Rationally Modified Antimicrobial Peptides from the N-Terminal Domain of Human RNase 3 Show Exceptional Serum Stability

Abstract

Multidrug resistance against conventional antibiotics poses an important threat to human health. In this context, antimicrobial peptides (AMPs) have been extensively studied for their antibacterial activity and promising results have been shown so far. However, AMPs tend to be rather vulnerable to protease degradation, which offsets their therapeutic appeal. Here, we demonstrate how replacing functional residues in the antimicrobial region of human RNase 3also named eosinophil cationic proteinby non-natural amino acids increases stability in human serum. These changes were also shown to reduce the hemolytic effect of the peptides in general terms, whereas the antimicrobial activity was reasonably preserved. Digestion profiles enabled us to design new peptides with superior stability and lower toxicity that could become relevant candidates to reach clinical stages.

In Vivo Evaluation of ECP Peptide Analogues for the Treatment of Acinetobacter baumannii Infection

“…Based on a target proteolysis study in serum, peptide analogues were synthesized with either all D-AA, Arg to Orn substitutions or blockage of peptide bond cleavage by D-AA introduction. Additionally, based on the previous analysis of the proteolysis digestion products [ 28 ], ECPep-2D-Orn was selected. ECPep-2D-Orn is the Orn-peptide analogue, which showed the highest half-life in serum and incorporates a D-AA at position 2 ( Figure 2 ).…”

“…Structural analysis in aqueous solution and in the presence of lipid dodecyl phosphocholine (DPC) micelles was previously assessed by NMR [ 28 ]. NMR indicated that the 30mer L-reference peptide adopts in aqueous solution a defined helix from residues 5 to 13 and tends to helical structuration within the 16–27 stretch.…”

“…In addition, a comparison of all-L and all-D peptides by CD confirmed that equivalent secondary structures are adopted by both enantiomers (Figure 3). Structural analysis in aqueous solution and in the presence of lipid dodecyl phosphocholine (DPC) micelles was previously assessed by NMR [28]. NMR indicated that the 30mer L-reference peptide adopts in aqueous solution a defined helix from residues 5 to 13 and tends to helical structuration within the 16-27 stretch.…”

“…Considering the outstanding performance of ECP native protein and derived peptides in vitro, we designed new ECP peptide analogues based on the 30mer lead template [ 27 ], that incorporate non-natural amino acids to limit proteolysis in human serum [ 28 ]. Next, two peptides were selected for further characterization and in vivo studies.…”

“…The first is the all-D version of the ECPep-L lead peptide (ECPep-D) [ 27 ], where all L-amino acids in the sequence have been replaced by D-amino acids. The second peptide incorporates Orn substitutions for Arg residues and includes a D-amino acid at position 2 (ECPep-2D-Orn), based on the previous identification of ECP peptide main proteolysis target sites [ 28 ]. In addition, recent work confirmed that Arg to Orn replacement ensured the peptide stability in serum in vitro up to more than 8 h and retained its antimicrobial properties [ 28 ].…”