Isoform-selective NADPH oxidase inhibitor panel for pharmacological target validation

Abstract Unphysiological reactive oxygen species (ROS) formation is considered an important pathomechanism for several disease phenotypes with high unmet medical need. Therapeutically, antioxidants have failed multiple times. Instead, focusing on only disease-relevant, enzymatic sources of ROS appears to be a more promising and highly validated approach. Here the family of five NADPH oxidases (NOX) stands out as drug targets. Validation has been restricted, however, mainly to genetically modified rodents and is lacking in other species including human. It is thus unclear whether the different NOX isoforms are sufficiently distinct to allow selective pharmacological modulation. Here we show for five of the most advanced NOX inhibitors that indeed isoform selectivity can be achieved. 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). Conditions need to be carefully controlled though as previously unrecognized non-specific antioxidant and assay artefacts may limit the interpretation of data and this included, surprisingly, one of the most advanced NOX inhibitors, GKT136901. As proof-of-principle that now also pharmacological and non-rodent target validation of different NOX isoforms is possible, we used a human blood-brain barrier model and NOX inhibitor panel at IC50 concentrations. The protective efficacy pattern of this panel confirmed the predominant role of NOX4 in stroke from previous genetic models. Our findings strongly encourage further lead optimization efforts for isoform-selective NOX inhibitors and clinical development and provide an experimental alternative when genetic validation of a NOX isoform is not an option.