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1.
Molecules ; 26(15)2021 Jul 29.
Artigo em Inglês | MEDLINE | ID: mdl-34361750

RESUMO

The purpose of this work is to investigate the protein kinase inhibitory activity of constituents from Acacia auriculiformis stem bark. Column chromatography and NMR spectroscopy were used to purify and characterize betulin from an ethyl acetate soluble fraction of acacia bark. Betulin, a known inducer of apoptosis, was screened against a panel of 16 disease-related protein kinases. Betulin was shown to inhibit Abelson murine leukemia viral oncogene homolog 1 (ABL1) kinase, casein kinase 1ε (CK1ε), glycogen synthase kinase 3α/ß (GSK-3 α/ß), Janus kinase 3 (JAK3), NIMA Related Kinase 6 (NEK6), and vascular endothelial growth factor receptor 2 kinase (VEGFR2) with activities in the micromolar range for each. The effect of betulin on the cell viability of doxorubicin-resistant K562R chronic myelogenous leukemia cells was then verified to investigate its putative use as an anti-cancer compound. Betulin was shown to modulate the mitogen-activated protein (MAP) kinase pathway, with activity similar to that of imatinib mesylate, a known ABL1 kinase inhibitor. The interaction of betulin and ABL1 was studied by molecular docking, revealing an interaction of the inhibitor with the ABL1 ATP binding pocket. Together, these data demonstrate that betulin is a multi-target inhibitor of protein kinases, an activity that can contribute to the anticancer properties of the natural compound and to potential treatments for leukemia.


Assuntos
Acacia/química , Antineoplásicos Fitogênicos/farmacologia , Regulação Leucêmica da Expressão Gênica/efeitos dos fármacos , Inibidores de Proteínas Quinases/farmacologia , Proteínas Proto-Oncogênicas c-abl/antagonistas & inibidores , Triterpenos/farmacologia , Antineoplásicos Fitogênicos/química , Antineoplásicos Fitogênicos/isolamento & purificação , Apoptose/efeitos dos fármacos , Apoptose/genética , Sítios de Ligação , Caseína Quinase Iépsilon/antagonistas & inibidores , Caseína Quinase Iépsilon/genética , Caseína Quinase Iépsilon/metabolismo , Proliferação de Células/efeitos dos fármacos , Proteínas de Fusão bcr-abl/antagonistas & inibidores , Proteínas de Fusão bcr-abl/genética , Proteínas de Fusão bcr-abl/metabolismo , Quinase 3 da Glicogênio Sintase/antagonistas & inibidores , Quinase 3 da Glicogênio Sintase/genética , Quinase 3 da Glicogênio Sintase/metabolismo , Humanos , Janus Quinase 3/antagonistas & inibidores , Janus Quinase 3/genética , Janus Quinase 3/metabolismo , Células K562 , Proteína Quinase 1 Ativada por Mitógeno/antagonistas & inibidores , Proteína Quinase 1 Ativada por Mitógeno/genética , Proteína Quinase 1 Ativada por Mitógeno/metabolismo , Proteína Quinase 3 Ativada por Mitógeno/antagonistas & inibidores , Proteína Quinase 3 Ativada por Mitógeno/genética , Proteína Quinase 3 Ativada por Mitógeno/metabolismo , Modelos Moleculares , Quinases Relacionadas a NIMA/antagonistas & inibidores , Quinases Relacionadas a NIMA/genética , Quinases Relacionadas a NIMA/metabolismo , Casca de Planta/química , Extratos Vegetais/química , Ligação Proteica , Conformação Proteica , Inibidores de Proteínas Quinases/química , Inibidores de Proteínas Quinases/isolamento & purificação , Proteínas Proto-Oncogênicas c-abl/química , Proteínas Proto-Oncogênicas c-abl/genética , Proteínas Proto-Oncogênicas c-abl/metabolismo , Transdução de Sinais , Triterpenos/química , Triterpenos/isolamento & purificação , Receptor 2 de Fatores de Crescimento do Endotélio Vascular/antagonistas & inibidores , Receptor 2 de Fatores de Crescimento do Endotélio Vascular/genética , Receptor 2 de Fatores de Crescimento do Endotélio Vascular/metabolismo
2.
Sci Rep ; 11(1): 10093, 2021 05 12.
Artigo em Inglês | MEDLINE | ID: mdl-33980892

RESUMO

Janus Kinases (JAKs) have emerged as an important drug target for the treatment of a number of immune disorders due to the central role that they play in cytokine signalling. 4 isoforms of JAKs exist in mammalian cells and the ideal isoform profile of a JAK inhibitor has been the subject of much debate. JAK3 has been proposed as an ideal target due to its expression being largely restricted to the immune system and its requirement for signalling by cytokine receptors using the common γ-chain. Unlike other JAKs, JAK3 possesses a cysteine in its ATP binding pocket and this has allowed the design of isoform selective covalent JAK3 inhibitors targeting this residue. We report here that mutating this cysteine to serine does not prevent JAK3 catalytic activity but does greatly increase the IC50 for covalent JAK3 inhibitors. Mice with a Cys905Ser knockin mutation in the endogenous JAK3 gene are viable and show no apparent welfare issues. Cells from these mice show normal STAT phosphorylation in response to JAK3 dependent cytokines but are resistant to the effects of covalent JAK3 inhibitors. These mice therefore provide a chemical-genetic model to study JAK3 function.


