Upadhyay_2025_Nat.Commun_16_5046

Staphylococcus aureus is a leading cause of bacteria-associated mortality worldwide. New tools are needed to both image and treat this pathogen. We previously identified a group of S. aureus serine hydrolases (Fphs), which regulate aspects of virulence and lipid metabolism. However, due to high structural and functional similarities, it remains challenging to distinguish the specific roles of members of this family. Here, we apply a high-throughput screening approach using a library of covalent electrophiles to identify inhibitors for FphB, FphE, and FphH. We identify selective covalent inhibitors for each target without the need for extensive medicinal chemistry optimization. Structural and biochemical analysis identify novel binding modes for several of the inhibitors. Functional studies using the inhibitors suggest that all three hydrolases likely play distinct functional roles in biofilm formation and virulence. This approach has the potential to be applied to target hydrolases in other diverse pathogens or higher eukaryotes.