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Open AccessArticle

Structure-Activity Study of an All-d Antimicrobial Octapeptide D2D

Department of Drug Design and Pharmacology, Faculty of Health and Medical Sciences, University of Copenhagen, Universitetsparken 2, 2100 Copenhagen, Denmark
Department of Science and Environment, Roskilde University, 4000 Roskilde, Denmark
Department of Clinical Microbiology, Rigshospitalet, Henrik Harpestrengs Vej 4A, 2100 Copenhagen, Denmark
Department of Biology, Section for functional Genomics, University of Copenhagen, Ole Maaløes Vej 5, 2200 Copenhagen, Denmark
Department of Chemistry, University of Oslo, Sem Sælands vei 26, 0371 Oslo, Norway
Department of Chemistry, University of Copenhagen, Universitetsparken 5, 2100 Copenhagen, Denmark
Author to whom correspondence should be addressed.
Academic Editor: Vincent Humblot
Molecules 2019, 24(24), 4571;
Received: 1 November 2019 / Revised: 6 December 2019 / Accepted: 11 December 2019 / Published: 13 December 2019
(This article belongs to the Special Issue Antimicrobial Peptides: From Synthesis to Application)
The increasing emergence of multi-drug resistant bacteria is a serious threat to public health worldwide. Antimicrobial peptides have attracted attention as potential antibiotics since they are present in all multicellular organisms and act as a first line of defence against invading pathogens. We have previously identified a small all-d antimicrobial octapeptide amide kk(1-nal)fk(1-nal)k(nle)-NH2 (D2D) with promising antimicrobial activity. In this work, we have performed a structure-activity relationship study of D2D based on 36 analogues aimed at discovering which elements are important for antimicrobial activity and toxicity. These modifications include an alanine scan, probing variation of hydrophobicity at lys5 and lys7, manipulation of amphipathicity, N-and C-termini deletions and lys-arg substitutions. We found that the hydrophobic residues in position 3 (1-nal), 4 (phe), 6 (1-nal) and 8 (nle) are important for antimicrobial activity and to a lesser extent cationic lysine residues in position 1, 2, 5 and 7. Our best analogue 5, showed MICs of 4 µg/mL against A. baumannii, E. coli, P. aeruginosa and S. aureus with a hemolytic activity of 47% against red blood cells. Furthermore, compound 5 kills bacteria in a concentration-dependent manner as shown by time-kill kinetics. Circular dichroism (CD) spectra of D2D and compounds 18 showed that they likely fold into α-helical secondary structure. Small angle x-ray scattering (SAXS) experiments showed that a random unstructured polymer-like chains model could explain D2D and compounds 1, 3, 4, 6 and 8. Solution structure of compound 5 can be described with a nanotube structure model, compound 7 can be described with a filament-like structure model, while compound 2 can be described with both models. Lipid interaction probed by small angle X-ray scattering (SAXS) showed that a higher amount of compound 5 (~50–60%) inserts into the bilayer compared to D2D (~30–50%). D2D still remains the lead compound, however compound 5 is an interesting antimicrobial peptide for further investigations due to its nanotube structure and minor improvement to antimicrobial activity compared to D2D. View Full-Text
Keywords: antimicrobial peptides; d-peptides; minimum inhibitory concentration; hemolytic activity; time-kill kinetics; circular dichroism; small angle X-ray scattering antimicrobial peptides; d-peptides; minimum inhibitory concentration; hemolytic activity; time-kill kinetics; circular dichroism; small angle X-ray scattering
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MDPI and ACS Style

Lone, A.; Thomsen, T.T.; Nielsen, J.E.; Thulstrup, P.W.; Klitgaard, R.N.; Løbner-Olesen, A.; Lund, R.; Jenssen, H.; Hansen, P.R. Structure-Activity Study of an All-d Antimicrobial Octapeptide D2D. Molecules 2019, 24, 4571.

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