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1.
Int J Mol Sci ; 20(17)2019 Aug 30.
Artigo em Inglês | MEDLINE | ID: mdl-31480368

RESUMO

THP-1 cells express high levels of native functional nucleotide-binding oligomerization domain 1 (NOD1), NOD2, and Toll-like receptor 4 (TLR4) receptors, and have often been used for investigating the immunomodulatory effects of small molecules. We postulated that they would represent an ideal cell-based model for our study, the aim of which was to develop a new in vitro tool for functional characterization of NOD antagonists. NOD antagonists were initially screened for their effect on NOD agonist-induced interleukin-8 (IL-8) release. Next, we examined the extent to which the selected NOD antagonists block the NOD-TLR4 synergistic crosstalk by measuring the effect of NOD antagonism on tumor necrosis factor-α (TNF-α) secretion from doubly activated THP-1 cells. Overall, the results obtained indicate that pro-inflammatory cytokine secretion from THP-1 provides a valuable, simple and reproducible in vitro tool for functional characterization of NOD antagonists.


Assuntos
Citocinas/biossíntese , Mediadores da Inflamação/metabolismo , Proteína Adaptadora de Sinalização NOD1/antagonistas & inibidores , Proteína Adaptadora de Sinalização NOD2/antagonistas & inibidores , Sobrevivência Celular , Humanos , Proteína Adaptadora de Sinalização NOD1/agonistas , Proteína Adaptadora de Sinalização NOD1/metabolismo , Proteína Adaptadora de Sinalização NOD2/agonistas , Proteína Adaptadora de Sinalização NOD2/metabolismo , Células THP-1 , Receptor 4 Toll-Like/metabolismo
2.
J Med Chem ; 61(4): 1519-1540, 2018 02 22.
Artigo em Inglês | MEDLINE | ID: mdl-29357251

RESUMO

Salutaxel (3) is a conjugate of docetaxel (7) and a muramyl dipeptide (MDP) analogue. Docetaxel (7) has been recognized as a highly active chemotherapeutic agent against various cancers. MDP and its analogues are powerful potentiators of the antitumor actions of various tumor-necrotizing agents. This article documents the discovery of compound 3 and presents pharmacological proof of its biological function in tumor-bearing mice. Drug candidate 3 was superior to compound 7 in its ability to prevent tumor growth and metastasis. Compound 3 suppressed myeloid-derived suppressor cell (MDSC) accumulation in the spleens of tumor-bearing mice and decreased various serum inflammatory cytokines levels. Furthermore, compound 3 antagonized the nucleotide-binding oligomerization domain-like receptor 1 (NOD1) signaling pathway both in vitro and in vivo.


Assuntos
Acetilmuramil-Alanil-Isoglutamina/química , Docetaxel/química , Metástase Neoplásica/tratamento farmacológico , Neoplasias/tratamento farmacológico , Pró-Fármacos/síntese química , Acetilmuramil-Alanil-Isoglutamina/análogos & derivados , Acetilmuramil-Alanil-Isoglutamina/uso terapêutico , Animais , Antineoplásicos/síntese química , Antineoplásicos/farmacologia , Linhagem Celular , Citocinas/sangue , Citocinas/efeitos dos fármacos , Docetaxel/uso terapêutico , Humanos , Camundongos , Células Supressoras Mieloides/efeitos dos fármacos , Neoplasias/patologia , Proteína Adaptadora de Sinalização NOD1/antagonistas & inibidores , Pró-Fármacos/uso terapêutico
3.
J Med Chem ; 60(12): 5162-5192, 2017 06 22.
Artigo em Inglês | MEDLINE | ID: mdl-28541685

RESUMO

Nucleotide-binding oligomerization domain-like receptors (NLRs) are intracellular sensors of pathogen-associated molecular patterns (PAMPs) and damage-associated molecular patterns (DAMPs). Previously, we reported nucleotide-binding oligomerization domain-containing protein 1 (NOD1) antagonists (11, 12) and a NOD2 antagonist (9) that sensitized docetaxel (DTX) or paclitaxel (PTX) treatment for breast or lung cancer. In this article, we describe for the first time a 1,4-benzodiazepine-2,5-dione (BZD) derivative (26bh) that acts as a dual NOD1/NOD2 antagonist and inhibits both nuclear factor κB (NF-κB) and mitogen-activated protein kinase (MAPK) inflammatory signaling, thereby sensitizing PTX to suppress Lewis lung carcinoma (LLC) growth. After investigation of the compound's cytotoxicity, a systematic structure-activity relationship (SAR) was completed and revealed several key factors that were necessary to maintain antagonistic ability. This study establishes the possibility for using adjuvant treatment to combat cancer by antagonizing both NOD1 and NOD2 signaling.


Assuntos
Protocolos de Quimioterapia Combinada Antineoplásica/farmacologia , Benzodiazepinas/química , Carcinoma Pulmonar de Lewis/tratamento farmacológico , Proteína Adaptadora de Sinalização NOD1/antagonistas & inibidores , Proteína Adaptadora de Sinalização NOD2/antagonistas & inibidores , Animais , Benzodiazepinas/farmacologia , Citocinas/metabolismo , Ensaios de Seleção de Medicamentos Antitumorais/métodos , Humanos , Macrófagos/efeitos dos fármacos , Macrófagos/metabolismo , Masculino , Camundongos Endogâmicos C57BL , Terapia de Alvo Molecular , Paclitaxel/administração & dosagem , Domínios Proteicos , Relação Estrutura-Atividade
4.
Bioorg Med Chem ; 24(21): 5221-5234, 2016 11 01.
Artigo em Inglês | MEDLINE | ID: mdl-27601373

RESUMO

NOD1 and NOD2 are important members of the pattern recognition receptor family and play a crucial role within the context of innate immunity. However, overactivation of NODs, especially of NOD1, has also been implicated in a number of diseases. Surprisingly, NOD1 remains a virtually unexploited target in this respect. To gain additional insight into the structure-activity relationships of NOD1 inhibitors, a series of novel analogs has been designed and synthesized and then screened for their NOD1-inhibitory activity. Selected compounds were also investigated for their NOD2-inhibitory activity. Two compounds 4 and 15, were identified as potent mixed inhibitors of NOD1 and NOD2, displaying a balanced inhibitory activity on both targets in the low micromolar range. The results obtained have enabled a deeper understanding of the structural requirements for NOD1 and NOD2 inhibition.


