Ultrashort Cationic Lipopeptides–Effect of N-Terminal Amino Acid and Fatty Acid Type on Antimicrobial Activity and Hemolysis

Abstract

Ultrashort cationic lipopeptides (USCLs) are promising antimicrobial agents that hypothetically may be alternatively used to combat pathogens such as bacteria and fungi. In general, USCLs consist of fatty acid chains and a few basic amino acid residues. The main shortcoming of USCLs is their relatively high cytotoxicity and hemolytic activity. This study focuses on the impact of the hydrophobic fatty acid chain, on both antimicrobial and hemolytic activities. To learn more about this region, a series of USCLs with different straight-chain fatty acids (C8, C10, C12, C14) attached to the tripeptide with two arginine residues were synthesized. The amino acid at the N-terminal position was exchanged for proteinogenic and non-proteinogenic amino acid residues (24 in total). Moreover, the branched fatty acid residues were conjugated to N-terminus of a dipeptide with two arginine residues. All USCLs had C-terminal amides. USCLs were tested against reference bacterial strains (including ESKAPE group) and Candida albicans. The hemolytic potential was tested on human erythrocytes. Hydrophobicity of the compounds was evaluated by RP-HPLC. Shortening of the fatty acid chain and simultaneous addition of amino acid residue at N-terminus were expected to result in more selective and active compounds than those of the reference lipopeptides with similar lipophilicity. Hypothetically, this approach would also be beneficial to other antimicrobial peptides where N-lipidation strategy was used to improve their biological characteristics.

Effect of Disulfide Cyclization of Ultrashort Cationic Lipopeptides on Antimicrobial Activity and Cytotoxicity

“…Our previous study showed that some disulfide-cyclized lipopeptides have higher antistaphylococcal activity when dissolved in aqueous solutions of acetic acid/BSA (0.01% and 0.2%, respectively) than in PBS (stock solution) [ 29 ]. Moreover, Gram-negative strains appeared to be more resistant to lipopeptides than Gram-positive bacteria and fungi, this being consistent with previous studies on lipopeptides [ 8 , 14 ]. Furthermore, cyclic analogs had a noticeably lower activity than the linear ones (even fourfold).…”

“…However, selectivity of USCL with four arginine (net charge +4) residues and hexadecanoic acid between hRBCs and bacteria/fungi based on previously determined activities is comparable to those estimated for linear USCLs used in this study [ 37 ]. In other studies, USCLs with arginine residues and shorter fatty acid chains (C14 and less) exhibited good selectivity between hRBCs, HaCaT cell line, and pathogens including bacterial strains and C. albicans [ 8 , 14 ].…”

“…The hemolysis assay was performed by using the method reported previously [ 8 , 60 ]. Fresh human red blood cells (hRBCs) with anticoagulant (EDTA) were rinsed three times with a PBS by centrifugation at 800× g for 10 min and diluted with PBS.…”

“…To solve this problem, researchers have attempted to design molecules with beneficial properties to balance between toxicity and antimicrobial activity. Undoubtedly, ultrashort (up to 7 amino acid residues) cationic lipopeptides (USCLs) have been claimed to be effective antimicrobial agents [ 5 , 6 , 7 , 8 ]. These molecules have a detergent-like mode of action and their membrane—cation interactions partially rely on the electrostatic attraction between positively charged amino acid residues and negatively charged membrane components.…”

“…Nevertheless, these properties make them perfect candidates for further studies and optimization of the structure. An appreciable antimicrobial activity of arginine- and lysine-based linear ultrashort cationic lipopeptides has been well documented [ 5 , 8 , 13 , 14 , 15 ]. A tetrapeptide containing four lysine residues and N -terminal hexadecenoic acid (palmitic acid, C 16 ) with proven high antimicrobial activity was used as a reference lipopeptide (C 16 -KKKK-NH 2 also known as Pal/Palm-KKKK-NH 2 ) [ 5 , 16 , 17 ].…”