Synthesis and biological evaluation of 3-substituted 5-benzylidene-1-methyl-2-thiohydantoins as potent NADPH oxidase (NOX) inhibitors.

We report the synthesis of novel 3-substituted 5-benzylidene-1-methyl-2-thiohydantoins 3, and their biological evaluation using NADPH oxidase (NOX) 1 and 4. Based on structural and pharmacophore analyses of known inhibitors such as hydroxypyrazole 2, we envisioned interesting 2-thiohydantoin compounds, 3-substituted 5-benzylidene-1-methyl-2-thiohydantoins 3 that would be expected to well match the structural features in 2. Efficient synthesis of eighteen target compounds 3 were achieved through the synthetic pathway of 4→11→3, established after consideration of several plausible synthetic pathways. The inhibitory activities of compounds 3 against NOX 1 and 4 were measured, with some of the target compounds showing similar or higher activities compared with reference 2; in particular, compounds 3bz, 3cz, and 3ez were found to be promising inhibitors of both NOX 1 and 4 with modest isozyme selectivities, which highlights the significance of the 2-thiohydantoin substructure for inhibition of NOX 1 and 4. This marks the first time these compounds have been applied to the inhibition of NOX enzymes.

A novel pyrazole derivative protects from ovariectomy-induced osteoporosis through the inhibition of NADPH oxidase.

Osteoclast cells (OCs) are differentiated from bone marrow-derived macrophages (BMMs) by activation of receptor activator of nuclear factor κB (NF-κB) ligand (RANKL). Activation of NADPH oxidase (Nox) isozymes is involved in RANKL-dependent OC differentiation, implicating Nox isozymes as therapeutic targets for treatment of osteoporosis. Here, we show that a novel pyrazole derivative, Ewha-18278 has high inhibitory potency on Nox isozymes. Blocking the activity of Nox with Ewha-18278 inhibited the responses of BMMs to RANKL, including reactive oxygen species (ROS) generation, activation of mitogen-activated protein (MAP) kinases and NF-κB, and OC differentiation. To evaluate the anti-osteoporotic function of Ewha-18278, the derivative was applied to estrogen-deficient ovariectomized (OVX) ddY mice. Oral administration of Ewha-18278 (10 mg/kg/daily, 4 weeks) into the mice recovered bone mineral density, trabecular bone volume, trabecular bone length, number and thickness, compared to control OVX ddY mice. Moreover, treatment of OVX ddY mice with Ewha-18278 increased bone strength by increasing cortical bone thickness. We provide that Ewha-18278 displayed Nox inhibition and blocked the RANKL-dependent cell signaling cascade leading to reduced differentiation of OCs. Our results implicate Ewha-18278 as a novel therapeutic agent for the treatment of osteoporosis.

NADPH Oxidase 1 Activity and ROS Generation Are Regulated by Grb2/Cbl-Mediated Proteasomal Degradation of NoxO1 in Colon Cancer Cells.

The generation of reactive oxygen species (ROS) is required for proper cell signaling, but must be tightly regulated to minimize deleterious oxidizing effects. Activation of the NADPH oxidases (Nox) triggers ROS production and, thus, regulatory mechanisms exist to properly control Nox activity. In this study, we report a novel mechanism in which Nox1 activity is regulated through the proteasomal degradation of Nox organizer 1 (NoxO1). We found that through the interaction between NoxO1 and growth receptor-bound protein 2 (Grb2), the Casitas B-lineage lymphoma (Cbl) E3 ligase was recruited, leading to decreased NoxO1 stability and a subsequent reduction in ROS generation upon epidermal growth factor (EGF) stimulation. Additionally, we show that EGF-mediated phosphorylation of NoxO1 induced its release from Grb2 and facilitated its association with Nox activator 1 (NoxA1) to stimulate ROS production. Consistently, overexpression of Grb2 resulted in decreased Nox1 activity, whereas knockdown of Grb2 led to increased Nox1 activity in response to EGF. CRISPR/Cas9-mediated NoxO1 knockout in human colon cancer cells abrogated anchorage-independent growth on soft agar and tumor-forming ability in athymic nude mice. Moreover, the expression and stability of NoxO1 were significantly increased in human colon cancer tissues compared with normal colon. Taken together, these results support a model whereby Nox1 activity and ROS generation are regulated by Grb2/Cbl-mediated proteolysis of NoxO1 in response to EGF, providing new insight into the processes by which excessive ROS production may promote oncogenic signaling to drive colorectal tumorigenesis.

Interaction of NADPH oxidase 1 with Toll-like receptor 2 induces migration of smooth muscle cells.