Assuntos
Janus Quinase 3/genética , Trifosfato de Adenosina/metabolismo , Animais , Sítios de Ligação , Técnicas de Introdução de Genes , Humanos , Isoenzimas/química , Isoenzimas/genética , Isoenzimas/metabolismo , Janus Quinase 3/química , Janus Quinase 3/metabolismo , Camundongos , Modelos Genéticos , Domínios Proteicos , Inibidores de Proteínas Quinases/química
3.
Commun Biol ; 4(1): 406, 2021 03 25.
Artigo em Inglês | MEDLINE | ID: mdl-33767339

RESUMO

Jak3 is the only non-promiscuous member of the Jak family of secondary messengers. Studies to date have focused on understanding and targeting the cell-autonomous role of Jak3 in immunity, while functional Jak3 expression outside the hematopoietic system remains largely unreported. We show that Jak3 is expressed in endothelial cells across hematopoietic and non-hematopoietic organs, with heightened expression in the bone marrow. The bone marrow niche is understood as a network of different cell types that regulate hematopoietic function. We show that the Jak3-/- bone marrow niche is deleterious for the maintenance of long-term repopulating hematopoietic stem cells (LT-HSCs) and that JAK3-overexpressing endothelial cells have increased potential to expand LT-HSCs in vitro. This work may serve to identify a novel function for a highly specific tyrosine kinase in the bone marrow vascular niche and to further characterize the LT-HSC function of sinusoidal endothelium.


Assuntos
Células Endoteliais/metabolismo , Hematopoese/genética , Células-Tronco Hematopoéticas/metabolismo , Janus Quinase 3/genética , Animais , Feminino , Técnicas de Introdução de Genes , Janus Quinase 3/metabolismo , Masculino , Camundongos
4.
Chem Biol Interact ; 333: 109316, 2021 Jan 05.
Artigo em Inglês | MEDLINE | ID: mdl-33285127

RESUMO

Streptomyces hygroscopicus UFPEDA 3370 was fermented in submerged cultivation and the biomass extract was partitioned, obtaining a fraction purified named EB1. After purification of EB1 fraction, nigericin free acid was obtained and identified. Nigericin presented cytotoxic activity against several cancer cell lines, being most active against HL-60 (human leukemia) and HCT-116 (human colon carcinoma) cell lines, presenting IC50 and (IS) values: 0.0014 µM, (30.0) and 0.0138 µM (3.0), respectively. On HCT-116, nigericin caused apoptosis and autophagy. In this study, nigericin was also screened both in vitro and in silico against a panel of cancer-related kinases. Nigericin was able to inhibit both JAK3 and GSK-3ß kinases in vitro and its binding affinities were mapped through the intermolecular interactions with each target in silico.


Assuntos
Antineoplásicos/farmacologia , Neoplasias Colorretais/patologia , Nigericina/farmacologia , Inibidores de Proteínas Quinases/farmacologia , Streptomyces/química , Antineoplásicos/química , Antineoplásicos/metabolismo , Apoptose/efeitos dos fármacos , Domínio Catalítico , Linhagem Celular Tumoral , Humanos , Janus Quinase 3/antagonistas & inibidores , Janus Quinase 3/química , Janus Quinase 3/metabolismo , Simulação de Acoplamento Molecular , Nigericina/química , Nigericina/metabolismo , Inibidores de Proteínas Quinases/química , Inibidores de Proteínas Quinases/metabolismo
5.
Eur J Med Chem ; 209: 112934, 2021 Jan 01.
Artigo em Inglês | MEDLINE | ID: mdl-33109396

RESUMO

In this study, a series of 3-(4-phenyl-1H-imidazol-2-yl)-1H-pyrazole derivatives were designed, synthesized, and evaluated for their biological activities. Upon performing kinase assays, most of the compounds exhibited potent inhibition against JAK2/3 and Aurora A/B with the IC50 values ranging from 0.008 to 2.52 µM. Among these derivatives, compound 10e expressed the most moderate inhibiting activities against all the four kinases with the IC50 values of 0.166 µM (JAK2), 0.057 µM (JAK3), 0.939 µM (Aurora A), and 0.583 µM (Aurora B), respectively. Moreover, most of the derived compounds exhibited potent cytotoxicity against human chronic myeloid leukemia cells K562 and human colon cancer cells HCT116, while compound 10e expressed antiproliferative activities against K562 (IC50=6.726  µM). According to western blot analysis, compound 10e down-regulated the phosphorylation of STAT3, STAT5, Aurora A, and Aurora B in a dose-dependent manner in K562 and HCT116 cells. Cell cycle analysis revealed that compound 10e inhibited the proliferation of cells by inducing cell cycle arrest in the G2 phase. The molecular modeling suggested that compound 10e could maintain a binding mode similar to the binding mode of AT9832, a common JAK 2/3 and Aurora A/B kinases multi-target kinase inhibitor. Therefore, compound 10e might be a potential agent for cancer therapy deserving further research.


