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1.
Clin Transl Sci ; 2019 Oct 24.
Artigo em Inglês | MEDLINE | ID: mdl-31654487

RESUMO

Bruton's tyrosine kinase (BTK) is a key regulator of B cell receptor and Fc receptor signaling, and a rational therapeutic target for autoimmune diseases. This first-in-human phase I, double-blind, placebo-controlled trial investigated the safety, tolerability, pharmacokinetics (PK), target occupancy, and effects on QT interval of evobrutinib, a highly selective, oral inhibitor of BTK, in healthy subjects. This dose escalation trial consisted of two parts. Part 1 included 48 subjects in 6 ascending dose cohorts (25, 50, 100, 200, 350, and 500 mg) randomized to a single dose of evobrutinib or placebo. Part 2 included 36 subjects in 3 ascending dose cohorts (25, 75, and 200 mg/day) randomized to evobrutinib or placebo once daily for 14 days. Safety and tolerability, as well as PK and target occupancy (total and free BTK in peripheral blood mononuclear cells), were assessed following single and multiple dosing. PK parameters were determined by noncompartmental methods. QT interval was obtained from 12-lead electrocardiogram recordings and corrected for heart rate by Fridericia's method (QTcF). Treatment-emergent adverse events (TEAEs) were mostly mild, occurring in 25% of subjects after single dosing, and 48.1% after multiple dosing. There was no apparent dose relationship regarding frequency or type of TEAE among evobrutinib-treated subjects. Absorption was rapid (time to reach maximum plasma concentration (Tmax ) ~ 0.5 hour), half-life short (~ 2 hours), and PK dose-proportional, with no accumulation or time dependency on repeat dosing. BTK occupancy was dose-dependent, reaching maximum occupancy of > 90% within ~ 4 hours after single doses ≥ 200 mg; the effect was long-lasting (> 50% occupancy at 96 hours with ≥ 100 mg). After multiple dosing, full BTK occupancy was achieved with 25 mg, indicating slow turnover of BTK protein in vivo. Concentration-QTcF analyses did not show any impact of evobrutinib concentration on corrected QT (QTc). In summary, evobrutinib was well-tolerated, showed linear and time-independent PK, induced long-lasting BTK inhibition, and was associated with no prolongation of QT/QTc interval in healthy subjects. Evobrutinib is, therefore, suitable for investigation in autoimmune diseases.

2.
J Med Chem ; 62(17): 7643-7655, 2019 Sep 12.
Artigo em Inglês | MEDLINE | ID: mdl-31368705

RESUMO

Bruton's tyrosine kinase (BTK) inhibitors such as ibrutinib hold a prominent role in the treatment of B cell malignancies. However, further refinement is needed to this class of agents, particularly in terms of adverse events (potentially driven by kinase promiscuity), which preclude their evaluation in nononcology indications. Here, we report the discovery and preclinical characterization of evobrutinib, a potent, obligate covalent inhibitor with high kinase selectivity. Evobrutinib displayed sufficient preclinical pharmacokinetic and pharmacodynamic characteristics which allowed for in vivo evaluation in efficacy models. Moreover, the high selectivity of evobrutinib for BTK over epidermal growth factor receptor and other Tec family kinases suggested a low potential for off-target related adverse effects. Clinical investigation of evobrutinib is ongoing in several autoimmune diseases, including multiple sclerosis, rheumatoid arthritis, and systemic lupus erythematosus.

3.
J Immunol ; 202(10): 2888-2906, 2019 05 15.
Artigo em Inglês | MEDLINE | ID: mdl-30988116

RESUMO

Because of its role in mediating both B cell and Fc receptor signaling, Bruton's tyrosine kinase (BTK) is a promising target for the treatment of autoimmune diseases such as rheumatoid arthritis (RA) and systemic lupus erythematosus (SLE). Evobrutinib is a novel, highly selective, irreversible BTK inhibitor that potently inhibits BCR- and Fc receptor-mediated signaling and, thus, subsequent activation and function of human B cells and innate immune cells such as monocytes and basophils. We evaluated evobrutinib in preclinical models of RA and SLE and characterized the relationship between BTK occupancy and inhibition of disease activity. In mouse models of RA and SLE, orally administered evobrutinib displayed robust efficacy, as demonstrated by reduction of disease severity and histological damage. In the SLE model, evobrutinib inhibited B cell activation, reduced autoantibody production and plasma cell numbers, and normalized B and T cell subsets. In the RA model, efficacy was achieved despite failure to reduce autoantibodies. Pharmacokinetic/pharmacodynamic modeling showed that mean BTK occupancy in blood cells of 80% was linked to near-complete disease inhibition in both RA and SLE mouse models. In addition, evobrutinib inhibited mast cell activation in a passive cutaneous anaphylaxis model. Thus, evobrutinib achieves efficacy by acting both on B cells and innate immune cells. Taken together, our data show that evobrutinib is a promising molecule for the chronic treatment of B cell-driven autoimmune disorders.


