In Vitro and In Vivo Investigations into the Potential of Quinazoline and Quinoline Derivatives as NorA Efflux Pump Inhibitors Against Resistant Staphylococcus aureus Strains

<b>Background:</b><i>Staphylococcus aureus</i> is a highly lethal Gram-positive bacterium that is responsible for over one million deaths annually. As a member of the ESKAPE pathogens, its methicillin-resistant strains (MRSA) are prevalent worldwide and exhibit significant antimicrobial resistance (AMR). Bacterial efflux pumps play a pivotal role in the development of AMR by facilitating the expulsion of a range of antimicrobial agents. <b>Methods</b>: The <i>S. aureus</i> strain SA-1199B, which overexpresses NorA and carries a GrlA mutation, was utilized to comprehensively profile the mechanism of the compounds <b>PQQ16P</b> and <b>PQK4F</b>. To assess the toxicity and genotoxicity of these compounds, RAW macrophages, HEK 293T, and HepG2 cell lines were utilized. Female BALB/c mice were utilized to assess the in vivo synergism of EPIs with CPX, <b>Results</b>: NorA efflux pump inhibitors (EPIs), <b>PQQ16P</b> and <b>PQK4F</b>, enhanced the efficacy of the antibacterial ciprofloxacin (CPX) against resistant <i>S. aureus</i> strains. The mechanism of EPIs involved the inhibition of NorA efflux pump, without compromising bacterial membrane permeability, ATP levels, or mammalian calcium channels. Moreover, the EPIs significantly augmented the bactericidal and post-antibiotic effects of CPX, elevating its mutation prevention concentration without manifesting substantial toxicity to human cells. Furthermore, the EPIs reduced <i>S. aureus</i> invasiveness in macrophages, indicating a role for NorA in bacterial virulence. Notably, the in vivo synergism of these EPIs with CPX was observed in a mouse infection model. <b>Conclusions</b>: This study provides substantial evidence for the potential of employing EPIs in a combination with CPX to counteract AMR, both in vitro and in vivo.