Topically Administered NOX4 Inhibitor, GLX7013114, Is Efficacious in Treating the Early Pathological Events of Diabetic Retinopathy.

NADPH oxidases (NOXs) are major players in generating reactive oxygen species (ROS) and are implicated in various neurodegenerative ocular pathologies. The aim of this study was to investigate the role of a NOX4 inhibitor (GLX7013114) in two in vivo, experimental streptozotocin (STZ) paradigms depicting the early events of diabetic retinopathy (DR). Animals in the diabetic treated group received GLX7013114 topically (20 µL/eye, 10 mg/mL, once daily) for 14 days (paradigm A: preventive) and 7 days (paradigm B: treated) at 48 h and 4 weeks after STZ injection, respectively. Several methodologies were used (immunohistochemistry, Western blot, real-time PCR, ELISA, pattern electroretinography [PERG]) to assess the diabetes-induced early events of DR, namely oxidative stress, neurodegeneration, and neuroinflammation, and the effect of GLX7013114 on the diabetic insults. GLX7013114, administered as eye drops (paradigms A and B), was beneficial in treating the oxidative nitrative stress, activation of caspase-3 and micro- and macroglia, and attenuation of neuronal markers. It also attenuated the diabetes-induced increase in vascular endothelial growth factor, Evans blue dye leakage, and proinflammatory cytokine (TNF-α protein, IL-1ß/IL-6 mRNA) levels. PERG amplitude values suggested that GLX7013114 protected retinal ganglion cell function (paradigm B). This study provides new findings regarding the pharmacological profile of the novel NOX4 inhibitor GLX7013114 as a promising therapeutic candidate for the treatment of the early stage of DR. ARTICLE HIGHLIGHTS: NADPH oxidases (NOXs) are implicated in the early pathological events of diabetic retinopathy (DR). The NOX4 inhibitor GLX7013114, topically administered, reduced oxidative damage and apoptosis in the rat streptozotocin model of DR. GLX7013114 protected retinal neurons and retinal ganglion cell function and reduced the expression of pro-inflammatory cytokines in the diabetic retina. GLX7013114 diminished the diabetes-induced increase in vascular endothelial growth factor levels and Evans blue dye leakage in retinal tissue. GLX7013114 exhibits neuroprotective, anti-inflammatory, and vasculoprotective properties that suggest it may have a role as a putative therapeutic for the early events of DR.

Diphenyleneiodonium efficiently inhibits the characteristics of a cancer stem cell model derived from induced pluripotent stem cells.

Diphenyleneiodonium (DPI) has long been evaluated as an anticancer drug inhibiting NADPH oxidase, the IC50 in several cancer cell lines was reported 10 µM, which is too high for efficacy. In this study, we employed miPS-Huh7cmP cells, which we previously established as a cancer stem cell (CSC) model from induced pluripotent stem cells, to reevaluate the efficacy of DPI because CSCs are currently one of the main foci of therapeutic strategy to treat cancer, but generally considered resistant to chemotherapy. As a result, the conventional assay for the cell growth inhibition by DPI accounted for an IC50 at 712 nM that was not enough to define the effectiveness as an anticancer drug. Simultaneously, the wound-healing assay revealed an IC50 of approximately 500 nM. Comparatively, the IC50 values shown on sphere formation, colony formation, and tube formation assays were 5.52, 12, and 8.7 nM, respectively. However, these inhibitory effects were not observed by VAS2780, also a reputed NADPH oxidase inhibitor. It is noteworthy that these three assays are evaluating the characteristic of CSCs and are designed in the three-dimensional (3D) culture methods. We concluded that DPI could be a suitable candidate to target mitochondrial respiration in CSCs. We propose that the 3D culture assays are more efficient to screen anti-CSC drug candidates and better mimic tumor microenvironment when compared to the adherent monolayer of 2D culture system used for a conventional assay, such as cell growth inhibition and wound-healing assays.

A small molecule inhibitor of Nox2 and Nox4 improves contractile function after ischemia-reperfusion in the mouse heart.

The NADPH oxidase enzymes Nox2 and 4, are important generators of Reactive oxygen species (ROS). These enzymes are abundantly expressed in cardiomyocytes and have been implicated in ischemia-reperfusion injury. Previous attempts with full inhibition of their activity using genetically modified animals have shown variable results, suggesting that a selective and graded inhibition could be a more relevant approach. We have, using chemical library screening, identified a new compound (GLX481304) which inhibits Nox 2 and 4 (with IC50 values of 1.25 µM) without general antioxidant effects or inhibitory effects on Nox 1. The compound inhibits ROS production in isolated mouse cardiomyocytes and improves cardiomyocyte contractility and contraction of whole retrogradely (Langendorff) perfused hearts after a global ischemia period. We conclude that a pharmacological and partial inhibition of ROS production by inhibition of Nox 2 and 4 is beneficial for recovery after ischemia reperfusion and might be a promising venue for treatment of ischemic injury to the heart.

A novel NADPH oxidase inhibitor targeting Nox4 in TGFß-induced lens epithelial to mesenchymal transition.

