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)-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 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.
Highlights
Multidrug-resistant (MDR) bacteria is a major global health problem [1]
In order to evaluate the role of each individual amino acid for the antimicrobial activity and toxicity of D2D, we performed alanine scan by systematically replacing all residues with d-alanine (Table 1). This identified four residues to be important for activity (compounds 3 (1-nal), 4, 6 (1-nal) and 8 (Table 1)). When these residues were replaced with d-alanine the MIC changed from 4–8 μg/mL to between 64 and >128 μg/mL for E. coli and S. aureus while P. aeruginosa and A
This is probably related to the concentration dependent killing, as the peptides get sequestered by killed cells leaving unaffected cells to regrow. Both compounds were shown to be capable of killing S. aureus with a 5 log reduction in viable cells at 5 × MIC. It seems that there could be a slight re-growth of S. aureus after 24 h, it is much less pronounced than it was for the Gram-negative species, indicating that bacterial killing probably was more pronounced
Summary
Multidrug-resistant (MDR) bacteria is a major global health problem [1]. Recently, WHO published a list of MDR bacteria which are of critical concern [2]. The stability of promising synthetic AMPs are typically improved by cyclization [10] or insertion of non-proteinogenic building blocks [11] These include d-amino acids [12], peptoids (N-substituted glycines) [13], peptidomimetics such as, α-peptide/β-peptides [14], lysine-based α-peptides/α-peptoids [15], lysine-based α-peptide/β-peptoids [16], and α/γ N-Acylated-N-Aminoethylpeptides (AApeptides) [17]. We have identified an all-d-peptide, D2D, which shows promising activity against clinical isolates of Methicillin Resistant Staphylococcus pseudintermedius and Pseudomonas aeruginosa. D2, was selected in a previous study after screening from a combinatorial library of de novo designed compounds for their activity against S. pseudintermedius and toxicity in vitro against red blood cells [19].
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