Cathelicidin-derived antimicrobial peptides as novel drugs against antibiotic-resistant pathogens

Antibiotics are essential for treating bacterial infections, but new alternatives are needed due to antimicrobial resistance (AMR). Among these, antimicrobial peptides (AMPs)—small molecules of the innate immune system—have shown great potential due to their antibacterial activity and lower risk of inducing resistance. Cathelicidins are a prominent AMP family, including α-helical peptides, which primarily disrupt bacterial membranes, and Proline-rich Antimicrobial Peptides (PrAMPs), which inhibit protein synthesis. Despite their promise, clinical application of cathelicidins is limited by cytotoxicity, protease instability, or narrow activity spectrum. This PhD project aimed to overcome such limitations by optimizing peptide structure and function to improve safety, stability and antimicrobial efficacy, while also deeper exploring their mechanisms of action. The work is structured into three studies: two published and one submitted. The first study focused on PMAP-36, a porcine α-helical cathelicidin with broad-spectrum activity but hindered by high synthesis costs, protease sensitivity, and cytotoxicity. Shorter PMAP-36 derivatives were designed to identify a pharmacophore and improve these properties. The central region (residues 12–24) emerged as a key antimicrobial core, leading to variants with retained activity and enhanced selectivity or stability. The second study investigated the synthetic 16-residue PrAMP B7-005, active mainly against Enterobacteriaceae. By extending its sequence with C-terminal regions from other PrAMPs (Bac7, Tur1A, Lip1), six novel chimeric peptides were generated, showing improved potency and broader spectra and maintaining low cytotoxicity and efficient translation inhibition with minimal membrane disruption. The third study validated BONCAT (bioorthogonal noncanonical amino acid tagging) as a new fluorescence-based method to study translation inhibition by AMPs in living bacteria, addressing traditional assays’ limitations. Using PrAMPs such as Bac7(1–35), Bac7(1–16), and B7-005, BONCAT confirmed protein synthesis inhibition in both E. coli and K. pneumoniae, distinguishing it from secondary membrane effects. It also enabled real-time kinetic analysis, showing nearly complete translation arrest by Bac7-based PrAMPs within 10 minutes. In conclusion, this thesis contributed to the design of safer and more effective cathelicidin-derived AMPs and established BONCAT as a valuable tool for investigating their antibacterial mechanisms.