Designing New Chimeric Proline-Rich Antimicrobial Peptides to Enhance Efficacy Toward the ESKAPE+E: Beyond Sequence Extension

Proline-rich antimicrobial peptides (PrAMPs) primarily exert their antimicrobial effects intracellularly, inhibiting protein synthesis. B7-005, a synthetic 16-amino acid PrAMP, has a broader antimicrobial spectrum compared to native counterparts, despite shorter PrAMPs typically exhibiting reduced activity. This study aimed to enhance B7-005’s potency by extending it with 6 or 11 amino acids derived from the C-terminal sequences of cetacean Tur1A and Lip1 PrAMPs, as well as bovine Bac7(1-35). Six chimeric derivatives were evaluated for antimicrobial and bactericidal potency, cytotoxicity, bacterial membrane permeabilization, and in vitro inhibition of protein synthesis. Extending B7-005 with sequences from other PrAMPs increased its activity against most ESKAPE+E pathogens, reducing minimum inhibitory concentration (MIC) values by 2- to 8-fold, with notable differences among bacterial species, without increasing cytotoxicity toward the A549 cell line. All chimeras retained the ability to inhibit protein synthesis in Escherichia coli and to modestly perturb the E. coli membranes like B7-005. These novel chimeric PrAMPs, particularly the 22-mer derivatives, hold promise for developing new antimicrobial agents. The study also highlights variability in bacterial responses to PrAMPs and underscores how minor sequence differences can significantly impact efficacy against specific microorganisms. PrAMPs thus represent a valuable scaffold to rationally design derivatives targeting high-priority pathogens.