Impact of Lysine-Mediated C-Terminal Dimerization of Anoplin on Killing Kinetics and Antibiotic Interaction Profiles

Antimicrobial resistance (AMR) limits the efficacy of conventional antibiotics, particularly in infections caused by multidrug-resistant bacteria. anoplin, a short membrane-active antimicrobial peptide, represents a promising scaffold for structural optimization. This study evaluated the antimicrobial performance of anoplin and its C-terminal lysine-mediated dimer, (anoplin)2K, against clinically relevant Gram-positive and Gram-negative bacteria, including carbapenemase-producing and non-producing Klebsiella pneumoniae strains. Minimum inhibitory concentration (MIC), minimum bactericidal concentration (MBC), time–kill kinetics, checkerboard interaction assays, and hemolytic activity were determined. While MIC values showed moderate differences between the peptides, (anoplin)2K demonstrated enhanced bactericidal kinetics, achieving complete eradication of Escherichia coli, Staphylococcus aureus, and Pseudomonas aeruginosa within 60 min at 256 µM. Additive effects predominated in combination assays, with selective synergism observed for sulfamethoxazole–trimethoprim. Reduced susceptibility was observed for carbapenemase-producing K. pneumoniae. Although the dimeric analogue exhibited increased hemolytic activity at higher concentrations, the results indicate that C-terminal dimerization modulates killing dynamics and enhances antimicrobial performance. These findings support further optimization of anoplin-derived dimers as potential adjunct strategies against multidrug-resistant pathogens.