Modulating Antimicrobial Activity and Structure of the Peptide Esc(1-21) via Site-Specific Isopeptide Bond Formation

Abstract

Antimicrobial peptides (AMPs) represent valid alternatives to conventional antibiotics primarily due to their mechanism of action, which consists of cytoplasmic membrane disruption. However, their clinical application is often limited by cytotoxicity at high concentrations and low intrinsic biostability. To address these limitations, various biochemical approaches have been explored. In recent years, the frog-skin derived AMP Esc(1-21) has been extensively characterized for its potent antimicrobial activity, especially against Gram-negative bacteria, both in vitro and in vivo. In this study, we designed and synthesized novel Esc(1-21) analogs in which a single isopeptide bond was introduced in place of a conventional peptide bond at specific positions within the sequence. The resulting five analogs were evaluated for their (i) chemical and structural properties, (ii) resistance to proteolytic degradation, (iii) antimicrobial and antibiofilm activities, (iv) hemolytic and cytotoxic effects, and (v) ability to perturb bacterial cytoplasmic membranes. Among these, Esc(1-21)ε20 showed the most promising features, maintaining antimicrobial and antibiofilm activities comparable to those of the parent peptide while exhibiting lower cytotoxicity towards eukaryotic cells at higher concentrations and greater resistance to enzymatic degradation. These findings highlight Esc(1-21)ε20 as an attractive lead candidate for the development of new antibiotic therapeutics.

FIGURE 1

Chemical structures of Esc(1‐21) peptide and its isopeptide bond analogs. Amino acid residues in black are L‐amino acids in α‐peptide bond. Amino acid residues involved in isopeptide bond formation are highlighted in red. In the analogs, lysine residues form covalent linkages via their ε‐amino groups to the carboxyl group of the adjacent amino acid, resulting in isopeptide bonds. (A) Esc(1‐21); (B) Esc(1‐21)ε5; (C) Esc(1‐21)ε9; (D) Esc(1‐21)ε10; (E) Esc(1‐21)ε12; (F) Esc(1‐21)ε20. The chemical structures of the peptides were generated using ChemDraw software.

FIGURE 2

Kinetics of cytoplasmic membrane permeabilization of E. coli ATCC 25922 induced by the addition (time = 0) of Esc(1‐21)ε5, Esc(1‐21)ε9, Esc(1‐21)ε10, Esc(1‐21)ε12, and Esc(1‐21)ε20 at different concentrations (from 1.56 to 50 μM) evaluated by the Sytox Green assay. Controls (Ctrl) are microbial cells without the addition of any peptide. The values are from one representative experiment out of three.

FIGURE 3

Kinetics of cytoplasmic membrane permeabilization of S. epidermidis ATCC 12228 induced by the addition (time = 0) of Esc(1‐21)ε5, Esc(1‐21)ε9, Esc(1‐21)ε10, Esc(1‐21)ε12, and Esc(1‐21)ε20 at different concentrations (from 1.56 to 50 μM) evaluated by the Sytox Green assay. Controls (Ctrl) are microbial cells without the addition of any peptide. The reported values are from one representative experiment out of three.

FIGURE 4

Activity of Esc(1‐21)ε12 and Esc(1‐21)ε20 against 20 h‐preformed biofilm of P. aeruginosa ATCC 15692 (A) and S. epidermidis ATCC 12228 (B) after 2 h of treatment. Biofilm viability was evaluated by measuring the reduction of the yellow MTT to its purple insoluble formazan and expressed as a percentage compared to untreated samples (bacterial biofilm not treated with the peptide, 100% viability). Data are the mean ± standard error of the mean (SEM) of three independent experiments. Statistical analysis was conducted using the two‐way ANOVA to determine the significance between the two peptides. *p < 0.05; ***p < 0.001; ****p < 0.0001; ns, not significant.

FIGURE 5

Effect of Esc(1‐21)ε5, Esc(1‐21)ε9, Esc(1‐21)ε10, Esc(1‐21)ε12, and Esc(1‐21)ε20 on mammalian RBCs after 30 min of peptide treatment at 37°C. The percentage of hemolysis was calculated with respect to the control (cells treated with vehicle). Data are the means ± SEM of three independent experiments.

FIGURE 6

Viability of HaCaT cells after 24 h treatment with two different concentrations of Esc(1‐21), Esc(1‐21)ε12, and Esc(1‐21)ε20. Cells not treated with peptides were used as controls. All data are the means of three replicates ± SEM. Statistical analysis was conducted using two‐way ANOVA to determine the significance between the peptides. *p < 0.05; ***p < 0.001; ****p < 0.0001; ns, not significant.

FIGURE 7

CD spectra of Esc(1–21)² and its analogues (A) in aqueous solution and (B) after LUVs addition (lipid concentration 1 mM, peptide concentration 20 μM).