Phage Selection of Cyclic Peptides Inhibiting Aminoglycoside Phosphotransferases to Control Resistant Bacteria

) enhanced peptide-APH interactions, suggesting a metal-dependent binding mechanism. Competitive fluorescence polarization assays revealed that these cyclic peptides primarily occupy the ATP-binding pocket of APH(3')-Ia, with representative candidate A-L3 demonstrating significant enzymatic inhibition. This study establishes a foundation for developing APH-targeted antibiotic adjuvants through (1) identification of novel cyclic peptide scaffolds with inhibitory potential, (2) elucidation of divalent metal ion effects on inhibitor binding, and (3) mechanistic insights into ATP-binding site competition. These findings provide critical structural and functional information to guide the rational design of next-generation antibiotic resistance breakers.