Assuntos
Indóis/farmacologia , Proteína Adaptadora de Sinalização NOD1/antagonistas & inibidores , Proteína Adaptadora de Sinalização NOD2/antagonistas & inibidores , Proliferação de Células/efeitos dos fármacos , Células Cultivadas , Relação Dose-Resposta a Droga , Desenho de Fármacos , Humanos , Indóis/síntese química , Indóis/química , Estrutura Molecular , NF-kappa B/antagonistas & inibidores , NF-kappa B/metabolismo , Proteína Adaptadora de Sinalização NOD1/genética , Proteína Adaptadora de Sinalização NOD2/genética , Relação Estrutura-Atividade
5.
Org Biomol Chem ; 14(3): 1013-23, 2016 Jan 21.
Artigo em Inglês | MEDLINE | ID: mdl-26631868

RESUMO

Peptidoglycan (PGN) is a major component of bacterial cell wall and is recognized as a potent immunostimulant. The PGN in the cell envelope of Mycobacterium Tuberculosis has been shown to possess several unique characteristics including the presence of N-glycolyl groups (in addition to N-acetyl groups) in the muramic acid residues, and amidation of the free carboxylic acid of d-Glu or of meso-DAP in the peptide chains. Using a newly developed, highly stereoselective, chemoenzymatic approach for the synthesis of meso-DAP in peptide stems, we successfully synthesized for the first time, a series of Mycobacterium PGN fragments that include both mono- and disaccharides of MurNGlyc or 1,6-anhydro-MurNGlyc, as well as peptide-amidated variants. The ability of these PGN fragments to stimulate the immune system through activation of human Nod1 and Nod2 was examined. The PGN fragments were found to modulate immune stimulation, specifically, amidation at the d-Glu and meso-DAP in the peptide stem strongly reduced hNod1 activation. This effect was dependent on modification position. Additionally, N-glycolyl (instead of acetyl) of muramic acid was associated with slightly reduced human Nod1 and Nod2 stimulatory capabilities.


Assuntos
Ácido Diaminopimélico/farmacologia , Imunidade Inata/efeitos dos fármacos , Imunidade Inata/imunologia , Mycobacterium tuberculosis/química , Peptidoglicano/imunologia , Ácido Diaminopimélico/química , Ácido Diaminopimélico/metabolismo , Células HEK293 , Humanos , Conformação Molecular , Mycobacterium tuberculosis/imunologia , Proteína Adaptadora de Sinalização NOD1/antagonistas & inibidores , Proteína Adaptadora de Sinalização NOD1/imunologia , Proteína Adaptadora de Sinalização NOD2/antagonistas & inibidores , Proteína Adaptadora de Sinalização NOD2/imunologia , Peptidoglicano/biossíntese , Peptidoglicano/química , Relação Estrutura-Atividade
6.
Chem Biol ; 22(9): 1174-84, 2015 Sep 17.
Artigo em Inglês | MEDLINE | ID: mdl-26320862

RESUMO

RIPK2 mediates pro-inflammatory signaling from the bacterial sensors NOD1 and NOD2, and is an emerging therapeutic target in autoimmune and inflammatory diseases. We observed that cellular RIPK2 can be potently inhibited by type II inhibitors that displace the kinase activation segment, whereas ATP-competitive type I inhibition was only poorly effective. The most potent RIPK2 inhibitors were the US Food and Drug Administration-approved drugs ponatinib and regorafenib. Their mechanism of action was independent of NOD2 interaction and involved loss of downstream kinase activation as evidenced by lack of RIPK2 autophosphorylation. Notably, these molecules also blocked RIPK2 ubiquitination and, consequently, inflammatory nuclear factor κB signaling. In monocytes, the inhibitors selectively blocked NOD-dependent tumor necrosis factor production without affecting lipopolysaccharide-dependent pathways. We also determined the first crystal structure of RIPK2 bound to ponatinib, and identified an allosteric site for inhibitor development. These results highlight the potential for type II inhibitors to treat indications of RIPK2 activation as well as inflammation-associated cancers.


