LL-37, acetylated, amidated

New peptides for lipopolysaccharide (LPS) and lipoteichoic acid (LTA) neutralization in upper respiratory tract infections were developed and evaluated in terms of efficacy and safety for application in humans. Based on the sequence of the human antimicrobial peptide LL-37 we developed and investigated length variants, substitution analogues and modifications to stabilize the peptides to prevent enzymatic degradation and to improve efficacy. The most promising peptide appears P60.4, a 24 amino acid peptide with similar efficacy as LL-37 in terms of LPS and LTA neutralization and lower pro-inflammatory activity. In addition, the acetylated and amidated version of this peptide shows no toxicity and displays higher or equal antimicrobial activity compared to LL-37.

Cathelicidin LL-37 belongs to the class of human defense peptides and is overexpressed in many cancers. Segments of LL-37 derived through biochemical processes have a wide range of activities. In this study, novel analogs of the 13-amino acid cathelicidin 17-29 amide segment F17 KRIV21 QR23 IK25 DF27 LR-NH2 were prepared and examined for their antimicrobial and hemolytic activities, as well as for their cytotoxicity on cancer bronchial epithelial cells. Selected substitutions were performed on residues R23 and K25 in the hydrophilic side, V21 and F27 in the hydrophobic side of the interphase, and F17 that interacts with cell membranes. Specific motifs IIKK and LLKKL with anticancer and antimicrobial activities isolated from animals were also inserted into the 17-29 fragment to investigate how they affect activity. Substitution of the amino-terminal positive charge by acetylation and replacement of lysine by the aliphatic leucine in the peptide analog Ac-FKRIVQRIL25 DFLR-NH2 resulted in significant cytotoxicity against A549 cancer cells with an IC50 value 3.90 μg/mL, with no cytotoxicity to human erythrocytes. The peptide Ac-FKRIVQI23 IKK26 FLR-NH2 , which incorporates the IIKK motif and the peptides FKRIVQL23 L24 KK26 L27 LR-NH2 and Ac-FKRIVQL23 L24 KK26 L27 LR-NH2 , which incorporate the LLKKL motif, displayed potent antimicrobial activity against gram-negative bacteria (MIC 3-7.5 μg/mL) and substantial cytotoxicity against bronchial epithelial cancer cells, (IC50 12.9-9.8 μg/mL), with no cytotoxic activity for human erythrocytes. The helical conformation of the synthetic peptides was confirmed by circular dichroism. Our study shows that appropriate substitutions, mainly in positions of the interphase, as well as the insertion of the motifs IIKK and LLKKL in the cathelicidin 17-29 segment, may lead to the preparation of effective biological compounds.

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.