AIMS: NADPH oxidase (Nox) isozymes that generate intracellular reactive oxygen species (ROS) and Toll-like receptor 2 (TLR2), an inflammatory mediator, are both involved in the development of atherosclerotic lesions. To identify the molecular connection between TLR2 and Nox isozymes in vascular remodelling, we analysed generation of ROS and pro-inflammatory cytokines in aortic smooth muscle cells from Nox1-deficient mice in response to the synthetic triacylated lipoprotein Pam3CSK, a TLR2 agonist. METHODS AND RESULTS: We showed that TLR2 signalling stimulates progression of the pro-inflammatory phenotype in mouse aortic smooth muscle cells (MASMCs) through activation of Nox1. We demonstrated the interaction of TLR2 with Nox1 using yeast two-hybrid and co-immunoprecipitation assays. MASMCs from Nox1-deficient mice failed to generate of ROS in response to Pam3CSK4, indicating that Nox1 is essential for TLR2-dependent production of ROS. We also found that Pam3CSK4 stimulated migration of MASMCs from wild-type mice in a Transwell system, but MASMCs from Nox1-deficient mice failed to show this response. Wild-type MASMCs produced matrix metalloprotease 2 in response to Pam3CSK4, whereas Nox1-deficient MASMCs failed to generate this protease. Moreover, stimulation of MASMCs with Pam3CSK4 resulted in increased expression of the pro-inflammatory cytokine macrophage inflammatory protein 2 in a Nox1-dependent manner, leading to enhanced monocyte-endothelial cell adhesion and trans-endothelial migration of U937 cells. CONCLUSION: These data suggest that Nox1 plays an important role in TLR2-mediated intracellular H2O2 generation, activation of matrix metalloprotease 2, and secretion of pro-inflammatory cytokines, which in turn stimulate MASMC migration and vascular remodelling.

Distinct TLR-mediated pathways regulate house dust mite-induced allergic disease in the upper and lower airways.

BACKGROUND: Allergic rhinitis (AR) and asthma are 2 entities of allergic airway diseases that frequently occur together, which is referred to as united airways. In contrast to this general concept, we hypothesized that innate immunity of the upper and lower airways is respectively distinctive, because the immunologic conditions of the nasal and lung mucosa as well as the functions of the immune cells within their epithelia are different. OBJECTIVE: We wanted to identify distinctive mechanisms of innate immunity in the nose and lung mucosa, which are responsible for house dust mite (HDM)-induced AR and allergic asthma (AA), respectively. METHODS: We constructed a mouse model of AR or AA induced by sensitization and consequent provocation with HDM extracts. RESULTS: HDM-derived ß-glucans, rather than LPS, were proven to be essential to activating innate immunity in the nasal mucosa and triggering AR, which depended on Toll-like receptor 2 (TLR2), but not on TLR4; however, the LPS/TLR4 signaling axis, rather than ß-glucans/TLR2, was critical to HDM-induced AA. These differences were attributed to the specific role of ß-glucans and LPS in inducing the surface expression of TLR2 and TLR4 and their translocation to lipid rafts in nasal and bronchial epithelial cells, respectively. We also showed that dual oxidase 2-generated reactive oxygen species mediate both ß-glucan-induced TLR2 activation and LPS-induced TLR4 activation. CONCLUSIONS: We describe a novel finding of distinctive innate immunity of the nose and lungs, respectively, which trigger AR and AA, by showing the critical role of HDM-induced TLR activation via dual oxidase 2-mediated reactive oxygen species.

Vitamin A deficiency induces fluid hyposecretion from the airway submucosal glands of mice.

Vitamin A deficiency (VAD) alters the phenotype of airway epithelium and attenuates the epithelial defense system, and many studies have reported the association of VAD with respiratory disease. In this study, we investigated changes in submucosal glands (SMG) in a mouse model of VAD. C57BL/6 mice were fed a vitamin A-devoid diet and the others were fed a control diet (1.2 mg retinol/kg). The areas of serous and mucous cells of SMG were measured in 4-, 8-, and 20-wk-old male mice. The volume and lysozyme concentration of glandular secretions were also measured. The 2 groups did not differ in body weight or general morbidity at 3-10 wk of age, although serum retinol concentrations were greater in the control mice than in the VAD mice after 4 wk. Upon histological evaluation, we found that the areal ratio of serous cells:total SMG cells was significantly lower after 8 wk in the VAD mice compared with the control mice, although the total area of SMG did not differ between groups throughout the 20-wk experiment. The number of secretory bubbles did not differ between the groups, but total secretion volume was reduced by 35% in 8-wk-old VAD mice compared with controls. Furthermore, the concentration of lysozyme in secretions from 8-wk-old VAD mice was also less than in controls, compounding the effect of diminished secretion volume. In this study, we found serous cell hypotrophy/hypoplasia and dysfunction in VAD mice, which may contribute to the susceptibility to airway infection linked to VAD.

Dual oxidase 2 is essential for the toll-like receptor 5-mediated inflammatory response in airway mucosa.

