Design of stapled antimicrobial peptides that are stable, nontoxic and kill antibiotic-resistant bacteria in mice

Abstract

The clinical translation of cationic alpha-helical antimicrobial peptides (AMPs) has been hindered by structural instability, proteolytic degradation, and in vivo toxicity from non-specific membrane lysis. Although analyses of hydrophobic content and charge distribution have informed the design of synthetic AMPs with increased potency and reduced in vitro hemolysis, non-specific membrane toxicity in vivo continues to impede AMP drug development. Here, we analyzed a 58-member library of stapled AMPs (StAMPs) based on Magainin-II, and applied the insights from structure-function-toxicity measurements to devise an algorithm for the design of stable, protease-resistant, potent, and nontoxic StAMP prototypes. We show that a lead double-stapled StAMP named Mag(i+4)1,15(A9K,B21A,N22K,S23K) can kill multidrug resistant Gram-negative pathogens, such as colistin-resistant Acinetobacter baumannii in a mouse peritonitis-sepsis model, without observed hemolysis or renal injury in murine toxicity studies. Inputting the amino acid sequences alone, we further generated membrane-selective StAMPs of pleurocidin, CAP18, and esculentin, highlighting the generalizability of our design platform.

Towards Robust Delivery of Antimicrobial Peptides to Combat Bacterial Resistance

“…Interestingly, they also noted that a single substitution of the peptide's l-lysine with d-lysine only slightly decreased the AMP's antimicrobial activity while both improving the peptide's stability and substantially decreasing its hemolytic activity [153]. It should be noted that d-peptide synthesis is quite costly [154], which hinders its straightforward translation into clinical practice.…”

A Novel Amphibian Antimicrobial Peptide, Phylloseptin-PV1, Exhibits Effective Anti-staphylococcal Activity Without Inducing Either Hepatic or Renal Toxicity in Mice

“…However, we admit that data for supporting such expectation is still preliminary, especially the considerable hemolytic activity of PPV1 could extensively compromise its potential clinical application. The further modification would be taken into consideration such as stapling the sequence to maintain the secondary structure to improve the cell selectivity (Mourtada et al, 2019), and encapsulation of AMPs within liposomes or nanoparticles (Ron-Doitch et al, 2016). Additionally, these data suggest that PPV1 exhibits an encouraging antimicrobial potential when applied by localized administration in vivo.…”

“…Mice were treated with peptide at 5 µg/g and vancomycin at 50 µg/g by intraperitoneal injection, respectively, at 2 h post-infection (10 mice for each group). Mice were monitored for 48 h and euthanized promptly if they became moribund (Mourtada et al, 2019). The livers and kidneys were then harvested and embedded in paraffin, sectioned, mounted on glass slides and stained with hematoxylin and eosin (H&E).…”

“…Another 10 mice treated with PBS only were employed as negative control. On day 9, blood was withdrawn for evaluation of RBC parameters and serum chemistries, and the animals were euthanized for necropsy and histologic analyses (Mourtada et al, 2019). Harvested tissues were embedded in paraffin, sectioned, mounted on glass slides and stained with H&E. All procedures involving animals were approved by the Animal Care and Use Committee of Nanjing University of Chinese Medicine and performed strictly according FIGURE 8 | In vivo antimicrobial activity of PPV1 in the treatment of infected mice.…”

Design, Engineering and Discovery of Novel α-Helical and β-Boomerang Antimicrobial Peptides against Drug Resistant Bacteria

“…Proteolytic degradation is likely to be the major cause of the short in vivo half-life of AMPs. Several strategies have been exploited for the generation of AMPs that are stable against proteases: e.g., cyclization of AMPs [56] and the introduction of disulfide bonds and covalent bonds between sidechains [57][58][59]. Alternative approaches to minimize proteolytic cleavage include producing synthetic AMPs that incorporate D-amino acids [60], β-amino acids [61], and other non-natural amino acids [62].…”