Different approaches to optimize the antimicrobial properties of cationic peptides: substitution by non-coded amino acids and conjugation to nanoparticles

Antimicrobial peptides are small, cationic and amphipathic peptides produced by virtually all living organisms as key components of the innate immune system. Importantly, they hold promise for the development of new broad-spectrum anti-infective agents. These are urgently needed, due to the growing emergence of microorganisms that are resistant to the available therapeutic agents. In recent years, research has focused on the characterization and optimization of these molecules. Albeit thousands of peptides have been isolated and characterized, only a reduced number of them has entered into clinical phases, because of some limitations. Among them: (i) the poor peptide bioavailability, (ii) the toxicity at high doses (iii) the high susceptibility to proteolytic degradation (iv) the inefficient peptide delivery to the target site at high concentration. Nowadays, thanks to advances in biochemical research, computational studies and nanotechnologies it has become possible to overcome some of these problems. Recently, a frog-skin derived AMP, named Esc(1-21), has been investigated for its potent activity against the Gram-negative bacterium Pseudomonas aeruginosa and immunomodulatory properties. However, it showed a weaker activity against Gram-positive bacteria and some cytotoxic effect on mammalian cells. In this thesis work, with the aim to optimize the biological properties of AMPs, e.g. Esc(1-21), three different approaches have been used: - Design and synthesis of an analog of Esc(1-21) carrying three alpha-aminoisobutyric acid (Aib) residues. When inserted into the primary structure of peptides, Aib residues are expected to increase the alpha-helical content of the peptides, due to their strong helicogenicity. Importantly, a stabilized alpha-helical structure is known to be a crucial parameter to improve the AMPs’ activity against Gram-positive bacteria but also to increase their toxicity against mammalian cells. The Aib-containing peptide has been studied for its structural and biological properties and compared with the parent peptide Esc(1-21). - Design and synthesis of a diastereomer of Esc(1-21), named Esc(1-21)-1c, by replacing two L-amino acids in the C-terminal portion, i.e. L-Leu14 and L-Ser17, with the corresponding D-enantiomers. Besides making a peptide more resistant to proteolytic degradation, the insertion of D- amino acids is expected to disrupt the peptide’s alpha-helical content and to reduce its cytotoxicity. The effect(s) of D-amino acids incorporation on the structural/biological properties of the peptide have been investigated. - Design and production of two different types of nanoparticles to protect the AMP from proteolytic degradation and to allow its delivery to the target site at high concentration without altering the antimicrobial properties: (i) inorganic gold nanoparticles (AuNPs) coated with Esc(1-21) and (ii) nano-embedded microparticles for pulmonary delivery of a model cationic antimicrobial peptide, i.e. colistin. As expected, the Aib-analog of Esc(1-21) resulted to be more active against Gram-positive bacteria, but also more cytotoxic against mammalian cells, due to its higher alpha-helical content in the secondary structure. Differently, the insertion of two D-amino acids provoked a disruption of the alpha-helix and, as a consequence, a significant reduction in the peptide’s cytotoxic effect. In addition, the diastereomer resulted to be more active against the biofilm form of P. aeruginosa and more stable in human serum. The conjugation of Esc(1-21) to AuNPs, led to an increase of the antimicrobial activity of the peptide and to a greater stability to proteolytic degradation, without interfering with the mechanism of membrane perturbation. In addition, the conjugation onto AuNPs did not affect the wound-healing properties of the free peptide and AuNPs@Esc(1-21) were not found to be toxic against human keratinocytes. Finally, the engineered biocompatible and biodegradable polymeric microparticles designed as a delivery system for an AMP model, i.e. colistin, resulted to have a prolonged activity against P. aeruginosa biofilm in comparison with the free peptide. This was likely due to their ability to penetrate into bacterial biofilm and to sustain colistin release inside it. The promising results obtained by these different approaches, have made it possible to take a step forward in the optimization of a cationic peptide for the development of potential new antibacterial drugs.