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Introduction: We reported increased spleen tyrosine kinase (SYK) expression in kidney biopsies of patients with IgA nephropathy (IgAN) and that inhibition of SYK reduces inflammatory cytokines production from IgA stimulated mesangial cells. Methods: This study was a double-blind, randomized, placebo-controlled phase 2 trial of fostamatinib (an oral SYK inhibitor) in 76 patients with IgAN. Patients were randomized to receive placebo, fostamatinib at 100 mg or 150 mg twice daily for 24 weeks on top of maximum tolerated dose of renin-angiotensin system inhibitors. The primary end point was reduction of proteinuria. Secondary end points included change from baseline in estimated glomerular filtration rate (eGFR) and kidney histology. Results: Although we could not detect significant reduction in proteinuria with fostamatinib overall, in a predetermined subgroup analysis, there was a trend for dose-dependent reduction in median proteinuria (from baseline to 24 weeks by 14%, 27%, and 36% in the placebo, fostamatinib 100 mg, and 150 mg groups, respectively) in patients with baseline urinary protein-to-creatinine ratios (UPCR) more than 1000 mg/g. Kidney function (eGFR) remained stable in all groups. Fostamatinib was well-tolerated. Side effects included diarrhea, hypertension, and increased liver enzymes. Thirty-nine patients underwent repeat biopsy showing reductions in SYK staining associated with therapy at low dose (-1.5 vs. 1.7 SYK+ cells/glomerulus in the placebo group, P < 0.05). Conclusions: There was a trend toward reduction in proteinuria with fostamatinib in a predefined analysis of high risk patients with IgAN despite maximal care, as defined by baseline UPCR greater than 1000 mg/g. Further study may be warranted.
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The fine equilibrium of bone homeostasis is maintained by bone-forming osteoblasts and bone-resorbing osteoclasts. Here, we show that TAM receptors MERTK and TYRO3 exert reciprocal effects in osteoblast biology: Osteoblast-targeted deletion of MERTK promotes increased bone mass in healthy mice and mice with cancer-induced bone loss, whereas knockout of TYRO3 in osteoblasts shows the opposite phenotype. Functionally, the interaction of MERTK with its ligand PROS1 negatively regulates osteoblast differentiation via inducing the VAV2-RHOA-ROCK axis leading to increased cell contractility and motility while TYRO3 antagonizes this effect. Consequently, pharmacologic MERTK blockade by the small molecule inhibitor R992 increases osteoblast numbers and bone formation in mice. Furthermore, R992 counteracts cancer-induced bone loss, reduces bone metastasis and prolongs survival in preclinical models of multiple myeloma, breast- and lung cancer. In summary, MERTK and TYRO3 represent potent regulators of bone homeostasis with cell-type specific functions and MERTK blockade represents an osteoanabolic therapy with implications in cancer and beyond.
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Proteínas Proto-Oncogênicas , Receptores Proteína Tirosina Quinases , Camundongos , Animais , c-Mer Tirosina Quinase/genética , c-Mer Tirosina Quinase/metabolismo , Receptores Proteína Tirosina Quinases/metabolismo , Proteínas Proto-Oncogênicas/metabolismo , Homeostase , Proteínas de TransporteRESUMO
Leukemia stem cells (LSCs) in chronic myeloid leukemia (CML) are quiescent, insensitive to BCR-ABL1 tyrosine kinase inhibitors (TKIs) and responsible for CML relapse. Therefore, eradicating quiescent CML LSCs is a major goal in CML therapy. Here, using a G0 marker (G0M), we narrow down CML LSCs as G0M- and CD27- double positive cells among the conventional CML LSCs. Whole transcriptome analysis reveals NF-κB activation via inflammatory signals in imatinib-insensitive quiescent CML LSCs. Blocking NF-κB signals by inhibitors of interleukin-1 receptor-associated kinase 1/4 (IRAK1/4 inhibitors) together with imatinib eliminates mouse and human CML LSCs. Intriguingly, IRAK1/4 inhibitors attenuate PD-L1 expression on CML LSCs, and blocking PD-L1 together with imatinib also effectively eliminates CML LSCs in the presence of T cell immunity. Thus, IRAK1/4 inhibitors can eliminate CML LSCs through inhibiting NF-κB activity and reducing PD-L1 expression. Collectively, the combination of TKIs and IRAK1/4 inhibitors is an attractive strategy to achieve a radical cure of CML.
