Serum stabilities of short tryptophan- and arginine-rich antimicrobial peptide analogs

Abstract

Background: Several short antimicrobial peptides that are rich in tryptophan and arginine residues were designed with a series of simple modifications such as end capping and cyclization. The two sets of hexapeptides are based on the Trp- and Arg-rich primary sequences from the "antimicrobial centre" of bovine lactoferricin as well as an antimicrobial sequence obtained through the screening of a hexapeptide combinatorial library.

Methodology/principal findings: HPLC, mass spectrometry and antimicrobial assays were carried out to explore the consequences of the modifications on the serum stability and microbicidal activity of the peptides. The results show that C-terminal amidation increases the antimicrobial activity but that it makes little difference to its proteolytic degradation in human serum. On the other hand, N-terminal acetylation decreases the peptide activities but significantly increases their protease resistance. Peptide cyclization of the hexameric peptides was found to be highly effective for both serum stability and antimicrobial activity. However the two cyclization strategies employed have different effects, with disulfide cyclization resulting in more active peptides while backbone cyclization results in more proteolytically stable peptides. However, the benefit of backbone cyclization did not extend to longer 11-mer peptides derived from the same region of lactoferricin. Mass spectrometry data support the serum stability assay results and allowed us to determine preferred proteolysis sites in the peptides. Furthermore, isothermal titration calorimetry experiments showed that the peptides all had weak interactions with albumin, the most abundant protein in human serum.

Conclusions/significance: Taken together, the results provide insight into the behavior of the peptides in human serum and will therefore aid in advancing antimicrobial peptide design towards systemic applications.

Figure 1. An example set of HPLC chromatograms from various time points of Lfc9 incubated in 25% human male serum at 37°C, showing the degradation of the intact peptide (∼13 mL ret. vol.) and appearance of its partially digested products (5–11 mL ret. vol.).

Figure 2. Serum stability profiles of the LfcinB- and Combi-based peptides. Relative peptide concentrations were determined by integration of the A215 peaks from RP-HPLC chromatograms.

A) Linear LfcinB hexamers showing individual and combined effects of C-terminal amidation and N-terminal acetylation, B) Linear Combi hexamers with end caps C) Linear and cyclized LfcinB peptides comparing the end-to-end disulfide bridging and head-to-tail backbone cyclization strategies to different sized peptides, and D) cyclized Combi peptides. Assays were performed in triplicate.