RESUMEN
Dysfunctional reactive oxygen species (ROS) signaling is considered an important disease mechanism. Therapeutically, non-selective scavenging of ROS by antioxidants, however, has failed in multiple clinical trials to provide patient benefit. Instead, pharmacological modulation of disease-relevant, enzymatic sources of ROS appears to be an alternative, more promising and meanwhile successfully validated approach. With respect to targets, the family of NADPH oxidases (NOX) stands out as main and dedicated ROS sources. Validation of the different NOX isoforms has been mainly through genetically modified rodent models and is lagging behind in other species. It is unclear whether the different NOX isoforms are sufficiently distinct to allow selective pharmacological modulation. Here we show for five widely used NOX inhibitors that isoform selectivity can be achieved, although individual compound specificity is as yet insufficient. NOX1 was most potently (IC50) targeted by ML171 (0.1 µM); NOX2, by VAS2870 (0.7 µM); NOX4, by M13 (0.01 µM) and NOX5, by ML090 (0.01 µM). In addition, some non-specific antioxidant and assay artefacts may limit the interpretation of data, which included, surprisingly, the clinically advanced NOX inhibitor, GKT136901. In a human ischemic blood-brain barrier hyperpermeability model where genetic target validation is not an option, we provide proof-of-principle that pharmacological target validation for different NOX isoforms is possible by applying an inhibitor panel at IC50 concentrations. Moreover, our findings encourage further lead optimization and development efforts for isoform-selective NOX inhibitors in different indications.