Our research is aimed at creating knowledge for the development of novel drugs based on host defense peptides, which act on cell membranes. Such cationic amphipathic peptides will target negatively charged lipids exposed on the outer leaflet of both bacterial and cancer cell membranes. Emphasis is on our concept that the inherent properties of lipids strongly influence the mode of action of membrane-active peptides, which needs to be considered in order to address lipid/peptide interactions adequately.

Antimicrobial antiseptic peptides

A series of about 150 peptides derived from the human protein lactoferrin was studied within the European RTD-project ANEPID ("Antimicrobial and endotoxin neutralizing peptides to combat infectious diseases", coordinated by K. Lohner) in order to design highly antimicrobial and antiseptic compounds, which are not antigenic or toxic to humans. Thereby, a stretch of 11 amino acid residues (LF11, FQWQRNIRKVR-NH2) corresponding to residues 21-31 of lactoferrin was taken as a parent peptide.

The arrangement of the amino acids of LF11 bound to negatively charged lipids emphasizes the importance of both electrostatic and hydrophobic interactions in a defined geometric arrangement. Specifically, the burial of the N-terminal hydrophobic amino acids represents a considerable free energy contribution in addition to electrostatic interactions. Thus, peptides were acylated at the N-terminus, which resulted in improved antimicrobial and especially endotoxin neutralizing activity. This strategy yields a number of highly potent peptide candidates for a novel drug that combines both antimicrobial and antiseptic activity (patent application, A1165/2006).

Furthermore, studies on the human multifunctional peptide LL-37 showed that this peptide exhibits distinctly different mechanism of membrane perturbation, strongly dependent not only on lipid headgroup charge but also on hydrocarbon chain length. In negatively charged DPPG1 model membranes LL-37 induces a peptide associated quasi-interdigitated phase, where the hydrocarbon chains are shielded from water by the peptide. In turn, LL-37 leads to a disintegration of the bilayer organization of zwitterionic DPPC into disk-like micelles (Fig. 18).

Interestingly, interdigitation was also observed for the longer-chain C18 and C20 PCs. The dual behavior of LL-37 can be attributed to a balance between electrostatic interactions reflected in different penetration depths of the peptide and hydrocarbon chain length. A phase diagram for PC and PG showing the different macroscopic lipid phases as a function of hydrocarbon chain length and LL-37 concentration was constructed. Very similar effects are also observed for melittin and the antimicrobial frog skin peptide PGLa, which suggests that this phase diagram is generally applicable to peptides, which are localized parallel to the membrane surface. This particularly emphasizes the role of the inherent properties of the lipids for peptide membrane interaction, which has been largely neglected in this research field.

Lipidomics based design of antibiotics against resistant bacteria

The main advantage of antimicrobial peptides, when considering bacterial resistance, is that they rapidly, within minutes, destroy bacteria and that due to the nature of their target, i.e. cell membrane rather than a specific receptor, bacterial resistance is less likely to occur. Nevertheless, a few instances of resistance to such cationic antimicrobial peptides have been reported, mostly due to modifications in the lipid headgroup. One example is the ?opportunistic? bacterium S. aureus, where a possible resistance mechanism could be based on an increased rate of esterification of PG with L-lysin, which confers a positive net charge to the otherwise negatively charged PG. Consequently this would result in a reduced affinity of cationic antimicrobial peptides. Thus, the aim of this project is to modify antimicrobial peptides accordingly to find more effective compounds. Further the lipid composition of various S. aureus strains will be investigated.

As a first step, extraction techniques of lipids from S. aureus were established using the strain ATCC 8325. Separation of the phospholipids was achieved on HPTLC-plates and identification using phosholipid standards and HPLC-MS. Latter has been performed in cooperation with F. Sinner (Joanneum Research). The lipids found in the extract were lysyl-PG, PG and traces of PE. Although the fatty acid composition is not completely analyzed yet in terms of acyl pairs, the lipids exhibit a high content of saturated, but no long hydrocarbon chains. Although the limited stability of lysyl-PG (most stable under acidic conditions) bears problems, microcalorimetry revealed that at physiological conditions lysyl-PG resembles the features of the parent PG lipid. However, in contrast to PG lowering the pH did not significantly affect the phase behavior of lysyl-PG, which can be explained by the differences in protonization due to the lysine group.

Antitumor peptides targeting PS

The aim of this project is to design novel antitumor peptides that selectively target PS exposed on the outside of cancer cells thereby lysing these cells without damaging healthy cells. Based on the knowledge gained within the ANEPID project and the fact that these cationic peptides specifically interact with negatively charged lipids, which are the target molecules in both bacterial and cancer cell membranes, an analogous strategy will be applied to enhance the interaction with PS.An initial screening on the interaction of PS (mimicking cancer cells) and PC (mimicking mammalian cells) liposomes with human lactoferricin derivatives from ANEPID, which were selected according to specific positions of the charged amino acid residues, revealed a number of peptides that discriminate between these two lipid model membranes. The most active peptides of these preliminary experiments will be used as templates for further studies.

Analysis of PS exposure on cancer cells (Fig. 19) will initially focus on cell lines that facilitate peptide application, e.g. melanoma for topical and kidney for liposomal application.

Liposomal Peptide Delivery Vehicles
Incorporation of membranolytic peptides into stable liposomal formulations was successfully shown during this reporting period (FWF-project P15657). Such delivery vehicles of defined lamellarity and size were spontaneously formed by a novel technique based on a ternary lipid mixture characterized by lipids of different molecular shape and by a defined charge density.

Selected references:
K. Lohner and S.E. Blondelle. Molecular mechanisms of membrane perturbation by antimicrobial peptides and the use of biophysical studies in the design of novel peptide antibiotics. Comb. Chem. High Throughput Screen., 8 (2005) 239-255.

B. Pozo Navas, K. Lohner, G. Deutsch, E. Sevcsik, K. A. Riske, R. Dimova, P. Garidel and G. Pabst. Composition dependence of vesicle morphology and mixing properties in a bacterial model membrane system. Biochim. Biophys. Acta, 1716, 1 (2005) 40-48.

O. Konovalov, S.M. O?Flaherty, E. Saint-Martin, G. Deutsch, E. Sevcsik and K. Lohner. The bending rigidity of phospholipid monolayers in presence of an antimicrobial frog peptide studied by X-ray grazing incidence diffraction. Physica B, 357, 1-2 (2005)185-189. 

W. Jing, E.J. Prenner, H.J. Vogel, A. Waring, R.I. Lehrer and K. Lohner. Headgroup structure and fatty acid chain length of the acidic phospholipids modulate the interaction of membrane mimetic vesicles with the antimicrobial peptide protegrin-1. J. Pept. Sci., 11 (2005) 735-743

J. Andrä, K. Lohner, S.E. Blondelle, R. Jerala, I. Moriyon, M.H.J. Koch, P. Garidel and K. Brandenburg. Enhancement of endotoxin neutralization by coupling of a C12-alkyl chain to a lactoferricin-derived peptide. Biochem. J. 385 (2005) 135-143.

D. Zweytick, G. Pabst, P.M. Abuja, A. Jilek, S.E. Blondelle, J. Andrä, R. Jerala, D. Monreal, G. Martinez de Tejada and K. Lohner. Influence of N-acylation of a peptide derived from human lactoferricin on membrane selectivity. Biochim. Biophys. Acta, 1758 (2006) 1426-1435.

B. Bechinger and K. Lohner. Detergent-like actions of linear amphipathic antimicrobial peptides. Biochim. Biophys. Acta, 1758 (2006) 1529-1539.