STRATEGIES FOR DEVELOPING NON-CYTOTOXIC ANTIMICROBIAL PEPTIDES: A FOCUS ON PHYSICOCHEMICAL ATTRIBUTES

Antimicrobial peptides (AMPs) are molecules capable of interacting with biomembranes, due to their physicochemical properties, such as positive charge, α-helical folding, amphipathicity and hydrophobicity. These features can be used to target pathogens membranes. However, imbalanced properties can cause issues like host cell membrane disruption. To develop non-cytotoxic AMPs, it is crucial to assess and balance these physicochemical properties. In this study, we synthesized 10 known bioactive peptides and 24 previously designed peptides using Fmoc solid-phase synthesis, purified by reverse-phase liquid chromatography and analyzed by MALDI-TOF mass spectrometry. Physicochemical properties values of the peptides were provided by Encrypted software, developed by our research group, and the results were submitted to the K-Means algorithm and WCSS method. The peptides were classified into four groups. Principal component analysis and ANOVA were employed to identify the critical physicochemical properties forming these groups, including basic or buried residues, alpha-helix, beta-sheet, and turn formations, volume, mass, and solvent accessibility to polar, non-polar, and main chain residues. In vitro MTT assays on mammalian cells confirmed that the most cytotoxic peptides belonged to group-1, which included other toxic peptides reported in previous studies. Notably, schistocinin-6 was non-toxic to murine macrophages within this group. Overall, our findings suggest that higher buried and basic residue content, along with increased volume, mass, alpha-helix, beta-sheet, and turn formations, and better solvent accessibility, contribute to greater cytotoxicity in RAW macrophages. These properties warrant further exploration for the design of new non-cytotoxic AMPs.