Assuntos
Proteína Adaptadora de Sinalização NOD1/antagonistas & inibidores , Proteína Adaptadora de Sinalização NOD2/antagonistas & inibidores , Inibidores de Proteínas Quinases/farmacologia , Proteína Serina-Treonina Quinase 2 de Interação com Receptor/antagonistas & inibidores , Sequência de Aminoácidos , Animais , Células Cultivadas , Humanos , Imidazóis/química , Imidazóis/farmacologia , Inflamação/metabolismo , Modelos Moleculares , Dados de Sequência Molecular , Proteína Adaptadora de Sinalização NOD1/metabolismo , Proteína Adaptadora de Sinalização NOD2/metabolismo , Ligação Proteica , Inibidores de Proteínas Quinases/química , Piridazinas/química , Piridazinas/farmacologia , Proteína Serina-Treonina Quinase 2 de Interação com Receptor/metabolismo , Células Sf9 , Transdução de Sinais/efeitos dos fármacos , Ubiquitinação/efeitos dos fármacos
7.
Cardiovasc Res ; 106(3): 375-86, 2015 Jun 01.
Artigo em Inglês | MEDLINE | ID: mdl-25824149

RESUMO

AIMS: Inflammation is a significant contributor to cardiovascular disease and its complications; however, whether the myocardial inflammatory response is harmonized after cardiac injury remains to be determined. Some receptors of the innate immune system, including the nucleotide-binding oligomerization domain-like receptors (NLRs), play key roles in the host response after cardiac damage. Nucleotide-binding oligomerization domain containing 1 (NOD1), a member of the NLR family, is expressed in the heart, but its functional role has not been elucidated. We determine whether selective NOD1 activation modulates cardiac function and Ca(2+) signalling. METHODS AND RESULTS: Mice were treated for 3 days with the selective NOD1 agonist C12-iE-DAP (iE-DAP), and cardiac function and Ca(2+) cycling were assessed. We found that iE-DAP treatment resulted in cardiac dysfunction, measured as a decrease in ejection fraction and fractional shortening. Cardiomyocytes isolated from iE-DAP-treated mice displayed a decrease in the L-type Ca(2+) current, [Ca(2+)]i transients and Ca(2+) load, and decreased expression of phospho-phospholamban, sarcoplasmic reticulum-ATPase, and Na(+)-Ca(2+) exchanger. Furthermore, iE-DAP prompted 'diastolic Ca(2+) leak' in cardiomyocytes, resulting from increased Ca(2+) spark frequency and RyR2 over-phosphorylation. Importantly, these iE-DAP-induced changes in Ca(2+) cycling were lost in NOD1(-/-) mice, indicating that iE-DAP exerts its actions through NOD1. Co-treatment of mice with iE-DAP and a selective inhibitor of NF-κB (BAY11-7082) prevented cardiac dysfunction and Ca(2+) handling impairment induced by iE-DAP. CONCLUSION: Our data provide the first evidence that NOD1 activation induces cardiac dysfunction associated with excitation-contraction coupling impairment through NF-κB activation and uncover a new pro-inflammatory player in the regulation of cardiovascular function.


Assuntos
Cálcio/metabolismo , Acoplamento Excitação-Contração , Mediadores da Inflamação/metabolismo , Contração Miocárdica , Miócitos Cardíacos/metabolismo , Proteína Adaptadora de Sinalização NOD1/metabolismo , Disfunção Ventricular Esquerda/metabolismo , Função Ventricular Esquerda , Animais , Anti-Inflamatórios/farmacologia , Canais de Cálcio Tipo L/metabolismo , Proteínas de Ligação ao Cálcio/metabolismo , Células Cultivadas , Acoplamento Excitação-Contração/efeitos dos fármacos , Mediadores da Inflamação/agonistas , Mediadores da Inflamação/antagonistas & inibidores , Masculino , Potenciais da Membrana , Camundongos Endogâmicos C57BL , Camundongos Knockout , Contração Miocárdica/efeitos dos fármacos , Miócitos Cardíacos/efeitos dos fármacos , NF-kappa B/metabolismo , Proteína Adaptadora de Sinalização NOD1/agonistas , Proteína Adaptadora de Sinalização NOD1/antagonistas & inibidores , Proteína Adaptadora de Sinalização NOD1/deficiência , Proteína Adaptadora de Sinalização NOD1/genética , Fosforilação , ATPases Transportadoras de Cálcio do Retículo Sarcoplasmático/metabolismo , Trocador de Sódio e Cálcio/metabolismo , Volume Sistólico , Disfunção Ventricular Esquerda/genética , Disfunção Ventricular Esquerda/fisiopatologia , Disfunção Ventricular Esquerda/prevenção & controle , Função Ventricular Esquerda/efeitos dos fármacos
8.
Rev. bras. enferm ; 68(2): 253-260, Mar-Apr/2015. tab
Artigo em Português | LILACS, BDENF - Enfermagem | ID: lil-752516

RESUMO

RESUMO Objetivo: construir e validar um instrumento para monitorar a qualidade dos registros de enfermagem no Programa de Assistência Domiciliar (PAD) em um hospital universitário. Método: estudo metodológico envolvendo a elaboração de um manual e submetido à validação de conteúdo por seis juízes sob consenso ≥ 80%. A coleta ocorreu em 2012 por meio de questionário contendo: evolução de enfermagem, diagnóstico e prescrição de enfermagem e normas para os registros da equipe de enfermagem preconizadas pelo Conselho Regional de Enfermagem-SP e pela instituição. Os itens do manual foram julgados de acordo com as variáveis - relevância, pertinência, clareza e simplicidade. Resultados: das 39 proposições 100% atingiram consenso ≥ 80% em relevância, pertinência e clareza; 92,3% em simplicidade. Os itens sono/repouso, mobilidade e checagem nas atividades prescritas não atingiram consenso mínimo favorável, sendo aprimorados pelas sugestões dos juízes. Conclusão: acreditamos que o instrumento possibilitará a melhoria dos processos de trabalho no PAD. .


