Enhanced Membrane Pore Formation through High-Affinity Targeted Antimicrobial Peptides

Many cationic antimicrobial peptides (AMPs) target the unique lipid composition of the prokaryotic cell membrane. However, the micromolar activities common for these peptides are considered weak in comparison to nisin, which follows a targeted, pore-forming mode of action. Here we show that AMPs can be modified with a high-affinity targeting module, which enables membrane permeabilization at low concentration. Magainin 2 and a truncated peptide analog were conjugated to vancomycin using click chemistry, and could be directed towards specific membrane embedded receptors both in model membrane systems and whole cells. Compared with untargeted vesicles, a gain in permeabilization efficacy of two orders of magnitude was reached with large unilamellar vesicles that included lipid II, the target of vancomycin. The truncated vancomycin-peptide conjugate showed an increased activity against vancomycin resistant Enterococci, whereas the full-length conjugate was more active against a targeted eukaryotic cell model: lipid II containing erythrocytes. This study highlights that AMPs can be made more selective and more potent against biological membranes that contain structures that can be targeted

Minimum inhibitory concentration (MIC, µg/mL) of vancomycin-magainin peptide derivatives.

a<p>compounds tested in conjunction with compounds reported in ref. <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0039768#pone.0039768-Arnusch2" target="_blank">[24]</a>^bV: vancomycin, values first published in ref. <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0039768#pone.0039768-Arnusch2" target="_blank">[24]</a>, ^cMRSA: methicillin resistant <i>S. aureus, ^d</i>VSE: vancomycin susceptible <i>Enterococci, ^eVRE:</i> vancomycin resistant <i>Enterococci.</i></p

Amount of hemolysis from erythrocytes with and without lipid II, after treatment with nisin (10 µM) compound 1 (10 µM), and compound 2 (50 µM).

<p>Amount of hemolysis from erythrocytes with and without lipid II, after treatment with nisin (10 µM) compound 1 (10 µM), and compound 2 (50 µM).</p

Synthesis of the vancomycin-targeted AMPs.

<p>Synthesis of the vancomycin-targeted AMPs.</p

Enhanced membrane pore formation by multimeric/oligomeric antimicrobial peptides

The pore-forming antibacterial peptide magainin 2 was made divalent, tetravalent, and octavalent via a copper(I)-mediated 1-3 dipolar cycloaddition reaction ("click" chemistry). This series of pore-forming compounds was tested in vitro for their ability to form pores in large unilamillar vesicles (LUVs). A large increase in the pore-forming capability was especially observed with the tetravalent and octavalent magainin compounds in the LUVs consisting of DOPC, and the octavalent magainin compound showed a marked increase with the DOPC/DOPG LUVs. Activity was observed in the low nanomolar range for these compounds. © 2007 American Chemical Society

Identification of peptide ligands for malignancy- and growth-regulating galectins using random phage-display and designed combinatorial peptide libraries

Peptide ligands were identified for the medicinally important galectins by screening on-bead with a synthetic solid-phase peptide library and by screening with a phage-display library. The first method yielded sequences binding at or near the carbohydrate-binding site while the latter yielded a sequence binding at a different site. Members of the galectin family of endogenous lectins are involved in tumor growth regulation and in establishing characteristics of the malignant phenotype via protein-carbohydrate and protein-protein interactions. To identify peptide ligands with the potential to modulate these tumor-relevant interactions beneficially, complementary screening methods were employed, that is, both phage-display and a combinatorial pentapeptide library with the key YXY tripeptide core. Three representative prototype galectins were selected. The search for high-affinity ligands among phage-displayed random heptamers yielded enrichment after five selection cycles of the nonglycomimetic CQSPSARSC peptide in the case of the chicken homologue of galectin-1 but not the human protein, an indication for specificity. The most active glycomimetic from the combinatorial library of 5832 pentamers was WYKYW. Identification of peptide ligands for galectins with and without glycomimetic properties is thus possible. Our study documents the potential to combine the two library-based approaches for structural optimization of lead peptides. © 2004 Elsevier Ltd. All rights reserved