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Quinases Associadas a Receptores de Interleucina-1/efeitos dos fármacos , Leucemia Mielogênica Crônica BCR-ABL Positiva/tratamento farmacológico , Leucemia Mieloide/tratamento farmacológico , Células-Tronco Neoplásicas/metabolismo , Animais , Antineoplásicos/farmacologia , Doença Crônica , Resistencia a Medicamentos Antineoplásicos/efeitos dos fármacos , Feminino , Proteínas de Fusão bcr-abl/metabolismo , Humanos , Mesilato de Imatinib/farmacologia , Inibidores de Checkpoint Imunológico/farmacologia , Quinases Associadas a Receptores de Interleucina-1/metabolismo , Leucemia Mielogênica Crônica BCR-ABL Positiva/genética , Leucemia Mielogênica Crônica BCR-ABL Positiva/metabolismo , Masculino , Camundongos , Pessoa de Meia-Idade , Inibidores de Proteínas Quinases/farmacologiaRESUMO
BACKGROUND: Patients with immune thrombocytopenia (ITP) are at risk of bleeding and, paradoxically, thromboembolic events (TEEs), irrespective of thrombocytopenia. The risk of thrombosis is increased by advanced age, obesity, and prothrombotic comorbidities: cancer, hyperlipidemia, diabetes, hypertension, coronary artery disease, and chronic kidney disease, among others. Certain ITP treatments further increase the risk of TEE, especially splenectomy and thrombopoietin receptor agonists. Spleen tyrosine kinase (SYK) is a key signaling molecule common to thromboembolic and hemostatic (in addition to inflammatory) pathways. Fostamatinib is an orally administered SYK inhibitor approved in the USA and Europe for treatment of chronic ITP in adults. METHODS: The phase III and extension studies included heavily pretreated patients with long-standing ITP, many of whom had risk factors for thrombosis prior to initiating fostamatinib. This report describes long-term safety and efficacy of fostamatinib in 146 patients with up to 5 years of treatment, a total of 229 patient-years, and assesses the incidence of thromboembolic events (by standardized MedDRA query). RESULTS: Platelet counts ⩾50,000/µL were achieved in 54% of patients and the safety profile was as described in the phase III clinical studies with no new toxicities observed over the 5 years of follow-up. The only TEE occurred in one patient (0.7%, or 0.44/100 patient-years), who experienced a mild transient ischemic attack. This is a much lower rate than might be expected in ITP patients. CONCLUSION: This report demonstrates durable efficacy and a very low incidence of TEE in patients receiving long-term treatment of ITP with the SYK inhibitor fostamatinib. CLINICALTRIALSGOV IDENTIFIERS: NCT02076399, NCT02076412, and NCT02077192.
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Using cultured human mast cells (CHMC) the optimization of 2,4-diaminopyrimidine compounds leading to 22, R406 is described. Compound 22 is a potent upstream inhibitor of mast cell degranulation and its mechanism of action is via inhibition of Syk kinase. Compound 22 has significant activity in inhibiting both IgE- and IgG-mediated activation of Fc receptor (FcR) in mast cells and basophils, and in addition inhibits Syk kinase-dependent activity of FcR-mediated activation of monocytes, macrophages, neutrophils, and B cell receptor (BCR)-mediated activation of B lymphocytes. Overall, the biological activity of 22 suggests that it has potential for application as a novel therapeutic for the treatment of an array of autoimmune maladies and hematological malignancies.
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Desenho de Fármacos , Imunoglobulina E/imunologia , Imunoglobulina G/imunologia , Pirimidinas/farmacologia , Receptores Fc/metabolismo , Transdução de Sinais/efeitos dos fármacosRESUMO
Controlled mechanical ventilation (CMV) is associated with the development of diaphragm atrophy and contractile dysfunction, and respiratory muscle weakness is thought to contribute significantly to delayed weaning of patients. Therefore, therapeutic strategies for preventing these processes may have clinical benefit. The aim of the current study was to investigate the role of the Janus kinase (JAK)/signal transducer and activator of transcription 3 (STAT3) signaling pathway in CMV-mediated diaphragm wasting and weakness in rats. CMV-induced diaphragm atrophy and contractile dysfunction coincided with marked increases in STAT3 phosphorylation on both tyrosine 705 (Tyr705) and serine 727 (Ser727). STAT3 activation was accompanied by its translocation into mitochondria within diaphragm muscle and mitochondrial dysfunction. Inhibition of JAK signaling during CMV prevented phosphorylation of both target sites on STAT3, eliminated the accumulation of phosphorylated STAT3 within the mitochondria, and reversed the pathologic alterations in mitochondrial function, reduced oxidative stress in the diaphragm, and maintained normal diaphragm contractility. In addition, JAK inhibition during CMV blunted the activation of key proteolytic pathways in the diaphragm, as well as diaphragm atrophy. These findings implicate JAK/STAT3 signaling in the development of diaphragm muscle atrophy and dysfunction during CMV and suggest that the delayed extubation times associated with CMV can be prevented by inhibition of Janus kinase signaling.-Smith, I. J., Godinez, G. L., Singh, B. K., McCaughey, K. M., Alcantara, R. R., Gururaja, T., Ho, M. S., Nguyen, H. N., Friera, A. M., White, K. A., McLaughlin, J. R., Hansen, D., Romero, J. M., Baltgalvis, K. A., Claypool, M. D., Li, W., Lang, W., Yam, G. C., Gelman, M. S., Ding, R., Yung, S. L., Creger, D. P., Chen, Y., Singh, R., Smuder, A. J., Wiggs, M. P., Kwon, O.-S., Sollanek, K. J., Powers, S. K., Masuda, E. S., Taylor, V. C., Payan, D. G., Kinoshita, T., Kinsella, T. M. Inhibition of Janus kinase signaling during controlled mechanical ventilation prevents ventilation-induced diaphragm dysfunction.