RESUMEN Objetivo: construir y validar un instrumento para monitorear la calidad del registros de enfermería en Programa de Atención Domiciliaria (PAD) de un Hospital Universitario. Metodo: estudio metodológico. Fue construido un manual y sometió a validación de contenido por seis jueces bajo el consenso ≥80%. La recogida currió en 2012, con un cuestionario que contiene: evolución de enfermería, diagnóstico y prescripción de enfermería y normas para los registros del personal de enfermaria estabelecidas por Consejo Regional de Enfermería-SP y por la institución. Los artículos del manual fueran juzgadso conforme las variables relevancia, pertinencia, claridad y sencillez. Resultados: de las 39 proposiciones 100% alcanzó consenso ≥ 80% en la relevancia, pertinencia y claridad; 92,3% en la simplicidad. Los itens sueño/resto, movilidad y verificar las actividades prescritas no alcanzó consenso favorable, siendo mejoradas por las sugerencias de los jueces. Conclusión: creemos que el instrumento permitirá la mejora de los procesos de trabajo en PAD. .


ABSTRACT Objective: to build and validate an instrument aimed at monitoring the quality of nursing records in the Home Care Program (HCP) of a university hospital. Method: methodological study involving the elaboration of a manual, whose content was later submitted to six experts for validation, reaching a ≥ 80% consensus. The data collection process was carried out in 2012 by means of a questionnaire comprised of the following issues: nursing evolution, nursing diagnosis, and nursing prescription, and standards for the nursing team recommended by the Regional Nursing Council of São Paulo and by the assessed institution. Manual items were judged according to the following variables: relevance, pertinence, clarity and simplicity. Results: of the 39 propositions, 100% achieved ≥ 80% agreement in the relevance, pertinence and clarity variables; 92.3% in the simplicity variable. Sleep/rest, Mobility and Check-out variables did not reach a favorable minimum consensus in the prescribed activities and were improved following suggestions from the experts. Conclusion: we believe that the instrument will enable the improvement of the HCP’s work process. .


Assuntos
Humanos , Actinas/metabolismo , Cofilina 1/metabolismo , Disenteria Bacilar/microbiologia , Proteína Adaptadora de Sinalização NOD1/metabolismo , Fosfoproteínas Fosfatases/metabolismo , Shigella flexneri/fisiologia , Actinas/química , Western Blotting , Células Cultivadas , Cofilina 1/genética , Ensaio de Imunoadsorção Enzimática , Técnica Indireta de Fluorescência para Anticorpo , Regulação da Expressão Gênica , Células HeLa , Ensaios de Triagem em Larga Escala , Técnicas Imunoenzimáticas , Imunoprecipitação , Inflamação , Mediadores da Inflamação/metabolismo , NF-kappa B/genética , NF-kappa B/metabolismo , Proteína Adaptadora de Sinalização NOD1/antagonistas & inibidores , Proteína Adaptadora de Sinalização NOD1/genética , Fosforilação , Fosfoproteínas Fosfatases/genética , Reação em Cadeia da Polimerase em Tempo Real , Reação em Cadeia da Polimerase Via Transcriptase Reversa , RNA Mensageiro/genética , RNA Interferente Pequeno/genética , Transdução de Sinais
9.
PLoS Pathog ; 10(9): e1004351, 2014 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-25187968

RESUMO

NOD1 is an intracellular pathogen recognition receptor that contributes to anti-bacterial innate immune responses, adaptive immunity and tissue homeostasis. NOD1-induced signaling relies on actin remodeling, however, the details of the connection of NOD1 and the actin cytoskeleton remained elusive. Here, we identified in a druggable-genome wide siRNA screen the cofilin phosphatase SSH1 as a specific and essential component of the NOD1 pathway. We show that depletion of SSH1 impaired pathogen induced NOD1 signaling evident from diminished NF-κB activation and cytokine release. Chemical inhibition of actin polymerization using cytochalasin D rescued the loss of SSH1. We further demonstrate that NOD1 directly interacted with SSH1 at F-actin rich sites. Finally, we show that enhanced cofilin activity is intimately linked to NOD1 signaling. Our data thus provide evidence that NOD1 requires the SSH1/cofilin network for signaling and to detect bacterial induced changes in actin dynamics leading to NF-κB activation and innate immune responses.


Assuntos
Actinas/metabolismo , Cofilina 1/metabolismo , Disenteria Bacilar/microbiologia , Proteína Adaptadora de Sinalização NOD1/metabolismo , Fosfoproteínas Fosfatases/metabolismo , Shigella flexneri/fisiologia , Actinas/química , Western Blotting , Células Cultivadas , Cofilina 1/genética , Ensaio de Imunoadsorção Enzimática , Técnica Indireta de Fluorescência para Anticorpo , Regulação da Expressão Gênica , Células HeLa , Ensaios de Triagem em Larga Escala , Humanos , Técnicas Imunoenzimáticas , Imunoprecipitação , Inflamação , Mediadores da Inflamação/metabolismo , NF-kappa B/genética , NF-kappa B/metabolismo , Proteína Adaptadora de Sinalização NOD1/antagonistas & inibidores , Proteína Adaptadora de Sinalização NOD1/genética , Fosfoproteínas Fosfatases/genética , Fosforilação , RNA Mensageiro/genética , RNA Interferente Pequeno/genética , Reação em Cadeia da Polimerase em Tempo Real , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Transdução de Sinais
10.
J Med Chem ; 57(16): 6897-918, 2014 Aug 28.
Artigo em Inglês | MEDLINE | ID: mdl-24707857

RESUMO

Dysregulation of nucleotide-binding oligomerization domains 1 and 2 (NOD1 and NOD2) has been implicated in the pathology of various inflammatory disorders, rendering them and their downstream signaling proteins potential therapeutic targets. Selective inhibition of NOD1 and NOD2 signaling could be advantageous in treating many acute and chronic diseases; therefore, harnessing the full potential of NOD inhibitors is a key topic in medicinal chemistry. Although they are among the best studied NOD-like receptors (NLRs), the therapeutic potential of pharmacological modulation of NOD1 and NOD2 is largely unexplored. This review is focused on the scientific progress in the field of NOD inhibitors over the past decade, including the recently reported selective inhibitors of NOD1 and NOD2. In addition, the potential approaches to inhibition of NOD signaling as well as the advantages and disadvantages linked with inhibition of NOD signaling are discussed. Finally, the potential directions for drug discovery are also discussed.


