Design, synthesis and evaluation of lysine- and leucine-rich hydrocarbon-stapled peptides as antibacterial agents

Abstract

To address the challenge of antimicrobial resistance, we investigated new antibacterial peptides based on lysine- and leucine-rich sequences. We stabilised their membrane-active secondary structures by applying hydrocarbon stapling at sequence positions i and i+4. Stapling improved peptide structural stability in both aqueous and lipid environments, regardless of the staple position. It also enhanced antibacterial efficiency against both gram-negative and gram-positive bacteria, including antibiotic-resistant strains, with minimum inhibitory concentrations (MICs) of 2 to 4 μM (2.5 to 5.5 μg/mL). The stapled peptides showed increased resistance to enzymatic degradation, particularly with staples incorporated near the N-terminus, and were not haemolytic or cytotoxic at their MICs. Molecular dynamics simulations revealed how stapling aids in (i) stabilising the membrane-active secondary structure of amphipathic peptides and (ii) accelerating their membrane insertion. Our results provide insight into peptide design for antimicrobial use. We show that hydrocarbon stapling of lysine- and leucine-rich short sequences may offer a pathway towards more stable and effective antibacterial agents.