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.
Reactive oxygen species (ROS) generated by Nox NADPH oxidases may play a critical role in the pathogenesis of diabetic nephropathy (DN). The efficacy of the Nox1/Nox4 inhibitor GKT137831 on the ...manifestations of DN was studied in OVE26 mice, a model of type 1 diabetes. Starting at 4-5 mo of age, OVE26 mice were treated with GKT137831 at 10 or 40 mg/kg, once-a-day for 4 wk. At both doses, GKT137831 inhibited NADPH oxidase activity, superoxide generation, and hydrogen peroxide production in the renal cortex from diabetic mice without affecting Nox1 or Nox4 protein expression. The increased expression of fibronectin and type IV collagen was reduced in the renal cortex, including glomeruli, of diabetic mice treated with GKT137831. GKT137831 significantly reduced glomerular hypertrophy, mesangial matrix expansion, urinary albumin excretion, and podocyte loss in OVE26 mice. GKT137831 also attenuated macrophage infiltration in glomeruli and tubulointerstitium. Collectively, our data indicate that pharmacological inhibition of Nox1/4 affords broad renoprotection in mice with preexisting diabetes and established kidney disease. This study validates the relevance of targeting Nox4 and identifies GKT137831 as a promising compound for the treatment of DN in type 1 diabetes.
Increased NADP reduced (NADPH) oxidase 4 (Nox4) and reduced expression of the nuclear hormone receptor peroxisome proliferator-activated receptor γ (PPARγ) contribute to hypoxia-induced pulmonary ...hypertension (PH). To examine the role of Nox4 activity in pulmonary vascular cell proliferation and PH, the current study used a novel Nox4 inhibitor, GKT137831, in hypoxia-exposed human pulmonary artery endothelial or smooth muscle cells (HPAECs or HPASMCs) in vitro and in hypoxia-treated mice in vivo. HPAECs or HPASMCs were exposed to normoxia or hypoxia (1% O(2)) for 72 hours with or without GKT137831. Cell proliferation and Nox4, PPARγ, and transforming growth factor (TGF)β1 expression were measured. C57Bl/6 mice were exposed to normoxia or hypoxia (10% O(2)) for 3 weeks with or without GKT137831 treatment during the final 10 days of exposure. Lung PPARγ and TGF-β1 expression, right ventricular hypertrophy (RVH), right ventricular systolic pressure (RVSP), and pulmonary vascular remodeling were measured. GKT137831 attenuated hypoxia-induced H(2)O(2) release, proliferation, and TGF-β1 expression and blunted reductions in PPARγ in HPAECs and HPASMCs in vitro. In vivo GKT137831 inhibited hypoxia-induced increases in TGF-β1 and reductions in PPARγ expression and attenuated RVH and pulmonary artery wall thickness but not increases in RVSP or muscularization of small arterioles. This study shows that Nox4 plays a critical role in modulating proliferative responses of pulmonary vascular wall cells. Targeting Nox4 with GKT137831 provides a novel strategy to attenuate hypoxia-induced alterations in pulmonary vascular wall cells that contribute to vascular remodeling and RVH, key features involved in PH pathogenesis.
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 CCl4 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. (HEPATOLOGY 2012)
Fibrosis describes a dysregulated tissue remodelling response to persistent cellular injury and is the final pathological consequence of many chronic diseases that affect the liver, kidney and lung. ...Nicotinamide adenine dinucleotide phosphate (NADPH)‐oxidase (NOX) enzymes produce reactive oxygen species (ROS) as their primary function. ROS derived from NOX1 and NOX4 are key mediators of liver, kidney and lung fibrosis. Setanaxib (GKT137831) is a first‐in‐class, dual inhibitor of NOX1/4 and is the first NOX inhibitor to progress to clinical trial investigation. The anti‐fibrotic effects of setanaxib in liver, kidney and lung fibrosis are supported by multiple lines of pre‐clinical evidence. However, despite advances in our understanding, the precise roles of NOX1/4 in fibrosis require further investigation. Additionally, there is a translational gap between the pre‐clinical observations of setanaxib to date and the applicability of these to human patients within a clinical setting. This narrative review critically examines the role of NOX1/4 in liver, kidney and lung fibrosis, alongside the available evidence investigating setanaxib as a therapeutic agent in pre‐clinical models of disease. We discuss the potential clinical translatability of this pre‐clinical evidence, which provides rationale to explore NOX1/4 inhibition by setanaxib across various fibrotic pathologies in clinical trials involving human patients.
The burden of oxidative stress is increased in chronic obstructive pulmonary disease (COPD). However, whether the intra-cellular mechanisms controlling the oxidant/anti-oxidant balance in structural ...airway cells such as airway smooth muscle in COPD is altered is unclear. We sought to determine whether the expression of the NADPH oxidase (NOX)-4 is increased in airway smooth muscle in COPD both in vivo and primary cells in vitro and its role in hydrogen peroxide-induced reactive oxygen species generation. We found that in vivo NOX4 expression was up-regulated in the airway smooth muscle bundle in COPD (n = 9) and healthy controls with >20 pack year history (n = 4) compared to control subjects without a significant smoking history (n = 6). In vitro NOX4 expression was increased in airway smooth muscle cells from subjects with COPD (n = 5) compared to asthma (n = 7) and upregulated following TNF-α stimulation. Hydrogen peroxide-induced reactive oxygen species generation by airway smooth muscle cells in COPD (n = 5) was comparable to healthy controls (n = 9) but lower than asthma (n = 5); and was markedly attenuated by NOX4 inhibition. Our findings demonstrate that NOX4 expression is increased in vivo and in vitro in COPD and although we did not observe an intrinsic increase in oxidant-induced reactive oxygen species generation in COPD, it was reduced markedly by NOX4 inhibition supporting a potential therapeutic role for NOX4 in COPD.