Assuntos
Proteína Adaptadora de Sinalização NOD1/antagonistas & inibidores , Proteína Adaptadora de Sinalização NOD1/metabolismo , Proteína Adaptadora de Sinalização NOD2/antagonistas & inibidores , Proteína Adaptadora de Sinalização NOD2/metabolismo , Transdução de Sinais , Anti-Inflamatórios não Esteroides/farmacologia , Asma/tratamento farmacológico , Asma/etiologia , Doença de Crohn/tratamento farmacológico , Doença de Crohn/metabolismo , Descoberta de Drogas/métodos , Humanos , Imunidade Inata/efeitos dos fármacos , Terapia de Alvo Molecular/métodos , Proteína Adaptadora de Sinalização NOD1/genética , Proteína Adaptadora de Sinalização NOD2/genética , Polimorfismo Genético
11.
PLoS One ; 7(9): e45260, 2012.
Artigo em Inglês | MEDLINE | ID: mdl-23028889

RESUMO

The innate immune system is responsible for the initial response of an organism to potentially harmful stressors, pathogens or tissue injury, and accordingly plays an essential role in the pathogenesis of many inflammatory processes, including some cardiovascular diseases. Toll like receptors (TLR) and nucleotide-binding oligomerization domain-like receptors (NLRs) are pattern recognition receptors that play an important role in the induction of innate immune and inflammatory responses. There is a line of evidence supporting that activation of TLRs contributes to the development and progression of cardiovascular diseases but less is known regarding the role of NLRs. Here we demonstrate the presence of the NLR member NOD1 (nucleotide-binding oligomerization domain containing 1) in the murine heart. Activation of NOD1 with the specific agonist C12-iEDAP, but not with the inactive analogue iE-Lys, induces a time- and dose-dependent cardiac dysfunction that occurs concomitantly with cardiac fibrosis and apoptosis. The administration of iEDAP promotes the activation of the NF-κB and TGF-ß pathways and induces apoptosis in whole hearts. At the cellular level, both native cardiomyocytes and cardiac fibroblasts expressed NOD1. The NLR activation in cardiomyocytes was associated with NF-κB activation and induction of apoptosis. NOD1 stimulation in fibroblasts was linked to NF-κB activation and to increased expression of pro-fibrotic mediators. The down-regulation of NOD1 by specific siRNAs blunted the effect of iEDAP on the pro-fibrotic TGF-ß pathway and cell apoptosis. In conclusion, our report uncovers a new pro-inflammatory target that is expressed in the heart, NOD1. The specific activation of this NLR induces cardiac dysfunction and modulates cardiac fibrosis and cardiomyocyte apoptosis, pathological processes involved in several cardiac diseases such as heart failure.


Assuntos
Fatores Biológicos/farmacologia , Fibrose Endomiocárdica/metabolismo , Insuficiência Cardíaca/metabolismo , Miocárdio/metabolismo , Miocárdio/patologia , Miócitos Cardíacos/metabolismo , Proteína Adaptadora de Sinalização NOD1/agonistas , Animais , Apoptose/efeitos dos fármacos , Relação Dose-Resposta a Droga , Fibrose Endomiocárdica/induzido quimicamente , Fibrose Endomiocárdica/complicações , Fibrose Endomiocárdica/patologia , Fibroblastos/efeitos dos fármacos , Fibroblastos/metabolismo , Fibroblastos/patologia , Regulação da Expressão Gênica/efeitos dos fármacos , Insuficiência Cardíaca/induzido quimicamente , Insuficiência Cardíaca/complicações , Insuficiência Cardíaca/patologia , Camundongos , Miócitos Cardíacos/efeitos dos fármacos , Miócitos Cardíacos/patologia , NF-kappa B/genética , NF-kappa B/metabolismo , Proteína Adaptadora de Sinalização NOD1/antagonistas & inibidores , Proteína Adaptadora de Sinalização NOD1/genética , RNA Interferente Pequeno/genética , Ratos , Transdução de Sinais/efeitos dos fármacos , Fator de Crescimento Transformador beta/genética , Fator de Crescimento Transformador beta/metabolismo
12.
Biosci Rep ; 32(6): 597-608, 2012 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-22908883

RESUMO

NOD1 {nucleotide-binding oligomerization domain 1; NLRC [NOD-LRR (leucine-rich repeat) family with CARD (caspase recruitment domain) 1]} and NOD2 (NLRC2) are among the most prominent members of the NLR (NOD-LRR) family -proteins that contain nucleotide-binding NACHT domains and receptor-like LRR domains. With over 20 members identified in humans, NLRs represent important components of the mammalian innate immune system, serving as intracellular receptors for pathogens and for endogenous molecules elaborated by tissue injury. NOD1 and NOD2 proteins operate as microbial sensors through the recognition of specific PG (peptidoglycan) constituents of bacteria. Upon activation, these NLR family members initiate signal transduction mechanisms that include stimulation of NF-κB (nuclear factor-κB), stress kinases, IRFs (interferon regulatory factors) and autophagy. Hereditary polymorphisms in the genes encoding NOD1 and NOD2 have been associated with an increasing number of chronic inflammatory diseases. In fact, potential roles for NOD1 and NOD2 in inflammatory disorders have been revealed by investigations using a series of animal models. In the present review, we describe recent experimental findings associating NOD1 and NOD2 with various autoimmune and chronic inflammatory disorders, and we discuss prospects for development of novel therapeutics targeting these NLR family proteins.