Assuntos
Antineoplásicos/síntese química , Aurora Quinase A/metabolismo , Aurora Quinase B/metabolismo , Janus Quinase 2/metabolismo , Janus Quinase 3/metabolismo , Inibidores de Proteínas Quinases/síntese química , Pirazóis/síntese química , Amidas/química , Antineoplásicos/farmacologia , Apoptose/efeitos dos fármacos , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Relação Dose-Resposta a Droga , Ensaios de Seleção de Medicamentos Antitumorais , Humanos , Modelos Moleculares , Inibidores de Proteínas Quinases/farmacologia , Pirazóis/farmacologia , Pirimidinas/química
6.
J Cell Physiol ; 236(7): 5193-5211, 2021 07.
Artigo em Inglês | MEDLINE | ID: mdl-33368247

RESUMO

Phospholipase D (PLD) isoforms PLD1 and PLD2 serve as the primary nodes where diverse signaling pathways converge. However, their isoform-specific functions remain unclear. We showed that PLD1 and PLD2 selectively couple to toll-like receptor 4 (TLR4) and interleukin 4 receptor (IL-4R) and differentially regulate macrophage polarization of M1 and M2 via the LPS-MyD88 axis and the IL-4-JAK3 signaling, respectively. Lipopolysaccharide (LPS) enhanced TLR4 or MyD88 interaction with PLD1; IL-4 induced IL-4R or JAK3 association with PLD2, indicating isozyme-specific signaling events. PLD1 and PLD2 are indispensable for M1 polarization and M2 polarization, respectively. Genetic and pharmacological targeting of PLD1 conferred protection against LPS-induced sepsis, cardiotoxin-induced muscle injury, and skin injury by promoting the shift toward M2; PLD2 ablation intensified disease severity by promoting the shift toward M1. Enhanced Foxp3+ regulatory T cell recruitment also influenced the anti-inflammatory phenotype of Pld1LyzCre macrophages. We reveal a previously uncharacterized role of PLD isoforms in macrophage polarization, signifying potential pharmacological interventions for macrophage modulation.


Assuntos
Macrófagos/fisiologia , Fosfolipase D/metabolismo , Cicatrização/fisiologia , Ferimentos e Lesões/prevenção & controle , Animais , Polaridade Celular/fisiologia , Inflamação/patologia , Inflamação/prevenção & controle , Janus Quinase 3/metabolismo , Lipopolissacarídeos , Macrófagos/imunologia , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Músculos/lesões , Fator 88 de Diferenciação Mieloide/metabolismo , Fosfolipase D/genética , Receptores de Interleucina-4/metabolismo , Sepse/imunologia , Linfócitos T Reguladores/imunologia , Receptor 4 Toll-Like/metabolismo , Ferimentos e Lesões/patologia
7.
Bioorg Chem ; 105: 104274, 2020 12.
Artigo em Inglês | MEDLINE | ID: mdl-33339080

RESUMO

New series of hexahydroquinoline and fused quinoline derivatives were designed and synthesized. The thirty seven new compounds were screened for in vitro antitumor activity against HepG2, HCT-116 and MCF-7 cancer cells. Results indicated that compounds 2e, 2h, 5b, 5c, 6a, 7d and 9b have the strongest potency against the three cancer cells, and they were further screened for in vitro cytotoxicity against A431 and H1975 cancer cells, as well as WI38 and WISH normal cells. Results revealed that 7d potently inhibited the growth of H1975 cells harboring EGFRT790M mutation (IC50 = 1.32 ± 0.2 µM) over A431 cells overexpressing EGFRWT (IC50 = 4.96 ± 0.3 µM). Moreover, the seven compounds displayed low cytotoxicity against the tested normal cells. The seven potent antitumor compounds were examined for their ability to inhibit the activity of EGFRWT. The attained data manifested that 7d has remarkable EGFRWT inhibitory activity (IC50 = 0.083 ± 0.002 µM) compared to erlotinib (IC50 = 0.067 ± 0.002 µM). Compound 7d was further studied for its enzymatic inhibitory activity against other eight human kinases, and it displayed outstanding inhibitory activity against EGFRL858R and EGFRT790M mutants (IC50 = 0.053 ± 0.002, 0.026 ± 0.001 µM, respectively), as well as JAK3 (IC50 = 0.069 ± 0.003 µM). Analysis of cell cycle evidenced that 7d induces cell cycle arrest in G2/M and pre-G1 phases in the tested cancer cells. In addition, cancer cell death induced by 7d was proved to take place via apoptosis supported by elevated Bax/Bcl-2 ratio in the tested cancer cells. Moreover, docking results confirmed the good binding interactions of 7d with EGFRWT, EGFRL858R, EGFRT790M and JAK3, which came in agreement with the results of in vitro enzyme assay. Further, 7d is predicted to have good oral absorption, good drug-likeness properties and low toxicity risks in human.


