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

Abstract

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)-NH 2 (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 lys 5 and lys 7 , 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 1-8 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.

Antimicrobial Peptide Nanomaterials

“…In order to overcome these problems, there has been an increasing on the development of non-native sequences which retain all the advantages of parent peptide sequences, whilst overcoming rapid proteolytic degradation. The stability of promising synthetic sequences is typically improved by cyclization or insertion of non-proteinogenic building blocks, including D-amino acids, peptoids (N-substituted glycines) and other peptidomimetics such as α-peptide/β-peptides (Molchanova et al, 2017, Lone et al, 2019. Approaches to achieve stability typically rely on post-synthetic modification of traditional sequences or the synthesis of modified sequences which recapitulate the desirable properties whilst enhancing stability.…”

“…It was postulated to have enhanced resistance to degradation owing to the use of non-native residues (Lone et al, 2019). Bactenecin is a 12-amino acid cyclic cationic antimicrobial peptide that contains one intramolecular disulfide bond.…”