Background/Objectives: The increasing prevalence of multidrug-resistant Staphylococcus aureus 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. Methods: Antimicrobial susceptibility assays were conducted against S. aureus 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. Results: 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). Conclusions: CMA4DMA effectively restored norfloxacin susceptibility in resistant S. aureus strains, highlighting its potential as a promising scaffold for developing novel efflux pump inhibitors and antibiotic adjuvants.

The emergence of efflux-mediated resistance in Staphylococcus aureus has limited the effectiveness of conventional antibiotics, highlighting the need for novel adjuvant compounds. This study investigated the synthetic chalcone CMA4DMA as a potential modulator of bacterial resistance associated with the NorA efflux pump. In vitro anti-microbial assays showed that CMA4DMA displayed no intrinsic antibacterial effect (MIC ≥ 1024 µg/mL) but significantly reduced the minimum inhibitory concentration of norfloxacin and ethidium bromide in both wild-type SA1199 and NorA-overexpressing SA1199B strains. Fluorescence assays demonstrated increased intracellular accumulation of ethidium bromide without affecting membrane permea-bility, supporting an efflux-inhibitory mechanism. Molecular docking analysis revealed that CMA4DMA binds to the same site as norfloxacin in the NorA protein, engaging in hydrophobic and hydrogen-bond interactions with key residues, consistent with direct inhibition. In silico ADMET predictions indicated favorable oral absorption and distri-bution, though possible rapid metabolism and risks of hepatotoxicity and cardiotoxici-ty warrant caution for further development. Overall, CMA4DMA effectively restored antibiotic susceptibility in resistant S. aureus strains and showed a consistent inhibitory effect when compared with studies involving other efflux systems, reinforcing its po-tential as a promising scaffold for the development of new bacterial resistance modu-lators.