Novel Cyclic Lipopeptide Antibiotics: Effects of Acyl Chain Length and Position

Abstract

Multidrug-resistant bacteria are a global health problem. One of the last-resort antibiotics against Gram-negative bacteria is the cyclic lipopeptide colistin, displaying a flexible linker with a fatty acid moiety. The aim of the present project was to investigate the effect on antimicrobial activity of introducing fatty acid moieties of different lengths and in different positions in a cyclic peptide, S3(B), containing a flexible linker. The lipidated analogues of S3(B) were synthesized by 9-fluorenylmethoxycarbonyl (Fmoc) solid-phase peptide synthesis. Following assembly of the linear peptide by Fmoc solid-phase peptide synthesis, on-resin head-to-tail cyclization and fatty acid acylation were performed. The antimicrobial activity was determined against the ESKAPE pathogens, Staphylococcus aureus, Klebsiella pneumoniae, Acinetobacter baumannii, Pseudomonas aeruginosa, and Escherichia coli. Furthermore, hemolytic activity was determined against human erythrocytes. A total of 18 cyclic lipopeptides were synthesized and characterized. It was found that introduction of fatty acids in positions next to the flexible linker was more strongly linked to antimicrobial activity. The fatty acid length altered the overall hydrophobicity, which was the driving force for both high antimicrobial and hemolytic activity. Peptides became highly hemolytic when carbon-chain length exceeded 10 (i.e., C₁₀), overlapping with the optimum for antimicrobial activity (i.e., C₈-C₁₂). The most promising candidate (C₈)⁵ showed antimicrobial activity corresponding to that of S3(B), but with an improved hemolytic profile. Finally, (C₈)⁵ was further investigated in a time-kill experiment.

Keywords: antimicrobial peptides; colistin; cyclic lipopeptides; fatty acid; hydrophobicity.

Figure 1

Structure of fatty acid S3(B) analogues studied in this paper.

Scheme 1

Strategy for synthesis of fatty acid S3(B) analogues (synthesis of cyclic lipopeptide 1 is shown).

Figure 2

Relationship between hydrophobicity and antimicrobial activity (MIC, μg/mL) and hemolytic activity (% hemolysis at 150 μM) of the (C_n)⁵ series of peptides. Data shown are obtained against P. aeruginosa and human erythrocytes. Arrows indicate the hydropmichobicity optima for MIC (minimum inhibitory concentration) and HA, the two optima being displaced.

Figure 3

Hemolysis as a function of peptide concentration. In the present study, the peptide (C₈)⁵ (blue curve) showed significant lower toxicity toward erythrocytes than S3(B) (red curve) in all tested concentrations (2.35–150 μM). The results are presented as mean ± standard deviations (n = 3).

Figure 4

Time-kill curves for (C₈)⁵ against P. aeruginosa at different concentrations; 1 × MIC and 5 × MIC. Data obtained for S3(B) and colistin are included as controls. The results are presented as mean ± standard deviations (n = 3).