Isoform-specific NADPH oxidase inhibition for pharmacological target validation

Unphysiological reactive oxygen species (ROS) formation is considered an important pathomechanism for several diseasephenotypes with high unmet medical need. After the clinical failure of antioxidants, inhibition of disease-relevant enzymatic sources of ROS appears to be the most promising alternative approach. With respect to most promising drug target, NADPH oxidases (NOXs) stand out, however, validation has been restricted mainly to genetically modified mice. Validation in other species including human is lacking and it is unclear whether the different NOX isoforms are sufficiently distinct for selective pharmacological inhibition. Here we show for the five most advanced NOX inhibitors that pharmacological 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). Of note, previously unrecognised non-specific antioxidant and assay artefacts may limit interpretations in some systems, which included, surprisingly, the clinically most advanced compound, GKT136901. As proof-of-principle for our pharmacological target validation approach, we used a human blood-brain barrier model and our NOX inhibitor panel at IC50 concentrations. Indeed, the protective efficacy pattern of this compound panel pointed towards a functional role NOX4 confirming previous genetic targeting. These findings strongly encourage further lead optimisation efforts for isoform-selective NOX inhibitors and their clinical development and provide already now an experimental alternative when genetic targeting of NOXs is not an option.