A subset of N-substituted phenothiazines inhibits NADPH oxidases.

NADPH oxidases (NOXs) constitute a family of enzymes generating reactive oxygen species (ROS) and are increasingly recognized as interesting drug targets. Here we investigated the effects of 10 phenothiazine compounds on NOX activity using an extensive panel of assays to measure production of ROS (Amplex red, WST-1, MCLA) and oxygen consumption. Striking differences between highly similar phenothiazines were observed. Two phenothiazines without N-substitution, including ML171, did not inhibit NOX enzymes, but showed assay interference. Introduction of an aliphatic amine chain on the N atom of the phenothiazine B ring (promazine) conferred inhibitory activity toward NOX2, NOX4, and NOX5 but not NOX1 and NOX3. Addition of an electron-attracting substituent in position 2 of the C ring extended the inhibitory activity to NOX1 and NOX3, with thioridazine being the most potent inhibitor. In contrast, the presence of a methylsulfoxide group at the same position (mesoridazine) entirely abolished NOX-inhibitory activity. A cell-free NOX2 assay suggested that inhibition by N-substituted phenothiazines was not due to competition with NADPH. A functional implication of NOX-inhibitory activity of thioridazine was demonstrated by its ability to block redox-dependent myofibroblast differentiation. Our results demonstrate that NOX-inhibitory activity is not a common feature of all antipsychotic phenothiazines and that substitution on the B-ring nitrogen is crucial for the activity, whereas that on the second position of the C ring modulates it. Our findings contribute to a better understanding of NOX pharmacology and might pave the path to discovery of more potent and selective NOX inhibitors.

Nicotinamide adenine dinucleotide phosphate oxidase in experimental liver fibrosis: GKT137831 as a novel potential therapeutic agent.

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.

Design, synthesis and biological activity of original pyrazolo-pyrido-diazepine, -pyrazine and -oxazine dione derivatives as novel dual Nox4/Nox1 inhibitors.

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.

NADPH oxidase (NOX) isoforms are inhibited by celastrol with a dual mode of action.

BACKGROUND: Celastrol is one of several bioactive compounds extracted from the medicinal plant Tripterygium wilfordii. Celastrol is used to treat inflammatory conditions, and shows benefits in models of neurodegenerative disease, cancer and arthritis, although its mechanism of action is incompletely understood. EXPERIMENTAL APPROACH: Celastrol was tested on human NADPH oxidases (NOXs) using a panel of experiments: production of reactive oxygen species and oxygen consumption by NOX enzymes, xanthine oxidase activity, cell toxicity, phagocyte oxidase subunit translocation, and binding to cytosolic subunits of NOX enzymes. The effect of celastrol was compared with diphenyleneiodonium, an established inhibitor of flavoproteins. KEY RESULTS: Low concentrations of celastrol completely inhibited NOX1, NOX2, NOX4 and NOX5 within minutes with concentration-response curves exhibiting higher Hill coefficients and lower IC50 values for NOX1 and NOX2 compared with NOX4 and NOX5, suggesting differences in their mode of action. In a cell-free system, celastrol had an IC50 of 1.24 and 8.4 µM for NOX2 and NOX5, respectively. Cytotoxicity, oxidant scavenging, and inhibition of p47(phox) translocation could not account for NOX inhibition. Celastrol bound to a recombinant p47(phox) and disrupted the binding of the proline rich region of p22(phox) to the tandem SH3 domain of p47(phox) and NOXO1, the cytosolic subunits of NOX2 and NOX1, respectively. CONCLUSIONS AND IMPLICATIONS: These results demonstrate that celastrol is a potent inhibitor of NOX enzymes in general with increased potency against NOX1 and NOX2. Furthermore, inhibition of NOX1 and NOX2 was mediated via a novel mode of action, namely inhibition of a functional association between cytosolic subunits and the membrane flavocytochrome.

First in class, potent, and orally bioavailable NADPH oxidase isoform 4 (Nox4) inhibitors for the treatment of idiopathic pulmonary fibrosis.

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.