RESUMEN
UNLABELLED: Nicotinamide adenine dinucleotide phosphate (NADPH) oxidase (NOX) generates reactive oxygen species (ROS) in hepatic stellate cells (HSCs) during liver fibrosis. In response to fibrogenic agonists, such as angiotensin II (Ang II), the NOX1 components form an active complex, including Ras-related botulinum toxin substrate 1 (Rac1). Superoxide dismutase 1 (SOD1) interacts with the NOX-Rac1 complex to stimulate NOX activity. NOX4 is also induced in activated HSCs/myofibroblast by increased gene expression. Here, we investigate the role of an enhanced activity SOD1 G37R mutation (SODmu) and the effects of GKT137831, a dual NOX1/4 inhibitor, on HSCs and liver fibrosis. To induce liver fibrosis, wild-type (WT) and SOD1mu mice were treated with CCl(4) or bile duct ligation (BDL). Then, to address the role of NOX-SOD1-mediated ROS production in HSC activation and liver fibrosis, mice were treated with a NOX1/4 inhibitor. Fibrosis and ROS generation was assessed by histology and measurement of thiobarbituric acid reactive substances and NOX-related genes. Primary cultured HSCs isolated from WT, SODmu, and NOX1 knockout (KO) mice were assessed for ROS production, Rac1 activity, and NOX gene expression. Liver fibrosis was increased in SOD1mu mice, and ROS production and Rac1 activity were increased in SOD1mu HSCs. The NOX1/4 inhibitor, GKT137831, attenuated liver fibrosis and ROS production in both SOD1mu and WT mice as well as messenger RNA expression of fibrotic and NOX genes. Treatment with GKT137831 suppressed ROS production and NOX and fibrotic gene expression, but not Rac1 activity, in SOD1mut and WT HSCs. Both Ang II and tumor growth factor beta up-regulated NOX4, but Ang II required NOX1. CONCLUSIONS: SOD1mu induces excessive NOX1 activation through Rac1 in HSCs, causing enhanced NOX4 up-regulation, ROS generation, and liver fibrosis. Treatment targeting NOX1/4 may be a new therapy for liver fibrosis.
Asunto(s)
Inhibidores Enzimáticos/uso terapéutico , Cirrosis Hepática/tratamiento farmacológico , Cirrosis Hepática/enzimología , NADH NADPH Oxidorreductasas/metabolismo , NADPH Oxidasas/metabolismo , Pirazoles/uso terapéutico , Piridinas/uso terapéutico , Superóxido Dismutasa/genética , Angiotensina II/farmacología , Animales , Inhibidores Enzimáticos/farmacología , Expresión Génica , Células Estrelladas Hepáticas/efectos de los fármacos , Cirrosis Hepática/genética , Masculino , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , NADH NADPH Oxidorreductasas/genética , NADPH Oxidasa 1 , NADPH Oxidasa 4 , NADPH Oxidasas/genética , Neuropéptidos/metabolismo , Pirazoles/farmacología , Pirazolonas , Piridinas/farmacología , Piridonas , ARN Mensajero/metabolismo , Especies Reactivas de Oxígeno/metabolismo , Superóxido Dismutasa-1 , Regulación hacia Arriba , Proteínas de Unión al GTP rac/metabolismo , Proteína de Unión al GTP rac1RESUMEN
Pyrazolo-pyrido-diazepine, -pyrazine and -oxazine dione derivatives are new chemical entities with good and attractive druglikeness properties. A series of pyrazolo-pyrido-diazepine dione analogs demonstrated to be particularly amenable to lead optimization through a couple of cycles in order to improve specificity for isoforms Nox4 and Nox1 and had excellent pharmacokinetic parameters by oral route. Several molecules such as compound 7c proved to be highly potent in in vitro assays on human lung fibroblasts differentiation as well as in curative murine models of bleomycin-induced pulmonary fibrosis with superior efficiency over Pirfenidone. Pyrazolo-pyrido-diazepine dione derivatives targeting Nox4 and Nox1 isoforms appear highly promising therapeutics for the treatment of idiopathic pulmonary fibrosis.
