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

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.