RESUMEN
As a result of emerging biological data suggesting that within the c-Jun N-terminal kinase (JNK) family, JNK1 and not JNK2 or JNK3 may be primarily responsible for fibrosis pathology, we sought to identify JNK inhibitors with an increased JNK1 bias relative to our previous clinical compound tanzisertib (CC-930). This manuscript reports the synthesis and structure-activity relationship (SAR) studies for a novel series of JNK inhibitors demonstrating an increased JNK1 bias. SAR optimization on a series of 2,4-dialkylamino-pyrimidine-5-carboxamides resulted in the identification of compounds possessing low nanomolar JNK inhibitory potency, overall kinome selectivity, and the ability to inhibit cellular phosphorylation of the direct JNK substrate c-Jun. Optimization of physicochemical properties in this series resulted in compounds that demonstrated excellent systemic exposure following oral dosing, enabling in vivo efficacy studies and the selection of a candidate for clinical development, CC-90001, which is currently in clinical trials (Phase II) in patients with idiopathic pulmonary fibrosis (NCT03142191).
Asunto(s)
Ciclohexilaminas/farmacología , Descubrimiento de Drogas , Proteínas Quinasas JNK Activadas por Mitógenos/antagonistas & inhibidores , Inhibidores de Proteínas Quinasas/farmacología , Pirimidinas/farmacología , Animales , Ciclohexilaminas/uso terapéutico , Humanos , Fibrosis Pulmonar Idiopática/tratamiento farmacológico , Fosforilación , Inhibidores de Proteínas Quinasas/química , Inhibidores de Proteínas Quinasas/uso terapéutico , Pirimidinas/uso terapéutico , Relación Estructura-Actividad , Especificidad por SustratoRESUMEN
Down syndrome (DS), trisomy of human chromosome 21 (HSA21), is characterized by lifelong cognitive impairments and the development of the neuropathological hallmarks of Alzheimer's disease (AD). The cellular and molecular modifications responsible for these effects are not understood. Here we performed single-nucleus RNA sequencing (snRNA-seq) employing both short- (Illumina) and long-read (Pacific Biosciences) sequencing technologies on a total of 29 DS and non-DS control prefrontal cortex samples. In DS, the ratio of inhibitory-to-excitatory neurons was significantly increased, which was not observed in previous reports examining sporadic AD. DS microglial transcriptomes displayed AD-related aging and activation signatures in advance of AD neuropathology, with increased microglial expression of C1q complement genes (associated with dendritic pruning) and the HSA21 transcription factor gene RUNX1 Long-read sequencing detected vast RNA isoform diversity within and among specific cell types, including numerous sequences that differed between DS and control brains. Notably, over 8,000 genes produced RNAs containing intra-exonic junctions, including amyloid precursor protein (APP) that had previously been associated with somatic gene recombination. These and related results illuminate large-scale cellular and transcriptomic alterations as features of the aging DS brain.
Asunto(s)
Envejecimiento/fisiología , Síndrome de Down/metabolismo , Isoformas de ARN/metabolismo , Enfermedad de Alzheimer/metabolismo , Precursor de Proteína beta-Amiloide/metabolismo , Encéfalo/metabolismo , Cromosomas Humanos Par 21 , Subunidad alfa 2 del Factor de Unión al Sitio Principal/metabolismo , Síndrome de Down/genética , Expresión Génica , Humanos , Microglía , ARN/metabolismo , Análisis de Secuencia de ARN , Regulación hacia ArribaRESUMEN
In this Article, the top label in Fig. 5d should read 'DISH 3/16' instead of 'DISH 3/17'. This error has been corrected online.
RESUMEN
The diversity and complexity of the human brain are widely assumed to be encoded within a constant genome. Somatic gene recombination, which changes germline DNA sequences to increase molecular diversity, could theoretically alter this code but has not been documented in the brain, to our knowledge. Here we describe recombination of the Alzheimer's disease-related gene APP, which encodes amyloid precursor protein, in human neurons, occurring mosaically as thousands of variant 'genomic cDNAs' (gencDNAs). gencDNAs lacked introns and ranged from full-length cDNA copies of expressed, brain-specific RNA splice variants to myriad smaller forms that contained intra-exonic junctions, insertions, deletions, and/or single nucleotide variations. DNA in situ hybridization identified gencDNAs within single neurons that were distinct from wild-type loci and absent from non-neuronal cells. Mechanistic studies supported neuronal 'retro-insertion' of RNA to produce gencDNAs; this process involved transcription, DNA breaks, reverse transcriptase activity, and age. Neurons from individuals with sporadic Alzheimer's disease showed increased gencDNA diversity, including eleven mutations known to be associated with familial Alzheimer's disease that were absent from healthy neurons. Neuronal gene recombination may allow 'recording' of neural activity for selective 'playback' of preferred gene variants whose expression bypasses splicing; this has implications for cellular diversity, learning and memory, plasticity, and diseases of the human brain.