Many of the small molecule-based inhibitors of NADPH oxidase activity are largely inadequate to substantiate broad claims, often exhibiting a lack of Nox-isoform-specificity, and sometimes only acting as scavengers of reactive oxygen species (ROS). In the present study, we use a newly developed highly selective Nox4 inhibitor, GLX7013114, to modulate TGFß-induced lens epithelial to mesenchymal transition (EMT). Rat lens epithelial explants were pre-treated with 0.3  µM of GLX7013114, and then treated with 200 pg/ml of TGF-ß2 to induce lens EMT. ROS production was visualized microscopically using the superoxide fluorogenic probe, dihydroethidium (DHE). The EMT process was documented using phase-contrast microscopy, and molecular EMT markers were immunolabeled. qPCR was also performed to observe changes in EMT-associated genes. TGFß-induced ROS was evident at 8 h of culture and its intensity was found to be significantly reduced when GLX7013114 was applied, comparable to ROS levels measured in untreated explants. Using phase-contrast microscopy to follow TGFß-induced EMT over 5 days in the presence of the inhibitor, lens epithelial cells in explants became myofibroblastic by day 2 and underwent progressive apoptosis to reveal a bare lens capsule by day 5. Explants treated with TGFß and GLX7013114 had some increased cell survival; however, these differences were not significant. For the first time, Nox4 inhibition by GLX7013114 was shown to reduce the TGFß-induced gene expression of α-smooth muscle actin (αSMA), collagen 1a and fibronectin. GLX7013114, given that it appears to block aspects of TGFß-induced EMT, including ROS production, may be a new useful Nox4-selective inhibitor for further studies.

The novel NADPH oxidase 4 selective inhibitor GLX7013114 counteracts human islet cell death in vitro.

It has been proposed that pancreatic beta-cell dysfunction in type 2 diabetes is promoted by oxidative stress caused by NADPH oxidase (Nox) over-activity. The aim of the present study was to evaluate the efficacy of novel Nox inhibitors as protective agents against cytokine- or high glucose + palmitate-induced human beta-cell death. The Nox2 protein was present mainly in the cytoplasm and was induced by cytokines. Nox4 protein immunoreactivity, with some nuclear accumulation, was observed in human islet cells, and was not affected by islet culture in the presence of cytokines or high glucose + palmitate. Nox inhibitors with partial or no isoform selectivity (DPI, dapsone, GLX351322, and GLX481372) all reduced ROS production of human islet cells exposed to high glucose + palmitate. This was paralleled by improved viability and reduced caspase 3 activation. The Nox1 selective inhibitor ML171 failed to reduce human islet cell death in response to both cytokines and high glucose + palmitate. The selective Nox2 inhibitor Phox-I2 also failed to protect against cytokines, but protected partially against high glucose + palmitate-induced cellular death. The highly selective Nox4 inhibitor GLX7013114 protected islet cells against both cytokines and high glucose + palmitate. However, as no osmotic control for high glucose was used, we cannot exclude the possibility that the high glucose effect was due to osmosis. It is concluded that Nox4 may participate in stress-induced islet cell death in human islets in vitro. We propose that Nox4 mediates pro-apoptotic effects in intact islets under stressful conditions and that selective Nox4-inhibition may be a therapeutic strategy in type 2 diabetes.

A 55 kDa hypothetical membrane protein is an iron-regulated virulence factor of Francisella tularensis subsp. novicida U112.

Iron is an important nutritional requirement for bacteria due to its conserved role in many essential metabolic processes. As a consequence of the lack of freely available iron in the mammalian host, bacteria upregulate a range of virulence factors during infection. Transcriptional analysis of Francisella tularensis subsp. novicida U112 grown in iron-deficient medium identified 21 genes upregulated in response to this condition, four of which were attributed to a siderophore operon. In addition, a novel iron-regulated gene, FTT0025, was identified which is part of this operon and encodes a 55 kDa hypothetical membrane protein. When grown on chrome azurol S agar, the F. tularensis subsp. novicida U112deltaFTT0025 mutant produced an increased reaction zone compared with the wild-type, suggesting that siderophore production was unaffected but that the bacteria may have a deficiency in their ability to re-sequester this iron-binding molecule. Furthermore, the deltaFTT0025 mutant was attenuated in a BALB/c mouse model of infection relative to wild-type F. tularensis subsp. novicida U112.

Diphenylene iodonium stimulates glucose uptake in skeletal muscle cells through mitochondrial complex I inhibition and activation of AMP-activated protein kinase.

NADPH oxidase inhibitors such as diphenylene iodonium (DPI) and apocynin lower whole body and blood glucose levels and improve diabetes when administered to rodents. Skeletal muscle has an important role in managing glucose homeostasis and we have used L6 cells, C(2)C(12) cells and primary muscle cells as model systems to investigate whether these drugs regulate glucose uptake in skeletal muscle cells. The data presented in this study show that apocynin does not affect glucose uptake in skeletal muscle cells in culture. Tat gp91ds, a chimeric peptide that inhibits NADPH oxidase activity, also failed to affect glucose uptake and we found no significant evidence of NADPH oxidase (subunits tested were Nox4, p22phox, gp91phox and p47phox mRNA) in skeletal muscle cells in culture. However, DPI increases basal and insulin-stimulated glucose uptake in L6 cells, C(2)C(12) cells and primary muscle cells. Detailed studies on L6 cells demonstrate that the increase of glucose uptake is via a mechanism independent of phosphoinositide-3 kinase (PI3K)/Akt but dependent on AMP-activated protein kinase (AMPK). We postulate that DPI through inhibition of mitochondrial complex 1 and decreases in oxygen consumption, leading to decreases of ATP and activation of AMPK, stimulates glucose uptake in skeletal muscle cells.