scholarly journals Identification of iNOS Inhibitors Using InteraX™

2009 ◽  
Vol 14 (3) ◽  
pp. 263-272 ◽  
Author(s):  
Philip R. Mallinder ◽  
Alan V. Wallace ◽  
Gary Allenby
Keyword(s):  
High Throughput Screening ◽  
Chimeric Proteins ◽  
Galactosidase Activity ◽  
Cytotoxicity Assays ◽  
A Cell ◽  
Inos Inhibitors ◽  
Oxygenase Domain ◽  
Oxide Synthase ◽  
Inducible Nitric Oxide ◽  
Dimerization Inhibitors

Inducible nitric oxide synthase (iNOS) is active as a homodimer. A cell-based assay suitable for high-throughput screening (HTS) was generated to identify inhibitors of iNOS dimerization using the InteraX™ enzyme complementation technology of Applied Biosystems. The cells contain 2 chimeric proteins of complementing deletion mutants of β-galactosidase, each fused to the oxygenase domain of human iNOS. The assay was characterized using known iNOS dimerization inhibitors, which gave a decrease in β-galactosidase activity. Surprisingly, the assay was also able to identify compounds that have the same profile as known inhibitors of fully formed dimeric iNOS by causing an increase in β-galactosidase activity. The iNOS InteraX™ assay was used to screen ~800,000 compounds in a 384-well format. After hit confirmation, 3359 compounds were taken forward for full IC50 determination in InteraX™ and cytotoxicity assays. Of these compounds 40.5% were confirmed as greater than 10-fold more active in InteraX™ compared to a cytotoxicity assay and were classified as potential iNOS dimerization inhibitors as they did not inhibit β-galactosidase alone. In the same primary screen, 901 compounds gave a significant increase in β-galactosidase activity. Many of these were known inhibitors of iNOS. After IC50 determination in InteraX™ and cytotoxicity assays, 182 novel compounds remained as potential arginine-competitive inhibitors of dimeric iNOS. ( Journal of Biomolecular Screening 2009:263-272)

scholarly journals Replication of Salmonella enterica serovar Typhimurium in RAW264.7 Phagocytes Correlates With Hypoxia and Lack of iNOS Expression

2020 ◽  
Vol 10 ◽  
Author(s):  
Marie Wrande ◽  
Kim Vestö ◽  
Speranta Puiac Banesaru ◽  
Naeem Anwar ◽  
Johan Nordfjell ◽  
...  
Keyword(s):  
Cell Culture ◽  
Salmonella Enterica ◽  
Salmonella Enterica Serovar Typhimurium ◽  
Inos Expression ◽  
Raw264.7 Cells ◽  
Serovar Typhimurium ◽  
A Cell ◽  
Infected Cells ◽  
Oxide Synthase ◽  
Inducible Nitric Oxide

Salmonella infection associates with tissue hypoxia, while inducible nitric oxide synthase (iNOS), relying for its activity on molecular oxygen, stands as a central host defence measure in murine salmonellosis. Here, we have detailed hypoxia and iNOS responses of murine macrophage-like RAW264.7 cells upon infection with Salmonella enterica serovar Typhimurium. We noted that only a proportion of the infected RAW264.7 cells became hypoxic or expressed iNOS. Heavily infected cells became hypoxic, while in parallel such cells tended not to express iNOS. While a proportion of the infected RAW264.7 cells revealed shutdown of protein synthesis, this was only detectable after 12 h post infection and after iNOS expression was induced in the cell culture. Our data implicate an intrinsic heterogeneity with regard to hypoxia and iNOS expression in a cell culture-based infection setting.

scholarly journals Bioactive α-Pyrone Derivatives from the Endophytic Fungus Diaporthe sp. CB10100 as Inducible Nitric Oxide Synthase Inhibitors

2021 ◽  
Vol 9 ◽  
Author(s):  
Hong Pu ◽  
Jianxin Liu ◽  
Yeji Wang ◽  
Yuhui Peng ◽  
Wanying Zheng ◽  
...  
Keyword(s):  
Nitric Oxide ◽  
Nitric Oxide Synthase ◽  
Inducible Nitric Oxide Synthase ◽  
Ellagic Acid ◽  
Endophytic Fungus ◽  
Dependent Manner ◽  
Protein Levels ◽  
Inos Inhibitors ◽  
Oxide Synthase ◽  
Inducible Nitric Oxide