Asunto(s)
Enfermedad de Alzheimer/genética , Enfermedad de Alzheimer/patología , Precursor de Proteína beta-Amiloide/genética , Variación Genética/genética , Neuronas/citología , Neuronas/patología , Recombinación Genética , Empalme Alternativo/genética , Animales , ADN Complementario/análisis , ADN Complementario/genética , ADN Polimerasa Dirigida por ADN/metabolismo , Exones/genética , Femenino , Humanos , Intrones/genética , Masculino , Ratones , Ratones Transgénicos , Neuronas/metabolismo , Especificidad de Órganos , Mutación Puntual/genética , ARN Mensajero/análisis , ARN Mensajero/genética , Análisis de Secuencia de ADN , Eliminación de Secuencia/genéticaRESUMEN
V(D)J recombination of Ig and TCR genes is strictly regulated in a lineage- and stage-specific manner by the accessibility of target gene chromatin to the recombinases RAG1 and RAG2. It has been shown that enforced expression of the basic helix-loop-helix protein, E2A, together with RAG1/2 in a nonlymphoid cell line BOSC23 can induce V(D)J recombination in endogenous Igκ and TCR loci by increasing chromatin accessibility of target gene segments. In this study, we demonstrate that ectopically expressed E2A proteins in BOSC23 cells have the ability to bind directly to the promoter and recombination signal sequence of Vκ genes and to recruit histone acetyltransferase CBP/p300. Overexpression of CBP/p300 in conjunction with E2A results in enhancement of E2A-induced histone acetylation, germline transcription, and Igκ rearrangement. Conversely, knockdown of endogenous CBP/p300 expression by small interfering RNA leads to a decrease in histone acetylation, germline transcription and Igκ rearrangement. Furthermore, analyses using a mouse pre-B cell line revealed that endogenous E2A proteins also bind to a distinct set of Vκ genes and regulatory regions in the mouse Igκ locus and act to increase histone acetylation by recruiting p300, confirming the similar findings observed with BOSC23 cells. These observations indicate that E2A plays critical roles in inducing Igκ rearrangement by directly binding to and increasing chromatin accessibility at target gene segments.
Asunto(s)
Factores de Transcripción con Motivo Hélice-Asa-Hélice Básico/fisiología , Cromatina/genética , Cadenas kappa de Inmunoglobulina/genética , Factores de Transcripción p300-CBP/fisiología , Animales , Factores de Transcripción con Motivo Hélice-Asa-Hélice Básico/biosíntesis , Factores de Transcripción con Motivo Hélice-Asa-Hélice Básico/genética , Línea Celular , Cromatina/metabolismo , Sinergismo Farmacológico , Elementos de Facilitación Genéticos/genética , Elementos de Facilitación Genéticos/inmunología , Células Germinativas/enzimología , Células Germinativas/inmunología , Células Germinativas/metabolismo , Histona Desacetilasas/metabolismo , Humanos , Cadenas kappa de Inmunoglobulina/metabolismo , Ratones , Recombinación V(D)J/genética , Factores de Transcripción p300-CBP/biosíntesis , Factores de Transcripción p300-CBP/genéticaRESUMEN
The human suppressor of morphogenesis in genitalia-1 (hSMG-1) protein kinase plays dual roles in mRNA surveillance and genotoxic stress response pathways in human cells. Here, we report that small interfering RNA-mediated depletion of hSMG-1, but not ATM, ATR, hUpf1, or hUpf2, in human U2OS osteosarcoma cells markedly increases the magnitude and accelerates the rate of apoptosis induced by tumor necrosis factor-alpha (TNFalpha) stimulation. The increase in TNFalpha-mediated cell killing observed in hSMG-1-depleted cells is not related to the suppression of nonsense-mediated mRNA decay or to the inhibition of TNFalpha-induced NF-kappaB activation. Rather, we observed that loss of hSMG-1 accelerates the degradation of the long form of the FLICE-inhibitory protein (FLIP(L)), an inhibitor of death-inducing signaling complex-mediated caspase-8 activation, in TNFalpha-treated cells. These results suggest that hSMG-1 plays an important role in cell survival during TNFalpha-induced stress.
