Balancing Cationicity and Hydrophobicity in Dermaseptin-A4 Generates a Selective Antimicrobial Peptide with Enhanced Therapeutic Potential

Background/Objectives: Antimicrobial peptides (AMPs) have emerged as promising alternatives to conventional antibiotics in response to the escalating global threat of antimicrobial resistance (AMR), owing to their potent antimicrobial activity and low propensity for resistance development. However, their clinical application remains limited by poor selectivity and undesirable toxicity toward mammalian cells. Methods: In this study, the naturally occurring frog-derived AMP Dermaseptin-A4 (A4) was selected as a template for rational design. Guided by the principle that optimising the balance between peptide hydrophobicity and cationicity could improve bacterial membrane targeting while reducing interactions with mammalian membranes, three analogues were designed through the targeted modulation of these physicochemical properties. Results: Among the designed analogues, A4-3 exhibited the best overall biological profile. A4-3 maintained a stable α-helical conformation in membrane-mimicking environments and displayed potent antimicrobial activity against tested Gram-positive and Gram-negative bacteria while exhibiting lower haemolytic and cytotoxic effects than the parent peptide. As a result, A4-3 showed improved selectivity, achieving a selectivity index of up to 34.5. A4-3 rapidly eradicated bacterial cells through a membrane-targeting mechanism, leading to membrane disruption and the loss of cellular integrity, and exhibited a low propensity for resistance development following prolonged exposure. A4-3 also retained its antimicrobial activity under physiologically relevant conditions. Conclusions: Collectively, these findings demonstrate that achieving an optimal balance between peptide hydrophobicity and cationicity is an effective strategy for enhancing antimicrobial selectivity without compromising antibacterial activity, highlighting A4-3 as a promising lead candidate for the development of novel antimicrobial therapeutics against drug-resistant bacterial infections.