Inducible nitric oxide synthase (iNOS) produces NO from l-arginine and plays critical roles in inflammation and immune activation. Selective and potent iNOS inhibitors may be potentially used in many indications, such as rheumatoid arthritis, pain, and neurodegeration. In the current study, five new compounds, including a dibenzo-α- pyrone derivative ellagic acid B (5) and four α-pyrones diaporpyrone A–D (9–12), together with three known compounds (6–8), were isolated from the endophytic fungus Diaporthe sp. CB10100. The structures of these new natural products were unambiguously elucidated using NMR, HRESIMS or electronic circular dichroism calculations. Ellagic acid B (5) features a tetracyclic 6/6/6/6 ring system with a fused 2H-chromene, which is different from ellagic acid (4) with a fused 2H-chromen-2-one. Both 2-hydroxy-alternariol (6) and alternariol (7) reduced the expression of iNOS at protein levels in a dose-dependent manner, using a lipopolysaccharide (LPS)-induced RAW264.7 cell models. Also, they decreased the protein expression levels of pro-inflammatory cytokines, such as tumor necrosis factor-α, interleukin-6 and monocyte chemotactic protein 1. Importantly, 6 and 7 significantly reduced the production of NO as low as 10 μM in LPS-induced RAW264.7 cells. Molecular docking of 6 and 7 to iNOS further suggests that both of them may interact with iNOS. Our study suggests that 6 and 7, as well as the alternariol scaffold may be further developed as potential iNOS inhibitors.

Effects of p38MAPK isoforms on renal mesangial cell inducible nitric oxide synthase expression

2004 ◽  
Vol 286 (1) ◽  
pp. C145-C152 ◽  
Author(s):  
Paul Lui ◽  
Chenbo Zeng ◽  
Stephen Acton ◽  
Steven Cok ◽  
Alison Sexton ◽  
...  
Keyword(s):  
Nitric Oxide ◽  
Nitric Oxide Synthase ◽  
Inducible Nitric Oxide Synthase ◽  
Mesangial Cell ◽  
Mesangial Cells ◽  
Inos Expression ◽  
Chimeric Proteins ◽  
Wild Type ◽  
Oxide Synthase ◽  
Inducible Nitric Oxide

Several related isoforms of p38MAPK have been identified and cloned in many species. Although they all contain the dual phosphorylation motif TGY, the expression of these isoforms is not ubiquitous. p38α and -β2 are ubiquitously expressed, whereas p38γ and -δ appear to have more restricted expression. Because there is evidence for selective activation by upstream kinases and selective preference for downstream substrates, the functions of these conserved proteins is still incompletely understood. We have demonstrated that the renal mesangial cell expresses the mRNA for all the isoforms of p38MAPK, with p38α mRNA expressed at the highest level, followed by p38γ and the lowest levels of expression by p38β2 and -δ. To determine the functional effects of these proteins on interleukin (IL)-1β-induced inducible nitric oxide synthase (iNOS) expression, we transduced TAT-p38 chimeric proteins into renal mesangial cells and assessed the effects of wild-type and mutant p38 isoforms on ligand induced iNOS expression. We show that whereas p38γ and -δ had minimal effects on iNOS expression, p38α and -β2 significantly altered its expression. p38α mutant and p38β2 wild-type dose dependently inhibited IL-1β-induced iNOS expression. These data suggest that p38α and β2 have reciprocal effects on iNOS expression in the mesangial cell, and these observations may have important consequences for the development of selective inhibitors targeting the p38MAPK family of proteins.

Withapubesides A–D: natural inducible nitric oxide synthase (iNOS) inhibitors from Physalis pubescens

2017 ◽  
Vol 15 (47) ◽  
pp. 10016-10023 ◽  
Author(s):  
Gui-Yang Xia ◽  
Tie Yao ◽  
Bing-Yang Zhang ◽  
Yang Li ◽  
Ning Kang ◽  
...  
Keyword(s):  
Nitric Oxide ◽  
Nitric Oxide Synthase ◽  
Inducible Nitric Oxide Synthase ◽  
Inos Inhibitors ◽  
Oxide Synthase ◽  
Inducible Nitric Oxide

Withapubesides A–D (1–4), candidates for the development of iNOS inhibitors, were isolated from Physalis pubescens.

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