With the continued upsurge of antibiotic resistance and reduced susceptibility to almost all frontline antibiotics, there is a pressing need for the development of new, effective, and safe alternatives. In this study, a scaffold-hopping strategy was utilized to develop a novel class of penicillin-binding protein 2a (PBP2a) inhibitors, centered around a 4H-chromen-4-one core structure. These newly designed compounds demonstrated strong antibacterial efficacy against methicillin-resistant Staphylococcus aureus (MRSA) and other drug-resistant gram-positive pathogens. Notably, compounds 16 and 18-20 exhibited significant potency against the tested Staphylococcal strains, with minimum inhibitory concentrations (MICs) ranging from 0.008 to 1 μg/mL, outperforming standard treatments such as vancomycin and linezolid, as well as the reference compound III. These derivatives also retained their activity against a range of clinically relevant multidrug-resistant gram-positive bacteria and showed no detectable cytotoxicity in human cell assays. Additionally, compounds 19 and 20 displayed synergistic effects when co-administered with β-lactam antibiotics against two MRSA strains. Finally, these derivatives exhibited excellent binding affinities to the allosteric site of PBP2a, with dissociation constants ranging from 13 to 23 μM, indicating that this novel class inhibits PBP2a as the primary target by binding non-covalently to its allosteric site, hence impairing cell-wall crosslinking, and resulting in cell death. Accordingly, the reported 4H-chromen-4-one-based class of inhibitors merit further investigation as potential therapeutic options for treatment of staphylococcal infections.