Assuntos
Tirosina Quinase da Agamaglobulinemia/antagonistas & inibidores , Artrite Reumatoide/tratamento farmacológico , Linfócitos B/imunologia , Lúpus Eritematoso Sistêmico/tratamento farmacológico , Ativação Linfocitária/efeitos dos fármacos , Piperidinas/farmacologia , Pirimidinas/farmacologia , Tirosina Quinase da Agamaglobulinemia/imunologia , Animais , Artrite Reumatoide/enzimologia , Artrite Reumatoide/imunologia , Artrite Reumatoide/patologia , Linfócitos B/enzimologia , Linfócitos B/patologia , Modelos Animais de Doenças , Feminino , Humanos , Lúpus Eritematoso Sistêmico/enzimologia , Lúpus Eritematoso Sistêmico/imunologia , Lúpus Eritematoso Sistêmico/patologia , Camundongos , Células U937
4.
J Am Acad Dermatol ; 81(1): 196-203, 2019 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-30926369

RESUMO

BACKGROUND: Interleukin 17 is involved in the pathogenesis of psoriasis, a chronic debilitating disease. OBJECTIVES: To evaluate the safety/tolerability, immunogenicity, pharmacokinetics/pharmacodynamics, and efficacy of M1095, an anti-interleukin 17A/F nanobody, in moderate-to-severe plaque psoriasis. METHODS: This multicenter, double-blind, placebo-controlled dose escalation phase 1 study randomized 44 patients 4:1 to treatment with subcutaneous M1095 (30, 60, 120, or 240 mg) or placebo biweekly for 6 weeks, in 4 ascending dose cohorts. RESULTS: The most frequent treatment-emergent adverse events with M1095 were pruritus (n = 4) and headache (n = 3); 2 patients withdrew owing to adverse events (injection site reaction and elevated liver enzyme levels). The terminal half-life of M1095 was 11 to 12 days. The area under the curve/maximum concentration was dose proportional. Of 10 M1095-treated patients positive for antidrug antibodies, 5 showed treatment-emergent antidrug antibody responses. There was no effect on M1095 exposure. Marked decreases in psoriasis inflammatory markers were observed with M1095. By day 85, 100% and 56% of patients receiving M1095, 240 mg, achieved psoriasis area and severity index 90 and 100, respectively. Improvements in static Physician's Global Assessment and affected body surface area were also seen. LIMITATIONS: Interpretation of efficacy data is limited by the small sample size. CONCLUSION: Multiple subcutaneous doses of M1095 showed a favorable safety profile with dose-dependent improvements in psoriasis.


Assuntos
Anticorpos Monoclonais/uso terapêutico , Interleucina-17/antagonistas & inibidores , Psoríase/diagnóstico , Psoríase/tratamento farmacológico , Adulto , Relação Dose-Resposta a Droga , Método Duplo-Cego , Esquema de Medicação , Feminino , Seguimentos , Humanos , Injeções Subcutâneas , Masculino , Dose Máxima Tolerável , Pessoa de Meia-Idade , Segurança do Paciente , Valores de Referência , Medição de Risco , Índice de Gravidade de Doença , Resultado do Tratamento , Adulto Jovem
5.
ChemMedChem ; 14(2): 217-223, 2019 01 22.
Artigo em Inglês | MEDLINE | ID: mdl-30521698

RESUMO

Bruton's tyrosine kinase (Btk) is an attractive target for the treatment of a wide array of B-cell malignancies and autoimmune diseases. Small-molecule covalent irreversible Btk inhibitors targeting Cys481 have been developed for the treatment of such diseases. In clinical trials, probe molecules are required in occupancy studies to measure the level of engagement of the protein by these covalent irreversible inhibitors. The result of this pharmacodynamic (PD) activity provides guidance for appropriate dosage selection to optimize inhibition of the drug target and correlation of target inhibition with disease treatment efficacy. This information is crucial for successful evaluation of drug candidates in clinical trials. Based on the pyridine carboxamide scaffold of a novel solvent-accessible pocket (SAP) series of covalent irreversible Btk inhibitors, we successfully developed a potent and selective affinity-based biotinylated probe 12 (2-[(4-{4-[5-(1-{5-[(3aS,4S,6aR)-2-oxo-hexahydro-1H-thieno[3,4-d]imidazol-4-yl]pentanamido}-3,6,9,12-tetraoxapentadecan-15-amido)pentanoyl]piperazine-1-carbonyl}phenyl)amino]-6-[1-(prop-2-enoyl)piperidin-4-yl]pyridine-3-carboxamide). Compound 12 has been used in Btk occupancy assays for preclinical studies to determine the therapeutic efficacy of Btk inhibition in two mouse lupus models driven by TLR7 activation and type I interferon.