Assuntos
Antineoplásicos/síntese química , Receptores ErbB/antagonistas & inibidores , Inibidores de Proteínas Quinases/síntese química , Quinolinas/síntese química , Antineoplásicos/farmacologia , Apoptose/efeitos dos fármacos , Pontos de Checagem do Ciclo Celular/efeitos dos fármacos , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Ensaios de Seleção de Medicamentos Antitumorais , Receptores ErbB/genética , Humanos , Janus Quinase 3/metabolismo , Simulação de Acoplamento Molecular , Estrutura Molecular , Mutação , Inibidores de Proteínas Quinases/farmacologia , Proteínas Proto-Oncogênicas c-bcl-2/metabolismo , Quinolinas/farmacocinética , Relação Estrutura-Atividade , Proteína X Associada a bcl-2/metabolismo
8.
Front Immunol ; 11: 597741, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-33329590

RESUMO

Keloids are disfiguring, fibroproliferative growths and their pathogenesis remains unclear, inhibiting therapeutic development. Available treatment options have limited efficacy and harbor safety concerns. Thus, there is a great need to clarify keloid pathomechanisms that may lead to novel treatments. In this study, we aimed to elucidate the profile of lesional and non-lesional keloid skin compared to normal skin. We performed gene (RNAseq, qRT-PCR) and protein (immunohistochemistry) expression analyses on biopsy specimens obtained from lesional and non-lesional skin of African American (AA) keloid patients compared to healthy skin from AA controls. Fold-change≥2 and false-discovery rate (FDR)<0.05 was used to define significance. We found that lesional versus normal skin showed significant up-regulation of markers of T-cell activation/migration (ICOS, CCR7), Th2- (IL-4R, CCL11, TNFSF4/OX40L), Th1- (CXCL9/CXCL10/CXCL11), Th17/Th22- (CCL20, S100As) pathways, and JAK/STAT-signaling (JAK3) (false-discovery rate [FDR]<0.05). Non-lesional skin also exhibited similar trends. We observed increased cellular infiltrates in keloid tissues, including T-cells, dendritic cells, mast cells, as well as greater IL-4rα+, CCR9+, and periostin+ immunostaining. In sum, comprehensive molecular profiling demonstrated that both lesional and non-lesional skin show significant immune alternations, and particularly Th2 and JAK3 expression. This advocates for the investigation of novel treatments targeting the Th2 axis and/or JAK/STAT-signaling in keloid patients.


Assuntos
Janus Quinase 3/metabolismo , Queloide/genética , Queloide/imunologia , Contagem de Linfócitos , Subpopulações de Linfócitos T/imunologia , Subpopulações de Linfócitos T/metabolismo , Transcriptoma , Adulto , Idoso , Biomarcadores , Feminino , Perfilação da Expressão Gênica , Sequenciamento de Nucleotídeos em Larga Escala , Humanos , Imuno-Histoquímica , Queloide/patologia , Ativação Linfocitária/imunologia , Pessoa de Meia-Idade , Células Th1/imunologia , Células Th1/metabolismo , Células Th17/imunologia , Células Th17/metabolismo , Células Th2/imunologia , Células Th2/metabolismo
9.
Eur J Med Chem ; 207: 112836, 2020 Dec 01.
Artigo em Inglês | MEDLINE | ID: mdl-32971426

RESUMO

Targeted covalent inhibitors represent a viable strategy to block protein kinases involved in different disease pathologies. Although a number of computational protocols have been published for identifying druggable cysteines, experimental approaches are limited for mapping the reactivity and accessibility of these residues. Here, we present a ligand based approach using a toolbox of fragment-sized molecules with identical scaffold but equipped with diverse covalent warheads. Our library represents a unique opportunity for the efficient integration of warhead-optimization and target-validation into the covalent drug development process. Screening this probe kit against multiple kinases could experimentally characterize the accessibility and reactivity of the targeted cysteines and helped to identify suitable warheads for designed covalent inhibitors. The usefulness of this approach has been confirmed retrospectively on Janus kinase 3 (JAK3). Furthermore, representing a prospective validation, we identified Maternal embryonic leucine zipper kinase (MELK), as a tractable covalent target. Covalently labelling and biochemical inhibition of MELK would suggest an alternative covalent strategy for MELK inhibitor programs.


Assuntos
Cisteína/metabolismo , Janus Quinase 3/metabolismo , Bibliotecas de Moléculas Pequenas/metabolismo , Transporte de Elétrons , Janus Quinase 3/antagonistas & inibidores , Ligantes , Inibidores de Proteínas Quinases/metabolismo , Inibidores de Proteínas Quinases/farmacologia , Bibliotecas de Moléculas Pequenas/farmacologia
10.
Bioorg Chem ; 104: 104258, 2020 11.
Artigo em Inglês | MEDLINE | ID: mdl-32956876

RESUMO

Five new nitrogen-containing compounds (1-3, 5, and 6), two compounds which was firstly isolated from natural origin (7 and 10), along with six known ones, were isolated from the ethanol extract of the whole bodies of Polyphaga plancyi. The structures of the new compounds including their absolute configurations at stereogenic centers were assigned on the basis of spectroscopic analyses and computational methods. Racemic 10 was separated by chiral HPLC. Biological activities of these isolates against extracellular matrix components in rat renal proximal tubular cells, EV71, COX-2, ROCK2, JAK3, and tuberculosis were evaluated. Importantly, 8 was found to be a selective Smad3 phosphorylation inhibitor.


