NADPH oxidase 4 (Nox4); The target of GLX351322 is NADPH oxidase 4 (NOX4) [1]

At an IC50 of 5 μM, GLX351322, an inhibitor of NADPH oxidase 4, prevents NOX4 overexpressing cells from producing hydrogen peroxide. GLX351322 exhibits little efficacy (IC50, 40 μM) against NOX2 in hPBMC cells.

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In isolated beating rat atria subjected to hypoxia, treatment with GLX351322 (10 μM) significantly blocked the hypoxia-induced upregulation of Sirt1 protein expression. This inhibition was associated with a reduction in hypoxia-stimulated atrial natriuretic peptide (ANP) secretion. Western blot analysis showed that GLX351322 attenuated the hypoxia-induced activation of signaling molecules downstream of NOX4, including Src, ERK1/2, Akt, and GATA4. Specifically, it decreased the phosphorylation levels of Src, ERK1/2, and Akt, as well as the nuclear translocation of GATA4, which is a key transcription factor for ANP gene expression [2]

GLX351322 (3.8 mg/kg/day, orally) can ameliorate hyperglycemia in rats generated by HF diet [1].
In type 2 diabetes, it has been proposed that pancreatic beta-cell dysfunction is promoted by oxidative stress caused by NADPH oxidase (NOX) overactivity. Five different NOX enzymes (NOX1-5) have been characterized, among which NOX1 and NOX2 have been proposed to negatively affect beta-cells, but the putative role of NOX4 in type 2 diabetes-associated beta-cell dysfunction and glucose intolerance is largely unknown. Therefore, we presently investigated the importance of NOX4 for high-fat diet or HFD-induced glucose intolerance using male C57BL/6 mice using the new NOX4 inhibitor GLX351322, which has relative NOX4 selectivity over NOX2. In HFD-treated male C57BL/6 mice a two-week treatment with GLX351322 counteracted non-fasting hyperglycemia and impaired glucose tolerance. This effect occurred without any change in peripheral insulin sensitivity. To ascertain that NOX4 also plays a role for the function of human beta-cells, we observed that glucose- and sodium palmitate-induced insulin release from human islets in vitro was increased in response to NOX4 inhibitors. In long-term experiments (1-3 days), high-glucose-induced human islet cell reactive oxygen species (ROS) production and death were prevented by GLX351322. We propose that while short-term NOX4-generated ROS production is a physiological requirement for beta-cell function, persistent NOX4 activity, for example, during conditions of high-fat feeding, promotes ROS-mediated beta-cell dysfunction. Thus, selective NOX inhibition may be a therapeutic strategy in type 2 diabetes [1].

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In C57BL/6 mice treated with a high-fat diet (HFD), oral administration of GLX351322 (30 mg/kg/day for 12 weeks) counteracts glucose intolerance. Compared to HFD-fed mice without the drug, those treated with GLX351322 show improved glucose tolerance, as evidenced by lower blood glucose levels during an oral glucose tolerance test (OGTT). Additionally, GLX351322 reduces HFD-induced increases in visceral fat mass and plasma insulin levels. It also normalizes HFD-induced upregulation of NOX4 mRNA expression in white adipose tissue (WAT) and skeletal muscle, and decreases the levels of lipid peroxidation products (e.g., 4-hydroxynonenal) in WAT, indicating reduced oxidative stress [1]

Enzyme-Linked Immunosorbent Assay [3]
Concentrations of IL-1β and IL-18 in cell culture medium or rat synovial fluid were measured with rat IL-1β enzyme-linked immunosorbent assay (ELISA) Kit and rat IL-18 ELISA Kit according to the supplier's protocols. Absorbance was measured at 450 nm wavelength using a PowerWave XS.

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In vitro Co-immunoprecipitation and Ubiquitination Assay [3]
Co-immunoprecipitation (Co-IP) was used to measure the interaction of USP7 and NOX4. Cell lysates extracted with RIPA buffer were incubated with anti-USP7, anti-NOX4, or normal IgG antibody at 4°C overnight, followed by incubation with protein A/G beads at 4°C for 2 h. The immunocomplexes were washed three times with lysis buffer on a magnetic rack and then examined by immunoblotting with anti-USP7 antibody, anti-NOX4 antibody, and anti-ubiquitin antibodies.

Proliferation Assay [3]
The proliferation of chondrocytes was assessed using Cell Counting Kit 8 following the manufacturer's protocol. Briefly, cells were seeded into 96-well plates and incubated for 24 h before treatment. Control or treated cells (90 μL) were mixed with CCK-8 reagent (10 μL) at 0, 24, 48, or 72 h. After incubating for 1 h, the optical density at 450 nm (OD 450) was measured using a reader.