Assuntos
Bioensaio/métodos , Piperazinas/química , Inibidores de Proteínas Quinases/análise , Piridinas/química , Tirosina Quinase da Agamaglobulinemia/antagonistas & inibidores , Animais , Biotina/química , Camundongos , Modelos Animais , Estrutura Molecular , Piperazinas/síntese química , Inibidores de Proteínas Quinases/metabolismo , Piridinas/síntese química , Relação Estrutura-Atividade
6.
Bioorg Med Chem Lett ; 28(21): 3419-3424, 2018 11 15.
Artigo em Inglês | MEDLINE | ID: mdl-30290988

RESUMO

Btk is an attractive target for the treatment of a range of Bcell malignancies as well as several autoimmune diseases such as murine lupus and rheumatoid arthritis. Several covalent irreversible inhibitors of Btk are currently in development including ibrutinib which was approved for treatment of B-cell malignancies. Herein, we describe our efforts using X-ray guided structure based design (SBD) to identify a novel chemical series of covalent Btk inhibitors. The resulting pyridine carboxamides were potent and selective inhibitors of Btk having excellent enzymatic and cellular inhibitory activity.


Assuntos
Tirosina Quinase da Agamaglobulinemia/antagonistas & inibidores , Inibidores de Proteínas Quinases/farmacologia , Piridinas/farmacologia , Pirimidinas/farmacologia , Administração Oral , Animais , Células CACO-2 , Humanos , Camundongos , Estrutura Molecular , Inibidores de Proteínas Quinases/administração & dosagem , Inibidores de Proteínas Quinases/síntese química , Inibidores de Proteínas Quinases/química , Pirazóis/farmacologia , Piridinas/administração & dosagem , Piridinas/síntese química , Piridinas/química , Pirimidinas/administração & dosagem , Pirimidinas/síntese química , Pirimidinas/química , Relação Estrutura-Atividade
7.
Bioorg Med Chem Lett ; 28(20): 3307-3311, 2018 11 01.
Artigo em Inglês | MEDLINE | ID: mdl-30243592

RESUMO

Bruton's tyrosine kinase (Btk) is a member of the Tec kinase family that is expressed in cells of hematopoietic lineage (e.g. B cells, macrophages, monocytes, and mast cells). Small molecule covalent irreversible Btk inhibitors targeting Cys481 within the ATP-binding pocket have been applied in the treatment of B-cell malignancies. Starting from a fragment, we discovered a novel series of potent covalent irreversible Btk inhibitors that bear N-linked groups occupying the solvent accessible pocket (SAP) of the active site of the Btk kinase domain. The hit molecules, however, displayed high P-gp mediated efflux ratio (ER) and poor A-B permeability in Caco-2 assay. By decreasing tPSA, installing steric hindrance and adjusting clogP, one top molecule 9 was discovered, which showed a 99% decrease in efflux ratio and a 90-fold increase in A-B permeability compared to hit molecule 1.


Assuntos
Tirosina Quinase da Agamaglobulinemia/antagonistas & inibidores , Niacinamida/farmacologia , Inibidores de Proteínas Quinases/farmacologia , Tirosina Quinase da Agamaglobulinemia/química , Animais , Células CACO-2 , Domínio Catalítico , Humanos , Camundongos , Estrutura Molecular , Niacinamida/análogos & derivados , Niacinamida/síntese química , Niacinamida/farmacocinética , Permeabilidade , Inibidores de Proteínas Quinases/síntese química , Inibidores de Proteínas Quinases/farmacocinética , Pirazóis/farmacologia , Pirimidinas/farmacologia
8.
Bioorg Med Chem Lett ; 28(17): 2939-2944, 2018 09 15.
Artigo em Inglês | MEDLINE | ID: mdl-30122225

RESUMO

Bruton's Tyrosine Kinase (BTK) is a member of the TEC kinase family that is expressed in cells of hematopoietic lineage (e.g., in B cells, macrophages, monocytes, and mast cells). Small molecule covalent irreversible BTK inhibitor targeting Cys481 within the ATP-binding pocket, for example ibrutinib, has been applied in the treatment of B-cell malignancies. Starting from a fragment hit, we discovered a novel series of potent covalent irreversible BTK inhibitors that occupy selectivity pocket of the active site of the BTK kinase domain. Guided by X-ray structures and a fragment-based drug design (FBDD) approach, we generated molecules showing comparable cellular potency to ibrutinib and higher kinome selectivity against undesirable off-targets like EGFR.


Assuntos
Tirosina Quinase da Agamaglobulinemia/antagonistas & inibidores , Descoberta de Drogas , Inibidores de Proteínas Quinases/farmacologia , Tirosina Quinase da Agamaglobulinemia/metabolismo , Cristalografia por Raios X , Relação Dose-Resposta a Droga , Humanos , Modelos Moleculares , Estrutura Molecular , Inibidores de Proteínas Quinases/síntese química , Inibidores de Proteínas Quinases/química , Relação Estrutura-Atividade
9.
Mol Pharmacol ; 91(3): 208-219, 2017 03.
Artigo em Inglês | MEDLINE | ID: mdl-28062735

