RESUMEN
Aberrant production of proinflammatory cytokines is linked to many autoimmune diseases, and their inhibition by small molecule compounds is considered beneficial. Here, we performed phenotypic screening in IFNγ/LPS-activated RAW264.7, mouse macrophage cells, and discovered AS2677131 and AS2795440 as novel and potent inhibitors of IL-12p40, a subunit of IL-23. Interestingly, these compounds exhibited unique pharmacological activities in their inhibition of the production of IL-12p40, IL-6 and IL-1ß but not TNFα in activated macrophages or dendritic cells, and expression of IgM-induced MHC class II on B cells. To reveal these mechanisms, we synthesized two different activity probes which were structurally related to the AS compounds, and identified probe-specific binding proteins, including PIKfyve, a Class III PI kinase. The AS compounds inhibited PIKfyve activity and mimicked the properties of PIKfyve-deficient cells, eventually validating PIKfyve as target molecule. Regarding mechanism, AS2677131 regulated DNA binding activity of c-Rel on IL-12p40 and IL-1ß promoter. As expected, a PIKfyve inhibitor prevented the development of arthritis in rats. Taken together, our findings of the novel and potent PIKfyve inhibitors AS2677131 and AS2795440 reveal the critical role of PIKfyve in proinflammatory cytokine production and B cell activation, and may indicate a potential new therapeutic option for treatment of inflammatory diseases.
Asunto(s)
Antiinflamatorios/farmacología , ADN/metabolismo , Inhibidores Enzimáticos/farmacología , Inhibidores de las Quinasa Fosfoinosítidos-3 , Proteínas Proto-Oncogénicas c-rel/metabolismo , Animales , Artritis/prevención & control , Linfocitos B/citología , Linfocitos B/efectos de los fármacos , Citocinas/biosíntesis , Femenino , Regulación de la Expresión Génica/efectos de los fármacos , Interferón gamma/farmacología , Subunidad p40 de la Interleucina-12/genética , Lipopolisacáridos/farmacología , Ratones , Fosfatidilinositol 3-Quinasas/metabolismo , Regiones Promotoras Genéticas/genética , Unión Proteica/efectos de los fármacos , Piridinas/farmacología , Células RAW 264.7 , RatasRESUMEN
Given the key role p38 mitogen-activated protein kinase (MAPK) plays in inflammatory responses through the production of cytokines and inflammatory mediators, its inhibition is considered a promising therapeutic strategy for chronic inflammatory diseases such as rheumatoid arthritis, psoriasis, inflammatory bowel disease, and chronic obstructive pulmonary disease. Here, we evaluated the anti-inflammatory effect and selectivity profile of the novel p38 MAPK inhibitor AS1940477. AS1940477 inhibited the enzymatic activity of recombinant p38α and ß isoforms but showed no effect against other 100 protein kinases including p38γ and δ isoforms. We also confirmed the selectivity of AS1940477 in the intracellular signaling pathway. In human peripheral blood mononuclear cells, AS1940477 inhibited lipopolysaccharide (LPS)- or phytohemagglutinin A (PHA)-induced production of proinflammatory cytokines, including TNFα, IL-1ß, and IL-6 at low concentrations (LPS/TNFα, IC(50)=0.45n M; PHA/TNFα, IC(50)=0.40 nM). In addition, equivalent concentrations of AS1940477 that inhibited cytokine production also inhibited TNFα- and IL-1 ß-induced production of IL-6, PGE(2), and MMP-3 in human synovial stromal cells. AS1940477 was also found to potently inhibit TNF production in whole blood (IC(50)=12 nM) and effectively inhibited TNFα production induced by systemically administered LPS in rats at less than 0.1mg/kg (ED(50)=0.053 mg/kg) with an anti-inflammatory effect lasting for 20h after oral administration. Overall, this study demonstrated that AS1940477 is a novel and potent p38 MAPK inhibitor and may be useful as a promising anti-inflammatory agent for treating inflammatory disorders.
