Screening of Forsythia suspensa-derived compounds identifies amentoflavone as a ciprofloxacin-potentiating candidate...

Methicillin-resistant Staphylococcus aureus (MRSA) remains a clinically important pathogen, and bacterial efflux systems can reduce intracellular antibiotic accumulation and compromise antibacterial efficacy. NorA, a well-studied multidrug efflux pump in S. aureus, is associated with the efflux of fluoroquinolones and fluorescent substrates such as ethidium bromide (EtBr), making it a potential target for antibiotic adjuvant discovery. In this study, 100 PubChem-identifiable Forsythia suspensa (F. suspensa)-derived small molecules were screened against NorA using CB-Dock2 with AutoDock Vina scoring. Amentoflavone (AME), a naturally occurring biflavonoid, was identified as the top-ranked candidate and displayed a predicted binding pose within the NorA transmembrane cavity, with multiple potential interactions involving residues such as Thr336, Arg310, and Ser133. Molecular dynamics simulation performed using GROMACS 2025.4 further supported the dynamic stability of the predicted AME-NorA complex in a membrane-embedded environment. Functionally, AME increased intracellular EtBr-associated fluorescence in MRSA, suggesting altered EtBr-associated intracellular accumulation or efflux-associated activity. Although AME alone showed no evident direct anti-MRSA activity at concentrations up to 512 μg/mL, its combination with ciprofloxacin enhanced ciprofloxacin-mediated inhibition and killing of MRSA. Specifically, AME reduced the minimum inhibitory concentration (MIC) of ciprofloxacin from 1 to 0.5 μg/mL and the minimum bactericidal concentration (MBC) from 2 to 1 μg/mL. These findings suggest that AME may serve as a F. suspensa-derived ciprofloxacin-potentiating candidate against MRSA, potentially associated with altered efflux-related activity. Further studies using NorA genetic models, membrane permeability assays, intracellular ciprofloxacin accumulation assays, and multiple clinical isolates are needed to clarify the target specificity and broader applicability of AME-mediated ciprofloxacin potentiation.