Setanaxib (GKT137831) is a potent, specific dual NADPH oxidase (NOX1/4) inhibitor.

NOX4:140 nM(Ki), NOX1:110 nM(Ki)

每天给长期低氧环境下的小鼠模型口服60 mg/kg GKT137831,减轻长期缺氧诱导的右心室肥大,血管重塑,肺细胞增殖以及低氧环境下肺PPARγ和TGF-β表达的改变.每天给糖尿病载脂蛋白E不足的小鼠口服60 mg/kg GKT137831可以减弱糖尿病加速的动脉粥样硬化.60 mg/kg i.g. GKT137831阻断WT和SOD1mut小鼠体内肝纤维化,并下调氧化应激,炎症和纤维化的发生.此外,GKT137831在注入angII的 c-hNox4Tg小鼠体内,消除氧化应激的增加,抑制Akt-mTOR 和 NF-κB信号通路,并减弱心脏重塑.

GKT137831可以预防人体主动脉内皮细胞对高血糖反应的氧化应激。GKT137831减弱HPAECs和HPASMCs中缺氧诱导的H2O2释放,细胞增殖和TGF-β1表达,并改善PPARγ表达减少。

Kinase Assay: IC50 of tyrosine kinase activity is measured by an enzyme-linked immunosorbent assay (ELISA) with recombinant catalytic domains of a panel of receptor and non-receptor tyrosine kinases (in some cases only part of the catalytic domain is used). Saracatinib dose ranges from 0.001-10 mM. Speci?city assays against a panel of serine/threonine kinases are performed using a ?lter capture assay with 32P. Brie?y, multidrop 384 plates containing 0.5 μL Saracatinib or controls (DMSO) alone or pH 3.0 buffer controls) are incubated with 15 μL of enzyme plus peptide/protein substrate for 5 min before the reaction is initiated by the addition of 10 μL of 20 mM Mg-ATP. For all enzymes the ?nal concentration is approximated to the Michaelis constant (Km). Assays are carried out for 30min at room temperature before termination by the addition of 5 μL orthophosphoric acid. After mixing, the well contents are harvested onto a P81 Uni?lter plate, using orthophosphoric acid as the wash buffer. Then IC50 is calculated.

Hypoxic HPASMC and HPAEC proliferation is determined using MTT assay, by Western blotting to detect proliferating cell nuclear antigen (PCNA) expression, or by manual cell counting after Trypan blue staining. Amplex Red Hydrogen Peroxide/Peroxidase Assay Kit is used to measure Water2 released into the culture media from HPAECs or HPASMCs. After exposure to control or hypoxic environments for 72 hours, Amplex Red reagent is added, and the cells are returned to control or hypoxic environments for an additional hour before fluorescence measurements. (Only for Reference)