Structure-Microbicidal Activity Relationship of Synthetic Fragments Derived from the Antibacterial α-Helix of Human Lactoferrin

Abstract

There is a need for new microbicidal agents with therapeutic potential due to antibiotic resistance in bacteria and fungi. In this study, the structure-microbicidal activity relationship of amino acid residues 14 to 31 (sequence 14-31) from the N-terminal end, corresponding to the antibacterial ␣-helix of human lactoferrin (LF), was investigated by downsizing, alanine scanning, and substitution of amino acids. Microbicidal analysis (99% killing) was performed by a microplate assay using Escherichia coli, Staphylococcus aureus, and Candida albicans as test organisms. Starting from the N-terminal end, downsizing of peptide sequence 14-31 showed that the peptide sequence 19-31 (KCFQWQRNMRKVR, HL9) was the optimal length for antimicrobial activity. Furthermore, HL9 bound to lipid A/lipopolysaccharide, as shown by neutralizing endotoxic activity in a Limulus assay. Alanine scanning of peptide sequence 20-31 showed that Cys20, Trp23, Arg28, Lys29, or Arg31 was important for expressing full killing activity, particularly against C. albicans. Substituting the neutral hydrophilic amino acids Gln24 and Asn26 for Lys and Ala (HLopt2), respectively, enhanced microbicidal activity significantly against all test organisms compared to the amino acids natural counterpart, also, in comparison with HL9, HLopt2 had more than 10-fold-stronger fungicidal activity. Furthermore, HLopt2 was less affected by metallic salts than HL9. The microbicidal activity of HLopt2 was slightly reduced only at pH 7.0, as tested in the pH range of 4.5 to 7.5. The results showed that the microbicidal activity of synthetic peptide sequences, based on the antimicrobial ␣-helix region of LF, can be significantly enhanced by optimizing the length and substitution of neutral amino acids at specific positions, thus suggesting a sequence lead with therapeutic potential.

Structure-Activity Relationship of Synthetic Variants of the Milk-Derived Antimicrobial Peptide α s2 -Casein f(183–207)

“…Only some degree of bacterial inhibition was observed in the presence of 1% NaCl for L. monocytogenes. These findings are in agreement with previous reports showing that AMPs are sensitive to high-salt environments (13,37). This is probably linked to the fact that most of the natural cationic peptides are strongly antagonized by physiological concentrations of mono-and divalent cations (38).…”

“…Template-based studies of peptide derivatives obtained through manipulation of the amino acid sequence have often been performed in order to identify properties that are important for AMP activity (12). Such studies have shed light on the importance of specific amino acids and residue positions in the activities of different peptides (13)(14)(15). However, it appears that the effect of particular manipulations is context dependent and varies according to the template sequence, in that analogous substitutions may have substantially different effects on different peptides or at different positions in the primary sequence (16).…”

Fungicidal activity of human lactoferrin-derived peptides based on the antimicrobial αβ region

“…Lactam formation between the side chains was achieved by treatment with a six-fold excess of benzotriazole-1-yl-oxy-trispyrrolidino-phosphoniumhexafluorophosphate-N-hydroxybenzotriazolediisopropylethylamine (PyBOP-HOBt-DIPEA) (1:1:2) in dimethylformamide (DMF) for 16 h. The resin was washed with DMF and dichloromethane and was dried under vacuum. The peptide was cleaved from the resin as described previously [11]. Lactam bridge formation induces some helical content in short peptide fragments that are random coiled.…”

“…hLF peptides were synthesised using a 9-fluoryl-mehoxycarbonyl continuous-flow strategy on a Pioneer TM automated peptide synthesizer (Applied Biosystems, Foster City, CA) and have been described previously [11].…”

“…In an earlier study on synthetic peptide fragments derived from the antibacterial -helix region of hLF, it was found that peptides comprising amino acid residues 17-31, 18-31, 19-31 and 20-31 from the N-terminal end expressed stronger microbicidal activity than the peptide fragment 21-31 [11]. By changing two neutral amino acid…”