Cyclization Increases the Antimicrobial Activity and Selectivity of Arginine- and Tryptophan-Containing Hexapeptides

Abstract

Arginine- and tryptophan-rich motifs have been identified in antimicrobial peptides with various secondary structures. We synthesized a set of linear hexapeptides derived from the sequence AcRRWWRF-NH(2) by substitution of tryptophan (W) by tyrosine (Y) or naphthylalanine (Nal) and by replacement of arginine (R) by lysine (K) to investigate the role of cationic charge and aromatic residues in membrane activity and selectivity. A second set of corresponding head-to-tail cyclic analogues was prepared to analyze the role of conformational constraints. The biological activity of the linear peptides followed the order Nal- >> W- > Y-containing compounds and slightly decreased upon R-K substitution. A pronounced activity-improving and bacterial selectivity-enhancing effect was found upon cyclization of the R- and W-bearing parent peptide, whereas the activity-modifying effect of cyclization of Y- and Nal-containing peptides was low. The analysis of the driving forces of peptide interaction with model membranes showed that the activities correlated with the partition coefficients and the depths of peptide insertion into neutral and negatively charged lipid bilayers. Spectroscopic studies, RP-HPLC, and titration calorimetry implied that the combination of cationic and aromatic amino acid composition and conformational rigidity afforded a membrane-active, amphipathic structure with a highly charged face opposed by a cluster of aromatic side chains. However, threshold values of low and high hydrophobicity seemed to exist beyond which the activity-enhancing effect of cyclization was negligible. The results suggest that cyclization of small peptides of an appropriate amino acid composition may serve as a promising strategy in the design of antimicrobial peptides.

Length Effects in Antimicrobial Peptides of the (RW)nSeries

“…Trp-rich AMPs preferentially bind to model membranes containing a negatively charged headgroup relative to those containing a zwitterionic headgroup (8,17,37). We recognize that the presence of multiple Trp residues does not allow for a simple interpretation of some of the spectroscopic data reported here (37).…”

“…Other studies suggest that the antimicrobial activity of peptides containing R is higher than those of peptides containing K (34,41), while peptides containing W are more potent than those with either F or Y (8,47,49). The guanidinium group of R has a more dispersed positive charge than the single amine of K, possibly enhancing electrostatic interactions between peptides and the negatively charged bacterial membrane surface (39,53).…”

“…Although some progress has been made in the recombinant expression of fusion proteins comprising multiple copies of AMPs (28), chemical peptide synthesis remains the preferred option for quality control purposes. Different approaches are being pursued in efforts to increase the effectiveness of AMPs, including alteration of sequences, inclusion of unnatural D-amino acids or beta-amino acids, cyclization of peptides, peptoid mimics, and synthesis of multivalent constructs of short peptides (8,25,26,33,35,51). At present, short AMPs that minimize damage to host cells or tissues would appear to be the most promising candidates for large-scale production (47).…”

In vitro assessment of the dual-targeting behavior of a peptide-based magnetic resonance imaging contrast agent

“…Dathe et al (25) reported that cyclic peptides display improved binding affinity to their cellular targets owing to minimal entropy costs for binding. Colomb et al (26) demonstrated that the use of disulfide-bridged cyclic NGR peptide led to a 10-fold increase in the efficacy of targeting to tumor sites compared to its linear analog.…”

Structures of cyclic, antimicrobial peptides in a membrane‐mimicking environment define requirements for activity

“…A replacement of tryptophan by tyrosine or arginine by lysine preserves the structure of the peptide but changes the activity considerably [6,8]. Since these findings point to the importance of tryptophan and arginine, the next step was the change in sequence while preserving the type of amino acid side chain.…”