Asunto(s)
Apoptosis/efectos de los fármacos , Citoprotección/efectos de los fármacos , Fosfatidilinositol 3-Quinasas/metabolismo , Factor de Necrosis Tumoral alfa/farmacología , Proteína Reguladora de Apoptosis Similar a CASP8 y FADD/metabolismo , Línea Celular Tumoral , Proteínas Adaptadoras de Señalización del Receptor del Dominio de Muerte/metabolismo , Humanos , FN-kappa B/metabolismo , Fosfatidilinositol 3-Quinasas/genética , Biosíntesis de Proteínas/genética , Proteínas Serina-Treonina Quinasas , ARN Mensajero/genética , ARN Interferente Pequeño/genéticaRESUMEN
OBJECTIVE: To evaluate the chemical composition and crystalline structures of Portland cement, gray ProRoot MTA (gray MTA), white ProRoot MTA (white MTA), and gray MTA-Angelus. STUDY DESIGN: X-ray diffraction analysis was used to identify and characterize crystalline phases, and energy dispersive x-ray spectrometer was used to determine the chemical composition of the test materials. Both powder form and set form were examined. RESULTS: The crystalline structure and chemical composition of gray and white MTA were similar except for the presence of iron in gray MTA. Both were composed mainly of bismuth oxide and calcium silicate oxide. Portland cement was composed mainly of calcium silicate oxide and did not contain bismuth oxide. Gray MTA-Angelus had a lower content of bismuth oxide than ProRoot MTA. There were no noticeable differences in the chemical composition and crystalline structures between the powder and set forms of any of the material tested. CONCLUSION: Portland cement differed from the MTA by the absence of bismuth ions and presence of potassium ions. Gray MTA contained a significant amount of iron when compared with white MTA. In addition, gray MTA-Angelus had a lower content of bismuth oxide than ProRoot MTA.
Asunto(s)
Materiales de Obturación del Conducto Radicular/química , Compuestos de Aluminio/química , Bismuto/análisis , Compuestos de Calcio/análisis , Compuestos de Calcio/química , Cristalización , Cristalografía por Rayos X , Cementos Dentales/química , Combinación de Medicamentos , Hierro/análisis , Óxidos/química , Potasio/análisis , Silicatos/análisis , Silicatos/química , Espectrometría por Rayos XRESUMEN
IKK-i and TBK1 were recently identified as IKK-related kinases that are activated by toll-like receptors TLR3 and TLR4. These kinases were identified as essential components of the virus-activated as well as LPS-MyD88 independent kinase complex that phosphorylates IRF3 and results in the production of cytokines involved in innate immunity. Both IKK-i and TBK1 have also been implicated in the activation of the NFkappaB pathway but the precise mechanism is not clear. Although the literature to date suggests that IKK-i and TBK1 play redundant roles in TLR3 and TLR4 signaling, recent data suggest that there may be subtle differences in the signaling pathways affected by these kinases. We have generated tetracycline-inducible stable cell lines that express a wild type or kinase-inactive mutant form of IKK-i. Our data suggest that expression of IKK-i can activate both NFkappaB and IRF3, leading to the production of several cytokines including interferon beta. IKK-i most likely acts upstream of IKK2 to activate NFkappaB in these cells since expression of the kinase-inactive version of IKK-i did not inhibit TNFalpha mediated production of inflammatory cytokines. The data suggest that IKK-i is not involved in TNF-alpha mediated signaling but instead could likely play a role in activating IKK2 downstream of Toll-like receptor signaling. We also identified STAT1, Tyk2, and JAK1 as secondary mediators of IKK-i signaling as a result of interferon beta production in these cells.
Asunto(s)
Citocinas/biosíntesis , Factor 3 Regulador del Interferón/fisiología , FN-kappa B/fisiología , Proteínas Serina-Treonina Quinasas/fisiología , Línea Celular , Quimiocina CCL5/biosíntesis , Quimiocina CXCL10 , Quimiocinas CXC/biosíntesis , Proteínas de Unión al ADN/metabolismo , Activación Enzimática , Humanos , Quinasa I-kappa B , Factor 3 Regulador del Interferón/metabolismo , Interferón beta/biosíntesis , Interleucina-8/biosíntesis , Janus Quinasa 1 , Mutación , Proteínas Serina-Treonina Quinasas/genética , Proteínas Tirosina Quinasas/metabolismo , Factor de Transcripción STAT1/metabolismo , Transducción de Señal , TYK2 Quinasa , Tetraciclina/farmacología , Factor de Necrosis Tumoral alfa/fisiologíaRESUMEN
Lymphocytes arise from hematopoietic stem cells through the coordinated action of transcription factors. The E proteins (E12, E47, HEB and E2-2) have emerged as key regulators of both B and T lymphocyte differentiation. This review summarizes the current data and examines the various functions of E proteins and their antagonists, Id2 and Id3, throughout lymphoid maturation. Beyond an established role in B and T lineage commitment, E proteins continue to be essential at subsequent stages of development. E protein activity regulates the expression of surrogate and antigen receptor genes, promotes Ig and TCR rearrangements, and coordinates cell survival and proliferation with developmental progression in response to TCR signaling. Finally, this review also discusses the role of E47 as a tumor suppressor.