Reactive oxygen species, ROS, are regulators of endothelial cell migration, proliferation and survival, events critically involved in angiogenesis. Different isoforms of ROS-generating NOX enzymes ...are expressed in the vasculature and provide distinct signaling cues through differential localization and activation. We show that mice deficient in NOX1, but not NOX2 or NOX4, have impaired angiogenesis. NOX1 expression and activity is increased in primary mouse and human endothelial cells upon angiogenic stimulation. NOX1 silencing decreases endothelial cell migration and tube-like structure formation, through the inhibition of PPARα, a regulator of NF-κB. Administration of a novel NOX-specific inhibitor reduced angiogenesis and tumor growth in vivo in a PPARα dependent manner. In conclusion, vascular NOX1 is a critical mediator of angiogenesis and an attractive target for anti-angiogenic therapies.
Background Bronchial epithelial ciliary dysfunction is an important feature of asthma. We sought to determine the role in asthma of neutrophilic inflammation and nicotinamide adenine dinucleotide ...phosphate (NADPH) oxidases in ciliary dysfunction. Methods Bronchial epithelial ciliary function was assessed by using video microscopy in fresh ex vivo epithelial strips from patients with asthma stratified according to their sputum cell differentials and in culture specimens from healthy control subjects and patients with asthma. Bronchial epithelial oxidative damage was determined by 8-oxo-dG expression. Nicotinamide adenine dinucleotide phosphate oxidase (NOX)/dual oxidase (DUOX) expression was assessed in bronchial epithelial cells by using microarrays, with NOX4 and DUOX1/2 expression assessed in bronchial biopsy specimens. Ciliary dysfunction following NADPH oxidase inhibition, using GKT137831, was evaluated in fresh epithelial strips from patients with asthma and a murine model of ovalbumin sensitization and challenge. Results Ciliary beat frequency was impaired in patients with asthma with sputum neutrophilia (n = 11) vs those without (n = 10) (5.8 0.6 Hz vs 8.8 0.5 Hz; P = .003) and was correlated with sputum neutrophil count ( r = –0.70; P < .001). Primary bronchial epithelial cells expressed DUOX1/2 and NOX4. Levels of 8-oxo-dG and NOX4 were elevated in patients with neutrophilic vs nonneutrophilic asthma, DUOX1 was elevated in both, and DUOX2 was elevated in nonneutrophilic asthma in vivo. In primary epithelial cultures, ciliary dysfunction did not persist, although NOX4 expression and reactive oxygen species generation was increased from patients with neutrophilic asthma. GKT137831 both improved ciliary function in ex vivo epithelial strips (n = 13), relative to the intensity of neutrophilic inflammation, and abolished ciliary dysfunction in the murine asthma model with no reduction in inflammation. Conclusions Ciliary dysfunction is increased in neutrophilic asthma associated with increased NOX4 expression and is attenuated by NADPH oxidase inhibition.
Ischemic retinal diseases such as retinopathy of prematurity are major causes of blindness due to damage to the retinal microvasculature. Despite this clinical situation, retinopathy of prematurity ...is mechanistically poorly understood. Therefore, effective preventative therapies are not available. However, hypoxic-induced increases in reactive oxygen species (ROS) have been suggested to be involved with NADPH oxidases (NOX), the only known dedicated enzymatic source of ROS. Our major aim was to determine the contribution of NOX isoforms (1, 2, and 4) to a rodent model of retinopathy of prematurity.
Using a genetic approach, we determined that only mice with a deletion of NOX1, but not NOX2 or NOX4, were protected from retinal neovascularization and vaso-obliteration, adhesion of leukocytes, microglial accumulation, and the increased generation of proangiogenic and proinflammatory factors and ROS. We complemented these studies by showing that the specific NOX inhibitor, GKT137831, reduced vasculopathy and ROS levels in retina. The source of NOX isoforms was evaluated in retinal vascular cells and neuro-glial elements. Microglia, the immune cells of the retina, expressed NOX1, 2, and 4 and responded to hypoxia with increased ROS formation, which was reduced by GKT137831.
Our studies are the first to identify the NOX1 isoform as having an important role in the pathogenesis of retinopathy of prematurity.
Our findings suggest that strategies targeting NOX1 have the potential to be effective treatments for a range of ischemic retinopathies.
Diabetes triggers peripheral nerve alterations at a structural and functional level, collectively referred to as diabetic peripheral neuropathy (DPN). This work highlights the role of the liver X ...receptor (LXR) signaling pathway and the cross talk with the reactive oxygen species (ROS)-producing enzyme NADPH oxidase-4 (Nox4) in the pathogenesis of DPN. Using type 1 diabetic (T1DM) mouse models together with cultured Schwann cells (SCs) and skin biopsies from patients with type 2 diabetes (T2DM), we revealed the implication of LXR and Nox4 in the pathophysiology of DPN. T1DM animals exhibit neurophysiological defects and sensorimotor abnormalities paralleled by defective peripheral myelin gene expression. These alterations were concomitant with a significant reduction in LXR expression and increase in Nox4 expression and activity in SCs and peripheral nerves, which were further verified in skin biopsies of patients with T2DM. Moreover, targeted activation of LXR or specific inhibition of Nox4 in vivo and in vitro to attenuate diabetes-induced ROS production in SCs and peripheral nerves reverses functional alteration of the peripheral nerves and restores the homeostatic profiles of MPZ and PMP22. Taken together, our findings are the first to identify novel, key mediators in the pathogenesis of DPN and suggest that targeting LXR/Nox4 axis is a promising therapeutic approach.
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