Lactam-cyclized, N-acylated FAKWAFKWLKK analog (Peptide 2) significantly boosts antibacterial potency and safety margin.

Antimicrobial resistance (AMR) represents a critical global health crisis, driving an urgent demand for novel antibiotics. Traditional antibiotics face increasing ineffectiveness, necessitating new therapeutic strategies. Antimicrobial peptides (AMPs) offer a promising alternative due to their broad-spectrum activity and distinct mechanisms of action, often involving membrane disruption. However, many linear AMPs suffer from low potency, narrow spectrum, and potential toxicity, limiting their clinical translation. Rational design strategies are crucial to overcome these limitations, focusing on structural modifications that enhance efficacy and safety while preserving their inherent antibacterial properties.

Researchers started with the natural amphipathic α-helical peptide FAKWAFKWLKK-NH2. They rationally designed a cyclized analog, Peptide 2, by introducing an i, i + 7 side-chain lactam bridge through mutating Ala2 to Lys and Leu9 to Glu, aiming to preserve the α-helical conformation. The team then further modified Peptide 2 with N-terminal C10 acylation. They evaluated the minimum inhibitory concentration (MIC) of the linear peptide, Peptide 2, and its acylated form against various bacterial strains, including Pseudomonas aeruginosa ATCC27853, Staphylococcus aureus ATCC25923, Acinetobacter baumannii BNCC337173, S. aureus ST9, and Listeria monocytogenes ATCC13932. Hemolysis assays (HC50) were also performed to assess cytotoxicity against red blood cells.

The lactam-cyclized Peptide 2 exhibited significantly improved antibacterial potency compared to its linear precursor. Against Pseudomonas aeruginosa ATCC27853, Peptide 2 achieved an MIC of 4 μg/mL, representing a >32-fold improvement over the linear peptide's MIC of >128 μg/mL. For Staphylococcus aureus ATCC25923, Peptide 2 showed an MIC of 2 μg/mL, an 8-fold improvement from the linear peptide's MIC of 16 μg/mL. This demonstrated potent activity against both Gram-negative and Gram-positive pathogens. N-terminal C10 acylation further enhanced potency: > The MIC against P. aeruginosa and A. baumannii was lowered to 2 μg/mL, while S. aureus ST9 and L. monocytogenes MICs were reduced to 4 μg/mL and 8 μg/mL, respectively. Hemolysis assays revealed that Peptide 2 had an HC50 of >100 μg/mL, resulting in a therapeutic index (HC50/MIC) of >25 against P. aeruginosa, indicating a favorable safety margin.