Asunto(s)
Azepinas/química , Inhibidores Enzimáticos/química , NADPH Oxidasas/antagonistas & inhibidores , Oxazinas/química , Pirazinas/química , Administración Oral , Animales , Azepinas/síntesis química , Azepinas/farmacología , Bleomicina/toxicidad , Modelos Animales de Enfermedad , Inhibidores Enzimáticos/síntesis química , Inhibidores Enzimáticos/farmacología , Humanos , Masculino , Ratones , Ratones Endogámicos C57BL , NADPH Oxidasa 1 , NADPH Oxidasa 4 , Oxazinas/síntesis química , Oxazinas/farmacología , Fibrosis Pulmonar/inducido químicamente , Fibrosis Pulmonar/tratamiento farmacológico , Pirazinas/síntesis química , Pirazinas/farmacología , Piridonas/farmacología , Relación Estructura-ActividadRESUMEN
We describe the design, synthesis, and optimization of first-in-class series of inhibitors of NADPH oxidase isoform 4 (Nox4), an enzyme implicated in several pathologies, in particular idiopathic pulmonary fibrosis, a life-threatening and orphan disease. Initially, several moderately potent pyrazolopyridine dione derivatives were found during a high-throughput screening campaign. SAR investigation around the pyrazolopyridine dione core led to the discovery of several double-digit nanomolar inhibitors in cell free assays of reactive oxygen species (ROS) production, showing high potency on Nox4 and Nox1. The compounds have little affinity for Nox2 isoform and are selective for Nox4/1 isoforms. The specificity of these compounds was confirmed in an extensive in vitro pharmacological profile, as well as in a counterscreening assay for potential ROS scavenging. Concomitant benefits are good oral bioavailability and high plasma concentrations in vivo, allowing further clinical trials for the potential treatment of fibrotic diseases, cancers, and cardiovascular and metabolic diseases.
Asunto(s)
NADPH Oxidasas/antagonistas & inhibidores , Fibrosis Pulmonar/tratamiento farmacológico , Pirazoles/síntesis química , Piridinas/síntesis química , Administración Oral , Animales , Disponibilidad Biológica , Línea Celular , Enfermedad Crónica , Cricetinae , Cricetulus , Depuradores de Radicales Libres/síntesis química , Depuradores de Radicales Libres/química , Depuradores de Radicales Libres/farmacología , Ensayos Analíticos de Alto Rendimiento , Humanos , Isoenzimas/antagonistas & inhibidores , Enfermedades Renales/tratamiento farmacológico , Masculino , Microsomas Hepáticos/metabolismo , NADPH Oxidasa 4 , Pirazoles/química , Pirazoles/farmacología , Piridinas/química , Piridinas/farmacología , Ratas , Ratas Sprague-Dawley , Especies Reactivas de Oxígeno/metabolismo , Relación Estructura-ActividadRESUMEN
We show that the allele-dependent expression of transcripts encoding soluble HLA-DQbeta chains is determined by branchpoint sequence (BPS) haplotypes in DQB1 intron 3. BPS RNAs associated with low inclusion of the transmembrane exon in mature transcripts showed impaired binding to splicing factor 1 (SF1), indicating that alternative splicing of DQB1 is controlled by differential BPS recognition early during spliceosome assembly. We also demonstrate that naturally occurring human BPS point mutations that alter splicing and lead to recognizable phenotypes cluster in BP and in position -2 relative to BP, implicating impaired SF1-BPS interactions in disease-associated BPS substitutions. Coding DNA variants produced smaller fluctuations of exon inclusion levels than random exonic substitutions, consistent with a selection against coding mutations that alter their own exonization. Finally, proximal splicing in this multi-allelic reporter system was promoted by at least seven SR proteins and repressed by hnRNPs F, H and I, supporting an extensive antagonism of factors balancing the splice site selection. These results provide the molecular basis for the haplotype-specific expression of soluble DQbeta, improve prediction of intronic point mutations and indicate how extraordinary, selection-driven DNA variability in HLA affects pre-mRNA splicing.