suggests a mechanistic rationale to promote regenerative repair in disease.
Asunto(s)
Proteínas Adaptadoras Transductoras de Señales/metabolismo , Anexina A2/antagonistas & inhibidores , Bibliotecas de Moléculas Pequeñas/farmacología , Factores de Transcripción/metabolismo , Administración Tópica , Células Madre Adultas/efectos de los fármacos , Células Madre Adultas/metabolismo , Animales , Anexina A2/metabolismo , Proliferación Celular/efectos de los fármacos , Relación Dosis-Respuesta a Droga , Ratones , Estructura Molecular , Bibliotecas de Moléculas Pequeñas/administración & dosificación , Bibliotecas de Moléculas Pequeñas/química , Proteínas Señalizadoras YAPRESUMEN
Activating NRF2-driven transcription with non-electrophilic small molecules represents an attractive strategy to therapeutically target disease states associated with oxidative stress and inflammation. In this study, we describe a campaign to optimize the potency and efficacy of a previously identified bis-sulfone based non-electrophilic ARE activator 2. This work identifies the efficacious analog 17, a compound with a non-cytotoxic profile in IMR32 cells, as well as ARE activators 18 and 22, analogs with improved cellular potency. In silico drug-likeness prediction suggested the optimized bis-sulfones 17, 18, and 22 will likely be of pharmacological utility.
Asunto(s)
Elementos de Respuesta Antioxidante , Antioxidantes , Antioxidantes/farmacología , Factor 2 Relacionado con NF-E2/metabolismo , Estrés OxidativoRESUMEN
The transcription factor NRF2 controls resistance to oxidative insult and is thus a key therapeutic target for treating a number of disease states associated with oxidative stress and aging. We previously reported CBR-470-1, a bis-sulfone which activates NRF2 by increasing the levels of methylglyoxal, a metabolite that covalently modifies NRF2 repressor KEAP1. Here, we report the design, synthesis, and structure activity relationship of a series of bis-sulfones derived from this unexplored chemical template. We identify analogs with sub-micromolar potencies, 7f and 7g, as well as establish that efficacious NRF2 activation can be achieved by non-toxic analogs 7c, 7e, and 9, a key limitation with CBR-470-1. Further efforts to identify non-covalent NRF2 activators of this kind will likely provide new insight into revealing the role of central metabolism in cellular signaling.
Asunto(s)
Antioxidantes/farmacología , Descubrimiento de Drogas , Tiofenos/farmacología , Antioxidantes/síntesis química , Antioxidantes/química , Supervivencia Celular/efectos de los fármacos , Relación Dosis-Respuesta a Droga , Humanos , Estructura Molecular , Relación Estructura-Actividad , Tiofenos/síntesis química , Tiofenos/químicaRESUMEN
The emerging use of covalent ligands as chemical probes and drugs would benefit from an expanded repertoire of cysteine-reactive electrophiles for efficient and diverse targeting of the proteome. Here we use the endogenous electrophile sensor of mammalian cells, the KEAP1-NRF2 pathway, to discover cysteine-reactive electrophilic fragments from a reporter-based screen for NRF2 activation. This strategy identified a series of 2-sulfonylpyridines that selectively react with biological thiols via nucleophilic aromatic substitution (SNAr). By tuning the electrophilicity and appended recognition elements, we demonstrate the potential of the 2-sulfonylpyridine reactive group with the discovery of a selective covalent modifier of adenosine deaminase (ADA). Targeting a cysteine distal to the active site, this molecule attenuates the enzymatic activity of ADA and inhibits proliferation of lymphocytic cells. This study introduces a modular and tunable SNAr-based reactive group for targeting reactive cysteines in the human proteome and illustrates the pharmacological utility of this electrophilic series.
Asunto(s)
Cisteína/química , Piridinas/química , Dióxido de Azufre/química , Línea Celular Tumoral , Teoría Funcional de la Densidad , Humanos , Estructura MolecularRESUMEN
The transcription factor nuclear factor erythroid 2-related factor 1 (NRF1) maintains proteostasis and promotes cellular resilience by stimulating the transcription of proteasomal subunits and a host of protective enzymes. Although NRF1 activation would likely be beneficial in a number of disease states, information regarding its ligandability and upstream regulation are lacking. Herein we report a high-throughput chemical screen that identified selective stimulators of NRF1-driven transcription, including unannotated inhibitors of the ubiquitin proteasome system (UPS) as well as two non-UPS-targeted compounds that synergistically activate NRF1 in the context of submaximal UPS inhibition. This work introduces a suite of tool molecules to study the NRF1 transcriptional response and to uncover the druggable components governing NRF1 activity in cells.
