The emergence of MDR Acinetobacter baumannii (Ab) represents a critical global health threat. We aimed to investigate the antibacterial properties of ENOblock, a compound identified through HTS of the EU-OPENSCREEN library, as potential treatment for MDR Ab.
In vitro assays were conducted to evaluate ENOblock’s antibacterial activity using microdilution, time-kill curves, and SYTOX Green membrane permeability tests. Bacterial cytological profiling (BCP) was employed to assess morphological changes after ENOblock treatment, shedding light on its mechanism of action (MoA). To identify the ENOblock molecular target, computational docking was performed to analyze its binding affinity to Ab enolase. Target validation was achieved using an enolase-deficient mutant (Δeno) by measuring changes in the MIC and bacterial growth. Synergistic effects of ENOblock combined with antibiotics were assessed in reference and colistin-resistant strains. Finally, ENOblock's efficacy was further evaluated in human epithelial and macrophage cells and in a Galleria mellonella model.
ENOblock demonstrated potent antibacterial activity with an MIC50 of 16 mg/L against clinical carbapenem- and colistin-resistant Ab strains, surpassing the MIC50 values of colistin and imipenem/meropenem. SYTOX Green assay revealed that ENOblock caused rapid and extensive membrane damage. BCP analysis revealed unique morphological changes not observed with clinically used antibiotics, suggesting a distinct ENOblock MoA. Computational docking indicated strong binding of ENOblock to enolase. The Δeno strain exhibited a four-fold increase in MIC and greater growth resistance to ENOblock, supporting enolase as its probable target. Importantly, ENOblock exhibited synergistic effects with colistin, enhancing its efficacy against both reference and colistin-resistant strains. Furthermore, ENOblock significantly reduced Ab adherence/invasion in epithelial and macrophage cells in vitro and improved survival rates in G. mellonella infected with Ab, demonstrating its therapeutic potential.
These findings highlight ENOblock as a promising candidate for antimicrobial development, with the potential to combat the critical threat posed by MDR Ab infections.