RESUMO

Bruton's tyrosine kinase (Btk) is expressed in a variety of hematopoietic cells. Btk has been demonstrated to regulate signaling downstream of the B-cell receptor (BCR), Fc receptors (FcRs), and toll-like receptors. It has become an attractive drug target because its inhibition may provide significant efficacy by simultaneously blocking multiple disease mechanisms. Consequently, a large number of Btk inhibitors have been developed. These compounds have diverse binding modes, and both reversible and irreversible inhibitors have been developed. Reported herein, we have tested nine Btk inhibitors and characterized on a molecular level how their interactions with Btk define their ability to block different signaling pathways. By solving the crystal structures of Btk inhibitors bound to the enzyme, we discovered that the compounds can be classified by their ability to trigger sequestration of Btk residue Y551. In cells, we found that sequestration of Y551 renders it inaccessible for phosphorylation. The ability to sequester Y551 was an important determinant of potency against FcεR signaling as Y551 sequestering compounds were more potent for inhibiting basophils and mast cells. This result was true for the inhibition of FcγR signaling as well. In contrast, Y551 sequestration was less a factor in determining potency against BCR signaling. We also found that Btk activity is regulated differentially in basophils and B cells. These results elucidate important determinants for Btk inhibitor potency against different signaling pathways and provide insight for designing new compounds with a broader inhibitory profile that will likely result in greater efficacy.


Assuntos
Inibidores de Proteínas Quinases/farmacologia , Proteínas Tirosina Quinases/antagonistas & inibidores , Receptores de Antígenos de Linfócitos B/metabolismo , Receptores Fc/metabolismo , Transdução de Sinais/efeitos dos fármacos , Tirosina/metabolismo , Tirosina Quinase da Agamaglobulinemia , Linhagem Celular Tumoral , Análise por Conglomerados , Cristalografia por Raios X , Ativação Enzimática/efeitos dos fármacos , Humanos , Modelos Moleculares , Proteínas Mutantes/metabolismo , Fosforilação/efeitos dos fármacos , Inibidores de Proteínas Quinases/química , Proteínas Tirosina Quinases/química , Proteínas Tirosina Quinases/metabolismo
10.
Clin Immunol ; 164: 65-77, 2016 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-26821304

RESUMO

Bruton's tyrosine kinase (Btk) is expressed in a variety of immune cells and previous work has demonstrated that blocking Btk is a promising strategy for treating autoimmune diseases. Herein, we utilized a tool Btk inhibitor, M7583, to determine the therapeutic efficacy of Btk inhibition in two mouse lupus models driven by TLR7 activation and type I interferon. In BXSB-Yaa lupus mice, Btk inhibition reduced autoantibodies, nephritis, and mortality. In the pristane-induced DBA/1 lupus model, Btk inhibition suppressed arthritis, but autoantibodies and the IFN gene signature were not significantly affected; suggesting efficacy was mediated through inhibition of Fc receptors. In vitro studies using primary human macrophages revealed that Btk inhibition can block activation by immune complexes and TLR7 which contributes to tissue damage in SLE. Overall, our results provide translational insight into how Btk inhibition may provide benefit to a variety of SLE patients by affecting both BCR and FcR signaling.


Assuntos
Lúpus Eritematoso Sistêmico/tratamento farmacológico , Inibidores de Proteínas Quinases/uso terapêutico , Proteínas Tirosina Quinases/antagonistas & inibidores , Tirosina Quinase da Agamaglobulinemia , Animais , Artrite/tratamento farmacológico , Artrite/patologia , Autoanticorpos/sangue , Modelos Animais de Doenças , Feminino , Articulações do Pé/efeitos dos fármacos , Articulações do Pé/patologia , Humanos , Imunossupressores , Interferon Tipo I/imunologia , Rim/efeitos dos fármacos , Rim/patologia , Lúpus Eritematoso Sistêmico/induzido quimicamente , Lúpus Eritematoso Sistêmico/imunologia , Lúpus Eritematoso Sistêmico/patologia , Macrófagos/efeitos dos fármacos , Macrófagos/imunologia , Masculino , Camundongos Endogâmicos C57BL , Camundongos Endogâmicos DBA , Nefrite/tratamento farmacológico , Nefrite/patologia , Inibidores de Proteínas Quinases/farmacocinética , Inibidores de Proteínas Quinases/farmacologia , Proteinúria/tratamento farmacológico , Proteinúria/patologia , Terpenos , Receptor 7 Toll-Like/imunologia
11.
Front Immunol ; 5: 233, 2014.
Artigo em Inglês | MEDLINE | ID: mdl-24904582

RESUMO

SLE is a complex autoimmune inflammatory disease characterized by pathogenic autoantibody production as a consequence of uncontrolled T-B cell activity and immune-complex deposition in various organs, including kidney, leading to tissue damage and function loss. There is a high unmet need for better treatment options other than corticosteroids and immunosuppressants. Phosphoinositol-3 kinase δ (PI3Kδ) is a promising target in this respect as it is essential in mediating B- and T-cell function in mouse and human. We report the identification of selective PI3Kδ inhibitors that blocked B-, T-, and plasmacytoid dendritic cell activities in human peripheral blood and in primary cell co-cultures (BioMAP(®)) without detecting signs of undesired toxicity. In an IFNα-accelerated mouse SLE model, our PI3Kδ inhibitors blocked nephritis development, whether administered at the onset of autoantibody appearance or the onset of proteinuria. Disease amelioration correlated with normalized immune cell numbers in the spleen, reduced immune-complex deposition as well as reduced inflammation, fibrosis, and tissue damage in the kidney. Improvements were similar to those achieved with a frequently prescribed drug for lupus nephritis, the potent immunosuppressant mycophenolate mofetil. Finally, we established a pharmacodynamics/pharmacokinetic/efficacy model that revealed that a sustained PI3Kδ inhibition of 50% is sufficient to achieve full efficacy in our disease model. These data demonstrate the therapeutic potential of PI3Kδ inhibitors in SLE and lupus nephritis.

