Evaluation of the antimicrobial and NorA and MepA efflux pump inhibitory activity of a hydrazone derivative of hydralazine against Staphylococcus aureus.

Bacterial resistance represents one of the major challenges in contemporary public health, compromising the effectiveness of conventional antibiotics. In this context, research focused on the discovery of new compounds and resistance modulators has gained increasing attention. The present study aimed to evaluate the antimicrobial activity and the antibiotic-modulating potential of a hydrazone derivative of hydralazine against Staphylococcus aureus strains harboring efflux pumps. The compound (E)-2-((2-(phthalazin-1-yl)hydrazineylidene)methyl)phenol (HDZH2OH) was synthesized from hydralazine and isoniazid and evaluated using the Minimum Inhibitory Concentration (MIC) assay by the broth microdilution method, as well as resistance modulation assays in association with specific antibiotics and ethidium bromide. The cell viability assay showed that HDZH2OH reduced macrophage viability in a concentration-dependent manner, with viability decreasing from 74.8 ± 3.0% at 3.25 µM to 27.25 ± 1.25% at 25 µM. MIC values ≥ 1024 µg/mL (3.88 mM) against both tested strains, indicating the absence of intrinsic antibacterial activity. However, in the S. aureus 1199B strain, a reduction in the MIC of norfloxacin from 256 µg/mL to 101.59 µg/mL (0.802 mM to 0.334 mM) was observed, suggesting a partial potentiating effect. In the K2068 strain, no potentiation of ciprofloxacin was observed; however, a significant reduction in the MIC of ethidium bromide from 8 µg/mL to 2 µg/mL (0.020 mM to 0.005 mM) was detected (p < 0.001). Molecular docking indicated that HDZH2OH strongly interacts with S. aureus efflux pumps, showing high affinity and selective binding. It overlaps with substrate sites in MepA and NorA, suggesting competitive inhibition. Its interactions, including hydrogen bonds, enhance stability, highlighting its potential to inhibit efflux activity and restore antimicrobial effectiveness. HDZH2OH shows favorable drug likeness consistent with Lipinski and Veber rules. It is mostly neutral at physiological pH (pKa 4.05), supporting good permeability and oral absorption. CNS MPO score (4.47) suggests potential CNS activity and BBB penetration. It has high plasma protein binding and moderate toxicity risk, with possible CYP inhibition and hepatotoxicity concerns. These findings indicate that the compound acts as a selective efflux pump modulator, with greater affinity for the MepA system, highlighting its potential as a therapeutic adjuvant in combating bacterial resistance.