Asunto(s)
Antiinflamatorios no Esteroideos/farmacología , Inhibidores de Proteínas Quinasas/farmacología , Piridazinas/farmacología , Pirimidinas/farmacología , Proteínas Quinasas p38 Activadas por Mitógenos/antagonistas & inhibidores , Animales , Antiinflamatorios no Esteroideos/uso terapéutico , Línea Celular , Endotoxinas/toxicidad , Femenino , Humanos , Inflamación/tratamiento farmacológico , Inflamación/metabolismo , Interleucina-1beta/metabolismo , Sistema de Señalización de MAP Quinasas/efectos de los fármacos , Inhibidores de Proteínas Quinasas/uso terapéutico , Piridazinas/uso terapéutico , Pirimidinas/uso terapéutico , Ratas , Especificidad por Sustrato , Factor de Necrosis Tumoral alfa/metabolismo , Proteínas Quinasas p38 Activadas por Mitógenos/metabolismoRESUMEN
The chromatin remodeling complex SWI/SNF is known to regulate the transcription of several genes by controlling chromatin structure in an ATP-dependent manner. SWI/SNF contains the Swi2p/Snf2p like ATPases BRG1 or BRM exclusively. We found that the expression of BRM gradually increases and that of BRG1 decreases as liver cells differentiate. Chromatin immunoprecipitation assays revealed that the ATPase subunits of SWI/SNF and tumor suppressor retinoblastoma (RB) family proteins bind to the promoter region of the albumin gene in hepatocytes, and that the replacement of BRG1 with BRM and pRB with p130 at this site occurs over the course of differentiation. Small interfering RNA experiments showed that blocking the expression of BRG1 and BRM in fetal and adult hepatocytes, respectively, causes a reduction in albumin expression. In luciferase reporter assays with a pREP4-based reporter plasmid that forms a chromatin structure, BRG1 showed activity stimulating the expression of the albumin promoter mediated by CCAAT/enhancer-binding protein alpha (C/EBPalpha). This enhancement was facilitated by the RB family members pRB and p130. ATPase assays showed that both pRB and C/EBPalpha proteins directly stimulate the ATPase activity of BRG1. Our findings suggest that the mechanism by which the activity of transcription factors is enhanced by RB family members and SWI/SNF includes an increase in the ATPase activity of the chromatin remodeling complex.
Asunto(s)
Albúminas/genética , Proteínas Cromosómicas no Histona/metabolismo , Hígado/crecimiento & desarrollo , Factores de Transcripción/metabolismo , Animales , Western Blotting , Proteína beta Potenciadora de Unión a CCAAT/metabolismo , Diferenciación Celular/fisiología , Células Cultivadas , Proteínas Cromosómicas no Histona/genética , ADN Helicasas/efectos de los fármacos , ADN Helicasas/genética , Regulación de la Expresión Génica/efectos de los fármacos , Hepatocitos/efectos de los fármacos , Hepatocitos/metabolismo , Hígado/citología , Hígado/metabolismo , Ratones , Proteínas Nucleares/efectos de los fármacos , Proteínas Nucleares/genética , ARN Interferente Pequeño/farmacología , Ratas , Ratas Sprague-Dawley , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa/métodos , Factores de Transcripción/efectos de los fármacos , Factores de Transcripción/genéticaRESUMEN
The chromatin remodeling complex, SWI/SNF, is known to regulate the transcription of several genes by altering the chromatin structure in an ATP-dependent manner. SWI/SNF exclusively contains BRG1 or BRM as an ATPase subunit. In the present study, we studied the role of SWI/SNF containing BRM or BRG1 in the expression of the liver-specific tryptophan oxygenase (TO) and tyrosine aminotransferase genes. Chromatin remodeling factors significantly repressed the expression of these genes induced by glucocorticoid receptor and dexamethasone. Since the repression was not reversed by trichostatin A treatment, it seemed to be independent of the well-known histone deacetylase pathway. Knock-down of BRG1 by small interfering RNA reversed the repression in primary fetal hepatocytes. These results support a model in which SWI/SNF containing BRG1 represses late stage-specific TO gene expression at an early stage of liver development.