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Evaluation of strategies for improving proteolytic resistance of antimicrobial peptides by using variants of EFK17, an internal segment of LL-37
Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Pharmacy, Department of Pharmacy.ORCID iD: 0000-0001-9070-6944
Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Pharmacy, Department of Pharmacy.
2009 (English)In: Antimicrobial Agents and Chemotherapy, ISSN 0066-4804, E-ISSN 1098-6596, Vol. 53, no 2, 593-602 p.Article in journal (Refereed) Published
Abstract [en]

Methods for increasing the proteolytic stability of EFK17 (EFKRIVQRIKDFLRNLV), a new peptide sequence with antimicrobial properties derived from LL-37, were evaluated. EFK17 was modified by four d-enantiomer or tryptophan (W) substitutions at known protease cleavage sites as well as by terminal amidation and acetylation. The peptide variants were studied in terms of proteolytic resistance, antibacterial potency, and cytotoxicity but also in terms their adsorption at model lipid membranes, liposomal leakage generation, and secondary-structure behavior. The W substitutions resulted in a marked reduction in the proteolytic degradation caused by human neutrophil elastase, Staphylococcus aureus aureolysin, and V8 protease but not in the degradation caused by Pseudomonas aeruginosa elastase. For the former two endoproteases, amidation and acetylation of the terminals also reduced proteolytic degradation but only when used in combination with W substitutions. The d-enantiomer substitutions rendered the peptides indigestible by all four proteases; however, those peptides displayed little antimicrobial potency. The W- and end-modified peptides, on the other hand, showed an increased bactericidal potency compared to that of the native peptide sequence, coupled with a moderate cytotoxicity that was largely absent in serum. The bactericidal, cytotoxic, and liposome lytic properties correlated with each other as well as with the amount of peptide adsorbed at the lipid membrane and the extent of helix formation associated with the adsorption. The lytic properties of the W-substituted peptides were less impaired by increased ionic strength, presumably by a combination of W-mediated stabilization of the largely amphiphilic helix conformation and a nonelectrostatic W affinity for the bilayer interface. Overall, W substitutions constitute an interesting means to reduce the proteolytic susceptibility of EFK17 while also improving antimicrobial performance.

Place, publisher, year, edition, pages
2009. Vol. 53, no 2, 593-602 p.
National Category
Pharmaceutical Sciences
URN: urn:nbn:se:uu:diva-88745DOI: 10.1128/AAC.00477-08ISI: 000262646500031PubMedID: 19029324OAI: oai:DiVA.org:uu-88745DiVA: diva2:158974
Available from: 2009-02-05 Created: 2009-02-05 Last updated: 2016-10-05
In thesis
1. Antimicrobial Peptide Interactions with Phospholipid Membranes: Effects of Peptide and Lipid Composition on Membrane Adsorption and Disruption
Open this publication in new window or tab >>Antimicrobial Peptide Interactions with Phospholipid Membranes: Effects of Peptide and Lipid Composition on Membrane Adsorption and Disruption
2009 (English)Doctoral thesis, comprehensive summary (Other academic)
Abstract [en]

The interactions between antimicrobial peptides and phospholipid membranes were investigated, in terms of lipid headgroup variations and the role of cholesterol, as well as peptide composition and structure. Also strategies for increasing proteolytic stability were evaluated. The interactions were studied on model membranes in the form of liposomes and supported bilayers, through a combination of ellipsometry, fluorescence spectroscopy, circular dichroism, dynamic light scattering, electrophoresis, electron cryomicroscopy, and bacterial/cell culture experiments.

The findings showed that membrane tolerance against the lytic activity of melittin, was increased on anionic membranes by electrostatic arrest in the headgroup region, and was reduced by hydration repulsion. The presence of cholesterol caused a reduction in melittin adsorption, while at the same time reducing membrane tolerance per adsorbed peptide. Differences in membrane leakage mechanisms were also attributed to cholesterol, where large scale structural effects contributed to the leakage, while other membranes followed the pore formation model.

Substituting specific amino acids for tryptophan on an LL-37 derivative, was shown to increase stability against bacterial proteases, while at the same time significantly increasing antibacterial properties. These substitutions, as well as terminal modifications, increased adsorption and membrane lytic properties in a way that was less dependent on electrostatics. Furthermore, by comparing short cationic peptides with oligotryptophan end-tagged versions, the lytic mechanism of end-tagged peptides, and the different contributions of arginine and lysine to membrane adsorption and disruption were demonstrated.

This thesis is a contribution to the development of antimicrobial peptides as therapeutic alternatives to conventional antibiotics.

Place, publisher, year, edition, pages
Uppsala: Acta Universitatis Upsaliensis, 2009. 61 p.
Digital Comprehensive Summaries of Uppsala Dissertations from the Faculty of Pharmacy, ISSN 1651-6192 ; 97
antimicrobial, peptide, adsorption, liposome, membrane, melittin, cholesterol, tryptophan, phospholipid, bilayer
National Category
Pharmaceutical Sciences
Research subject
Pharmaceutical Physical Chemistry
urn:nbn:se:uu:diva-100966 (URN)978-91-554-7520-8 (ISBN)
Public defence
2009-05-20, B42, BMC, Husargatan 3, Uppsala, 13:15 (English)
Available from: 2009-04-29 Created: 2009-04-15 Last updated: 2009-05-12Bibliographically approved

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