Elucidating the Molecular Determinants of High-Affinity Binding and Inhibition of Staphyllococcus aureus NorA Efflux Pump: Its Implication for Drug Design

Antimicrobial resistance (AMR) poses an important global health risk by diminishing the efficacy of existing antibiotics and complicating the treatment of bacterial infections. A key resistance mechanism involves bacterial efflux pumps, such as NorA, which expel diverse antimicrobial agents including fluoroquinolones, lowering intracellular drug concentrations and reducing activity. Efflux pump inhibitors (EPIs) can restore antibiotic potency by blocking these pumps. Although reserpine is a known NorA inhibitor, its clinical use is limited by adverse effects such as depression and neurotoxicity. Understanding molecular interactions that confer high-binding affinity to NorA is essential for designing potent and less toxic EPIs. The 3D crystal structure of NorA efflux pump was retrieved from Protein Data Bank (PDB ID: 7LO8). The protein was prepared for docking using BIOVIA Discovery Studio and AutoDockTools (ADT) following standard protein preparation protocol for molecular docking with AutoDockVina. In a similar way, ligands were retrieved from PubChem in SDF (Structure Data File) format and prepared for docking. They were energy-minimized on Avogadro, and Gasteiger charges were computed and torsions were defined on ADT. Data was analyzed based on binding affinity and interactions with relevant residues in the binding pocket of NorA. Reserpine demonstrated a significantly stronger binding affinity of -9.5 kcal/mol compared to -8.9kcal/mol for oleandomycin. Ciprofloxacin exhibited the lowest binding affinity of -8.3 kcal/mol. Reserpine formed extensive interactions with the pocket residues and with key residues (Glu 222, Asp 307 and Arg 310). The study revealed key interactions that confer high-affinity binding to the pocket of NorA efflux pump, offering a basis for designing more potent and safer EPIs.