12.
Proc Natl Acad Sci U S A ; 110(39): 15776-81, 2013 Sep 24.
Artigo em Inglês | MEDLINE | ID: mdl-24019486

RESUMO

E26 transformation-specific sequence 1 (Ets-1), the prototype of the ETS family of transcription factors, is critical for the expression of IL-2 by murine Th cells; however, its mechanism of action is still unclear. Here we show that Ets-1 is also essential for optimal production of IL-2 by primary human Th cells. Although Ets-1 negatively regulates the expression of Blimp1, a known suppressor of IL-2 expression, ablation of B lymphocyte-induced maturation protein 1 (Blimp1) does not rescue the expression of IL-2 by Ets-1-deficient Th cells. Instead, Ets-1 physically and functionally interacts with the nuclear factor of activated T-cells (NFAT) and is required for the recruitment of NFAT to the IL-2 promoter. In addition, Ets-1 is located in both the nucleus and cytoplasm of resting Th cells. Nuclear Ets-1 quickly exits the nucleus in response to calcium-dependent signals and competes with NFAT proteins for binding to protein components of noncoding RNA repressor of NFAT complex (NRON), which serves as a cytoplasmic trap for phosphorylated NFAT proteins. This nuclear exit of Ets-1 precedes rapid nuclear entry of NFAT and Ets-1 deficiency results in impaired nuclear entry, but not dephosphorylation, of NFAT proteins. Thus, Ets-1 promotes the expression of IL-2 by modulating the activity of NFAT.


Assuntos
Núcleo Celular/metabolismo , Interleucina-2/genética , Fatores de Transcrição NFATC/metabolismo , Regiões Promotoras Genéticas , Proteína Proto-Oncogênica c-ets-1/metabolismo , Animais , Sequência de Bases , Cálcio/metabolismo , Técnicas de Inativação de Genes , Humanos , Interleucina-2/biossíntese , Camundongos , Dados de Sequência Molecular , Complexos Multiproteicos/metabolismo , Fator 1 de Ligação ao Domínio I Regulador Positivo , Ligação Proteica/genética , Transporte Proteico , Proteína Proto-Oncogênica c-ets-1/deficiência , Transdução de Sinais , Células Th1/metabolismo , Fatores de Transcrição/deficiência , Fatores de Transcrição/metabolismo
13.
J Immunol ; 188(5): 2244-53, 2012 Mar 01.
Artigo em Inglês | MEDLINE | ID: mdl-22266280

RESUMO

IL-10 is a multifunctional cytokine that plays a crucial role in immunity and tolerance. IL-10 is produced by diverse immune cell types, including B cells and subsets of T cells. Although Th1 produce IL-10, their expression levels are much lower than Th2 cells under conventional stimulation conditions. The potential role of E26 transformation-specific 1 (Ets-1) transcription factor as a negative regulator for Il10 gene expression in CD4(+) T cells has been implicated previously. In this study, we investigated the underlying mechanism of Ets-1-mediated Il10 gene repression in Th1 cells. Compared with wild type Th1 cells, Ets-1 knockout Th1 cells expressed a significantly higher level of IL-10, which is comparable with that of wild type Th2 cells. Upregulation of IL-10 expression in Ets-1 knockout Th1 cells was accompanied by enhanced chromatin accessibility and increased recruitment of histone H3 acetylation at the Il10 regulatory regions. Reciprocally, Ets-1 deficiency significantly decreased histone deacetylase 1 (HDAC1) enrichment at the Il10 regulatory regions. Treatment with trichostatin A, an inhibitor of HDAC family, significantly increased Il10 gene expression by increasing histone H3 acetylation recruitment. We further demonstrated a physical interaction between Ets-1 and HDAC1. Coexpression of Ets-1 with HDAC1 synergistically repressed IL-10 transcription activity. In summary, our data suggest that an interaction of Ets-1 with HDAC1 represses the Il10 gene expression in Th1 cells.


