Rational Engineering of a Brevinin-2 Peptide: Decoupling Potency from Toxicity Through C-Terminal Truncation and N-Terminal Chiral Substitution.

: The clinical potential of antimicrobial peptides (AMPs) against dual threats like antimicrobial resistance (AMR) and cancer is often limited by their high host cell toxicity. Here, we focused on brevinin-2OS (B2OS), a novel peptide from the skin of with potent haemolytic activity. The objective was to study the structure-activity relationship and optimise the safety via targeted modifications. A dual-modification strategy involving C-terminal truncation and subsequent N-terminal D-amino acid substitution was employed. The bioactivities and safety profiles of the resulting analogues were evaluated using antimicrobial, haemolysis, and cytotoxicity assays. Removal of the rana box in B2OS(1-22)-NH substantially reduced haemolysis while maintaining bioactivities. Remarkably, the D-leucine substitution in [D-Leu]B2OS(1-22)-NH displayed a superior HC value of 118.1 µM, representing a more than ten-fold improvement compared to its parent peptide (HC of 10.44 µM). This optimised analogue also demonstrated faster bactericidal kinetics and enhanced membrane permeabilisation, leading to a greater than 22-fold improvement in its therapeutic index against Gram-positive bacteria. The C-terminal rana box is a primary determinant of toxicity rather than a requirement for activity in the B2OS scaffold. The engineered peptide [D-Leu]B2OS(1-22)-NH emerges as a promising lead compound, and this dual-modification strategy provides a powerful design principle for developing safer, more effective peptide-based therapeutics.