Assuntos
Antituberculosos/farmacologia , Produtos Biológicos/farmacologia , Besouros/química , Ciclo-Oxigenase 2/metabolismo , Inibidores Enzimáticos/farmacologia , Janus Quinase 3/antagonistas & inibidores , Tuberculose/tratamento farmacológico , Animais , Antituberculosos/química , Antituberculosos/isolamento & purificação , Produtos Biológicos/química , Produtos Biológicos/isolamento & purificação , Células Cultivadas , Teoria da Densidade Funcional , Relação Dose-Resposta a Droga , Inibidores Enzimáticos/química , Inibidores Enzimáticos/isolamento & purificação , Janus Quinase 3/metabolismo , Estrutura Molecular , Ratos , Relação Estrutura-Atividade
11.
Cell Rep ; 32(11): 108158, 2020 09 15.
Artigo em Inglês | MEDLINE | ID: mdl-32937124

RESUMO

The traditional Chinese medicinal herb Notopterygium incisum Ting ex H.T. Chang has anti-rheumatism activity, and a mass spectrometry assay of patients' serum after administration of the herb revealed that notopterol is the most abundant component enriched. However, the functions of notopterol and its molecular target in rheumatoid arthritis (RA) treatment remain unknown. Here, we show in different RA mouse strains that both oral and intraperitoneal administration of notopterol result in significant therapeutic effects. Mechanistically, notopterol directly binds Janus kinase (JAK)2 and JAK3 kinase domains to inhibit JAK/signal transducers and activators of transcription (JAK-STAT) activation, leading to reduced production of inflammatory cytokines and chemokines. Critically, combination therapy using both notopterol and tumor necrosis factor (TNF) blocker results in enhanced therapeutic effects compared to using TNF blocker alone. We demonstrate that notopterol ameliorates RA pathology by targeting JAK-STAT signaling, raising the possibility that notopterol could be effective in treating other diseases characterized by aberrant JAK-STAT signaling pathway.


Assuntos
Artrite Reumatoide/patologia , Cumarínicos/farmacologia , Inflamação/patologia , Janus Quinase 2/antagonistas & inibidores , Janus Quinase 3/antagonistas & inibidores , Animais , Artrite Experimental/tratamento farmacológico , Artrite Experimental/enzimologia , Artrite Experimental/patologia , Artrite Experimental/prevenção & controle , Artrite Reumatoide/tratamento farmacológico , Artrite Reumatoide/enzimologia , Produtos Biológicos/administração & dosagem , Produtos Biológicos/química , Produtos Biológicos/farmacologia , Produtos Biológicos/uso terapêutico , Quimiocinas/metabolismo , Cumarínicos/administração & dosagem , Cumarínicos/química , Cumarínicos/uso terapêutico , Etanercepte/farmacologia , Inflamação/tratamento farmacológico , Inflamação/enzimologia , Mediadores da Inflamação/metabolismo , Interferon gama/farmacologia , Janus Quinase 2/química , Janus Quinase 3/metabolismo , Lipopolissacarídeos , Macrófagos/efeitos dos fármacos , Macrófagos/metabolismo , Camundongos Endogâmicos C57BL , Camundongos Endogâmicos DBA , Domínios Proteicos , Fatores de Transcrição STAT/metabolismo , Fator de Necrose Tumoral alfa/farmacologia
12.
PLoS One ; 15(7): e0235766, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32639993

RESUMO

High-grade serous ovarian carcinoma (HGSOC) remains the deadliest form of epithelial ovarian cancer and despite major efforts little improvement in overall survival has been achieved. Identification of recurring "driver" genetic lesions has the potential to enable design of novel therapies for cancer. Here, we report on a study to find such new therapeutic targets for HGSOC using exome-capture sequencing approach targeting all kinase genes in 127 patient samples. Consistent with previous reports, the most frequently mutated gene was TP53 (97% mutation frequency) followed by BRCA1 (10% mutation frequency). The average mutation frequency of the kinase genes mutated from our panel was 1.5%. Intriguingly, after BRCA1, JAK3 was the most frequently mutated gene (4% mutation frequency). We tested the transforming properties of JAK3 mutants using the Ba/F3 cell-based in vitro functional assay and identified a novel gain-of-function mutation in the kinase domain of JAK3 (p.T1022I). Importantly, p.T1022I JAK3 mutants displayed higher sensitivity to the JAK3-selective inhibitor Tofacitinib compared to controls. For independent validation, we re-sequenced the entire JAK3 coding sequence using tagged amplicon sequencing (TAm-Seq) in 463 HGSOCs resulting in an overall somatic mutation frequency of 1%. TAm-Seq screening of CDK12 in the same population revealed a 7% mutation frequency. Our data confirms that the frequency of mutations in kinase genes in HGSOC is low and provides accurate estimates for the frequency of JAK3 and CDK12 mutations in a large well characterized cohort. Although p.T1022I JAK3 mutations are rare, our functional validation shows that if detected they should be considered as potentially actionable for therapy. The observation of CDK12 mutations in 7% of HGSOC cases provides a strong rationale for routine somatic testing, although more functional and clinical characterization is required to understand which nonsynonymous mutations alterations are associated with homologous recombination deficiency.


