Developing Inhibitors of the p47phox–p22phox Protein–Protein Interaction by Fragment-Based Drug Discovery

Nicotinamide adenine dinucleotide phosphate oxidase isoform 2 is an enzyme complex, which generates reactive oxygen species and contributes to oxidative stress. The p47phox–p22phox interaction is critical for the activation of the catalytical NOX2 domain, and p47phox is a potential target for therapeutic intervention. By screening 2500 fragments using fluorescence polarization and a thermal shift assay and validation by surface plasmon resonance, we found eight hits toward the tandem SH3 domain of p47phox (p47phoxSH3A–B) with KD values of 400–600 μM. Structural studies revealed that fragments 1 and 2 bound two separate binding sites in the elongated conformation of p47phoxSH3A–B and these competed with p22phox for binding to p47phoxSH3A–B. Chemical optimization led to a dimeric compound with the ability to potently inhibit the p47phoxSH3A–B–p22phox interaction (Ki of 20 μM). Thereby, we reveal a new way of targeting p47phox and present the first report of drug-like molecules with the ability to bind p47phox and inhibit its interaction with p22phox.