Investigations into the membrane activity of arenicin antimicrobial peptide AA139

• Published in: Biochimica et biophysica acta. General subjects Vol. 1866; no. 8; p. 130156
• Main Authors: Edwards, Ingrid A., Henriques, Sónia T., Blaskovich, Mark A.T., Elliott, Alysha G., Cooper, Matthew A.
• Format: Journal Article
• Language: English
• Published: Netherlands Elsevier B.V 01.08.2022
• Subjects: Antimicrobial peptide; Arenicin-3; Lipid bilayer; Membrane permeabilization; Model membranes; Peptide-membrane interactions; Membrane permeabilization; Model membranes; RBC; AMP; MIC; Lipid bilayer; HEK-293; Arenicin-3; Peptide-membrane interactions; ATCC; Antimicrobial peptide
• ISSN: 0304-4165; 1872-8006
• DOI: 10.1016/j.bbagen.2022.130156

Arenicin-3 is an amphipathic β-hairpin antimicrobial peptide that is produced by the lugworm Arenicola marina. In this study, we have investigated the mechanism of action of arenicin-3 and an optimized synthetic analogue, AA139, by studying their effects on lipid bilayer model membranes and Escherichia coli bacterial cells. The results show that simple amino acid changes can lead to subtle variations in their interaction with membranes and therefore alter their pre-clinical potency, selectivity and toxicity. While the mechanism of action of arenicin-3 is primarily dependent on universal membrane permeabilization, our data suggest that the analogue AA139 relies on more specific binding and insertion properties to elicit its improved antibacterial activity and lower toxicity, as exemplified by greater selectivity between lipid composition when inserting into model membranes i.e. the N-terminus of AA139 seems to insert deeper into lipid bilayers than arenicin-3 does, with a clear distinction between zwitterionic and negatively charged lipid bilayer vesicles, and AA139 demonstrates a cytoplasmic permeabilization dose response profile that is consistent with its greater antibacterial potency against E. coli cells compared to arenicin-3. [Display omitted] •Arenicin-3, a cationic β-hairpin disulfide-constrained antimicrobial peptide (AMP) isolated from the sandworm Arenicola marina, is highly attractive due to its broad and potent spectrum of antimicrobial activity. A structure-activity-relationship campaign of arenicin-3 gave rise to the more potent and less toxic analogue AA139. We recently published the in vitro and in vivo efficacy of lead drug candidate AA139 in Nature Communications, Elliott et al 2020 11 (1), 3184. In Elliott et al., we showed AA139 to be highly effective in infection models of peritonitis, urinary tract and pneumonia, and like many other AMPs is membrane active. Simple amino acid changes can lead to subtle variations in the peptide interaction with membranes and therefore alter their pre-clinical potency, selectivity and toxicity. In this current study we show while the mechanism of action of arenicin-3 is primarily dependent on universal membrane permeabilization, our data suggest that the analogue AA139 relies on more specific binding and insertion properties to elicit its improved antibacterial activity and lower toxicity, as exemplified by greater selectivity between lipid composition when inserting into model membranes i.e. the N-terminus of AA139 seems to insert deeper into lipid bilayers than arenicin-3 does, with a clear distinction between zwitterionic and negatively charged lipid bilayer vesicles, and AA139 demonstrates a cytoplasmic permeabilization dose response profile that is consistent with its greater antibacterial potency against Escherichia coli cells compared to arenicin-3.•The use of lipid bilayer model membranes and bacterial cells, allowed us to propose a model for the mechanism of action of AA139 (1) binding to the outer membrane through electrostatic interactions, (2) insertion into the hydrophobic core of the outer membrane creating partial permeabilization, (3) access to, and permeabilization of the cytoplasmic membrane ultimately leading to cell death.•We now have a better understanding of the mechanism of action of arenicin peptides and how the mutation of only three amino acids has led to a significant increase in the therapeutic value of the peptide. Natural AMPs offer great potential for the fight against resistant bacteria. The redesign of AMPs can lead to improved pre-clinical candidates. The differences seen in the membrane-binding and -disrupting properties of arenicin-3 and AA139 may provide a pathway to better design and screen for optimized peptide antibiotics in the future.