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Diafragma/metabolismo , Janus Quinases/metabolismo , Respiração Artificial/efeitos adversos , Transdução de Sinais/fisiologia , Animais , Interleucina-6/metabolismo , Masculino , Mitocôndrias/metabolismo , Debilidade Muscular/metabolismo , Atrofia Muscular/metabolismo , Estresse Oxidativo/fisiologia , Fosforilação/fisiologia , Proteólise , Ratos , Ratos Sprague-Dawley , Fator de Transcrição STAT3/metabolismo , Serina/metabolismo , Tirosina/metabolismoRESUMO
Allogeneic hematopoietic cell transplantation (HCT) is the most effective therapy for hematopoietic malignancies through T-cell-mediated graft-vs-leukemia (GVL) effects but often leads to severe graft-vs-host disease (GVHD). Given that protein kinase Cθ (PKCθ), in cooperation with PKCα, is essential for T-cell signaling and function, we have evaluated PKCθ and PKCα as potential therapeutic targets in allogeneic HCT using genetic and pharmacologic approaches. We found that the ability of PKCα(-/-)/θ(-/-) donor T cells to induce GVHD was further reduced compared with PKCθ(-/-) T cells in relation with the relevance of both isoforms to allogeneic donor T-cell proliferation, cytokine production, and migration to GVHD target organs. Treatment with a specific inhibitor for both PKCθ and PKCα impaired donor T-cell proliferation, migration, and chemokine/cytokine production and significantly decreased GVHD in myeloablative preclinical murine models of allogeneic HCT. Moreover, pharmacologic inhibition of PKCθ and PKCα spared T-cell cytotoxic function and GVL effects. Our findings indicate that PKCα and θ contribute to T-cell activation with overlapping functions essential for GVHD induction while less critical to the GVL effect. Thus, targeting PKCα and PKCθ signaling with pharmacologic inhibitors presents a therapeutic option for GVHD prevention while largely preserving the GVL activity in patients receiving HCT.
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Doença Enxerto-Hospedeiro/prevenção & controle , Efeito Enxerto vs Leucemia/efeitos dos fármacos , Isoenzimas/antagonistas & inibidores , Proteína Quinase C-alfa/antagonistas & inibidores , Proteína Quinase C/antagonistas & inibidores , Animais , Separação Celular , Modelos Animais de Doenças , Inibidores Enzimáticos/farmacologia , Citometria de Fluxo , Doença Enxerto-Hospedeiro/enzimologia , Transplante de Células-Tronco Hematopoéticas/efeitos adversos , Leucemia/terapia , Ativação Linfocitária/efeitos dos fármacos , Linfoma/terapia , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos C57BL , Camundongos Knockout , Proteína Quinase C-theta , Linfócitos T/efeitos dos fármacos , Linfócitos T/imunologiaRESUMO
Antibody-mediated glomerulonephritis, including that resulting from immune complexes, is an important cause of renal failure and is in need of more specific and effective treatment. Binding of antibody or immune complexes to Fc receptors activates intracellular signal transduction pathways, including spleen tyrosine kinase (Syk), leading to the production of inflammatory cytokines. We examined the effect of R788 (fostamatinib disodium), an oral prodrug of the selective Syk inhibitor R406, in nephrotoxic nephritis in Wistar-Kyoto rats. Treatment with R788 reduced proteinuria, tissue injury, glomerular macrophage and CD8+ cell numbers, and renal monocyte chemoattractant protein-1 (MCP-1) and IL-1beta, even when we started treatment after the onset of glomerulonephritis. When we administered R788 from days 4 to 10, glomerular crescents reduced by 100% (P < 0.01) compared with the vehicle group. When we administered R788 treatment from days 7 to 14, established glomerular crescents reversed (reduced by 21%, P < 0.001), and renal function was better than the vehicle group (P < 0.001). In vitro, R406 downregulated MCP-1 production from mesangial cells and macrophages stimulated with aggregated IgG. These results suggest that Syk is an important therapeutic target for the treatment of glomerulonephritis.
