New NADPH Oxidase 2 Inhibitors Display Potent Activity against Oxidative Stress by Targeting p22phox-p47phox Interactions

Abstract

NADPH oxidase (NOX2) is responsible for reactive oxygen species (ROS) production in neutrophils and has been recognized as a key mediator in inflammatory and cardiovascular pathologies. Nevertheless, there is a lack of specific NOX2 pharmacological inhibitors. In medicinal chemistry, heterocyclic compounds are essential scaffolds for drug design, and among them, indole is a very versatile pharmacophore. We tested the hypothesis that indole heteroaryl-acrylonitrile derivatives may serve as NOX2 inhibitors by evaluating the capacity of 19 of these molecules to inhibit NOX2-derived ROS production in human neutrophils (HL-60 cells). Of these compounds, C6 and C14 exhibited concentration-dependent inhibition of NOX2 (IC50~1 µM). These molecules also reduced NOX2-derived oxidative stress in cardiomyocytes and prevented cardiac damage induced by ischemia-reperfusion. Compound C6 significantly reduced the membrane translocation of p47phox, a cytosolic subunit that is required for NOX2 activation. Molecular docking analyses of the binding modes of these molecules with p47phox indicated that C6 and C14 interact with specific residues in the inner part of the groove of p47phox, the binding cavity for p22phox. This combination of methods showed that novel indole heteroaryl acrylonitriles represent interesting lead compounds for developing specific and potent NOX2 inhibitors.

Targeting NOX2 with Bivalent Small-Molecule p47phox–p22phox Inhibitors

“…30 After a successful identification of the favorable linking positions and linker lengths, we turned our focus to the exploration of other types of linkers with similar length as PEG 2 . We replaced PEG 2 linked at C5 with an alkyl chain attached via oxygen (23) and an "NPEG" linker in which the middle oxygen of the PEG linker was replaced by nitrogen (24). Also, the NPEG nitrogen was exploited as a handle for introducing methyl, p-toluenesulfonyl (Tosyl, Ts), and phenyl (25,26,27) substituents directly onto the linker.…”

“…White solid (0.17 g, 59%). 1 (23). This previously unreported compound was synthesized according to general procedure C using 83 (0.64 g, 1.64 mmol), tert-butylamine (1.20 g, 16.4 mmol), Ts 2 O (2.14 g, 6.56 mmol) and TFA (8.2 mL).…”

“…Several NOX2 inhibitors including both peptides and small molecules have been reported. − However, most of these have issues with off-target binding, isoform selectivity, oxidative effects, chemical liabilities, or assay-interference properties, with GSK2795039 and its analogue NCATS-SM7270 as potential exceptions to this. , Small-molecule p47phox–p22phox inhibitors have also been reported, for example LMH001, CPP11G, C6, and ebselen . However, ebselen is nonspecific in its action and even shown to induce aggregation of p47phox via a covalent mechanism, and LMH001 is chemically very unstable. , CPP11G (or CPP11H) and C6 have not been shown to directly bind p47phox using biophysical experiments, but this has been demonstrated for two other small-molecule series: Garsi et al recently reported some peptide-derived triproline mimetics developed by structure-based drug discovery, which showed binding to p47phox by surface plasmon resonance (SPR) .…”

“…15−19 However, most of these have issues with off-target binding, isoform selectivity, oxidative effects, chemical liabilities, or assay-interference properties, with GSK2795039 and its analogue NCATS-SM7270 as potential exceptions to this. 6,20 Small-molecule p47phox−p22phox inhibitors have also been reported, for example LMH001, 21 CPP11G, 22 C6, 23 and ebselen. 24 However, ebselen is nonspecific in its action and even shown to induce aggregation of p47phox via a covalent mechanism, and LMH001 is chemically very unstable.…”