Assuntos
Proteína BRCA1/genética , Cistadenocarcinoma Seroso/genética , Janus Quinase 3/genética , Mutação , Neoplasias Ovarianas/genética , Proteínas Quinases/genética , Proteína Supressora de Tumor p53/genética , Proteína BRCA1/metabolismo , Estudos de Casos e Controles , Estudos de Coortes , Cistadenocarcinoma Seroso/metabolismo , Cistadenocarcinoma Seroso/patologia , Feminino , Sequenciamento de Nucleotídeos em Larga Escala , Humanos , Janus Quinase 3/metabolismo , Neoplasias Ovarianas/metabolismo , Neoplasias Ovarianas/patologia , Proteínas Quinases/metabolismo , Proteína Supressora de Tumor p53/metabolismo
13.
Mol Cell Biol ; 40(18)2020 08 28.
Artigo em Inglês | MEDLINE | ID: mdl-32631903

RESUMO

Precursor B cell acute lymphoblastic leukemia (B-ALL) is caused by genetic lesions in developing B cells that function as drivers for the accumulation of additional mutations in an evolutionary selection process. We investigated secondary drivers of leukemogenesis in a mouse model of B-ALL driven by PU.1/Spi-B deletion (Mb1-CreΔPB). Whole-exome-sequencing analysis revealed recurrent mutations in Jak3 (encoding Janus kinase 3), Jak1, and Ikzf3 (encoding Aiolos). Mutations with a high variant-allele frequency (VAF) were dominated by C→T transition mutations that were compatible with activation-induced cytidine deaminase, whereas the majority of mutations, with a low VAF, were dominated by C→A transversions associated with 8-oxoguanine DNA damage caused by reactive oxygen species (ROS). The Janus kinase (JAK) inhibitor ruxolitinib delayed leukemia onset, reduced ROS and ROS-induced gene expression signatures, and altered ROS-induced mutational signatures. These results reveal that JAK mutations can alter the course of leukemia clonal evolution through ROS-induced DNA damage.


Assuntos
Dano ao DNA , Janus Quinase 1/genética , Janus Quinase 1/metabolismo , Leucemia de Células B/metabolismo , Animais , Linfócitos B/metabolismo , Linhagem Celular Tumoral , Proliferação de Células , Humanos , Janus Quinase 3/metabolismo , Leucemia de Células B/genética , Leucemia de Células B/patologia , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Mutação , Leucemia-Linfoma Linfoblástico de Células Precursoras/genética , Leucemia-Linfoma Linfoblástico de Células Precursoras/metabolismo , Leucemia-Linfoma Linfoblástico de Células Precursoras/patologia , Proteínas Proto-Oncogênicas/genética , Proteínas Proto-Oncogênicas/metabolismo , Proteínas Proto-Oncogênicas c-ets/genética , Proteínas Proto-Oncogênicas c-ets/metabolismo , Espécies Reativas de Oxigênio/metabolismo , Transativadores/genética , Transativadores/metabolismo
14.
Exp Biol Med (Maywood) ; 245(15): 1395-1403, 2020 09.
Artigo em Inglês | MEDLINE | ID: mdl-32664806

RESUMO

IMPACT STATEMENT: Accumulating evidence suggests that vascular remodeling due to immoderate proliferation and migration of SMCs is a common process occurring in APE. In this work, we tried to find a breakthrough in the pathological mechanism to alleviate the prognosis of APE by improving SMCs proliferation and explored the effect of JANEX-1 on PDGF-induced proliferation-related molecules in PVSMCs and assessed the therapeutic potential of JAK3 for vascular remodeling in APE mice. We demonstrated that JANEX-1, blocking JAK3 expression or activity, reduced JAK3/STAT3 signaling pathway, VEGF expression and FAK activation, and PDGF-induced proliferation of PVSMCs. Moreover, JANEX-1 inhibited the thrombus-induced intimal hyperplasia and the expression of VEGF and FAK activation in neointimal SMCs of APE mice. The data are helpful to elucidate the pharmacological mechanism and potential therapeutic effect of JANEX-1 in APE.


Assuntos
Proteína-Tirosina Quinases de Adesão Focal/metabolismo , Miócitos de Músculo Liso/patologia , Artéria Pulmonar/patologia , Embolia Pulmonar/tratamento farmacológico , Embolia Pulmonar/patologia , Quinazolinas/uso terapêutico , Fator A de Crescimento do Endotélio Vascular/metabolismo , Doença Aguda , Animais , Proliferação de Células/efeitos dos fármacos , Modelos Animais de Doenças , Janus Quinase 3/metabolismo , Pulmão/efeitos dos fármacos , Pulmão/patologia , Pulmão/fisiopatologia , Masculino , Camundongos Endogâmicos C57BL , Miócitos de Músculo Liso/efeitos dos fármacos , Miócitos de Músculo Liso/metabolismo , Fosforilação/efeitos dos fármacos , Fator de Crescimento Derivado de Plaquetas/farmacologia , Embolia Pulmonar/fisiopatologia , Quinazolinas/farmacologia , Fator de Transcrição STAT3/metabolismo , Remodelação Vascular/efeitos dos fármacos
15.
Bioorg Chem ; 102: 104086, 2020 09.
Artigo em Inglês | MEDLINE | ID: mdl-32688114

RESUMO

Pipajiains H-J (1-3), three new phenolic derivatives with an unusual sulfone group, pipajiamides A-C (4-6), three new amide derivatives, pipajiaine A (7), one new imidazole analogue, and pipajiaine B (8), a pair of new pyrrolidine derivatives, along with three known compounds were isolated from the insect Blaps japanensis. Their structures were identified by spectroscopic and computational methods. Chiral HPLC was used to separate the (-)- and (+)-antipodes of 4 and 8. Biological activities of all the new compounds against extracellular matrix in rat renal proximal tubular cells, human cancer cells (A549, Huh-7, and K562), COX-2, ROCK1, and JAK3 were evaluated. The results show that compounds 2, (+)-4, and (-)-4 are active against kidney fibrosis, whereas, compound 9 is active toward human cancer cells, inflammation, and JAK3 kinase.