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Glomerulonefrite/metabolismo , Glomerulonefrite/fisiopatologia , Peptídeos e Proteínas de Sinalização Intracelular/antagonistas & inibidores , Oxazinas/farmacologia , Proteínas Tirosina Quinases/antagonistas & inibidores , Piridinas/farmacologia , Índice de Gravidade de Doença , Aminopiridinas , Animais , Células Cultivadas , Quimiocina CCL2/metabolismo , Modelos Animais de Doenças , Glomerulonefrite/patologia , Interleucina-1beta/metabolismo , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Macrófagos/efeitos dos fármacos , Macrófagos/metabolismo , Macrófagos/patologia , Células Mesangiais/efeitos dos fármacos , Células Mesangiais/metabolismo , Células Mesangiais/patologia , Morfolinas , Proteínas Tirosina Quinases/metabolismo , Pirimidinas , Ratos , Ratos Endogâmicos WKY , Transdução de Sinais/fisiologia , Quinase SykRESUMO
BACKGROUND: Urogenital abnormalities are among the most common of all human birth defects. In developmental toxicity studies with the Syk kinase inhibitor R788, a spectrum of findings, including renal agenesis, were observed. R788 has also been found to inhibit the receptor tyrosine kinase Ret. Ret kinase is known to be an essential component in the signaling pathway required for renal organogenesis and ureteric duct formation. Previously known is that mutant mice without the c-ret gene, develop urogenital malformations including renal agenesis. METHODS: In GLP developmental toxicity studies, gravid rabbits were treated orally with R788 at doses of 0, 10, 22, and 50 mg/kg/day (gestation days 7-19) and gravid rats received 0, 5, 12.5, and 25 mg/kg/day (gestation days 6-17) by the same route. The activity of R406 against Ret kinase was assessed in biochemical and cell-based assays. RESULTS: A dose-dependent increase in malformations, including renal and ureteric agenesis and a specific major vessel anomaly, retroesophageal right subclavian artery, was observed in both the rat and rabbit. R788 proved to be a potent inhibitor of Ret kinase. CONCLUSIONS: R788 promoted a spectrum of developmental toxicity, including renal and ureteric agenesis and a specific major vessel abnormality, retroesophageal right subclavian artery, in two different species. These effects are likely the result of inhibition of Ret kinase given its importance in the normal ontogeny of the urogenital and cardiovascular systems across species.
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Anormalidades Induzidas por Medicamentos/patologia , Oxazinas/toxicidade , Proteínas Proto-Oncogênicas c-ret/antagonistas & inibidores , Piridinas/toxicidade , Testes de Toxicidade , Aminopiridinas , Animais , Células Cultivadas , Desenvolvimento Embrionário/efeitos dos fármacos , Inibidores Enzimáticos/toxicidade , Feminino , Infertilidade/induzido quimicamente , Infertilidade/epidemiologia , Masculino , Morfolinas , Gravidez , Efeitos Tardios da Exposição Pré-Natal/induzido quimicamente , Efeitos Tardios da Exposição Pré-Natal/patologia , Pirimidinas , Coelhos , Ratos , Reprodução/efeitos dos fármacos , Testes de Toxicidade/métodosRESUMO
Small-molecule library screening to find compounds that inhibit TNFalpha-induced, but not interleukin 1beta (IL-1beta)-induced, intercellular adhesion molecule 1 (ICAM-1) expression in lung epithelial cells identified a class of triazoloquinoxalines. These compounds not only inhibited the TNFalpha-induced nuclear factor kappaB (NFkappaB) survival pathway but also blocked death-pathway activation. Such dual activity makes them unique against other known NFkappaB-pathway inhibitors that inhibit only a subset of TNFalpha signals leading to increased TNFalpha-induced cytotoxicity. Interestingly, these compounds inhibited association of TNFalpha receptor (TNFalphaR) I with TNFalphaR-associated death domain protein (TRADD) and receptor interacting protein 1 (RIP1), the initial intracellular signaling event following TNFalpha stimulation. Further study showed that they blocked ligand-dependent internalization of the TNFalpha-TNFalphaR complex, thereby inhibiting most of the TNFalpha-induced cellular responses. Thus, compounds with a triazoloquinoxaline scaffold could be a valuable tool to investigate small molecule-based anti-TNFalpha therapies.
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Apoptose/efeitos dos fármacos , Inibidores Enzimáticos/farmacologia , Molécula 1 de Adesão Intercelular/metabolismo , Quinoxalinas/farmacologia , Proteína Serina-Treonina Quinases de Interação com Receptores/metabolismo , Receptores Tipo I de Fatores de Necrose Tumoral/metabolismo , Proteína de Domínio de Morte Associada a Receptor de TNF/metabolismo , Triazóis/farmacologia , Fator de Necrose Tumoral alfa , Apoptose/fisiologia , Membrana Celular/química , Membrana Celular/metabolismo , Células Epiteliais/citologia , Células Epiteliais/patologia , Regulação da Expressão Gênica/genética , Regulação da Expressão Gênica/fisiologia , Molécula 1 de Adesão Intercelular/genética , Pulmão/citologia , Pulmão/patologia , Proteína Serina-Treonina Quinases de Interação com Receptores/genética , Receptores Tipo I de Fatores de Necrose Tumoral/genética , Transdução de Sinais/genética , Transdução de Sinais/fisiologia , Bibliotecas de Moléculas Pequenas , Proteína de Domínio de Morte Associada a Receptor de TNF/genéticaRESUMO
Spleen tyrosine kinase (Syk) is a novel pharmaceutical target for treatment of allergic, autoimmune, and neoplastic disorders. Previous studies have indicated that Syk signaling plays critical roles in regulating the lymphohematopoietic system. These observations prompted us to investigate whether inhibition of Syk would promote immunotoxicity. In a series of studies, rats were treated orally with R406, at dose levels up to and including 100 mg/kg/day (or its prodrug R788 at dose levels up to and including 100 mg/kg/day, reduced to 50 mg/kg/day for females as MTD was exceeded), a potent Syk inhibitor, twice daily for 28 days. In addition to standard toxicological assessments, immunophenotyping by flow cytometric analysis, and a study of humoral immune response measuring anti-KLH IgM and IgG levels, were undertaken. Other immunotoxicity studies included three host resistance models in female Balb/c mice to further ascertain effects of R406 on innate and acquired immunity. Following R406 treatment, expected immunomodulating effects (e.g., decreased thymic and spleen weight, hypocellularity of bone marrow, and reduced lymphocyte counts, including T and B cells) were observed in the rat studies. These changes essentially resolved during a 14-day treatment-free recovery period. A KLH challenge in rats demonstrated no adverse effects on IgG or IgM response. R788/406, administered orally at dose levels up to and including 80 mg/kg/day for 28 days, did not affect bacterial or viral clearance in the Listeria, Streptococcal, or Influenza host resistance mouse models, respectively. This correlated with previous in vitro macrophage and neutrophil function assays (assessing migration, phagocytosis, oxidative burst and microbicidal activity), which revealed that R406 did not adversely affect macrophage or neutrophil function in innate immune responses. Collectively, these results demonstrate that R406 has minimal functional immunotoxicity notwithstanding its lymphocytopenic effect, suggesting that inhibition of Syk might not lead to unacceptable mechanism-based adverse effects.