Asunto(s)
Factor Nuclear 1 de Respiración/metabolismo , Bibliotecas de Moléculas Pequeñas/farmacología , Activación Transcripcional/efectos de los fármacos , Supervivencia Celular/efectos de los fármacos , Células Hep G2 , Ensayos Analíticos de Alto Rendimiento , Humanos , Leupeptinas/farmacología , Factor Nuclear 1 de Respiración/agonistas , Factor Nuclear 1 de Respiración/genética , Complejo de la Endopetidasa Proteasomal/química , Complejo de la Endopetidasa Proteasomal/metabolismo , Bibliotecas de Moléculas Pequeñas/química , Ubiquitina/antagonistas & inhibidores , Ubiquitina/metabolismoRESUMEN
Recent studies show that histone deacetylase 6 (HDAC6) has important roles in the human brain, especially in the context of a number of nervous system disorders. Animal models of neurodevelopmental, neurodegenerative, and neuropsychiatric disorders show that HDAC6 modulates important biological processes relevant to disease biology. Pan-selective histone deacetylase (HDAC) inhibitors had been studied in animal behavioral assays and shown to induce synaptogenesis in rodent neuronal cultures. While most studies of HDACs in the nervous system have focused on class I HDACs located in the nucleus (e.g., HDACs 1,2,3), recent findings in rodent models suggest that the cytoplasmic class IIb HDAC, HDAC6, plays an important role in regulating mood-related behaviors. Human studies suggest a significant role for synaptic dysfunction in the prefrontal cortex (PFC) and hippocampus in depression. Studies of HDAC inhibitors (HDACi) in human neuronal cells show that HDAC6 inhibitors (HDAC6i) increase the acetylation of specific lysine residues in proteins involved in synaptogenesis. This has led to the hypothesis that HDAC6i may modulate synaptic biology not through effects on the acetylation of histones, but by regulating acetylation of non-histone proteins.
Asunto(s)
Histona Desacetilasa 6/metabolismo , Neuronas/citología , Diferenciación Celular/efectos de los fármacos , Hipocampo/citología , Hipocampo/metabolismo , Histona Desacetilasa 6/antagonistas & inhibidores , Inhibidores de Histona Desacetilasas/farmacología , Humanos , Neuronas/efectos de los fármacos , Transducción de Señal/efectos de los fármacosRESUMEN
We undertook an unbiased metabolite profiling of fibroblasts from schizophrenia patients and healthy controls to identify metabolites and pathways that are dysregulated in disease, seeking to gain new insights into the disease biology of schizophrenia and to discover potential disease-related biomarkers. We measured polar and nonpolar metabolites in the fibroblasts under normal conditions and under two stressful physiological perturbations: growth in low-glucose media and exposure to the steroid hormone dexamethasone. We found that metabolites that were significantly different between schizophrenia and control subjects showed separation of the two groups by partial least-squares discriminant analysis methods. This separation between schizophrenia and healthy controls was more robust with metabolites identified under the perturbation conditions. The most significant individual metabolite differences were also found in the perturbation experiments. Metabolites that were significantly different between schizophrenia and healthy controls included a number of plasmalogens and phosphatidylcholines. We present these results in the context of previous reports of metabolic profiling of brain tissue and plasma in schizophrenia. These results show the applicability of metabolite profiling under stressful perturbations to reveal cellular pathways that may be involved in disease biology.
Asunto(s)
Fibroblastos/metabolismo , Metaboloma , Fosfatidilcolinas/metabolismo , Plasmalógenos/metabolismo , Esquizofrenia/metabolismo , Estrés Fisiológico , Adulto , Antipsicóticos/uso terapéutico , Biomarcadores/metabolismo , Estudios de Casos y Controles , Medios de Cultivo/farmacología , Dexametasona/farmacología , Análisis Discriminante , Femenino , Fibroblastos/efectos de los fármacos , Fibroblastos/patología , Glucocorticoides/farmacología , Glucosa/deficiencia , Glucosa/farmacología , Humanos , Análisis de los Mínimos Cuadrados , Masculino , Persona de Mediana Edad , Cultivo Primario de Células , Esquizofrenia/tratamiento farmacológico , Esquizofrenia/patologíaRESUMEN
Schizophrenia and bipolar disorder are complex psychiatric disorders that present unique challenges in the study of disease biology. There are no objective biological phenotypes for these disorders, which are characterized by complex genetics and prominent roles for gene-environment interactions. The study of the neurobiology underlying these severe psychiatric disorders has been hindered by the lack of access to the tissue of interest - neurons from patients. The advent of reprogramming methods that enable generation of induced pluripotent stem cells (iPSCs) from patient fibroblasts and peripheral blood mononuclear cells has opened possibilities for new approaches to study relevant disease biology using iPSC-derived neurons. While early studies with patient iPSCs have led to promising and intriguing leads, significant hurdles remain in our attempts to capture the complexity of these disorders in vitro. We present here an overview of studies to date of schizophrenia and bipolar disorder using iPSC-derived neuronal cells and discuss potential future directions that can result in the identification of robust and valid cellular phenotypes that in turn can lay the groundwork for meaningful clinical advances.