Assuntos
Imunidade Inata , Proteína Adaptadora de Sinalização NOD1/imunologia , Proteína Adaptadora de Sinalização NOD2/imunologia , Animais , Anti-Inflamatórios/farmacologia , Infecções Bacterianas/tratamento farmacológico , Infecções Bacterianas/genética , Infecções Bacterianas/imunologia , Fenômenos Fisiológicos Bacterianos , Regulação da Expressão Gênica , Interações Hospedeiro-Patógeno , Humanos , Imunidade Inata/efeitos dos fármacos , Inflamação/tratamento farmacológico , Inflamação/genética , Inflamação/imunologia , Proteína Adaptadora de Sinalização NOD1/antagonistas & inibidores , Proteína Adaptadora de Sinalização NOD1/genética , Proteína Adaptadora de Sinalização NOD2/antagonistas & inibidores , Proteína Adaptadora de Sinalização NOD2/genética
13.
Chem Biol ; 18(7): 825-32, 2011 Jul 29.
Artigo em Inglês | MEDLINE | ID: mdl-21802003

RESUMO

NLR family proteins play important roles in innate immune response. NOD1 (NLRC1) activates various signaling pathways including NF-κB in response to bacterial ligands. Hereditary polymorphisms in the NOD1 gene are associated with asthma, inflammatory bowel disease, and other disorders. Using a high throughput screening (HTS) assay measuring NOD1-induced NF-κB reporter gene activity, followed by multiple downstream counter screens that eliminated compounds impacting other NF-κB effectors, 2-aminobenzimidazole compounds were identified that selectively inhibit NOD1. Mechanistic studies of a prototypical compound, Nodinitib-1 (ML130; CID-1088438), suggest that these small molecules cause conformational changes of NOD1 in vitro and alter NOD1 subcellular targeting in cells. Altogether, this inaugural class of inhibitors provides chemical probes for interrogating mechanisms regulating NOD1 activity and tools for exploring the roles of NOD1 in various infectious and inflammatory diseases.


Assuntos
Benzimidazóis/química , Benzimidazóis/farmacologia , Proteína Adaptadora de Sinalização NOD1/antagonistas & inibidores , Transdução de Sinais/efeitos dos fármacos , Linhagem Celular , Células Cultivadas , Células Dendríticas/efeitos dos fármacos , Avaliação Pré-Clínica de Medicamentos , Genes Reporter/efeitos dos fármacos , Ensaios de Triagem em Larga Escala , Humanos , NF-kappa B/genética , Proteína Adaptadora de Sinalização NOD1/imunologia
14.
J Biol Chem ; 285(14): 10553-62, 2010 Apr 02.
Artigo em Inglês | MEDLINE | ID: mdl-20118240

RESUMO

The cytosolic nucleotide-binding oligomerization domain 1 (NOD1)/CARD4 and NOD2/CARD15 proteins are members of NOD-like receptors recognizing specific motifs within peptidoglycans of both Gram-negative and Gram-positive bacteria. NOD1 and NOD2 signal via the downstream adaptor serine/threonine kinase RIP2/CARDIAK/RICK to initiate NF-kappaB activation and the release of inflammatory cytokines/chemokines. In this report, we show that 5,6-dimethylxanthenone-4-acetic acid (DMXAA), a cell-permeable, small molecule that has anti-tumor activity, can also activate NOD1 and NOD2. This was demonstrated: 1) by using human embryonic kidney epithelial (HEK) 293 cells transfected with a NF-kappaB reporter plasmid in combination with NOD1 or NOD2 expression plasmids; 2) by inhibiting DMXAA-induced chemokine (CXCL10) mRNA and protein production in the AB12 mesothelioma cell line using a pharmacological inhibitor of RICK kinase, SB20358; and 3) by using small interfering RNA to knock down NOD2 and lentiviral short hairpin RNA to knock down RICK. These findings expand the potential ligands for the NOD-like receptors, suggesting that other xanthone compounds may act similarly and could be developed as anti-tumor agents. This information also expands our knowledge on the mechanisms of action of the anti-tumor agent DMXAA (currently in clinical trials) and may be important for its biological activity.


Assuntos
Antineoplásicos/farmacologia , NF-kappa B/metabolismo , Proteína Adaptadora de Sinalização NOD1/metabolismo , Proteína Adaptadora de Sinalização NOD2/metabolismo , Nucleotídeos/metabolismo , Transdução de Sinais/efeitos dos fármacos , Xantonas/farmacologia , Células Cultivadas , Quimiocina CXCL10/genética , Quimiocina CXCL10/metabolismo , Inibidores Enzimáticos/farmacologia , Humanos , Imidazóis/farmacologia , Immunoblotting , Rim/citologia , Rim/metabolismo , Luciferases/metabolismo , Mesotelioma/genética , Mesotelioma/metabolismo , Mesotelioma/patologia , NF-kappa B/genética , Proteína Adaptadora de Sinalização NOD1/antagonistas & inibidores , Proteína Adaptadora de Sinalização NOD1/genética , Proteína Adaptadora de Sinalização NOD2/antagonistas & inibidores , Proteína Adaptadora de Sinalização NOD2/genética , Piridinas/farmacologia , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , RNA Interferente Pequeno/farmacologia , Proteína Serina-Treonina Quinase 2 de Interação com Receptor/antagonistas & inibidores , Proteína Serina-Treonina Quinase 2 de Interação com Receptor/genética , Proteína Serina-Treonina Quinase 2 de Interação com Receptor/metabolismo , Reação em Cadeia da Polimerase Via Transcriptase Reversa
15.
J Immunol ; 183(12): 8099-109, 2009 Dec 15.
Artigo em Inglês | MEDLINE | ID: mdl-20007577