Assuntos
Besouros/química , Compostos de Nitrogênio/farmacologia , Inibidores de Proteínas Quinases/farmacologia , Enxofre/farmacologia , Animais , Células Cultivadas , Teoria da Densidade Funcional , Relação Dose-Resposta a Droga , Fibrose/tratamento farmacológico , Humanos , Janus Quinase 3/antagonistas & inibidores , Janus Quinase 3/metabolismo , Estrutura Molecular , Compostos de Nitrogênio/química , Compostos de Nitrogênio/isolamento & purificação , Inibidores de Proteínas Quinases/química , Inibidores de Proteínas Quinases/isolamento & purificação , Ratos , Relação Estrutura-Atividade , Enxofre/química , Enxofre/isolamento & purificação , Quinases Associadas a rho/antagonistas & inibidores , Quinases Associadas a rho/metabolismo
16.
J Cell Mol Med ; 24(13): 7427-7438, 2020 07.
Artigo em Inglês | MEDLINE | ID: mdl-32558259

RESUMO

Gain- or loss-of-function mutations in Janus kinase 3 (JAK3) contribute to the pathogenesis of various haematopoietic malignancies and immune disorders, suggesting that aberrant JAK3 signalling is an attractive therapeutic target to treat these disorders. In this study, we performed structure-based computational database screening using the 3D structure of the JAK3 kinase domain and the National Cancer Institute diversity set and identified tubulosine as a novel JAK3 inhibitor. Tubulosine directly blocked the catalytic activity of JAK3 by selective interacting with the JAK3 kinase domain. Consistently, tubulosine potently inhibited persistently activated and interleukin-2-dependent JAK3, and JAK3-mediated downstream targets. Importantly, it did not affect the activity of other JAK family members, particularly prolactin-induced JAK2/signal transducer and activator of transcription 5 and interferon alpha-induced JAK1-TYK2/STAT1. Tubulosine specifically decreased survival and proliferation of cancer cells, in which persistently active JAK3 is expressed, by inducing apoptotic and necrotic/autophagic cell death without affecting other oncogenic signalling. Collectively, tubulosine is a potential small-molecule compound that selectively inhibits JAK3 activity, suggesting that it may serve as a promising therapeutic candidate for treating disorders caused by aberrant activation of JAK3 signalling.


Assuntos
Trifosfato de Adenosina/metabolismo , Emetina/análogos & derivados , Janus Quinase 3/antagonistas & inibidores , Transdução de Sinais , Apoptose/efeitos dos fármacos , Autofagia/efeitos dos fármacos , Sítios de Ligação , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Sobrevivência Celular/efeitos dos fármacos , Emetina/química , Emetina/farmacologia , Humanos , Janus Quinase 3/metabolismo , Modelos Biológicos , Necrose , Oncogenes , Fator de Transcrição STAT5/metabolismo , Transdução de Sinais/efeitos dos fármacos
17.
Int Immunopharmacol ; 86: 106706, 2020 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-32570038

RESUMO

Kidney renal clear cell carcinoma (KIRC) is one of the most common cancers globally, with an overall poor prognosis. The Janus kinase (JAK) family plays an essential role in cellular mechanisms such as proliferation, metastasis, invasion, and immunity. In our study, various web-portals were used to explore the expression and clinical significance of JAK3 in KIRC. JAK3 expression was significantly up-regulated in KIRC tissues. Patients with KIRC having high JAK3 levels displayed a substantially decreased disease-free survival rate and overall survival rate. Significant correlations were obtained between JAK3 expression and the abundance of immune cells and immune biomarker sets. Enrichment function analysis revealed that gene function significantly correlated with JAK3, which was primarily associated with the immune response, JAK-STAT signaling pathway, Ras signaling pathway via several cancer-related kinases, miRNAs, and transcription factors. Moreover, we also identified several kinase, miRNA or transcription factor targets of JAK3 in KIRC. The hub genes (JAK3, FCHO1, INSl3, DEF6, and GPR132) were associated with the activation or inhibition of several famous cancer related pathways. Our results demonstrated that JAK3 is a potential biomarker and associated with immune infiltration in KIRC.


