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Alanine and Lysine Scans of the LL-37-Derived Peptide Fragment KR-12 Reveal Key Residues for Antimicrobial Activity
Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Pharmacy, Department of Medicinal Chemistry, Farmakognosi.
Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Pharmacy, Department of Medicinal Chemistry, Farmakognosi.
Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Pharmacy, Department of Medicinal Chemistry, Farmakognosi.ORCID iD: 0000-0001-9070-6944
Univ Queensland, Sch Biomed Sci, Brisbane, Qld 4072, Australia.
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2018 (English)In: ChemBioChem (Print), ISSN 1439-4227, E-ISSN 1439-7633, Vol. 19, no 9, p. 931-939Article in journal (Refereed) Published
Abstract [en]

The human host defence peptide LL-37 is a broad-spectrum antibiotic with immunomodulatory functions. Residues 18-29 in LL-37 have previously been identified as a minimal peptide (KR-12) that retains antibacterial activity with decreased cytotoxicity. In this study, analogues of KR-12 were generated by Ala and Lys scans to identify key elements for activity. These were tested against a panel of human pathogens and for membrane permeabilisation on liposomes. Replacements of hydrophobic and cationic residues with Ala were detrimental for antibiotic potency. Substitutions by Lys increased activity, as long as the increase in cationic density did not disrupt the amphiphilic disposition of the helical structure. Importantly, substitutions showed differential effects against different organisms. Replacement of Gln5 with Lys and Asp9 with Ala or Lys improved the broad-spectrum activity most, each resulting in up to an eightfold increase in potency against Staphylococcus aureus, Pseudomonas aeruginosa and Candida albicans. The improved analogues displayed no significant toxicity against human cells, and thus, KR-12 is a tuneable template for antibiotic development.

Place, publisher, year, edition, pages
WILEY-V C H VERLAG GMBH , 2018. Vol. 19, no 9, p. 931-939
Keywords [en]
antibiotics, cytotoxicity, drug discovery, peptides, structure-activity relationships
National Category
Biochemistry and Molecular Biology
Identifiers
URN: urn:nbn:se:uu:diva-356391DOI: 10.1002/cbic.201700599ISI: 000431625100008PubMedID: 29430821OAI: oai:DiVA.org:uu-356391DiVA, id: diva2:1235466
Funder
Swedish Research Council, 2011-3403Carl Tryggers foundation , CTS 10: 126Carl Tryggers foundation , CTS 11: 169Swedish Society of Medicine, SLS-254511Available from: 2018-07-25 Created: 2018-07-25 Last updated: 2020-02-18Bibliographically approved
In thesis
1. LL-37-derived cyclic antimicrobial drug leads: Design, synthesis, activity and different ways of creating them 
Open this publication in new window or tab >>LL-37-derived cyclic antimicrobial drug leads: Design, synthesis, activity and different ways of creating them 
2019 (English)Doctoral thesis, comprehensive summary (Other academic)
Abstract [en]

In an era where last-line antibiotics are failing, one of the powerful approaches to develop novel therapeutic agents is to turn back to nature in order to identify possible drug candidates. Among the potential candidates, antimicrobial peptides (AMPs) have garnered much attention as an antimicrobial. These are broad spectrum host defense molecules produced by all living organisms. LL-37 is such a multitask human defense peptide that mediates various host immune responses and also exerts antimicrobial activity. However, the direct use of this 37-amino acid long α-helical peptide is hampered by protease susceptibility, in particular for antimicrobial applications. A small 12-residues peptide, referred as KR-12, derived from LL-37, has been reported to have selective toxic effect on bacteria. 

Analogues of KR-12 were generated in the form of Alanine and Lysine scans to find out the positions important for improved activity and selectivity. Backbone-cyclised dimers based on KR-12 and KR-12 analogues, tethered by linkers of two to four amino acid residues, were synthesised to explore the concept of cyclisation, dimerisation and cross-linking as means to enhance peptide stability and activity. Antimicrobial activities of the linear peptides and cyclic dimers were assayed against human pathogens, in buffer and/or physiological conditions. Proteolytic stability, permeabilisation efficacy on microbial membranes and, their structures were also characterised.  

From Ala and Lys scans, it was possible to identify two key positions for the enhanced broad-spectrum antibacterial activity: replacement of Gln5 with Lys, and Asp9 with either Ala or Lys. In serum stability assay, KR-12 and analogues were found to be unstable. The backbone-cyclised KR-12 dimers showed improved antimicrobial activity and increased stability compared to monomeric KR-12. KR-12 monomers adopt a well-defined α-helical structure in membrane-mimicking environment, while cyclised dimers were unstructured in solution judged by NMR. The KR-12 (Q5K, D9A) cyclised dimers retained antimicrobial activity in physiological conditions. Circular dichroism showed that the cyclic dimer, cd4-PP, had 77% helical content when bound to lyso-phosphatidylglycerol micelles.

Moreover, the limits of cyanobactin-macrocyclase PatGmac were explored to cyclise peptides larger than their natural substrates, namely the PawS derived peptide Sunflower Trypsin Inhibitor-1 (SFTI-1) and the cyclotide kalata B1. PatGmac was used very efficiently to cyclise SFTI-1. In addition, semi-pure butelase 1, isolated from Clitoria ternatea seeds, was immobilised on NHS column. The immobilised column was then used to produce substrates ranging from 16 to 34 varying length.

Place, publisher, year, edition, pages
Uppsala: Acta Universitatis Upsaliensis, 2019. p. 65
Series
Digital Comprehensive Summaries of Uppsala Dissertations from the Faculty of Pharmacy, ISSN 1651-6192 ; 281
Keywords
antimicrobial peptide, host defense, antimicrobial, LL-37, KR-12, peptide cyclisation, peptide dimerisation, croos-linking, cyanobactin-macrocyclase, PatGmac, butelase 1, enzymatic cyclisation, immobilisation
National Category
Medical and Health Sciences
Research subject
Pharmacognosy
Identifiers
urn:nbn:se:uu:diva-397191 (URN)978-91-513-0813-5 (ISBN)
Public defence
2019-12-19, A1:107a, Biomedical Centrum (BMC), Uppsala, 09:15 (English)
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Supervisors
Available from: 2019-11-27 Created: 2019-11-18 Last updated: 2020-01-13Bibliographically approved

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Gunasekera, SunithiMuhammad, TajStrömstedt, Adam A.Göransson, Ulf

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