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Isolated rat atria were prepared and maintained in an oxygenated physiological solution. After stabilization, the atria were divided into groups: normoxia, hypoxia, and hypoxia + GLX351322 (10 μM). Hypoxia was induced by exposing the atria to a gas mixture with reduced oxygen content for a specified duration. GLX351322 was added to the incubation medium 30 minutes before the start of hypoxia. At the end of the treatment period, the culture medium was collected to measure ANP secretion using radioimmunoassay. Atrial tissues were homogenized, and protein extracts were analyzed by western blotting to detect the expression levels of Sirt1, and the phosphorylation status of Src, ERK1/2, Akt, and GATA4 nuclear translocation [2]

All rats were randomly divided into: control, hypoxia only, BQ123/BQ788/BQ123 + BQ788 + hypoxia/ET-1, GLX351322 + hypoxia, ET-1 only, varespladib + hypoxia/ET-1, CAY10650 + hypoxia/ET-1, NAC + hypoxia, Src inhibitor 1 + hypoxia, PD98059 + hypoxia, and LY294002 + hypoxia groups (n = 6 per group). Each atrium was perfused for 60 min to stabilize ANP secretion and atrial dynamics parameters. After two control cycles (12-min experimental cycle), O2 was replaced with N2 and a hypoxic buffer was infused for four periods to observe changes in the atrial dynamics and ANP levels of the perfusates. Under a controlled temperature of 4°C, perfusates were collected every 2 min to measure ANP levels. Immediately after perfusion, atrial tissue was frozen and stored at –80°C for western blotting. Subsequently, another series of experiments were performed to investigate the mechanism of hypoxia-induced ANP secretion. After one control period, one treatment cycle was followed by four cycles of infusion of the treatment agent plus hypoxia. Treatment agents were as follows: BQ123 (0.3 µM), BQ788 (0.3 µM), ET-1 (3.0 nM) GLX351322 (35.0 µM), varespladib (5.0 µM), CAY10650 (120.0 nM), NAC (15.0 mM), Src inhibitor 1 (1.0 µM), PD98059 (30.0 µM), and LY294002 (30.0 µM).[2]

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C57BL/6 mice are randomly divided into three groups: a control group fed a normal diet, an HFD group fed a high-fat diet, and an HFD + GLX351322 group fed a high-fat diet and administered GLX351322. GLX351322 is dissolved in drinking water at a concentration that allows a daily intake of 30 mg/kg body weight, and is administered continuously for 12 weeks. During the study, body weight is measured weekly. After 11 weeks, an OGTT is performed: mice are fasted overnight, given glucose (2 g/kg) by oral gavage, and blood glucose levels are measured at 0, 15, 30, 60, and 120 minutes. At the end of the 12-week treatment, mice are euthanized, and tissues (including WAT, skeletal muscle, liver) and blood samples are collected for further analysis [1]

[1]. The novel NADPH oxidase 4 inhibitor GLX351322 counteracts glucose intolerance in high-fat diet-treated C57BL/6 mice. Free Radic Res. 2015;49(11):1308-18.

[2]. NOX4/Src regulates ANP secretion through activating ERK1/2 and Akt/GATA4 signaling in beating rat hypoxic atria. Korean J Physiol Pharmacol. 2021 Mar 1;25(2):159–166.

[3]. USP7 Inhibition Alleviates H2O2-Induced Injury in Chondrocytes via Inhibiting NOX4/NLRP3 Pathway. Front Pharmacol . 2021 Jan 29:11:617270.

GLX351322 is a novel NADPH oxidase 4 inhibitor. By inhibiting NOX4, it reduces oxidative stress, which is associated with the improvement of glucose intolerance in HFD-induced obese mice. This suggests its potential role in managing metabolic disorders related to insulin resistance and obesity [1]
Osteoarthritis (OA), the most common form of arthritis, is a very common joint disease that often affects middle-aged to elderly people. However, current treatment options for OA are predominantly palliative. Thus, understanding its pathological process and exploring its potential therapeutic approaches are of great importance. Rat chondrocytes were isolated and exposed to hydrogen peroxide (H2O2) to mimic OA. The effects of H2O2 on ubiquitin-specific protease 7 (USP7) expression, reactive oxygen species (ROS) levels, proliferation, inflammatory cytokine release, and pyroptosis were measured. USP7 was knocked down (KD) or overexpressed to investigate the role of USP7 in OA. Co-immunoprecipitation (Co-IP) was used to study the interaction between USP7 and NAD(P)H oxidases (NOX)4 as well as NOX4 ubiquitination. NOX4 inhibitor was applied to study the involvement of NOX4 in USP7-mediated OA development. USP7 inhibitor was given to OA animals to further investigate the role of USP7 in OA in vivo. Moreover, H2O2 treatment significantly increased USP7 expression, enhanced ROS levels, and inhibited proliferation in rat chondrocytes. The overexpression of USP7 enhanced pyroptosis, ROS production, interleukin (IL)-1β and IL-18 levels, and the expression level of NLRP3, GSDMD-N, active caspase-1, pro-caspase-1, matrix metalloproteinases (MMP) 1, and MMP13, which was abolished by ROS inhibition. The USP7 KD protected rat chondrocytes against H2O2-induced injury. Co-IP results showed that USP7 interacted with NOX4, and USP7 KD enhanced NOX4 ubiquitinylation. The inhibition of NOX4 blocked the pro-OA effect of USP7. Moreover, the USP7 inhibitor given to OA animals suppressed OA in vivo. USP7 inhibited NOX4 ubiquitination for degradation which leads to elevated ROS production. ROS subsequently activates NLPR3 inflammasome, leading to enhanced production of IL-1β and IL-18, GSDMD-N-dependent pyroptosis, and extracellular matrix remodeling. Thus, UPS7 contributes to the progression of OA via NOX4/ROS/NLPR3 axis. [3]

