Peptide-Lipid Interaction (Margitta Dathe)


Small cyclic antimicrobial peptides

Small antimicrobial peptides (AMPs) have a promising potential as lead structures for the development of new antibiotic compounds: there is hardly any resistance mechanisms among microorganisms, they are cost-effective in production and can be designed to be proteolytically stable which impedes their degradation during medical application. Most of these peptides interact with lipid components of biological membranes and thereby exert their antimicrobial activity. Among the structurally diverse AMPs, small sequences rich in arginine (R) and tryptophan (W) are of particular interest.

Our work has been focused on a small cyclic peptide which is highly active against Gram positive and Gram negative bacteria while no toxicity towards eukaryotic cells was detected. Optimizing the activity and bacterial selectivity of antimicrobial peptides (AMPs) requires an understanding of their mechanism of action. With its amphipathic structure and high content of arginine residues, the synthetic cyclic hexapeptide cWFW (cyclo(RRRWFW)) is highly membrane-active. However, in contrast to most antimicrobial peptides, cWFW neither permeabilizes the membrane nor translocates into the cytoplasm of bacteria [1]. In cooperation with the Centre for Bacterial Cell Biology, Newcastle University, UK, we showed that cWFW instead triggers a rapid reduction of membrane fluidity. This effect is accompanied by the formation of distinct membrane domains which differ in local membrane fluidity and which severely disturb protein organization by segregating peripheral and integral membrane proteins into domains of different rigidity [2]. We consider these major membrane disturbances as key events that cause specific inhibition of bacterial cell wall synthesis. Additionally, the peptide was found to be non-toxic against eukaryotic cells (HELA) and to translocate into their cytoplasm using an endocytotic uptake route.
The novel antibacterial mode of action carries a low risk of inducing bacterial resistance and with its membrane-translocating ability the peptide provides a valuable basis for the design of new synthetic antimicrobial compounds for the treatment of intracellular pathogens.
Ongoing research activities at the Stellenbosch University (M. Rautenbach, SA) are focused upon optimizing the high antifungal activity of our small cyclic compounds.

cWFW-triggered formation of lipid domains and segregation of membrane proteins
(a) Phase contrast, GFP-protein fluorescence, Nile red staining of lipid domains and fluorescent color overlays for cells expressing different integral membrane proteins (upper panels) and different peripheral membrane proteins (lower panels) in the presence of cWFW (20 min incubation with 12 μM). Whereas there is uniform membrane fluorescence of GFP-proteins and Nile red in non-treated cells (not shown), the formation of domains of different fluidity is observed after incubation with cWFW. Integral proteins accumulate in Nile red-free regions whereas peripheral proteins co-localize with Nile red, the regions in which cWFW also accumulates. Strains used: (a) B. 816 subtilis BS23 (AtpA-GFP), B. subtilis HS41 (YhaP-GFP), B. subtilis HS64 (WALP23-GFP), 817 (b) B. subtilis KR318 (SpoVM-GFP), B. subtilis HS65 (GFP-MinDMTS) and B. subtilis HS208 818 (SepFMTS-GFP)

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  1. [1] Scheinpflug, K. et al. (2013) Pharmaceuticals 6, 1130-1144.
         Scheinpflug, K. et al. (2015) PlosONE 10(4) e0125056.
  2. [2] Scheinpflug, K. et al. (2017) Scientific Reports (7) 44332.

Peptide-modified micellar and liposomal nanocarriers

Lipid-based particles equipped with cell-targeting and uptake-mediating peptides have attracted much attention as means of overcoming cellular barriers. We introduced a dipalmitoylated sequence (LRKLRKRLLR)2, named P2A2, as an uptake-mediating compound. The peptide exhibits characteristics of cell-penetrating peptides (CPPs), comprises specific binding sites for the low density lipoprotein receptor (LDLr) and binds non-specifically to cell-surface heparan sulfate proteoglycans (HSPGs). The lipopeptide self-assembles into micelles and rapidly incorporates into liposomes. These properties provided the potential for the activation of different cellular uptake modes and for the generation of different carrier systems [1].

In cooperation with the University Hospital Münster (H. Traupe) we developed a peptide-tagged liposomal preparation with encapsulated recombinant human transglutaminase 1 (rhTG1) as a basis for a causative therapy of (TG1)-deficient ichthyosis (ARCI), a rare and severe skin disease [2]. In a skin-humanized mouse model the treatment with rhTG1-liposomes results in considerable improvement of the ichthyosis phenotype and in normalization of the regenerated ARCI skin.  In 2013 “Orphan Drug” designation was granted by the European Commission for “rhTG1 encapsulated into liposomes for the treatment of TG1-deficient autosomal recessive congenital ichthyosis“.  In 2015, the therapeutic modality was honored with the Leibniz Drug of the Year Prize.  
Ongoing activities with industrial partners to advance the preclinical development of the preparation underline the huge therapeutic and economic potential of the liposomal TG1 formulation.
Further studies using liposomes decorated with an oligo-lysine lipopeptide showed that they are highly suitable for transporting  Corneodesmosin to its site of action, the membrane of skin keratinocytes, and provide a promising basis for the development of a pathogenesis-based therapy for Peeping Skin Disease (in cooperation with University Hospital Münster, V. Oji).

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Chemical exchange saturation transfer with hyperpolarized xenon nuclei (Hyper-CEST) allows sensitive detection of supramolecular cages such as cryptophane-A (CrA) in non-invasive Magnetic Resonance Imaging (MRI). We generated a lipopeptide, PCrAA2, with a covalently attached CrA. Studies in cooperation with L. Schröder (FMP) confirmed cell selectivity of PCrAA2 micelles and allowed us to distinguish blood brain barrier (BBB) endothelial cells from control aortic endothelial cells based on high local cage concentration and reliable quantification of the signal molecule.
Also poly-arginine lipopeptides proved to be very promising targeting and uptake-mediating candidates addressing BBB cells independent of particle size and peptide density on the surface of the carrier system [3].  Liposomes loaded with CrA circumvent chemical modification and biocompatibility issues of the signal molecule and Xe Hyper-CEST MRI studies demonstrated efficient delivery of CrA into BBB cells [4].
Both, the PCrAA2 micelles  and P2Rn-tagged, CrA-loaded liposomes combine a high selectivity for human brain capillary endothelial cells with the great sensitivity of Xe Hyper-CEST MRI and might be a potential tool in MRI-based brain diagnostics.

Graphical abstract of peptide-modified carriers: Micelles and liposomes varying in size and lipopeptide surface density and lipopeptide based micelles. R-rich lipopeptides promote pronounced uptake into brain capillary endothelial cells independent of the size and peptide loading of the carrier system. The pixel histogram of the Hyper-CEST effect of human aortic endothelial cells ( HAoEC) and human brain capillary endothelial cells (HBMEC) incubated with PCrAA2 micelles confirms the selectivity.

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  1. [1] Leupold  E. et al. (2009) Biochim. Biophys. Acta 1788, 442-449.
  2. [2] Aufenvenne K et al. (2013) Am J Hum Genetics 93, 620-630.
  3. [3] Sydow, K. et al. (2016) Euro J Pharm Biopharm  109, 130-139.
  4. [4] Schnurr M. et al. (2015) Adv Healthcare Mat  4, 40-45.

Leibniz-Forschungsinstitut für Molekulare Pharmakologie im Forschungsverbund Berlin e.V. (FMP)
Campus Berlin-Buch
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