We demonstrated GKT137831 to be specific for NADPH oxidases over other flavoprotein-containing oxidases and also excluded the possibility that GKT137831 is a general ROS scavenger: GKT137831 was further tested in a xanthine oxidase (XO) assay using similar ROS production methodology as in our proprietary NOX assays and with the same readout. Whereas DPI showed high affinity (Ki = 50 nM) consistent with its nonspecific mechanism of action, GKT137831 demonstrated no affinity

for XO (Ki > 100 μM) (Fig. 2B,C) as well as the inability to scavenge superoxide (O2.−), the common endproduct of Nox proteins and XO. To further demonstrate the specificity of GKT137831 for Nox enzymes, our candidate drug was subjected to an extensive in vitro off-target AZD8055 order pharmacological profile on 170 different proteins, including ROS-producing and redox-sensitive enzymes, as well as representative proteins of well-recognized drug target families, such as G-protein-coupled receptors, kinases, ion channels, and other enzymes.25 GKT137831, when tested at 10 μM, did not show any significant inhibition of any tested target protein, demonstrating the excellent specificity of this compound

(see Supporting Table 2). To investigate the role of SOD1 and the effect of NOX1/4 inhibition on liver fibrosis, liver fibrosis was induced in SOD1mu (with increased catalytic activity) and WT mice by 12 consecutive CCl4 injections over a 6-week period. During the last half of CCl4 injections, some mice were treated with GKT137831 daily. CCl4-induced liver fibrosis was more pronounced in SOD1mu, compared to selleck compound WT, mice. Liver fibrosis in both SOD1mu and WT mice was attenuated by GKT137831

treatment. The NOX1/4 inhibitor reduced the levels of hepatic collagen deposition in CCl4-induced fibrosis in SOD1mu and WT mice to the same low level, as assessed by Sirius Red staining and its quantification (Fig. 3A,B). mafosfamide Hepatic α-SMA expression, a marker for HSC activation, was enhanced in SOD1mu mice after CCl4 injections, compared to WT mice, as assessed by IHC and immunoblotting. The increased hepatic α-SMA expression was markedly decreased in SOD1mu mice treated with GKT137831 to a level similar to that of WT mice given the NOX1/4 inhibitor (Fig. 3C,D). The mRNAs of fibrogenic markers, including collagen α1(I), tissue inhibitor of metalloprotease 1 (TIMP-1), and TGF-β were increased in SOD1mu mice to higher levels than in WT mice after CCl4 injections, but treatment with GKT137831 reduced the induction of those genes to the same lower levels (Fig. 3E). Similarly, BDL-induced hepatic fibrosis in both WT and SOD1mu mice was decreased by treatment with GK137831 (Supporting Fig. 1). Thus, both hepatotoxin (CCl4)- (this study) and cholestasis (BDL)-induced (this study and REFA) liver fibrosis is suppressed by blocking NOX1 and NOX4. To investigate the role of SOD1 and the effect of NOX1/4 inhibition on liver inflammation, macrophage infiltration and activation were evaluated.