Derivatives of esculentin-1a: promising frog-skin peptides for the development of new antipseudomonal drugs with expanding properties

Naturally-occurring antimicrobial peptides (AMPs) represent an interesting class of molecules for the development of new anti-infective agents with multiple properties. The discovery of new antimicrobials is highly demanded, due to the increasing number of microorganisms that are resistant to the currently-used drugs. Remarkably, one of the most feared opportunistic pathogens, especially in the lungs of cystic fibrosis (CF) sufferers is the gram-negative bacterium Pseudomonasaeruginosa,becauseofitsintrinsiclowsusceptibilitytoantibioticsandabilitytoformbiofilm-communities that are resistant to a variety of environmental insults. Recently, we discovered that a derivative of the frog-skin AMP esculentin-1a, esculentin-1a(1-21)NH2 [Esc(1-21) GIFSKLAGKKIKNLLISGLKG-NH2 had potent antimicrobial activity against both free-living and biofilm forms ofP. aeruginosa with minimal concentrations inhibiting microbial growth (MICs) ranging from 4 to 8 µM (1). Subsequently, a diastereomer of Esc(1-21), Esc(1-21)-1c, carrying two D-amino acids was designed. It resulted to be slightly weaker than the all-L peptide against the planktonic form ofP. aeruginosa (with a concentration causing 99.9% killing of bacterial cells equal to 4 µMversus 1 µM of the parent peptide). However, it showed a higher bactericidal activity against the more dangerous biofilm phenotype; a lower cytotoxicity and higher biostability (2). In this work, further biological properties were investigated. Among them: (i) the ability of Esc(1-21) and its diastereomer to induce bacterial resistance after multiple cycles of treatment at sub-MICs in comparison with conventional antibiotics; (ii) the peptides' effect on the motility of the planktonic form of CF clinical isolates of P. aeruginosa and (iii) the effect of the diastereomer on preformed Pseudomonas biofilm in the presence of artificial sputum medium (ASM) that better simulates the lung environment (i.e. sputum composition) of CF patients. The results of these experiments have indicated that in contrast with aztreonam, colistin, tobramycin and ciprofloxacin, both peptides do not induce resistance after a prolonged exposure (~ 1 month) of the bacterial culture to them, as pointed out by the invariant MIC. In addition, the diastereomer was found to have a higher ability than the all-L Esc(1-21) to inhibit the bacterium's motility and presumably its ability to reach and colonize a biological surface (e.g. airways epithelium) switching to the biofilm form. Finally, when the diastereomer was added to Pseudomonas biofilm in ASM it was found to cause 20 % killing of the biofilm after 6 h treatment at the MIC, while only 2% reduction of viable biofilm was found for the conventional antibiotic aztreonam. All these data suggest that the two frog-skin derived AMPs represent very interesting candidates for the development of new antipseudomonal drugs for treatment of lung infections.