Asunto(s)
Trastorno Bipolar/patología , Técnicas de Cultivo de Célula/métodos , Células Madre Pluripotentes Inducidas/citología , Esquizofrenia/patología , Trastorno Bipolar/genética , Trastorno Bipolar/metabolismo , Humanos , Células Madre Pluripotentes Inducidas/metabolismo , Modelos Biológicos , Fenotipo , Medicina de Precisión/métodos , Esquizofrenia/genética , Esquizofrenia/metabolismoRESUMEN
The AKT family of serine-threonine kinases functions downstream of phosphatidylinositol 3-kinase (PI3K) to transmit signals by direct phosphorylation of a number of targets, including the mammalian target of rapamycin (mTOR), glycogen synthase kinase 3ß (GSK3ß), and ß-catenin. AKT binds to phosphatidylinositol (3,4,5)-triphosphate (PIP3) generated by PI3K activation, which results in its membrane localization and subsequent activation through phosphorylation by phosphoinositide-dependent protein kinase 1 (PDK1). Together, the PI3K-AKT signaling pathway plays pivotal roles in many cellular systems, including in the central nervous system where it governs both neurodevelopment and neuroplasticity. Recently, lysine residues (Lys14 and Lys20) on AKT, located within its pleckstrin homology (PH) domain that binds to membrane-bound PIP3, have been found to be acetylated under certain cellular contexts in various cancer cell lines. These acetylation modifications are removed by the enzymatic action of the class III lysine deacetylases, SIRT1 and SIRT2, of the sirtuin family. The extent to which reversible acetylation regulates AKT function in other cell types remains poorly understood. We report here that AKT kinase activity is modulated by a class IIb lysine deacetylase, histone deacetylase 6 (HDAC6), in human neural progenitor cells (NPCs). We find that HDAC6 and AKT physically interact with each other in the neuronal cells, and in the presence of selective HDAC6 inhibition, AKT is acetylated at Lys163 and Lys377 located in the kinase domain, two novel sites distinct from the acetylation sites in the PH-domain modulated by the sirtuins. Measurement of the functional effect of HDAC6 inhibition on AKT revealed decreased binding to PIP3, a correlated decrease in AKT kinase activity, decreased phosphorylation of Ser552 on ß-catenin, and modulation of neuronal differentiation trajectories. Taken together, our studies implicate the deacetylase activity of HDAC6 as a novel regulator of AKT signaling and point to novel mechanisms for regulating AKT activity with small-molecule inhibitors of HDAC6 currently under clinical development.
Asunto(s)
Histona Desacetilasa 6/química , Histona Desacetilasa 6/metabolismo , Lisina/metabolismo , Células-Madre Neurales/enzimología , Proteínas Proto-Oncogénicas c-akt/metabolismo , Acetilación , Animales , Diferenciación Celular , Activación Enzimática , Humanos , Lisina/química , Ratones , Estructura Molecular , Células-Madre Neurales/citología , Isoformas de Proteínas/química , Isoformas de Proteínas/metabolismoRESUMEN
Many studies suggest the presence of aberrations in cellular metabolism in bipolar disorder. We studied the metabolome in bipolar disorder to gain insight into cellular pathways that may be dysregulated in bipolar disorder and to discover evidence of novel biomarkers. We measured polar and nonpolar metabolites in fibroblasts from subjects with bipolar I disorder and matched healthy control subjects, under normal conditions and with two physiologic perturbations: low-glucose media and exposure to the stress-mediating hormone dexamethasone. Metabolites that were significantly different between bipolar and control subjects showed distinct separation by principal components analysis methods. The most statistically significant findings were observed in the perturbation experiments. The metabolite with the lowest p value in both the low-glucose and dexamethasone experiments was α-aminoadipate, whose intracellular level was consistently lower in bipolar subjects. Our study implicates α-aminoadipate as a possible biomarker in bipolar disorder that manifests under cellular stress. This is an intriguing finding given the known role of α-aminoadipate in the modulation of kynurenic acid in the brain, especially as abnormal kynurenic acid levels have been implicated in bipolar disorder.
