Tryptophan-containing lipopeptide antibiotics derived from polymyxin B with activity against Gram positive and Gram negative bacteria

• New polymyxin analogs with low MICs for E. coli and S. aureus are reported.
• Key mutations include Arg, D(Trp) and NLeu residues, and a C10-C12 alkyl chain.
• Analogs perturb phosphatidilglycerol vesicles causing leakage and fusion.
• Perturbation is favored by phosphatidilethanolamine, abundant in Gram negatives.
• Flow cytometry in bacterial cells is consistent with biophysical experiments.

Resistance to all known antibiotics is a growing concern worldwide, and has renewed the interest in antimicrobial peptides, a structurally diverse class of amphipathic molecules that essentially act on the bacterial membrane. Propelled by the antimicrobial potential of this compound class, we have designed three new lipopeptides derived from polymyxin B, sp-34, sp-96 and sp-100, with potent antimicrobial activity against both Gram positive and Gram negative bacteria. The three peptides bind with high affinity to lipopolysaccharide as demonstrated by monolayer penetration and dansyl-displacement. The interaction with the cytoplasmic membrane has been elucidated by biophysical experiments with model membranes of POPG or POPE/POPG (6:4), mimicking the Gram positive and Gram negative bacterial membrane. Trp-based fluorescence experiments including steady-state, quenching, anisotropy and FRET, reveal selectivity for anionic phospholipids and deep insertion into the membrane. All three lipopeptides induce membrane fusion and leakage from anionic vesicles, a process that is favored by the presence of POPE. The molecules bind to zwitterionic POPC vesicles, a model of the eukaryotic membrane, but in a different way, with lower affinity, less penetration into the bilayer and no fusion or permeabilization of the membrane. Results in model membranes are consistent with flow cytometry experiments in Escherichia coli and Staphylococcus aureus using a membrane potential sensitive dye (bis-oxonol) and a nucleic acid dye (propidium iodide), suggesting that the mechanism of action is based on membrane binding and collapse of membrane integrity by depolarization and permeabilization.

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