Rational design and N-terminal acylation of a lactam-cyclized 11-residue antimicrobial peptide for improved...

Linear antimicrobial peptides frequently suffer from low potency and narrow spectrum, restricting their clinical utility. Starting from the natural amphipathic α-helical peptide FAKWAFKWLKK-NH₂, we rationally introduced an i, i + 7 side-chain lactam bridge by mutating Ala² and Leu⁹ to Lys and Glu, respectively, while preserving the predicted α-helical conformation. Cyclized peptide 2 displayed a minimum inhibitory concentration (MIC) of 4 μg/mL against Pseudomonas aeruginosa ATCC27853 (>32-fold improvement over the linear peptide, MIC >128 μg/mL) and 2 μg/mL against Staphylococcus aureus ATCC25923 (8-fold improvement; linear peptide MIC 16 μg/mL). Peptide 2 also showed potent activity against Acinetobacter baumannii BNCC337173 (MIC = 8 μg/mL), S. aureus ST9 (MIC = 32 μg/mL), and Listeria monocytogenes ATCC13932 (MIC = 32 μg/mL), indicating activity against both Gram-negative and Gram-positive pathogens. N-terminal C10 acylation further improved potency, lowering the MIC against P. aeruginosa and A. baumannii to 2 μg/mL, and against S. aureus ST9 and L. monocytogenes to 4 and 8 μg/mL, respectively. Hemolysis assays showed that peptide 2 had an HC₅₀ >100 μg/mL, yielding a therapeutic index (HC₅₀/MIC) >25 against P. aeruginosa and a favorable safety margin. These findings support a focused stepwise rational design strategy for this peptide scaffold, combining natural template selection, lactam cyclization, and N-terminal lipophilic modification.