Evaluation of Strategies for Improving Proteolytic Resistance of Antimicrobial Peptides by Using Variants of EFK17, an Internal Segment of LL-37

Abstract

Methods for increasing the proteolytic stability of EFK17 (EFKRIVQRIKDFLRNLV), a new peptide sequence with antimicrobial properties derived from LL-37, were evaluated. EFK17 was modified by four d-enantiomer or tryptophan (W) substitutions at known protease cleavage sites as well as by terminal amidation and acetylation. The peptide variants were studied in terms of proteolytic resistance, antibacterial potency, and cytotoxicity but also in terms their adsorption at model lipid membranes, liposomal leakage generation, and secondary-structure behavior. The W substitutions resulted in a marked reduction in the proteolytic degradation caused by human neutrophil elastase, Staphylococcus aureus aureolysin, and V8 protease but not in the degradation caused by Pseudomonas aeruginosa elastase. For the former two endoproteases, amidation and acetylation of the terminals also reduced proteolytic degradation but only when used in combination with W substitutions. The d-enantiomer substitutions rendered the peptides indigestible by all four proteases; however, those peptides displayed little antimicrobial potency. The W-and end-modified peptides, on the other hand, showed an increased bactericidal potency compared to that of the native peptide sequence, coupled with a moderate cytotoxicity that was largely absent in serum. The bactericidal, cytotoxic, and liposome lytic properties correlated with each other as well as with the amount of peptide adsorbed at the lipid membrane and the extent of helix formation associated with the adsorption. The lytic properties of the W-substituted peptides were less impaired by increased ionic strength, presumably by a combination of W-mediated stabilization of the largely amphiphilic helix conformation and a nonelectrostatic W affinity for the bilayer interface. Overall, W substitutions constitute an interesting means to reduce the proteolytic susceptibility of EFK17 while also improving antimicrobial performance.The cathelicidins are a major family of antibacterial peptides in mammals (66). The human cathelicidin hCAP-18 is released by several types of epithelial and immune cells, notably neutrophils, in response to inflammation and subsequently cleaved to generate the active C-terminal LL-37 peptide (18, 52). LL-37 is an extensively investigated peptide displaying strong antibacterial effects also at a high ionic strength (57). It also displays a range of other interesting properties, e.g., relating to lipopolysaccharide neutralization, immune cell stimulation and attraction, reepithelization, and wound closure (13). Although LL-37 displays some cytotoxicity against human cells, possibly originating from a hydrophobic interaction with cell membranes (31), this is largely absent in human serum due to interactions with apolipoproteins and other serum factors (61). It was previously found that reducing the number of residues from the N terminus of LL-37 decreases its cytotoxicity while retaining the bactericidal properties, although such truncations may also result in an increased proteolyt...

Boosting Antimicrobial Peptides by Hydrophobic Oligopeptide End Tags

“…Similarly, Li et al (46) investigated aurein 1.2 analogs and found the Phe residues in these peptides to penetrate 2-5 Å below the polar headgroup region. Through this interaction with the phospholipid membrane, Trp/Phe residues are able to insert into the membrane, acting as an anchor for the peptide (48,50) and resulting in increased bactericidal effects (49,51,52) and salt resistance (48). In contrast to these previous investigations, which report on positionspecific single tryptophan substitutions in AMPs, however, the present work describes a generally applicable approach for boosting potency of essentially all (highly charged) AMPs without interfering with the AMP sequence.…”

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

“…Although a number of hydrophobic amino acids may be used as end tags, Trp and Phe end tags have emerged as particularly potent choices (30). Through interaction with the phospholipid membrane, Trp/Phe residues are able to insert into the membrane, acting as an anchor for the peptide and resulting in increased bactericidal effects (31,32). Our findings show that hydrophobic end tagging of the ␣ s2 -casein f(193-203) and ␣ s2 -casein f(197-207) peptides with multiple Trp or Phe residues significantly increased their activity against L. monocytogenes, but this effect was not observed for C. sakazakii.…”

Helical antimicrobial polypeptides with radial amphiphilicity

“…Another drawback of most AMP-derived antibiotics is their poor stability in biological systems. LL-37 and magainin, for example, can be degraded by proteases in several minutes in blood circulation and lose their antimicrobial activities (16,17). Finally, an important drawback of AMPs is related to their FA structure with an exposed hydrophobic helix face (Fig.…”