RESUMO

Helicobacter pylori rapidly activates MAPKs and transcription factors, NF-kappaB and AP-1, in gastric epithelial cells following host attachment. Activation of these signal transducers is largely dependent on the cag pathogenicity island (cagPAI)-encoded Type IV Secretion System. H. pylori was shown to translocate peptidoglycan through the Type IV Secretion System, which is recognized by the pathogen recognition molecule, NOD1, thus resulting in NF-kappaB activation. The mechanisms of H. pylori-induced MAPK and AP-1 activation, however, are less well defined and therefore, we assessed the contribution of NOD1 to their activation. For this, we used gastric epithelial cell lines, stably expressing siRNA to either NOD1 or a control gene. In siNOD1-expressing cells stimulated with cagPAI(+) H. pylori, we observed significant reductions in p38 and ERK phosphorylation (p < 0.05), whereas the levels of Jnk phosphorylation remained unchanged. Consistent with a previous report, however, we were able to demonstrate NOD1-dependent Jnk phosphorylation by the invasive pathogen Shigella flexneri, highlighting pathogen-specific host responses to infection. We also show that NOD1 was essential for H. pylori induction of not only NF-kappaB, but also AP-1 activation, implying that NOD1 induces robust proinflammatory responses, in an attempt to rapidly control infection. Pharmacological inhibition of p38 and ERK activity significantly reduced IL-8 production in response to H. pylori, further emphasizing the importance of MAPKs in innate immune responses to the pathogen. Thus, for the first time we have shown the important role for NOD1 in MAPK and AP-1 activation in response to cagPAI(+) H. pylori.


Assuntos
MAP Quinases Reguladas por Sinal Extracelular/metabolismo , Helicobacter pylori/imunologia , Proteína Adaptadora de Sinalização NOD1/fisiologia , Fator de Transcrição AP-1/metabolismo , Antígenos de Bactérias/fisiologia , Proteínas da Membrana Bacteriana Externa/metabolismo , Proteínas da Membrana Bacteriana Externa/fisiologia , Proteínas de Bactérias/fisiologia , Linhagem Celular , Linhagem Celular Tumoral , Ativação Enzimática/imunologia , MAP Quinases Reguladas por Sinal Extracelular/fisiologia , Marcação de Genes , Humanos , Mediadores da Inflamação/metabolismo , Mediadores da Inflamação/fisiologia , MAP Quinase Quinase 4/metabolismo , Proteína Adaptadora de Sinalização NOD1/antagonistas & inibidores , Proteína Adaptadora de Sinalização NOD1/genética , Fosforilação/imunologia , Vesículas Secretórias/metabolismo , Transdução de Sinais/imunologia
16.
J Dent Res ; 88(8): 762-7, 2009 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-19734466

RESUMO

Pulp fibroblasts express various pro-inflammatory mediators leading to marked infiltration of inflammatory cells in the progression of pulpitis. We hypothesized that pulp fibroblasts play roles in the recognition of invaded caries-related bacteria and the subsequent innate immune responses. We found clear expressions of TLR2, NOD1, and NOD2 and a faint expression of TLR4 in human dental pulp fibroblasts (HDPF) by RT-PCR and flow cytometry. We also observed that various pro-inflammatory mediators, including cytokines, chemokines, adhesion molecules, prostaglandin E(2) and its key enzyme COX-2, not iNOS or caspase-1, were markedly up-regulated by stimulation with these TLR and NOD agonists. More over, the NOD2 agonist acted synergistically with the TLR2, not the TLR4, agonist to stimulate the production of pro-inflammatory mediators in HDPF. These findings indicate that TLR2, TLR4, NOD2, and NOD1 in HDPF are functional receptors, and NOD2 is a modulator of signals transmitted through TLR2 in pulpal immune responses, leading to progressive pulpitis.


Assuntos
Apoptose/imunologia , Polpa Dentária/imunologia , Fibroblastos/imunologia , Proteína Adaptadora de Sinalização NOD1/análise , Proteína Adaptadora de Sinalização NOD2/análise , Receptor 2 Toll-Like/análise , Receptor 4 Toll-Like/análise , Acetilmuramil-Alanil-Isoglutamina/farmacologia , Células Cultivadas , Quimiocina CCL2/análise , Quimiocina CXCL10/análise , Ciclo-Oxigenase 2/imunologia , Polpa Dentária/citologia , Ácido Diaminopimélico/análogos & derivados , Dinoprostona/análise , Escherichia coli , Humanos , Mediadores da Inflamação/imunologia , Interleucina-6/análise , Interleucina-8/análise , Lipopeptídeos/farmacologia , Lipopolissacarídeos/farmacologia , Proteína Adaptadora de Sinalização NOD1/antagonistas & inibidores , Proteína Adaptadora de Sinalização NOD2/antagonistas & inibidores , Pulpite/imunologia , Transdução de Sinais/imunologia , Streptococcus mutans/imunologia , Receptor 2 Toll-Like/antagonistas & inibidores , Receptor 4 Toll-Like/antagonistas & inibidores , Molécula 1 de Adesão de Célula Vascular/análise
17.
PLoS One ; 4(4): e5396, 2009.
Artigo em Inglês | MEDLINE | ID: mdl-19401779