Assuntos
Regulação para Baixo/imunologia , Regulação da Expressão Gênica/imunologia , Histona Desacetilase 1/fisiologia , Interleucina-10/antagonistas & inibidores , Interleucina-10/biossíntese , Proteína Proto-Oncogênica c-ets-1/fisiologia , Células Th1/imunologia , Células Th1/metabolismo , Animais , Diferenciação Celular/genética , Diferenciação Celular/imunologia , Células Cultivadas , Regulação para Baixo/genética , Células HEK293 , Histona Desacetilase 1/antagonistas & inibidores , Histona Desacetilase 1/metabolismo , Humanos , Interleucina-10/genética , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos C57BL , Camundongos Knockout , Proteína Proto-Oncogênica c-ets-1/deficiência , Proteína Proto-Oncogênica c-ets-1/metabolismo , Células Th1/citologia , Regulação para Cima/genética , Regulação para Cima/imunologia
14.
J Immunol ; 186(2): 969-76, 2011 Jan 15.
Artigo em Inglês | MEDLINE | ID: mdl-21148801

RESUMO

The expression of CD127, the IL-7-binding subunit of the IL-7 R, is tightly regulated during the development and activation of T cells and is reduced during chronic viral infection. However, the molecular mechanism regulating the dynamic expression of CD127 is still poorly understood. In this study, we report that the transcription factor Ets-1 is required for maintaining the expression of CD127 in murine peripheral T cells. Ets-1 binds to and activates the CD127 promoter, and its absence leads to reduced CD127 expression, attenuated IL-7 signaling, and impaired IL-7-dependent homeostatic proliferation of T cells. The expression of CD127 and Ets-1 is strongly correlated in human T cells. Both CD127 and Ets-1 expression are decreased in CD8(+) T cells during HIV infection. In addition, HIV-associated loss of CD127 is only observed in Ets-1(low) effector memory and central memory but not in Ets-1(high) naive CD8(+) T cells. Taken together, our data identify Ets-1 as a critical regulator of CD127 expression in T cells.


Assuntos
Interleucina-7/biossíntese , Proteína Proto-Oncogênica c-ets-1/fisiologia , Subpopulações de Linfócitos T/imunologia , Subpopulações de Linfócitos T/metabolismo , Animais , Células Cultivadas , Feminino , HIV-1/imunologia , Humanos , Interleucina-7/genética , Subunidade alfa de Receptor de Interleucina-7/biossíntese , Subunidade alfa de Receptor de Interleucina-7/genética , Subunidade alfa de Receptor de Interleucina-7/metabolismo , Masculino , Camundongos , Camundongos Knockout , Regiões Promotoras Genéticas/imunologia , Ligação Proteica/imunologia , Proteína Proto-Oncogênica c-ets-1/deficiência , Proteína Proto-Oncogênica c-ets-1/genética , Proteína Proto-Oncogênica c-ets-1/metabolismo , Subpopulações de Linfócitos T/virologia
15.
Eur J Immunol ; 40(4): 1174-84, 2010 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-20127678

RESUMO

The function of transcription factors can be critically regulated by SUMOylation. c-Maf, the cellular counterpart of v-maf oncogene, is a potent transactivator of the IL-4 gene in Th2 cells. We found in a yeast two-hybrid screen that c-Maf can interact with Ubc9 and PIAS1, two key enzymes of the SUMOylation pathway. In this study, we report that c-Maf co-localized with these two SUMO (small ubiquitin-like modifier) ligases in the nucleus and that c-Maf can be SUMOylated in vitro and also in primary Th2 cells. We also demonstrated that lysine-33 is the dominant, if not the only, SUMO acceptor site of c-Maf. SUMOylation of c-Maf attenuated its transcriptional activity. Reciprocally, a SUMOylation resistant c-Maf was more potent than WT-c-Maf in driving IL-4 production in c-Maf-deficient Th2 cells. Furthermore, we showed that ablation of the SUMO site did not alter the subcellular localization or the stability of c-Maf protein but instead enhanced its recruitment to the Il4-promoter. We conclude that SUMOylation at lysine-33 is a functionally critical post-translational modification event of c-Maf in Th cells.


Assuntos
Interleucina-4/biossíntese , Proteínas Inibidoras de STAT Ativados/fisiologia , Processamento de Proteína Pós-Traducional , Proteínas Proto-Oncogênicas c-maf/fisiologia , Proteínas Modificadoras Pequenas Relacionadas à Ubiquitina/fisiologia , Células Th2/metabolismo , Enzimas de Conjugação de Ubiquitina/fisiologia , Sequência de Aminoácidos , Animais , Linhagem Celular , Células Cultivadas/metabolismo , Humanos , Interleucina-4/genética , Rim , Lisina/química , Camundongos , Dados de Sequência Molecular , Proteínas Inibidoras de STAT Ativados/química , Proteínas Inibidoras de STAT Ativados/isolamento & purificação , Mapeamento de Interação de Proteínas , Proteínas Proto-Oncogênicas c-maf/química , Proteínas Recombinantes de Fusão/fisiologia , Alinhamento de Sequência , Homologia de Sequência de Aminoácidos , Proteínas Modificadoras Pequenas Relacionadas à Ubiquitina/química , Proteínas Modificadoras Pequenas Relacionadas à Ubiquitina/genética , Proteínas Modificadoras Pequenas Relacionadas à Ubiquitina/isolamento & purificação , Transcrição Genética , Técnicas do Sistema de Duplo-Híbrido , Enzimas de Conjugação de Ubiquitina/química
16.
J Immunol ; 184(3): 1309-16, 2010 Feb 01.
Artigo em Inglês | MEDLINE | ID: mdl-20038639

