Development of a first-in-class homolog-selective, covalent NOX4 inhibitor.

The NADPH oxidase (NOX) family in humans includes seven enzymes whose main function is to produce reactive oxygen species. Because of their role in oxidative damage, these enzymes are being explored as targets for new therapies against oxidative stress-related diseases. However, their conserved catalytic core poses selective inhibition challenging. Current inhibitors suffer from low potency, cytotoxicity, assay interference, and poor selectivity. VAS2870, a covalent NOX inhibitor targeting a cysteine in the dehydrogenase (DH) domain, does not present assay interference but still shows limited selectivity, solubility, and safety. This study aimed to develop improved VAS2870-based targeted covalent inhibitors (TCIs) by designing and screening a library of cysteine-targeting compounds against NOX1, NOX2, NOX4, and NOX5. A hit compound, compound 2, was optimized, yielding a potent, homolog-selective NOX4 inhibitor, compound 18. Its mechanism of action was validated, demonstrating covalent binding to a cysteine residue and showing that compound binding is hindered by the presence of FAD in the DH domain.