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Formação de Anticorpos/efeitos dos fármacos , Inibidores Enzimáticos/toxicidade , Imunocompetência/efeitos dos fármacos , Peptídeos e Proteínas de Sinalização Intracelular/efeitos dos fármacos , Oxazinas/toxicidade , Proteínas Tirosina Quinases/efeitos dos fármacos , Piridinas/toxicidade , Transdução de Sinais/efeitos dos fármacos , Animais , Infecções Bacterianas/imunologia , Medula Óssea/efeitos dos fármacos , Relação Dose-Resposta a Droga , Feminino , Imunidade Inata/efeitos dos fármacos , Imunotoxinas/toxicidade , Leucócitos/efeitos dos fármacos , Macrófagos/efeitos dos fármacos , Masculino , Dose Máxima Tolerável , Camundongos , Camundongos Endogâmicos BALB C , Pró-Fármacos/farmacologia , Ratos , Ratos Sprague-Dawley , Fatores Sexuais , Quinase SykRESUMO
Recent compelling evidence has lead to renewed interest in the role of antibodies and immune complexes in the pathogenesis of several autoimmune disorders, such as rheumatoid arthritis. These immune complexes, consisting of autoantibodies to self-antigens, can mediate inflammatory responses largely through binding and activating the immunoglobulin Fc receptors (FcRs). Using cell-based structure activity relationships with cultured human mast cells, we have identified the small molecule R406 [N4-(2,2-dimethyl-3-oxo-4H-pyrid[1,4]oxazin-6-yl)-5-fluoro-N2-(3,4,5-trimethoxyphenyl)-2,4-pyrimidinediamine] as a potent inhibitor of immunoglobulin E (IgE)- and IgG-mediated activation of Fc receptor signaling (EC(50) for degranulation = 56-64 nM). Here we show that the primary target for R406 is the spleen tyrosine kinase (Syk), which plays a key role in the signaling of activating Fc receptors and the B-cell receptor (BCR). R406 inhibited phosphorylation of Syk substrate linker for activation of T cells in mast cells and B-cell linker protein/SLP65 in B cells. R406 bound to the ATP binding pocket of Syk and inhibited its kinase activity as an ATP-competitive inhibitor (K(i) = 30 nM). Furthermore, R406 blocked Syk-dependent FcR-mediated activation of monocytes/macrophages and neutrophils and BCR-mediated activation of B lymphocytes. R406 was selective as assessed using a large panel of Syk-independent cell-based assays representing both specific and general signaling pathways. Consistent with Syk inhibition, oral administration of R406 to mice reduced immune complex-mediated inflammation in a reverse-passive Arthus reaction and two antibody-induced arthritis models. Finally, we report a first-inhuman study showing that R406 is orally bioavailable, achieving exposures capable of inhibiting Syk-dependent IgE-mediated basophil activation. Collectively, the results show R406 potential for modulating Syk activity in human disease.