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Osteoarthritis (OA), the most common form of arthritis, is a very common joint disease that often affects middle-aged to elderly people. However, current treatment options for OA are predominantly palliative. Thus, understanding its pathological process and exploring its potential therapeutic approaches are of great importance. Rat chondrocytes were isolated and exposed to hydrogen peroxide (H2O2) to mimic OA. The effects of H2O2 on ubiquitin-specific protease 7 (USP7) expression, reactive oxygen species (ROS) levels, proliferation, inflammatory cytokine release, and pyroptosis were measured. USP7 was knocked down (KD) or overexpressed to investigate the role of USP7 in OA. Co-immunoprecipitation (Co-IP) was used to study the interaction between USP7 and NAD(P)H oxidases (NOX)4 as well as NOX4 ubiquitination. NOX4 inhibitor was applied to study the involvement of NOX4 in USP7-mediated OA development. USP7 inhibitor was given to OA animals to further investigate the role of USP7 in OA in vivo. Moreover, H2O2 treatment significantly increased USP7 expression, enhanced ROS levels, and inhibited proliferation in rat chondrocytes. The overexpression of USP7 enhanced pyroptosis, ROS production, interleukin (IL)-1β and IL-18 levels, and the expression level of NLRP3, GSDMD-N, active caspase-1, pro-caspase-1, matrix metalloproteinases (MMP) 1, and MMP13, which was abolished by ROS inhibition. The USP7 KD protected rat chondrocytes against H2O2-induced injury. Co-IP results showed that USP7 interacted with NOX4, and USP7 KD enhanced NOX4 ubiquitinylation. The inhibition of NOX4 blocked the pro-OA effect of USP7. Moreover, the USP7 inhibitor given to OA animals suppressed OA in vivo. USP7 inhibited NOX4 ubiquitination for degradation which leads to elevated ROS production. ROS subsequently activates NLPR3 inflammasome, leading to enhanced production of IL-1β and IL-18, GSDMD-N-dependent pyroptosis, and extracellular matrix remodeling. Thus, UPS7 contributes to the progression of OA via NOX4/ROS/NLPR3 axis.[2]

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In the context of hypoxic rat atria, GLX351322 acts as a NOX4 inhibitor to suppress the NOX4/Src-mediated signaling pathway, thereby reducing ANP secretion stimulated by hypoxia. This indicates that GLX351322 can modulate NOX4-dependent cellular responses in cardiac atrial tissue under hypoxic conditions [2]

C21H25N3O5S

431.15

431.151

C, 58.45; H, 5.84; N, 9.74; O, 18.54; S, 7.43

835598-94-2

2697686

White to off-white solid powder

1.3±0.1 g/cm3

665.5±55.0 °C at 760 mmHg

356.3±31.5 °C

0.0±2.0 mmHg at 25°C

1.625

4.1

1

7

7

30

655

0

KEVHLTCEMHIJTQ-UHFFFAOYSA-N

InChI=1S/C21H25N3O5S/c1-2-28-21(27)18-14-5-3-7-16(14)30-19(18)22-17(25)13-23-8-10-24(11-9-23)20(26)15-6-4-12-29-15/h4,6,12H,2-3,5,7-11,13H2,1H3,(H,22,25)

2934.99.9001

Powder      -20°C    3 years

                     4°C     2 years

In solvent   -80°C    6 months

                  -20°C    1 month

Room temperature (This product is stable at ambient temperature for a few days during ordinary shipping and time spent in Customs)

Solubility in Formulation 1: 2.08 mg/mL (4.82 mM) in 10% DMSO + 40% PEG300 + 5% Tween80 + 45% Saline (add these co-solvents sequentially from left to right, and one by one), suspension solution; with sonication.
For example, if 1 mL of working solution is to be prepared, you can add 100 μL of 20.8 mg/mL clear DMSO stock solution to 400 μL PEG300 and mix evenly; then add 50 μL Tween-80 to the above solution and mix evenly; then add 450 μL normal saline to adjust the volume to 1 mL.
Preparation of saline: Dissolve 0.9 g of sodium chloride in 100 mL ddH₂ O to obtain a clear solution.

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Solubility in Formulation 2: ≥ 2.08 mg/mL (4.82 mM) (saturation unknown) in 10% DMSO + 90% Corn Oil (add these co-solvents sequentially from left to right, and one by one), clear solution.
For example, if 1 mL of working solution is to be prepared, you can add 100 μL of 20.8 mg/mL clear DMSO stock solution to 900 μL of corn oil and mix evenly.

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 (Please use freshly prepared in vivo formulations for optimal results.)