Peptide inhibitors of bacterial protein synthesis with broad spectrum and 2SbmA-independent bactericidal activity against clinical pathogens.

Proline-rich antimicrobial peptides (PrAMPs) are promising agents to combat multi-drug resistant pathogens due to a high antimicrobial activity, yet low cytotoxicity. A library of derivatives of the PrAMP Bac5(1-17) was synthesized and screened to identify which residues are relevant for its activity. In this way, we discovered that two central motifs -PIRXP-cannot be modified, while residues at N-and C-termini tolerated some variations. We found five Bac5(1-17) derivatives bearing 1-5 substitutions, with an increased number of arginine and/or tryptophan residues, exhibiting improved antimicrobial activity and broader spectrum of activity while retaining low cytotoxicity toward eukaryotic cells. Transcription/translation and bacterial membrane permeabilization assays showed that these new derivatives still retained the ability to strongly inhibit bacterial protein synthesis, but also acquired permeabilizing activity to different degrees. These new Bac5(1-17) derivatives therefore show a dual mode of action which could hinder the selection of bacterial resistance against these molecules.

The spread of antibiotic‐resistant pathogens has boosted the search for new antimicrobial drugs. Proline‐rich antimicrobial peptides are promising lead compounds for the development of next‐generation antibiotics, given their very low cytotoxicity and their good antimicrobial activity targeting the bacterial ribosome. Bac5(1–25) is an N‐terminal fragment of the bovine proline‐rich antimicrobial peptide Bac5, whose mode of action has been recently described. In this work we tested a number of Bac5(1–25) fragments, and we characterized their antimicrobial activity against Escherichia coli, Acinetobacter baumannii, Klebsiella pneumoniae, Staphylococcus aureus, Salmonella enterica, and Pseudomonas aeruginosa. We evaluated their cytotoxicity toward human cells and their efficacy in inhibiting bacterial protein synthesis. This allowed us to identify some shorter fragments of Bac5(1–25) with a good balance between antibacterial efficacy, protein synthesis inhibition, and ease/cost‐effectiveness of synthesis, suitable as lead compounds to develop new antibacterials.

Proline-rich antimicrobial peptides (PrAMPs) may be a valuable weapon against multi-drug resistant pathogens, combining potent antimicrobial activity with low cytotoxicity. We have identified novel PrAMPs from five cetacean species (cePrAMPs), and characterized their potency, mechanism of action and in vitro cytotoxicity. Despite the homology between the N-terminal of cePrAMPs and the bovine PrAMP Bac7, some differences emerged in their sequence, activity spectrum and mode of action. CePrAMPs with the highest similarity with the Bac7(1-35) fragment inhibited bacterial protein synthesis without membrane permeabilization, while a second subgroup of cePrAMPs was more membrane-active but less efficient at inhibiting bacterial translation. Such differences may be ascribable to differences in presence and positioning of Trp residues and of a conserved motif seemingly required for translation inhibition. Unlike Bac7(1-35), which requires the peptide transporter SbmA for its uptake, the activity of cePrAMPs was mostly independent of SbmA, regardless of their mechanism of action. Two peptides displayed a promisingly broad spectrum of activity, with minimal inhibiting concentration MIC ≤ 4 µM against several bacteria of the ESKAPE group, including Pseudomonas aeruginosa and Enterococcus faecium. Our approach has led us to discover several new peptides; correlating their sequences and mechanism of action will provide useful insights for designing optimized future peptide-based antibiotics.

The Front Cover highlights the discovery of derivatives of the proline‐rich antimicrobial peptide Bac5 that have acquired a dual mode of action to permeabilize the cell membrane of bacteria and inhibit their ribosomes. Many of the derivatives display improved antimicrobial activity as well as a broader spectrum of action to target not only pathogenic Escherichia coli strains but also Acinetobacter baumannii and to a lesser extent Pseudomonas aeruginosa. Importantly, the improvements in antimicrobial potency and selectivity are not followed by a corresponding increase in toxicity towards eukaryotic cells. More information can be found in the Full Paper by Mario Mardirossian, Daniel N. Wilson, Marco Scocchi et al. on page 2025 in Issue 24, 2019 (DOI: 10.1002/cmdc.201900465).