Enantiomeric optimization markedly increased safety and efficacy of a cationic peptide against multidrug-resistant pathogens.

Pseudomonas aeruginosa is a pathogenic bacterium known to cause serious human infections, especially in immune‐compromised patients. This is due to its unique ability to transform from a drug‐tolerant planktonic to a more dangerous and treatment‐resistant sessile life form, called biofilm. Recently, two derivatives of the frog skin antimicrobial peptide esculentin‐1a, i.e. Esc(1‐21) and its D‐amino acids containing diastereomer Esc(1‐21)‐1c, were characterized for their powerful anti‐Pseudomonal activity against both forms. Prevention of biofilm formation already in its early stages could be even more advantageous for counteracting infections induced by this bacterium. In this work, we studied how the diastereomer Esc(1‐21)‐1c can inhibit Pseudomonas biofilm formation in comparison to the parent peptide and two clinically‐used conventional antibiotics, i.e. colistin and aztreonam, when applied at dosages below the minimal growth inhibitory concentration. Biofilm prevention was correlated to the peptides’ ability to inhibit Pseudomonas motility and to reduce the production of virulent metabolites, for example, pyoverdine and rhamnolipids. Furthermore, the molecular mechanism underlying these activities was evaluated by studying the peptides’ effect on the expression of key genes involved in the virulence and motility of bacteria, as well as by monitoring the peptides’ binding to the bacterial signaling nucleotide ppGpp. Our results demonstrate that the presence of only two D‐amino acids in Esc(1‐21)‐1c is sufficient to downregulate ppGpp‐mediated expression of biofilm‐associated genes, presumably as a result of higher peptide stability and therefore prolonged interaction with the nucleotide. Overall, these studies should assist efficient design and optimization of new anti‐infective agents with multiple pharmacologically beneficial properties.

In recent years, we have discovered Esc(1-21) and its diastereomer (Esc peptides) as valuable candidates for the treatment of Pseudomonas lung infection, especially in patients with cystic fibrosis (CF). Furthermore, engineered poly(lactide-co-glycolide) (PLGA) nanoparticles (NPs) were revealed to be a promising pulmonary delivery system of antimicrobial peptides. However, the “ad hoc” development of novel therapeutics requires consideration of their stability, tolerability, and safety. Hence, by means of electrophysiology experiments and preclinical studies on healthy mice, we demonstrated that neither Esc peptides or Esc-peptide-loaded PLGA NPs significantly affect the integrity of the lung epithelium, nor change the global gene expression profile of lungs of treated animals compared to those of vehicle-treated animals. Noteworthy, the Esc diastereomer endowed with the highest antimicrobial activity did not provoke any pulmonary pro-inflammatory response, even at a concentration 15-fold higher than the efficacy dosage 24 h after administration in the free or encapsulated form. The therapeutic index was ≥70, and the peptide was found to remain available in the bronchoalveolar lavage of mice, after two days of incubation. Overall, these studies should open an avenue for a new up-and-coming pharmacological approach, likely based on inhalable peptide-loaded NPs, to address CF lung disease.