Towards Profiles of Resistance Development and Toxicity for the Small Cationic Hexapeptide RWRWRW-NH2 - PubMed (original) (raw)

Towards Profiles of Resistance Development and Toxicity for the Small Cationic Hexapeptide RWRWRW-NH2

Michaela Wenzel et al. Front Cell Dev Biol. 2016.

Abstract

RWRWRW-NH2 (MP196) is an amphipathic hexapeptide that targets the bacterial cytoplasmic membrane and inhibits cellular respiration and cell wall synthesis. In previous studies it showed promising activity against Gram-positive bacteria and no significant cytotoxicity or hemolysis. MP196 is therefore used as lead structure for developing more potent antibiotic derivatives. Here we present a more comprehensive study on the parent peptide MP196 with regard to clinically relevant parameters. We found that MP196 acts rapidly bactericidal killing 97% of initial CFU within 10 min at two times MIC. We were unable to detect resistance in standard 24 and 48 h resistance frequency assays. However, MP196 was effective against some but not all MRSA and VISA strains. Serum binding of MP196 was intermediate and we confirmed its low toxicity against mammalian cell lines. MP196 did neither induce NFκB activation nor cause an increase in IL8 levels at 250 μg/mL, and no IgE-dependent activation of basophil granulocytes was detected at 500 μg/mL. Yet, MP196 demonstrated acute toxicity in mice upon injection into the blood stream. Phase contrast microscopy of mouse blood treated with MP196 revealed a shrinking of erythrocytes at 250 μg/mL and severe morphological changes and lysis of erythrocytes at 500 μg/mL. These data suggest that MP196 derivatization directed at further lowering hemolysis could be instrumental in overcoming acute toxicity. The assessment of hemolysis is a critical step in the evaluation of the clinical potential of promising antimicrobial peptides and should be accompanied by microscopy-based morphological analysis of blood cells.

Keywords: acute; antimicrobial cationic peptides; antimicrobial peptide; antimicrobial peptide resistance; toxicity mechanisms; toxicity tests.

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Figures

Figure 1

Figure 1

Activation of Nf-κB in RT4 bladder cells at concentrations up to 25 μg/mL (A) and at 250 and 500 μg/mL (B).

Figure 2

Figure 2

IL8 levels in RT4 bladder cells challenged with MP196 and in DMSO and flagellin-treated controls.

Figure 3

Figure 3

IgE-dependent basophil activation measured by FACS. Anti-IgE (PE) reacts with human IgE and is used for the characterization of basophilic granulocytes. Anti-gp53 (FITC) recognizes a glycoprotein expressed on activated basophils. The chemotactic peptide N-formyl-Met-Leu-Phe served as positive, DMSO as negative control.

Figure 4

Figure 4

Phase contrast images of whole mouse blood mixed with MP196. Five percent DMSO is the maximum DMSO concentration reached by diluting MP196 from the stock solution. Scale bar represents 20 μm.

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