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Complexo Antígeno-Anticorpo/fisiologia , Inibidores Enzimáticos/farmacologia , Inflamação/tratamento farmacológico , Oxazinas/farmacologia , Proteínas Tirosina Quinases/antagonistas & inibidores , Piridinas/farmacologia , Receptores Fc/antagonistas & inibidores , Transdução de Sinais/efeitos dos fármacos , Baço/enzimologia , Animais , Artrite Experimental/patologia , Reação de Arthus/fisiopatologia , Linfócitos B/efeitos dos fármacos , Linfócitos B/fisiologia , Basófilos/efeitos dos fármacos , Western Blotting , Células Cultivadas , Cristalografia , Método Duplo-Cego , Inibidores Enzimáticos/farmacocinética , Imunoensaio de Fluorescência por Polarização , Humanos , Imunoglobulina G/biossíntese , Imunoglobulina G/imunologia , Inflamação/patologia , Lipopolissacarídeos/farmacologia , Macrófagos/efeitos dos fármacos , Mastócitos/efeitos dos fármacos , Camundongos , Camundongos Endogâmicos BALB C , Oxazinas/farmacocinética , Agregação Plaquetária/efeitos dos fármacos , Piridinas/farmacocinética , Estimulação Química , Acetato de Tetradecanoilforbol/farmacologiaRESUMO
RATIONALE: Spleen tyrosine kinase (Syk) is important for Fc and B-cell receptor-mediated signaling. OBJECTIVE: To determine the activity of a specific Syk inhibitor (R406) on mast cell activation in vitro and on the development of allergen-induced airway hyperresponsiveness (AHR) and inflammation in vivo. METHODS: AHR and inflammation were induced after 10 d of allergen (ovalbumin [OVA]) exposure exclusively via the airways and in the absence of adjuvant. This approach was previously established to be IgE, FcepsilonRI, and mast cell dependent. Alternatively, mice were passively sensitized with OVA-specific IgE, followed by limited airway challenge. In vitro, the inhibitor was added to cultures of IgE-sensitized bone marrow-derived mast cells (BMMCs) before cross-linking with allergen. RESULTS: The inhibitor prevented OVA-induced degranulation of passively IgE-sensitized murine BMMCs and inhibited the production of interleukin (IL)-13, tumor necrosis factor alpha, IL-2, and IL-6 in these sensitized BMMCs. When administered in vivo, R406 inhibited AHR, which developed in BALB/c mice exposed to aerosolized 1% OVA for 10 consecutive d (20 min/d), as well as pulmonary eosinophilia and goblet cell metaplasia. A similar inhibition of AHR was demonstrated in mice passively sensitized with OVA-specific IgE and exposed to limited airway challenge. CONCLUSION: This study delineates a functional role for Syk in the development of mast cell- and IgE-mediated AHR and airway inflammation, and these results indicate that inhibition of Syk may be a target in the treatment of allergic asthma.
Assuntos
Hiper-Reatividade Brônquica/imunologia , Peptídeos e Proteínas de Sinalização Intracelular/antagonistas & inibidores , Mastócitos/imunologia , Inibidores de Proteínas Quinases/farmacologia , Proteínas Tirosina Quinases/antagonistas & inibidores , Animais , Linfócitos B/imunologia , Células Cultivadas , Modelos Animais de Doenças , Feminino , Peptídeos e Proteínas de Sinalização Intracelular/imunologia , Mastócitos/efeitos dos fármacos , Camundongos , Camundongos Endogâmicos BALB C , Proteínas Tirosina Quinases/imunologia , Receptores Fc/imunologia , Transdução de Sinais , Quinase SykRESUMO
We evaluated the role of Syk, using an inhibitor, on allergen-induced airway hyperresponsiveness (AHR) and airway inflammation in a system shown to be B cell- and mast cell-independent. Sensitization of BALB/c mice with ovalbumin (OVA) and alum after three consecutive OVA challenges resulted in AHR to inhaled methacholine and airway inflammation. The Syk inhibitor R406 (30 mg/kg, administered orally, twice daily) prevented the development of AHR, increases in eosinophils and lymphocytes and IL-13 levels in bronchoalveolar lavage (BAL) fluid, and goblet cell metaplasia when administered after sensitization and before challenge with OVA. Levels of IL-4, IL-5, and IFN-gamma in BAL fluid and allergen-specific antibody levels in serum were not affected by treatment. Because many of these responses may be influenced by dendritic cell function, we investigated the effect of R406 on bone marrow-derived dendritic cell (BMDC) function. Co-culture of BMDC with immune complexes of OVA and IgG anti-OVA together with OVA-sensitized spleen mononuclear cells resulted in increases in IL-13 production. IL-13 production was inhibited if the BMDCs were pretreated with the Syk inhibitor. Intratracheal transfer of immune complex-pulsed BMDCs (but not nonpulsed BMDCs) to naive mice before airway allergen challenge induced the development of AHR and increases in BAL eosinophils and lymphocytes. All of these responses were inhibited if the transferred BMDCs were pretreated with R406. These results demonstrate that Syk inhibition prevents allergen-induced AHR and airway inflammation after systemic sensitization and challenge, at least in part through alteration of DC function.
