Targeting Staphylococcus aureus with potent de novo-designed proline-core short antimicrobial lipopeptides

Antimicrobial peptides (AMPs) show great therapeutic potential due to their unique mechanism of action that guarantees broad-spectrum efficacy and limits bacterial antibiotic resistance. However, challenges such as limited stability and cytotoxicity toward host cells still limit their clinical translation, highlighting the need for new approaches, such as size reduction and lipid conjugation, to enhance their efficacy, cell penetration, stability, and safety. Herein, we report the de novo design of a library of ultra-short lipopeptides based on a rigid L-Arg–L-Pro–L-Arg core, conceived to control conformational restriction and amphiphilic organization rather than mimicking longer natural AMPs. The compounds were synthesized and preliminarily evaluated in vitro against 3 Gram-negative and 3 Gram-positive strains. Systematic modulation of lipid positioning and linker orientation in this minimal scaffold led to the identification of promising candidates displaying MIC values in the low-μM range against both gram-negative and gram-positive bacteria. Of the newly developed compounds, 15 exhibited optimal lipophilicity, excellent human-serum stability and a favourable safety profile, showing only low to moderate toxicity toward renal, hepatic, and red blood cells. Additionally, 15 proved effective in reducing S. aureus biofilm formation and showed strong activity against five clinical isolates. It acts as a bacteriostatic agent by perturbing bacterial membrane integrity, positioning it as a promising starting point for the development of a new class of chemotypes that could offer an alternative strategy for treating infections caused by this gram-positive pathogen.