Evaluation of the Activity of Amino Chalcone Against Staphylococcus Strains Harboring Efflux Pumps

<b>Background/Objectives:</b> The increasing prevalence of multidrug-resistant <i>Staphylococcus aureus</i> represents a major clinical challenge, primarily driven by the acquisition of multiple resistance mechanisms. Among these, efflux pumps such as NorA play a pivotal role in quinolone resistance by promoting active drug extrusion and reducing intracellular antibiotic levels. This study evaluated the synthetic chalcone CMA4DMA as a potential NorA efflux pump inhibitor and modulator of bacterial resistance. <b>Methods:</b> Antimicrobial susceptibility assays were conducted against <i>S. aureus</i> SA1199 (wild-type) and SA1199B (NorA-overexpressing) strains. The minimum inhibitory concentration (MIC) of CMA4DMA and its modulatory effects on norfloxacin and ethidium bromide were determined. Efflux inhibition was assessed by ethidium bromide accumulation and SYTOX Green assays. Molecular docking and in silico ADMET analyses were performed to predict binding affinity and pharmacokinetic parameters. <b>Results:</b> CMA4DMA exhibited no intrinsic antibacterial activity (MIC ≥ 1024 µg/mL) but reduced the MIC of norfloxacin from 32 to 8 µg/mL and that of ethidium bromide from 32 to 8 µg/mL in SA1199. In SA1199B, reductions from 64 to 16 µg/mL and from 64 to 32 µg/mL were observed, respectively. Fluorescence increased by 15% without affecting membrane integrity. Docking revealed a binding affinity of -7.504 kcal/mol, stronger than norfloxacin (-7.242 kcal/mol), involving key residues Leu218, Ile309, Arg310, and Ile313. ADMET data indicated high intestinal absorption (88.76%) and permeability (Papp = 1.38 × 10^-5 cm/s). <b>Conclusions:</b> CMA4DMA effectively restored norfloxacin susceptibility in resistant <i>S. aureus</i> strains, highlighting its potential as a promising scaffold for developing novel efflux pump inhibitors and antibiotic adjuvants.