Synthesis and preliminary studies on an esculentin analog carrying Aib residues

Esculentins-1 are a family of frog skin antimicrobial peptides (AMPs), characterized by a 46 amino acids primary structure and a broad range of antimicrobial activity. Studies on the mode of action of amphibian AMPs evidenced that bacterial membranes are their major target. Esculentins possess the basic features common to most linear AMPs, that is an overall positive charge at neutral pH and a considerable proportion of hydrophobic residues. These properties allow the peptides to bind to the negatively-charged microbial membranes by folding into an amphiphilic structure, thus perturbing membrane permeability. Esculentin 1-21 [Esc(1-21)], consists of the first 20 aminoacids of the naturally occurring Esculentin 1a, with a glycinamide residue at the C-terminus: H-Gly-Ile-Phe-Ser-Lys-Leu-Ala-Gly-Lys-Gly-Leu-Lys-Asn-Leu-Leu-Ile-Ser-Gly-Leu-Lys-Gly-NH2. It was previously demonstrated that Esc(1-21) is highly potent against the most common mastitis-causing microbes in dairy cattle (e.g. Streptococcus agalactiae) both in vitro and in vivo. Furthermore, recent studies have indicated that this peptide can rapidly kill both planktonic and biofilm forms of the opportunist pathogen Pseudomonas aeruginosa and improve survival in a murine model of Pseudomonas lung infection upon its topical administration. These properties make Esc(1-21) an attractive starting point for the development of novel antimicrobial agents able to cure mastitis in dairy cows and to treat Pseudomonas-related infections. Current therapies in clinical and veterinary medicine suffer from drawbacks that include the increased emergence of multidrug-resistant pathogens, and the production of milk contaminated with antibiotics has become a serious threat in the livestock. In this connection, we thought significant to explore the effects of the incorporation of ?-aminoisobutyric acid (Aib) residues into the sequence of Esc(1-21). When inserted into peptides, this strongly helicogenic, non-coded, C?-tetrasubstituted ?-amino acid might increase the population and stability of helical structures and also potentially confer resistance against enzymatic degradation. In this work we report on the synthesis, the characterization, a preliminary conformational study in different environments by CD and 2D-NMR techniques, and the biological activity of an analog of Esc(1-21) bearing three residues of ?-aminoisobutyric acid (Aib) at sequence position 1, 10 and 18.