AIMS: Airway mucosa is constantly exposed to various airborne microbes, and epithelial host defense requires a robust innate immunity. Recently, it has been suggested that NADPH oxidase (NOX) isozymes serve functional roles in toll-like receptor (TLR)-mediated innate immune responses. However, the molecular mechanism between TLR and NOX-mediated reactive oxygen species (ROS) production in human airway mucosa has been poorly understood. RESULTS: Here, we show that flagellin-induced ROS generation is dependent on dual oxidase 2 (DUOX2) activation, which is regulated by [Ca(2+)](i) mobilization in primary normal human nasal epithelial (NHNE) cells. Interestingly, we observed that silencing of DUOX2 expression in NHNE cells and nasal epithelium of Duox2 knockout mice failed to trigger mucin and MIP-2? production upon challenging flagellin. INNOVATION: Our observation in this study reveals that flagellin-induced hydrogen peroxide (H(2)O(2)) generation is critical for TLR5-dependent innate immune responses, including IL-8 production and MUC5AC expression in the nasal epithelium. Furthermore, DUOX2-mediated H(2)O(2) generation activated by the flagellin-TLR5 axis might serve as a novel therapeutic target for infectious inflammation diseases in the airway tract. CONCLUSION: Taken together, we propose that DUOX2 plays pivotal roles in TLR5-dependent inflammatory response of nasal airway epithelium.

Crosstalk between platelet-derived growth factor-induced Nox4 activation and MUC8 gene overexpression in human airway epithelial cells.

Reactive oxygen species (ROS) contribute to chronic airway inflammation, and NADPH oxidase (Nox) is an important source of ROS. However, little is known of the role that ROS play in chronic upper respiratory tract inflammation. We investigated the mechanism of ROS generation and its association with mucin gene overexpression in the nasal epithelium. The level of platelet-derived growth factor (PDGF) expression was increased in sinusitis mucosa, and high-level PDGF expression induced intracellular ROS, followed by MUC8 gene overexpression in normal human nasal epithelial cells. Knockdown of Nox4 expression with Nox4 siRNA decreased PDGF-induced intracellular ROS and MUC8 expression. Infection with an adenovirus containing Nox4 cDNA resulted in Nox4 overexpression and increased intracellular levels of ROS and MUC8 expression. PDGF and Nox4 overexpression are essential components of intracellular ROS generation and may contribute to chronic inflammation in the nasal epithelium through induction of MUC8 overexpression.

α-Melanocyte-stimulating hormone inhibits tumor necrosis factor α-stimulated MUC5AC expression in human nasal epithelial cells.

Mucin hypersecretion is an important clinical feature of several respiratory diseases, including asthma, cystic fibrosis, nasal allergy, rhinitis, and sinusitis. It has been shown that α-melanocyte-stimulating hormone (α-MSH), a proopiomelanocortin (POMC)-derived peptide, has immunomodulatory activities by inhibiting NF-κB activation induced by proinflammatory cytokines such as TNF-α. Because MUC5AC expression is known to be up-regulated by TNF-α via NF-κB activation, we evaluated the inhibitory effect of α-MSH on MUC5AC gene expression induced by TNF-α in normal human nasal epithelial (NHNE) cells. Melanocortin-1-receptor (MC-1R) was detected by RT-PCR, Western blotting, and immunofluorescent labeling in NHNE cells. α-MSH suppressed NF-κB/p65 phosphorylation induced by TNF-α as well as IkB-α degradation in a dose-dependent manner, as assessed by Western blotting. In addition, α-MSH inhibited TNF-α-induced nuclear translocation of NF-κB and NF-κB luciferase activity. Real-time quantitative PCR data showed that α-MSH inhibited TNF-α-induced expression of MUC5AC, and this effect of α-MSH was neutralized by knockdown of MC-1R using MC-1R shRNA lentivirus. Analyses using RT-PCR and Western blotting showed the expression of POMC and two key enzymes in the POMC processing, proprotein convertases (PC)1 and PC2, and 7B2, which is required for enzymatic activity of PC2, in normal human nasal mucosa. We conclude that α-MSH down-regulates MUC5AC expression by inhibiting TNF-α-induced NF-κB activity through MC-1R stimulation in NHNE cells and that normal human nasal mucosa possesses the POMC processing machinery. Therefore, α-MSH may be a promising candidate to decrease mucin overproduction initiated by NF-κB activation.

Auxin-induced reactive oxygen species production requires the activation of phosphatidylinositol 3-kinase.

We recently reported that production of reactive oxygen species (ROS) is essential for auxin-induced gravitropic signaling. Here, we investigated the role of phosphatidylinositol 3-kinase and its product, PtdIns(3)P, in auxin-mediated ROS production and the root gravitropic response. Pretreatment with LY294002, an inhibitor of PtdIns 3-kinase activity, blocked auxin-mediated ROS generation, and reduced the sensitivity of root tissue to gravistimulation. The amount of PtdIns(3)P increased in response to auxin, and this effect was abolished by pretreatment with LY294002. In addition, sequestration of PtdIns(3)P by transient expression of the endosome binding domain in protoplasts abrogated IAA-induced ROS accumulation. These results indicate that activation of PtdIns 3-kinase and its product PtdIns(3)P are required for auxin-induced production of ROS and root gravitropism.