In Vitro and In Silico Study of the Inhibitory Potential of Riparins I, II, III, and IV on the NorA Efflux Pump of Staphylococcus aureus

Abstract

AbstractThe NorA efflux protein, encoded by Staphylococcus aureus, plays a critical role in antimicrobial resistance by actively extruding a broad range of antibiotics, including fluoroquinolones such as norfloxacin and ciprofloxacin. Inhibiting this mechanism is considered a promising strategy to restore the efficacy of conventional antibiotics. Natural products and their derivatives have emerged as valuable sources of efflux pump inhibitors (EPIs),, however, identifying effective and safe candidates remains a considerable challenge. The present study evaluated riparins N‐(4‐methoxyphenethyl)benzamide (I), 2‐hydroxy‐N‐[2‐(4‐methoxyphenyl)ethyl]benzamide (II), 2,6‐dihydroxy‐N‐[2‐(4‐methoxyphenyl)ethyl]benzamide (III), and 3,4,5‐trimethoxy‐N‐[2‐(4‐methoxyphenethyl)]benzamide (IV), benzamide derivatives obtained from Aniba riparia, as potential NorA efflux pump inhibitors using a comprehensive in vitro and in silico approach. Microbiological assays were performed against S. aureus strains 1199 (wild type) and 1199B (NorA overexpressing). When combined at subinhibitory concentrations with norfloxacin and ethidium bromide (EtBr), both recognized NorA substrates, the riparins significantly reduced the minimum inhibitory concentrations (MICs) of the antibiotics. Riparin III was particularly noteworthy, reducing the MIC of norfloxacin from 128 to 2 µg/mL (64‐fold) in the 1199 strain and from 128 to 20.16 µg/mL (6‐fold) in the 1199B strain. All riparins increased the fluorescence emission of EtBr, indicating inhibition of NorA‐mediated efflux, and riparins I and IV promoted dose‐dependent cytoplasmic membrane permeabilization. Molecular docking analyses revealed strong and stable interactions between the riparins and key residues within the NorA binding site, such as Glu222 and Phe140, with binding energies more favorable than those observed for chlorpromazine and norfloxacin. These interactions suggest a high binding affinity and stability, surpassing even the known NorA inhibitor chlorpromazine. This is the first report to explore the NorA efflux pump inhibitory potential of riparins I, II, III, and IV through a multidimensional strategy involving microbiological assays, membrane integrity evaluation, fluorescence‐based accumulation tests, and computational modeling. The findings indicate that these compounds, particularly riparin III, may serve as promising antibiotic adjuvants capable of mitigating NorA‐mediated antimicrobial resistance. Taken together, the results contribute to the ongoing search for natural product‐derived EPIs and highlight the strategic relevance of efflux inhibition in overcoming infections caused by multidrug‐resistant S. aureus. Further studies are warranted to optimize the pharmacokinetic properties and therapeutic applicability of riparins in combination therapies.