Alanine and Lysine Scans of the LL‐37‐Derived Peptide Fragment KR‐12 Reveal Key Residues for Antimicrobial Activity

Abstract

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.

Backbone Cyclization and Dimerization of LL-37-Derived Peptides Enhance Antimicrobial Activity and Proteolytic Stability

“…In this case the dimers are just 2-3 times as potent as the monomers. The 23-fold difference in EC50 for KR-12 in either liposome system is not mirrored in the antimicrobial assay ( Table 2), and supports the previous proposition that membrane permeabilization is not the sole mechanism of action for the KR-12 monomer on bacteria (Gunasekera et al, 2018).…”

“…Substitutions with Lys increased activity as long as the resulting increase in cationic density did not disrupt the amphiphilic disposition of the helical structure. Key positions were identified as Gln5 and Asp9, which when replaced with Ala or Lys increased broad-spectrum activity up to 8-fold against S. aureus, P. aeruginosa, and C. albicans (Gunasekera et al, 2018). In line with previous work (Wang, 2008;Wang et al, 2008), KR-12 showed weak cytotoxic activity with only a 13% loss of human cell line viability at the maximum concentration of 80 µM.…”

“…In our hands, KR-12 displayed a MIC of 2.5 µM against Escherichia coli and 10 µM against other tested pathogens, i.e., Pseudomonas aeruginosa, Staphylococcus aureus, and Candida albicans (Gunasekera et al, 2018). In that work, Ala and Lys scans of KR-12 showed that replacement of hydrophobic and cationic residues with Ala were detrimental for antimicrobial potency.…”

“…In comparison, LL-37 displayed distinct cytotoxicity in this assay, having an IC50 of 10 µM. Importantly, the beneficial mutations of Gln5 and Asp9 were well tolerated, and neither of the double substituted analogues Q5A,D9A and Q5K,D9K showed any substantial cytotoxicity within the concentration interval used (Gunasekera et al, 2018). All the above indicated that KR-12 was an attractive starting point for peptide engineering.…”

“…To examine the intact peptide after serum incubation, aliquots of each incubation mixture were taken at each time point for LC-MS analysis (Figure 5). In our previous studies we have shown that Ala and Lys analogs break down in 10 min (Gunasekera et al, 2018). In comparison to KR-12 and its derivatives, about 20% of retro-KR-12 peptide remained after 10 min, but it could not be detected after 20 min; R1, R11, and K13 were identified as cleavage sites by the analysis of degradation products in serum.…”