RESUMO

The Th2 cytokine gene locus has emerged as a remarkable example of coordinated gene expression, the regulation of which seems to be rooted in an extensive array of cis-regulatory regions. Using a hypothesis-generating computational approach that integrated multispecies (n = 11) sequence comparisons with algorithm-based transcription factor binding-site predictions, we sought to identify evolutionarily conserved noncoding regions (ECRs) and motifs shared among them, which may underlie coregulation. Twenty-two transcription factor families were predicted to have binding sites in at least two Th2 ECRs. The ranking of these shared motifs according to their distribution and relative frequency pointed to a regulatory hierarchy among the transcription factor families. GATA sites were the most prevalent and widely distributed, consistent with the known role of GATA3 as a Th2 master switch. Unexpectedly, sites for ETS-domain proteins were also predicted within several Th2 ECRs and the majority of these sites were found to support Ets-1 binding in vitro and in vivo. Of note, the expression of all three Th2 cytokines (IL-5, -13, and -4) was significantly and selectively decreased in Th2 cells generated from Ets-1-deficient mice. Collectively, these data suggest that Ets-1 contributes to Th2 cytokine gene regulation by interacting with multiple cis-regulatory regions throughout the Th2 locus.


Assuntos
Citocinas/biossíntese , Citocinas/genética , Regulação da Expressão Gênica/imunologia , Loci Gênicos/imunologia , Filogenia , Proteína Proto-Oncogênica c-ets-1/fisiologia , Células Th2/imunologia , Células Th2/metabolismo , Animais , Bovinos , Galinhas , Sequência Conservada , Cães , Evolução Molecular , Feminino , Humanos , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Proteína Proto-Oncogênica c-ets-1/biossíntese , Proteína Proto-Oncogênica c-ets-1/deficiência , Proteína Proto-Oncogênica c-ets-1/genética , Ratos
17.
J Exp Med ; 206(12): 2685-99, 2009 Nov 23.
Artigo em Inglês | MEDLINE | ID: mdl-19917777

RESUMO

The transcription factor Ets1 contributes to the differentiation of CD8 lineage cells in the thymus, but how it does so is not understood. In this study, we demonstrate that Ets1 is required for the proper termination of CD4 expression during the differentiation of major histocompatability class 1 (MHC I)-restricted thymocytes, but not for other events associated with their positive selection, including the initiation of cytotoxic gene expression, corticomedullary migration, or thymus exit. We further show that Ets1 promotes expression of Runx3, a transcription factor important for CD8 T cell differentiation and the cessation of Cd4 gene expression. Enforced Runx3 expression in Ets1-deficient MHC I-restricted thymocytes largely rescued their impaired Cd4 silencing, indicating that Ets1 is not required for Runx3 function. Finally, we document that Ets1 binds at least two evolutionarily conserved regions within the Runx3 gene in vivo, supporting the possibility that Ets1 directly contributes to Runx3 transcription. These findings identify Ets1 as a key player during CD8 lineage differentiation and indicate that it acts, at least in part, by promoting Runx3 expression.


Assuntos
Antígenos CD4/imunologia , Linfócitos T CD8-Positivos/imunologia , Diferenciação Celular/imunologia , Subunidade alfa 3 de Fator de Ligação ao Core/imunologia , Proteína Proto-Oncogênica c-ets-1/imunologia , Timo/imunologia , Regulação para Cima/imunologia , Animais , Antígenos CD4/genética , Linfócitos T CD8-Positivos/citologia , Diferenciação Celular/genética , Subunidade alfa 3 de Fator de Ligação ao Core/genética , Antígenos de Histocompatibilidade Classe I/genética , Antígenos de Histocompatibilidade Classe I/imunologia , Camundongos , Camundongos Knockout , Proteína Proto-Oncogênica c-ets-1/genética , Elementos de Resposta/genética , Elementos de Resposta/imunologia , Timo/citologia , Transcrição Genética/genética , Transcrição Genética/imunologia , Regulação para Cima/genética
18.
Eur J Immunol ; 38(6): 1700-5, 2008 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-18465773

RESUMO

The transcription factor Ets-1 critically regulates differentiation and function of T helper (Th) cells. In vitro studies have demonstrated that DNA binding and transcriptional activity of Ets-1 are regulated by phosphorylation. Depending on the site of phosphorylation, Ets-1 function can either be increased or inhibited. In addition, a splice variant lacking several inhibitory phosphorylation sites has been identified, raising the possibility that this splice variant may function differently from the full-length Ets-1. However, it is unclear how the activating and inhibitory phosphorylation events of Ets-1 are coordinated during Th cell activation. Furthermore, the biological consequences of Ets-1 phosphorylation and alternative splicing in regulating the function of Th cells are unknown. We report here that both activating and inhibitory phosphorylation events of Ets-1 occur simultaneously and independently of each other during Th cell activation. We further demonstrate that the effect of Ets-1 phosphorylation is very modest and that full-length Ets-1 and its splice variant are functionally interchangeable in the regulation of cytokine production in Th cells.


