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Abstract
Antimicrobial resistance (AMR) causes an estimated 4.95 million deaths annually, with the WHO identifying ESKAPE pathogens, including Staphylococcus aureus, as highly resistant to most antibiotics. A major AMR mechanism in S. aureus is the NorA efflux pump, a multidrug transporter that expels antibiotics, reducing intracellular concentrations to subtherapeutic levels. In this study, we used molecular dynamics (MD) simulations to examine the pH-dependent behavior of NorA. Ciprofloxacin, a known substrate, was docked to NorA with binding affinities ranging from –7.4 to –6.0 kcal/mol, and the complex was subjected to MD simulations. The NorA structure, modeled with AlphaFold and simulated under acidic (pH 5), neutral (pH 7), and slightly basic (pH 7.5) conditions using CHARMM36, was analyzed via RMSD, RMSF, radius of gyration (Rg), free energy landscapes, principal component analysis, and binding energy estimation in GROMACS. At pH 5, ciprofloxacin was unstable and exited the binding cavity, whereas at pH 7 and 7.5 it remained stably bound, approaching the central cavity. These conditions also stabilized NorA, minimized structural energy, widened the pore, and enhanced water permeation. Our findings suggest that neutral to slightly basic environments favor antibiotic stabilization in the catalytic domain, potentially influencing drug uptake and efflux efficiency and guiding the design of potent NorA inhibitors.
Keywords: Antimicrobial resistance, NorA efflux pump, Staphylococcus aureus, ciprofloxacin, molecular dynamics simulations, pH-dependent stability, fluoroquinolones.