RESUMO

Helicobacter spp. represent a proportionately small but significant component of the normal intestinal microflora of animal hosts. Several of these intestinal Helicobacter spp. are known to induce colitis in mouse models, yet the mechanisms by which these bacteria induce intestinal inflammation are poorly understood. To address this question, we performed in vitro co-culture experiments with mouse and human epithelial cell lines stimulated with a selection of Helicobacter spp., including known pathogenic species as well as ones for which the pathogenic potential is less clear. Strikingly, a member of the normal microflora of rodents, Helicobacter muridarum, was found to be a particularly strong inducer of CXC chemokine (Cxcl1/KC, Cxcl2/MIP-2) responses in a murine intestinal epithelial cell line. Time-course studies revealed a biphasic pattern of chemokine responses in these cells, with H. muridarum lipopolysaccharide (LPS) mediating early (24-48 h) responses and live bacteria seeming to provoke later (48-72 h) responses. H. muridarum LPS per se was shown to induce CXC chemokine production in HEK293 cells stably expressing Toll-like receptor 2 (TLR2), but not in those expressing TLR4. In contrast, live H. muridarum bacteria were able to induce NF-kappaB reporter activity and CXC chemokine responses in TLR2-deficient HEK293 and in AGS epithelial cells. These responses were attenuated by transient transfection with a dominant negative construct to NOD1, and by stable expression of NOD1 siRNA, respectively. Thus, the data suggest that both TLR2 and NOD1 may be involved in innate immune sensing of H. muridarum by epithelial cells. This work identifies H. muridarum as a commensal bacterium with pathogenic potential and underscores the potential roles of ill-defined members of the normal flora in the initiation of inflammation in animal hosts. We suggest that H. muridarum may act as a confounding factor in colitis model studies in rodents.


Assuntos
Helicobacter/patogenicidade , Intestinos/microbiologia , Proteína Adaptadora de Sinalização NOD1/metabolismo , Receptor 2 Toll-Like/metabolismo , Sequência de Aminoácidos , Animais , Sequência de Bases , Linhagem Celular , Quimiocina CXCL1/biossíntese , Quimiocina CXCL2/biossíntese , Quimiocinas CXC/biossíntese , Técnicas de Cocultura , Colite/etiologia , Colite/imunologia , Colite/metabolismo , Colite/microbiologia , Primers do DNA/genética , Modelos Animais de Doenças , Células Epiteliais/imunologia , Células Epiteliais/metabolismo , Células Epiteliais/microbiologia , Flagelina/genética , Helicobacter/genética , Helicobacter/imunologia , Helicobacter/isolamento & purificação , Infecções por Helicobacter/etiologia , Infecções por Helicobacter/metabolismo , Infecções por Helicobacter/microbiologia , Humanos , Imunidade Inata , Intestinos/imunologia , Lipopolissacarídeos/toxicidade , Camundongos , Dados de Sequência Molecular , NF-kappa B/metabolismo , Proteína Adaptadora de Sinalização NOD1/antagonistas & inibidores , Proteína Adaptadora de Sinalização NOD1/genética , Homologia de Sequência de Aminoácidos , Especificidade da Espécie , Receptor 2 Toll-Like/genética , Receptor 4 Toll-Like/genética , Receptor 4 Toll-Like/metabolismo
18.
Cell Microbiol ; 10(3): 682-95, 2008 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-18042252

RESUMO

Bacterial infections trigger the activation of innate immunity through the interaction of pathogen-associated molecular patterns (PAMPs) with pattern recognition molecules (PRMs). The nucleotide-binding oligomerization domain (Nod) proteins are intracellular PRMs that recognize muramylpeptides contained in peptidoglycan (PGN) of bacteria. It is still unclear how Nod1 physically interacts with PGN, a structure internal to the Gram-negative bacterial envelope. To contribute to the understanding of this process, we demonstrate that, like Escherichia coli, Bordetella pertussis and Neisseria gonorrheae, the Gram-negative pathogen Shigella spontaneously releases PGN fragments and that this process can be increased by inactivating either ampG or mppA, genes involved in PGN recycling. Both Shigella mutants, but especially the strain carrying the mppA deletion, trigger Nod1-mediated NF-kappaB activation to a greater extent than the wild-type strain. Likewise, muramylpeptides spontaneously shed by Shigella are able per se to trigger a Nod1-mediated response consistent with the relative amount. Finally, we found that qualitative changes in muramylpeptide shedding can alter in vivo host responses to Shigella infection. Our findings support the idea that muramylpeptides released by pathogens during infection could modulate the immune response through Nod proteins and thereby influence the outcome of disease.


Assuntos
Peptidoglicano/metabolismo , Shigella flexneri/imunologia , Shigella flexneri/metabolismo , Animais , Fusão Gênica Artificial , Proteínas de Bactérias/genética , Proteínas de Transporte/genética , Contagem de Colônia Microbiana , Citocinas/análise , Disenteria Bacilar , Feminino , Deleção de Genes , Inativação Gênica , Genes Reporter , Humanos , Fígado/microbiologia , Fígado/patologia , Luciferases de Vaga-Lume/genética , Luciferases de Vaga-Lume/metabolismo , Pulmão/química , Pulmão/microbiologia , Pulmão/patologia , Proteínas de Membrana Transportadoras/genética , Camundongos , Camundongos Endogâmicos BALB C , NF-kappa B/imunologia , NF-kappa B/metabolismo , Proteína Adaptadora de Sinalização NOD1/antagonistas & inibidores , Proteína Adaptadora de Sinalização NOD1/imunologia , Proteína Adaptadora de Sinalização NOD1/metabolismo , Baço/microbiologia , Baço/patologia
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