Assuntos
Carcinoma de Células Renais/genética , Carcinoma de Células Renais/imunologia , Janus Quinase 3/genética , Janus Quinase 3/imunologia , Neoplasias Renais/genética , Neoplasias Renais/imunologia , Biomarcadores Tumorais/genética , Biomarcadores Tumorais/metabolismo , Biologia Computacional , Bases de Dados Genéticas , Intervalo Livre de Doença , Regulação Neoplásica da Expressão Gênica/genética , Regulação Neoplásica da Expressão Gênica/imunologia , Humanos , Janus Quinase 3/metabolismo , Neoplasias Renais/diagnóstico , Linfócitos do Interstício Tumoral , MicroRNAs/metabolismo , Fosfotransferases/metabolismo , Mapas de Interação de Proteínas , Fatores de Transcrição STAT/genética , Fatores de Transcrição STAT/metabolismo , Transdução de Sinais , Regulação para Cima
18.
AAPS PharmSciTech ; 21(5): 175, 2020 Jun 17.
Artigo em Inglês | MEDLINE | ID: mdl-32556636

RESUMO

Wound-healing is a very complex and evolutionary process that involves a great variety of dynamic steps. Although different pharmaceutical agents have been developed to hasten the wound-healing process, the existing agents are still far from optimal. The present work aimed to prepare and evaluate the wound-healing efficacy of phenytoin-loaded copper nanoparticles (PHT-loaded CuNPs). CuNPs were biosynthesized using licorice aqueous extract. The prepared CuNPs were loaded with PHT by adsorption, characterized, and evaluated for wound-healing efficiency. Results showed that both plain and PHT-loaded CuNPs were monodisperse and exhibited a cubic and hexagonal morphology. The mechanism by which PHT was adsorbed on the surface of CuNPs was best fit by the Langmuir model with a maximum loaded monolayer capacity of 181 mg/g. The kinetic study revealed that the adsorption reaction followed the pseudo-second order while the thermodynamic parameters indicated that the adsorption process was physical in nature and endothermic, and occurred spontaneously. Moreover, the in vivo wound-healing activity of PHT-loaded CuNP impregnated hydroxypropylmethyl cellulose (HPMC) gel was carried out using an excisional wound model in rats. Data showed that PHT-loaded CuNPs accelerated epidermal regeneration and stimulated granulation and tissue formation in treated rats compared to controls. Additionally, quantitative real-time polymerase chain reaction (RT-PCR) analysis showed that lesions treated with PHT-loaded CuNPs were associated with a marked increase in the expression of dermal procollagen type I and a decrease in the expression of the inflammatory JAK3 compared to control samples. In conclusion, PHT-loaded CuNPs are a promising platform for effective and rapid wound-healing.


Assuntos
Cobre/farmacologia , Nanopartículas , Fenitoína/farmacologia , Cicatrização/efeitos dos fármacos , Animais , Colágeno Tipo I/metabolismo , Janus Quinase 3/metabolismo , Masculino , Ratos , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Pele/patologia
20.
FASEB J ; 34(7): 9120-9140, 2020 07.
Artigo em Inglês | MEDLINE | ID: mdl-32433819

RESUMO

Homeostasis between pro- and anti- inflammatory responses induced by bacteria is critical for the maintenance of health. In the oral cavity, pro-inflammatory mechanisms induced by pathogenic bacteria are well-established; however, the anti-inflammatory responses that act to restrain innate responses remain poorly characterized. Here, we demonstrate that infection with the periodontal pathogen Porphyromonas gingivalis enhances the activity of Janus kinase 3 (JAK3) in innate immune cells, and subsequently phospho-inactivates Nedd4-2, an ubiquitin E3 ligase. In turn, Wingless-INT (Wnt) 3 (Wnt3) ubiquitination is decreased, while total protein levels are enhanced, leading to a reduction in pro-inflammatory cytokine levels. In contrast, JAK3 or Wnt3a inhibition robustly enhances nuclear factor kappa-light-chain-enhancer of activated B cells activity and the production of pro-inflammatory cytokines in P. gingivalis-stimulated innate immune cells. Moreover, using gain- and loss-of-function approaches, we demonstrate that downstream molecules of Wnt3a signaling, including Dvl3 and ß-catenin, are responsible for the negative regulatory role of Wnt3a. In addition, using an in vivo P. gingivalis-mediated periodontal disease model, we show that JAK3 inhibition enhances infiltration of inflammatory cells, reduces expression of Wnt3a and Dvl3 in P. gingivalis-infected gingival tissues, and increases disease severity. Together, our results reveal a new anti-inflammatory role for JAK3 in innate immune cells and show that the underlying signaling pathway involves Nedd4-2-mediated Wnt3a ubiquitination.


Assuntos
Infecções por Bacteroidaceae/complicações , Reabsorção Óssea/prevenção & controle , Inflamação/prevenção & controle , Janus Quinase 3/metabolismo , Doenças Periodontais/prevenção & controle , Substâncias Protetoras , Proteína Wnt3A/metabolismo , Animais , Infecções por Bacteroidaceae/microbiologia , Reabsorção Óssea/etiologia , Reabsorção Óssea/metabolismo , Reabsorção Óssea/patologia , Inflamação/etiologia , Inflamação/metabolismo , Inflamação/patologia , Janus Quinase 3/genética , Camundongos , Camundongos Endogâmicos C57BL , Doenças Periodontais/etiologia , Doenças Periodontais/metabolismo , Doenças Periodontais/patologia , Porphyromonas gingivalis/patogenicidade , Transdução de Sinais , Proteína Wnt3A/genética
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