RESUMEN
We examined the effects of isoform-specific histone deacetylase (HDAC) inhibitors on ß-catenin posttranslational modifications in neural progenitor cells (NPCs) derived from human induced pluripotent stem cells (iPSCs). ß-catenin is a multifunctional protein with important roles in the developing and adult central nervous system. Activation of the Wnt pathway results in stabilization and nuclear translocation of ß-catenin, resulting in activation of multiple target genes. In addition, ß-catenin forms a complex with cadherins at the plasma membrane as part of the adherens junctions. The N-terminus of ß-catenin has phosphorylation, ubiquitination, and acetylation sites that regulate its stability and signaling. In the absence of a Wnt signal, Ser33, Ser37, and Thr41 are constitutively phosphorylated by glycogen synthase kinase 3ß (GSK3ß). ß-Catenin phosphorylated at these sites is recognized by ß-transducin repeat-containing protein (ßTrCP), which results in ubiquitination and degradation by the ubiquitin-proteasome pathway. The N-terminal regulatory domain of ß-catenin also includes Ser45, a phosphorylation site for Casein Kinase 1α (CK1α) and Lys49, which is acetylated by the acetyltransferase p300/CBP-associated factor (PCAF). The relevance of Lys49 acetylation and Ser45 phosphorylation to the function of ß-catenin is an active area of investigation. We find that HDAC6 inhibitors increase Lys49 acetylation and Ser45 phosphorylation but do not affect Ser33, Ser37, and Thr41 phosphorylation. Lys49 acetylation results in decreased ubiquitination of ß-catenin in the presence of proteasome inhibition. While increased Lys49 acetylation does not affect total levels of ß-catenin, it results in increased membrane localization of ß-catenin.
Asunto(s)
Membrana Celular/efectos de los fármacos , Inhibidores de Histona Desacetilasas/farmacología , Histona Desacetilasas/metabolismo , Neuronas/efectos de los fármacos , Procesamiento Proteico-Postraduccional , beta Catenina/metabolismo , Acetilación , Sitios de Unión , Diferenciación Celular , Membrana Celular/metabolismo , Membrana Celular/ultraestructura , Células Cultivadas , Expresión Génica , Inhibidores de Histona Desacetilasas/química , Histona Desacetilasas/genética , Humanos , Células Madre Pluripotentes Inducidas/citología , Células Madre Pluripotentes Inducidas/metabolismo , Isoenzimas/genética , Isoenzimas/metabolismo , Cinética , Lisina/química , Lisina/metabolismo , Modelos Moleculares , Neuronas/metabolismo , Neuronas/ultraestructura , Fosforilación , Complejo de la Endopetidasa Proteasomal/efectos de los fármacos , Complejo de la Endopetidasa Proteasomal/metabolismo , Estructura Secundaria de Proteína , Estructura Terciaria de Proteína , Transducción de Señal , Relación Estructura-Actividad , Ubiquitinación , beta Catenina/química , beta Catenina/genéticaRESUMEN
Novel therapeutic approaches are urgently required for multiple myeloma (MM). We used a phenotypic screening approach using co-cultures of MM cells with bone marrow stromal cells to identify compounds that overcome stromal resistance. One such compound, BRD9876, displayed selectivity over normal hematopoietic progenitors and was discovered to be an unusual ATP non-competitive kinesin-5 (Eg5) inhibitor. A novel mutation caused resistance, suggesting a binding site distinct from known Eg5 inhibitors, and BRD9876 inhibited only microtubule-bound Eg5. Eg5 phosphorylation, which increases microtubule binding, uniquely enhanced BRD9876 activity. MM cells have greater phosphorylated Eg5 than hematopoietic cells, consistent with increased vulnerability specifically to BRD9876's mode of action. Thus, differences in Eg5-microtubule binding between malignant and normal blood cells may be exploited to treat multiple myeloma. Additional steps are required for further therapeutic development, but our results indicate that unbiased chemical biology approaches can identify therapeutic strategies unanticipated by prior knowledge of protein targets.