Discovery of NOX3 inhibitors for the prevention of acquired hearing loss.

The accumulation of reactive oxygen species (ROS) in cells and tissues contributes to the development and the progression of numerous diseases such as cancers, metabolic syndromes, or sensorineural disorders. NAPDH oxidases, a family of enzymes which sole function is to produce reactive oxygen species (ROS), appeared as relevant therapeutic targets in the treatment of oxidant-mediated pathologies. More specifically, the NOX3 isoform is only expressed in the inner ear and, although its physiological role in the cochlea is not known, there is increasing evidence that NOX3 is involved in different forms of acquired hearing loss. Thus, the inhibition of NOX3 would provide an efficient otoprotec-tive strategy, notably by preventing ROS-induced damages to the auditory synapse. This project aims at discovering NOX3 small molecule inhibitors for the prevention of acquired sensorineural hearing loss. We developed a cell-based high-throughput screen using an inducible system allowing the expression of NOX3 upon 24 h treatment with tetracy-cline. NOX3 activity was assessed through the detection of generated extracellular superoxide radical anion (O2*-) using the colorimetric assay WST-1. The non-specific NOX inhibitor diphenyleneiodonium chloride (DPI) was used as a reference compound for maximal inhibition. Among the 15,511 compounds screened, 115 showed an inhibitory activity on NOX3 equal to or higher than 50% and were considered as hits. These hits were further tested in dose-response using WST-1 and validated using orthogonal assays detecting hydrogen peroxide (Amplex Red/HRP and CBA fluorometric assays) and cytotoxicity. The specificity of the validated hits for NOX3 over the 6 other isoforms was also assessed. This critical early drug discovery step paves the way to the development of new small molecule therapeutics for the prevention of sensorineural hearing loss. [ABSTRACT FROM AUTHOR]