Paola Saporito:
Synthesis and activity of antimicrobial peptides

Date: 02-03-2018    Supervisor: Anders Løbner Olesen

The emergence of bacterial resistance to antibiotics is a major concern for therapeutic medicine worldwide. The ability of bacteria to form biofilms constitutes one of the possible defense mechanisms from antimicrobial agents. Biofilms formation on medical devices, as indwelling catheters, and in infected wounds leads to chronic and life-threatening infections that are recalcitrant to antibiotics treatment. Therefore, it is urgent to develop novel antimicrobial agents able to prevent and treat biofilms associated infections. Antimicrobial peptides (AMPs) are considered promising tools for defeating both acute and chronic bacterial infections and have a lower rate of resistance development than the antibiotics. They are widely expressed in most living organisms as effectors of innate immunity to resist microbial infections and are useful templates for the synthesis of novel antimicrobial agents. A main drawback of antimicrobial peptides is their proneness to proteolytic degradation. Peptoids have been suggested as alternative antimicrobial peptidomimetics, resistant to proteolysis due to their peculiar backbone.

The aim of this PhD project was to identify shorter fragments of the human AMP cathelicidin LL-37 which retained the previously reported antimicrobial, antibiofilm and wound healing activity of the parent peptide LL-37 while reduced its cytotoxicity to the host. We identified two 12-mer peptides directly derived by fragmentation of LL-37, namely FK-12 and KR-12, and the single mutated peptide VQ-1226V as valid antimicrobials able to prevent and eradicate S. epidermidis biofilms and less cytotoxic than LL-37. In addition, the peptides KR-12 and VQ-1226V demonstrated wound healing potential in vitro. The study enabled to identify the gain of antimicrobial activities correlated to the single substitution aspartate to valine in residue-26 and led to a second study where the effect of this substitutions where further investigated. A new set of peptides was synthetized to evaluate the effect of similar aspartate to valine substitutions in inactive antimicrobial peptides (IK-12 and DF-12) and to investigate the relevance of the phenylalanine residue in position-27 for the antimicrobial activity of fragments KR-12 and VQ-1226V. In addition, the effect of charge and hydrophobicity of residue-26 in peptides FK-12 and KR-12 on their antibiofilm activity toward S. epidermidis, E. coli and P. aeruginosa as well as on cytotoxicity and wound healing potential was investigated. This study demonstrated that the valine substitution did not improve the antimicrobial activities of peptides IK-12, DF-12, suggested that the phenyl ring in the residue 27 is essential for antimicrobial activity and enabled to identify the more potent antimicrobial peptide KR-1227Y. Moreover, the anti-biofilm activity of peptides FK-12 and KR-12 were enhanced by the charge and -to less extent- by hydrophobicity of residue-26, whereas the antimicrobial activity was not affected by the substitutions. In addition, in FK-12 all the substitutions increased cytotoxicity toward human keratinocytes while only the aspartate to arginine substitution improved the wound healing potential. In contrast, all the substitutions did not affect neither the cytotoxicity or wound healing activity of KR-12.

An additional aim of the PhD project was to compare antimicrobial and antibiofilm properties of analogues peptides and peptoids toward E. coli. The study demonstrated that peptides and peptoids modelled on the previously in silico identified peptide GN-2, have different antimicrobial properties: the peptides are more potent antimicrobials toward planktonic E. coli, whereas analogues peptoids are more active against E. coli biofilms. The study enabled to correlate the antimicrobial activity of peptides with their hydrophobicity and LPS binding potential but not with their amphiphilicity. In contrast, the study demonstrated that the anti-biofilm activity of peptoids is inversely correlated to hydrophobicity. In addition, the substitution of lysine-3 with arginine was demonstrated to be detrimental for antimicrobial, anti-biofilm and LPS binding activities of the peptides tested.

Overall, these studies suggested possible substitutions in natural and synthetic AMPs and peptidomimetics that could be used to optimize the antimicrobial activities toward planktonic and sessile bacteria as well as their wound healing properties. However, these results warrant for further and deeper investigations on the mode of action of these antimicrobial agents as well as for in vivo studies.