Cyclic Diguanylate G-Quadruplex Inducer–Siderophore Hybrids as Bifunctional Antibiofilm Agents against <i>Pseudomonas aeruginosa</i>

Biofilm formation is a crucial determinant of both pathogenicity and enhanced antibiotic resistance in <i>Pseudomonas aeruginosa</i>. Sustainable iron utilization in the bacterial growth environments and intracellular c-di-GMP signaling molecules are key factors promoting bacterial biofilm development. Capitalizing on our previous findings that c-di-GMP G-quadruplex inducers exhibit potent antibiofilm activity and that 3-hydroxypyridin-4(<i>1H</i>)-ones act as effective iron chelators, we designed and synthesized dual-functional hybrid molecules incorporating both pharmacophores. The lead compound <b>11f</b>, which features a benzothiazole-pyridinone scaffold, demonstrated potent biofilm inhibition against both wild-type <i>P. aeruginosa</i> PAO1 and the hyper-biofilm-forming PAO1-Δ<i>wspF</i> mutant. Mechanistic investigations revealed that <b>11f</b> operates through dual pathways: facilitating c-di-GMP G-quadruplex formation and disrupting iron acquisition systems. Notably, in <i>Galleria mellonella</i> infection models, <b>11f</b> exhibited significant synergistic effects with ciprofloxacin and tobramycin while maintaining an excellent safety profile, highlighting its potential as an antibacterial adjuvant.