Assuntos
Hiper-Reatividade Brônquica/fisiopatologia , Células Dendríticas/enzimologia , Inflamação/enzimologia , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Proteínas Tirosina Quinases/metabolismo , Sistema Respiratório/fisiopatologia , Alérgenos , Animais , Linfócitos B/fisiologia , Células da Medula Óssea/efeitos dos fármacos , Células da Medula Óssea/metabolismo , Hiper-Reatividade Brônquica/imunologia , Hiper-Reatividade Brônquica/patologia , Células Cultivadas , Técnicas de Cocultura , Citocinas/biossíntese , Células Dendríticas/fisiologia , Ativação Enzimática , Feminino , Células Caliciformes/patologia , Inflamação/imunologia , Inflamação/patologia , Interleucina-13/biossíntese , Peptídeos e Proteínas de Sinalização Intracelular/antagonistas & inibidores , Mastócitos/fisiologia , Metaplasia , Cloreto de Metacolina , Camundongos , Camundongos Endogâmicos BALB C , Ovalbumina/imunologia , Oxazinas/farmacologia , Proteínas Tirosina Quinases/antagonistas & inibidores , Piridinas/farmacologia , Sistema Respiratório/efeitos dos fármacos , Sistema Respiratório/imunologia , Quinase SykRESUMO
TRAC-1 (T cell RING (really interesting new gene) protein identified in activation screen) is a novel E3 ubiquitin ligase identified from a retroviral vector-based T cell surface activation marker screen. The C-terminal truncated TRAC-1 specifically inhibited anti-TCR-mediated CD69 up-regulation in Jurkat cells, a human T leukemic cell line. In this study, we show that TRAC-1 is a RING finger ubiquitin E3 ligase with highest expression in lymphoid tissues. Point mutations that disrupt the Zn(2+)-chelating ability of its amino-terminal RING finger domain abolished TRAC-1's ligase activity and the dominant inhibitory effect of C-terminal truncated TRAC-1 on TCR stimulation. The results of in vitro biochemical studies indicate that TRAC-1 can stimulate the formation of both K48- and K63-linked polyubiquitin chains and therefore could potentially activate both degradative and regulatory ubiquitin-dependent pathways. Antisense oligonucleotides to TRAC-1 specifically reduced TRAC-1 mRNA levels in Jurkat and primary T cells and inhibited their activation in response to TCR cross-linking. Collectively, these results indicate that the E3 ubiquitin ligase TRAC-1 functions as a positive regulator of T cell activation.
Assuntos
Proteínas de Ciclo Celular/fisiologia , Ativação Linfocitária/imunologia , Proteínas Nucleares/fisiologia , Linfócitos T/enzimologia , Linfócitos T/imunologia , Ubiquitina-Proteína Ligases/fisiologia , Regulação para Cima/imunologia , Motivos de Aminoácidos , Sequência de Aminoácidos , Sequência de Bases , Catálise , Proteínas de Ciclo Celular/biossíntese , Proteínas de Ciclo Celular/genética , Proteínas de Ciclo Celular/isolamento & purificação , Linhagem Celular , Linhagem Celular Tumoral , Proteínas de Ligação a DNA , Humanos , Células Jurkat , Ativação Linfocitária/genética , Tecido Linfoide/citologia , Tecido Linfoide/enzimologia , Tecido Linfoide/imunologia , Dados de Sequência Molecular , Proteínas Nucleares/biossíntese , Proteínas Nucleares/genética , Proteínas Nucleares/isolamento & purificação , Correpressor 2 de Receptor Nuclear , Fragmentos de Peptídeos/genética , Fragmentos de Peptídeos/metabolismo , Poliubiquitina/metabolismo , Receptores de Antígenos de Linfócitos T/fisiologia , Proteínas Repressoras , Proteínas de Saccharomyces cerevisiae/metabolismo , Transdução de Sinais/genética , Transdução de Sinais/imunologia , Linfócitos T/metabolismo , Fatores de Transcrição/metabolismo , Enzimas de Conjugação de Ubiquitina/metabolismo , Ubiquitina-Proteína Ligases/biossíntese , Ubiquitina-Proteína Ligases/genética , Ubiquitina-Proteína Ligases/isolamento & purificação , Regulação para Cima/genéticaRESUMO
To identify novel components of the TCR signaling pathway, a large-scale retroviral-based functional screen was performed using CD69 expression as a marker for T cell activation. In addition to known regulators, two truncated forms of p21-activated kinase 2 (PAK2), PAK2DeltaL(1-224) and PAK2DeltaS(1-113), both lacking the kinase domain, were isolated in the T cell screen. The PAK2 truncation, PAK2DeltaL, blocked Ag receptor-induced NFAT activation and TCR-mediated calcium flux in Jurkat T cells. However, it had minimal effect on PMA/ionomycin-induced CD69 up-regulation in Jurkat cells, on anti-IgM-mediated CD69 up-regulation in B cells, or on the migratory responses of resting T cells to chemoattractants. We show that PAK2 kinase activity is increased in response to TCR stimulation. Furthermore, a full-length kinase-inactive form of PAK2 blocked both TCR-induced CD69 up-regulation and NFAT activity in Jurkat cells, demonstrating that kinase activity is required for PAK2 function downstream of the TCR. We also generated a GFP-fused PAK2 truncation lacking the Cdc42/Rac interactive binding region domain, GFP-PAK2(83-149). We show that this construct binds directly to the kinase domain of PAK2 and inhibits anti-TCR-stimulated T cell activation. Finally, we demonstrate that, in primary T cells, dominant-negative PAK2 prevented anti-CD3/CD28-induced IL-2 production, and TCR-induced CD40 ligand expression, both key functions of activated T cells. Taken together, these results suggest a novel role for PAK2 as a positive regulator of T cell activation.