Assuntos
Proteína Proto-Oncogênica c-ets-1/metabolismo , Linfócitos T Auxiliares-Indutores/metabolismo , Células Th1/metabolismo , Animais , Anticorpos/imunologia , Anticorpos/farmacologia , Apresentação do Antígeno/imunologia , Western Blotting , Complexo CD3/imunologia , Interferon gama/metabolismo , Interleucina-10/metabolismo , Interleucina-2/metabolismo , Ionomicina/farmacologia , Cinética , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Ovalbumina/imunologia , Fosforilação/efeitos dos fármacos , Isoformas de Proteínas/genética , Isoformas de Proteínas/metabolismo , Proteína Proto-Oncogênica c-ets-1/genética , Receptores de Antígenos de Linfócitos T/genética , Transdução de Sinais/efeitos dos fármacos , Linfócitos T Auxiliares-Indutores/efeitos dos fármacos , Acetato de Tetradecanoilforbol/farmacologia , Células Th1/efeitos dos fármacos , Células Th2/efeitos dos fármacos , Células Th2/metabolismo , Transfecção
19.
J Exp Med ; 204(12): 2825-35, 2007 Nov 26.
Artigo em Inglês | MEDLINE | ID: mdl-17967903

RESUMO

IL-17 is a proinflammatory cytokine that plays a role in the clearance of extracellular bacteria and contributes to the pathology of many autoimmune and allergic conditions. IL-17 is produced mainly by a newly characterized subset of T helper (Th) cells termed Th17. Although the role of Th17 cells in the pathology of autoimmune diseases is well established, the transcription factors regulating the differentiation of Th17 cells remain poorly characterized. We report that Ets-1-deficient Th cells differentiated more efficiently to Th17 cells than wild-type cells. This was attributed to both low IL-2 production and increased resistance to the inhibitory effect of IL-2 on Th17 differentiation. The resistance to IL-2 suppression was caused by a defect downstream of STAT5 phosphorylation, but was not caused by a difference in the level of RORgamma t. Furthermore, Ets-1-deficient mice contained an abnormally high level of IL-17 transcripts in their lungs and exhibited increased mucus production by airway epithelial cells in an IL-17-dependent manner. Based on these observations, we report that Ets-1 is a negative regulator of Th17 differentiation.


Assuntos
Proteína Proto-Oncogênica c-ets-1/genética , Linfócitos T Auxiliares-Indutores/imunologia , Animais , Diferenciação Celular , Éxons , Interleucina-17/genética , Ionomicina/farmacologia , Camundongos , Camundongos Knockout , Proteína Proto-Oncogênica c-ets-1/deficiência , Linfócitos T Auxiliares-Indutores/citologia , Linfócitos T Auxiliares-Indutores/efeitos dos fármacos , Acetato de Tetradecanoilforbol/farmacologia , Células Th1/efeitos dos fármacos , Células Th1/imunologia , Células Th2/efeitos dos fármacos , Células Th2/imunologia
20.
J Immunol ; 176(9): 5161-6, 2006 May 01.
Artigo em Inglês | MEDLINE | ID: mdl-16621979

RESUMO

Among the many factors regulating Th cell differentiation, some nuclear hormone receptors are emerging as important players. The retinoid X receptor (RXR) functions as heterodimerization partner for a variety of nuclear hormone receptors. We show in this study that RXR is critical for Th2-mediated immunity. An RXR antagonist inhibited Th2 differentiation, resulting in reduced production of IL-4, IL-10, and IL-13, whereas IFN-gamma production was enhanced. This effect was dependent on the presence of APCs. In addition, IL-5 production was blocked directly in Th cells. In vivo, inhibition of RXR prevented experimentally induced allergic lung inflammation. Th1-mediated inflammation was not affected. Its specific role in Th2-mediated inflammation makes RXR a promising target for the development of therapies against diseases such as allergic asthma and atopic dermatitis.


Assuntos
Diferenciação Celular/efeitos dos fármacos , Hipersensibilidade/prevenção & controle , Pneumonia/patologia , Pneumonia/prevenção & controle , Receptores X Retinoide/antagonistas & inibidores , Células Th2/citologia , Células Th2/efeitos dos fármacos , Animais , Células Apresentadoras de Antígenos/citologia , Células Apresentadoras de Antígenos/efeitos dos fármacos , Células Cultivadas , Hipersensibilidade/metabolismo , Hipersensibilidade/patologia , Interferon gama/deficiência , Interferon gama/genética , Interferon gama/metabolismo , Interleucina-12/deficiência , Interleucina-12/genética , Interleucina-12/metabolismo , Interleucina-5/biossíntese , Camundongos , Camundongos Knockout , Estrutura Molecular , Pneumonia/metabolismo , Receptores X Retinoide/metabolismo , Linfócitos T/efeitos dos fármacos , Linfócitos T/metabolismo
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