Rational Framework for the Design of Trp- and Arg-Rich Peptide Antibiotics Against Multidrug-Resistant Bacteria

Abstract

The threat of antibiotic resistance warrants the discovery of agents with novel antimicrobial mechanisms. Antimicrobial peptides (AMPs) directly disrupting bacterial membranes may overcome resistance to traditional antibiotics. AMP development for clinical use has been mostly limited to topical application to date. We developed a rational framework for systematically addressing this challenge using libraries composed of 86 novel Trp- and Arg-rich engineered peptides tested against clinical strains of the most common multidrug-resistant bacteria known as ESKAPE pathogens. Structure-function correlations revealed minimum lengths (as low as 16 residues) and Trp positioning for maximum antibacterial potency with mean minimum inhibitory concentration (MIC) of 2–4 μM and corresponding negligible toxicity to mammalian cells. Twelve peptides were selected based on broad-spectrum activity against both gram-negative and -positive bacteria and <25% toxicity to mammalian cells at maximum test concentrations. Most of the selected PAX remained active against the colistin-resistant clinical strains. Of the selected peptides, the shortest (the 16-residue E35) was further investigated for antibacterial mechanism and proof-of-concept in vivo efficacy. E35 killed an extensively-resistant isolate of Pseudomonas aeruginosa (PA239 from the CDC, also resistant to colistin) by irreversibly disrupting the cell membranes as shown by propidium iodide incorporation, using flow cytometry and live cell imaging. As proof of concept, in vivo toxicity studies showed that mice tolerated a systemic dose of up to 30 mg/kg peptide and were protected with a single 5 mg/kg intravenous (IV) dose against an otherwise lethal intraperitoneal injection of PA239. Efficacy was also demonstrated in an immune-compromised Klebsiella pneumoniae infection model using a daily dose of 4mg/kg E35 systemically for 2 days. This framework defines the determinants of efficacy of helical AMPs composed of only cationic and hydrophobic amino acids and provides a path for a potential departure from the restriction to topical use of AMPs toward systemic application.

Novel Helical Trp‐ and Arg‐Rich Antimicrobial Peptides Locate Near Membrane Surfaces and Rigidify Lipid Model Membranes

“…Notwithstanding, the % RBC lysis and WBC toxicity are <25% in all cases, which is an important criterion for AMP selection. [45] In an attempt to understand which physical properties are most important for both bacterial killing efficiency and toxicity to eukaryotic membranes, we plotted μH or H versus MIC or versus % toxicity (Figure S1, Supporting Information), and μH/H versus MIC or versus % toxicity (Figure S2, Supporting Information). See the Supporting Information for these results and discussion.…”

“…Determination of Toxicity to Mammalian Cells: Toxicity to primary cells was examined using human RBCs and peripheral mononuclear cells (PBMCs or WBCs) as previously described. [45,81] Briefly, RBCs and WBCs were separated by histopaque differential centrifugation using blood anonymously obtained from the Central Blood Bank (Pittsburgh, PA). For the RBC lysis assay, the isolated RBCs were resuspended in PBS at a concentration of 5%.…”

“…We obtain the secondary structure of these six AMPs in G(-) (Gram-negative), G(þ) (Gram-positive) bacterial, and Euk33 (eukaryotic with 33 mol% cholesterol) cell lipid model membranes (LMMs), and the peptide/lipid interactions of a subset (E2-05, E2-35, and E2-35K). The peptides were rationally designed, which varies the length, hydrophobicity (H), hydrophobic moment (μH), helicity, overall net charge, and position of W. [45,46] Circular dichroism (CD) measurements were used to study AMP secondary structures and explore correlations with activity. Antibacterial activity and cytotoxicity were determined by in vitro microbiological assays.…”

Recent advances in therapeutic targets identification and development of treatment strategies towards Pseudomonas aeruginosa infections

“…Peptides are becoming increasingly important for many therapeutic areas [106], due to their capacity to permeate tissues and membranes and the low rate of resistance emergence towards them. The de novoengineered cationic peptide antibiotic E35, for example, irreversibly damages cell membranes and kills the extensively-resistant isolate PA239 [107].…”

Novel non-helical antimicrobial peptides insert into and fuse lipid model membranes

“…LE-53 and LE-55 were initially screened for antibacterial potency against an MDR panel of Gram negative (G(À)) and Gram positive (G(+)) bacterial isolates from UPMC. The MIC is measured by a horizontal growth curve taken every hour; 61 these MIC values are listed in Table 2. The MICs represent the average of different strains of each type of bacteria.…”