Study on the Structure-Activity Relationship and Effect Evaluation of Benzo -γ -Pyrones Anti-S. aureus Hla Inhibitors

Benzo-γ-pyrone derivatives are widely utilized in both the food industry and pharmaceutical research. From a constructed Benzo-γ-pyrone Natural Compounds Database (BNCD), we screened 18 compounds with the core benzo-γ-pyrone skeleton against Staphylococcus aureus α-hemolysin (Hla) and categorized them into five classes (A-E). We identified 10 effective inhibitors by hemolytic assay that could be pooled with the use of a dose-response model to generate an IC 50 curve. Dose-response analysis yielded IC 50 values, which, together with molecular docking results for the Hla secretion regulators (AgrC, AgrA, SarR, SarA), revealed a key pharmacophore. The optimal structure is characterized by a benzo-γ-pyrone backbone featuring a C2-linked B-ring, an essential 5,7-dihydroxy motif on the A-ring, and a B-ring substituted with either a 4′-hydroxyl or a 3′,4′-catechol group. The minimum inhibitory concentrations (MICs) of morin, apigenin, (+/-)-naringenin, and liquiritigenin were all > 2000 μg/mL. Subsequent cytotoxicity assessments (CCK-8 assay) revealed no significant cytotoxicity (P > 0.05) for morin and apigenin up to 2000 μg/mL, for naringenin up to 500 μg/mL, and for liquiritigenin up to 250 μg/mL in bovine mammary epithelial cells. Furthermore, these compounds conferred concentration-dependent protection against S. aureus -induced damage in both in vitro Raw264.7 macrophage and in vivo Galleria mellonella larval infection models. In conclusion, our study identifies specific dietary benzo-γ-pyranones as promising non-antibiotic, anti-virulence agents targeting S. aureus Hla. These findings provide a rational basis for designing functional foods and developing novel anti-virulence therapeutics.