Towards the design of peptide antibiotics: new insights from synthetic analogues and from natural sources

Resistance to antibiotics is spreading worldwide across bacteria generating an increasing number of multi- and even pan-drug resistant pathogens. Antibiotics currently on the market hardly could keep this threat at bay. In order to overcome this problem, novel classes of drugs with different mechanisms of action are extremely necessary. High hopes are pointed towards antimicrobial peptides (AMPs), natural molecules with multiple defensive roles produced by eukaryotes and bacteria. Among AMPs, proline-rich antimicrobial peptides (PrAMPs) share a generally low cytotoxicity thanks to an intracellular mode of action based on inhibition of protein synthesis but also a relatively narrow spectrum of activity. This work was aimed to optimize the antimicrobial activity of two peptides derived from the natural cathelicidin-derived PrAMPs Bac7 and Bac5, to characterize them along with seven novel PrAMPs discovered in cetaceans. First, we characterized the antimicrobial activity, toxicity and mode of action of eight fragments of Bac5, discovering that Bac5(1-17) was the shortest fragment retaining appreciable antimicrobial and translation-inhibiting activity against E. coli. Subsequently, we screened libraries of mutants of Bac5(1-17) and Bac7(1-16), another known PrAMP fragment with antimicrobial activity. This led us to select ten novel (optimized) PrAMPs derived from of Bac7(1-16) and Bac5(1-17), with single or multiple amino acid substitutions, to be further characterized for activity/toxicity. In parallel, we searched and characterized orthologs of the bovine PrAMP Bac7 in cetacean species, finding five novel cetacean PrAMPs (cePrAMPs). These five novel molecules, along with the two recently discovered cePrAMPs Tur1A and Tur1B, and our ten optimized PrAMP fragments, were characterized for antimicrobial activity, mode of action, cytotoxicity and stability in serum and salty media. Among the selected optimized PrAMP fragments, some displayed wider activity spectrum or increased activity compared to the original PrAMPs. Two of them showed MIC = 1-8 µM against reference strains of Escherichia coli, Salmonella typhimurium, Klebsiella pneumoniae, and Acinetobacter baumannii, without cytoxicity against eukaryotic cell line MEC-1 up to 64 µM. One Bac7 derivative was effective against E. coli (MIC ≤ 4µM) also in presence of 10% human serum, and another peptide retained a MIC = 16 µM in presence of 2.7% NaCl. Analysis of natural cePrAMPs identified two peptides, Bal1 and Lip1, that showed excellent spectrum of activity, with MIC ≤ 2 µM and MIC ≤ 8 µM against reference strains of six Gram-negative and two Gram-positive species, respectively. The two cePrAMPs showed somewhat cell-dependent cytotoxicity but no hemolytic activity and kept their antibacterial activity also in 10%HS and 2.7% NaCl. Regarding the mechanism of action, cePrAMps may be divided into two subgroups. Four of them showed scarce translational-inhibiting effectiveness but significant membrane-perturbing activity while the other three peptides showed to be excellent translational inhibitors, similarly to Bac7(1-35). It is worth noting that some cePrAMP fragments were also found to have a mechanism consisting in both inhibition of protein synthesis and perturbation of E. coli membranes. Importantly, three cePrAMPs and the best optimized peptide share a sequence motif present in other PrAMPs, which was already proposed as consensus for translation inhibition. The best optimized peptides and CePrAMPs were used as starting points to design six chimeric PrAMPs. Preliminary results , however, suggest that no futher improvements have been obtained. Results of this work let to gain hints for the design of novel peptide antibiotics displaying a wider spectrum of activity and retaining low toxicity but acquiring a dual mode of action not restricted to protein synthesis inhibition.