Assuntos
Ativação Linfocitária , Proteínas Nucleares , Proteínas Serina-Treonina Quinases/imunologia , Linfócitos T/imunologia , Antígenos CD/análise , Antígenos CD/biossíntese , Antígenos de Diferenciação de Linfócitos T/análise , Antígenos de Diferenciação de Linfócitos T/biossíntese , Linfócitos B/metabolismo , Biomarcadores/análise , Linhagem Celular Tumoral , Proteínas de Ligação a DNA/metabolismo , Humanos , Lectinas Tipo C , Mutação , Fatores de Transcrição NFATC , Proteínas Serina-Treonina Quinases/genética , Proteínas Serina-Treonina Quinases/fisiologia , Estrutura Terciária de Proteína , Receptores de Antígenos de Linfócitos T/fisiologia , Transdução de Sinais , Linfócitos T/metabolismo , Fatores de Transcrição/metabolismo , Quinases Ativadas por p21RESUMO
BACKGROUND: The activation of T cells, mediated by the T-cell receptor (TCR), activates a battery of specific membrane-associated, cytosolic and nuclear proteins. Identifying the signaling proteins downstream of TCR activation will help us to understand the regulation of immune responses and will contribute to developing therapeutic agents that target immune regulation. RESULTS: In an effort to identify novel signaling molecules specific for T-cell activation we undertook a large-scale dominant effector genetic screen using retroviral technology. We cloned and characterized 33 distinct genes from over 2,800 clones obtained in a screen of 7 x 108 Jurkat T cells on the basis of a reduction in TCR-activation-induced CD69 expression after expressing retrovirally derived cDNA libraries. We identified known signaling molecules such as Lck, ZAP70, Syk, PLC gamma 1 and SHP-1 (PTP1C) as truncation mutants with dominant-negative or constitutively active functions. We also discovered molecules not previously known to have functions in this pathway, including a novel protein with a RING domain (found in a class of ubiquitin ligases; we call this protein TRAC-1), transmembrane molecules (EDG1, IL-10R alpha and integrin alpha2), cytoplasmic enzymes and adaptors (PAK2, A-Raf-1, TCPTP, Grb7, SH2-B and GG2-1), and cytoskeletal molecules (moesin and vimentin). Furthermore, using truncated Lck, PLC gamma 1, EDG1 and PAK2 mutants as examples, we showed that these dominant immune-regulatory molecules interfere with IL-2 production in human primary lymphocytes. CONCLUSIONS: This study identified important signal regulators in T-cell activation. It also demonstrated a highly efficient strategy for discovering many components of signal transduction pathways and validating them in physiological settings.
Assuntos
Ativação Linfocitária/fisiologia , Proteínas/fisiologia , Antígenos CD/fisiologia , Antígenos de Diferenciação de Linfócitos T/fisiologia , Linhagem Celular Tumoral , Regulação da Expressão Gênica , Genes/fisiologia , Humanos , Células Jurkat/química , Células Jurkat/metabolismo , Lectinas Tipo C , Receptores de Antígenos de Linfócitos T/fisiologia , Projetos de Pesquisa , Transdução de Sinais/fisiologia , Linfócitos T/fisiologia , Regulação para Cima/fisiologiaRESUMO
Inteins are polypeptide sequences found in a small set of primarily bacterial proteins that promote the splicing of flanking pre-protein sequences to generate mature protein products. Inteins can be engineered in a "split and inverted" configuration such that the protein splicing product is a cyclic polypeptide consisting of the sequence linking two intein subdomains. We have engineered a split intein into a retroviral expression system to enable the intracellular delivery of a library of random cyclic peptides in human cells. Cyclization of peptides could be detected in cell lysates using mass spectrometry. A functional genetic screen to identify 5-amino acid-long cyclic peptides that block interleukin-4 mediated IgE class switching in B cells yielded 13 peptides that selectively inhibited germ line epsilon transcription. These results demonstrate the generation of cyclic peptide libraries in human cells and the power of functional screening to rapidly identify biologically active peptides.
Assuntos
Linfócitos B/imunologia , Proteínas de Bactérias , Interleucina-4/antagonistas & inibidores , Biblioteca de Peptídeos , Peptídeos Cíclicos/química , Transdução de Sinais/fisiologia , Linfócitos B/efeitos dos fármacos , Cianobactérias/genética , DNA Helicases/genética , DNA Helicases/metabolismo , DnaB Helicases , Vetores Genéticos , Humanos , Splicing de RNA , Retroviridae , Transdução de Sinais/efeitos dos fármacos , TransfecçãoRESUMO
The regulation of SNARE complex assembly likely plays an important role in governing the specificity as well as the timing of membrane fusion. Here we identify a novel brain-enriched protein, amisyn, with a tomosyn- and VAMP-like coiled-coil-forming domain that binds specifically to syntaxin 1a and syntaxin 4 both in vitro and in vivo, as assessed by co-immunoprecipitation from rat brain. Amisyn is mostly cytosolic, but a fraction co-sediments with membranes. The amisyn coil domain can form SNARE complexes of greater thermostability than can VAMP2 with syntaxin 1a and SNAP-25 in vitro, but it lacks a transmembrane anchor and so cannot act as a v-SNARE in this complex. The amisyn coil domain prevents the SNAP-25 C-terminally mediated rescue of botulinum neurotoxin E inhibition of norepinephrine exocytosis in permeabilized PC12 cells to a greater extent than it prevents the regular exocytosis of these vesicles. We propose that amisyn forms nonfusogenic complexes with syntaxin 1a and SNAP-25, holding them in a conformation ready for VAMP2 to replace it to mediate the membrane fusion event, thereby contributing to the regulation of SNARE complex formation.