In vivo efficacy of anuran trypsin inhibitory peptides against staphylococcal skin infection and the impact of peptide cyclization

Abstract

Staphylococcus aureus is a virulent pathogen that is responsible for a wide range of superficial and invasive infections. Its resistance to existing antimicrobial drugs is a global problem, and the development of novel antimicrobial agents is crucial. Antimicrobial peptides from natural resources offer potential as new treatments against staphylococcal infections. In the current study, we have examined the antimicrobial properties of peptides isolated from anuran skin secretions and cyclized synthetic analogues of these peptides. The structures of the peptides were elucidated by nuclear magnetic resonance (NMR) spectroscopy, revealing high structural and sequence similarity with each other and with sunflower trypsin inhibitor 1 (SFTI-1). SFTI-1 is an ultrastable cyclic peptide isolated from sunflower seeds that has subnanomolar trypsin inhibitory activity, and this scaffold offers pharmaceutically relevant characteristics. The five anuran peptides were nonhemolytic and noncytotoxic and had trypsin inhibitory activities similar to that of SFTI-1. They demonstrated weak in vitro inhibitory activities against S. aureus, but several had strong antibacterial activities against S. aureus in an in vivo murine wound infection model. pYR, an immunomodulatory peptide from Rana sevosa, was the most potent, with complete bacterial clearance at 3 mg · kg(-1). Cyclization of the peptides improved their stability but was associated with a concomitant decrease in antimicrobial activity. In summary, these anuran peptides are promising as novel therapeutic agents for treating infections from a clinically resistant pathogen.

FIG 1

Hα NMR secondary shift analysis of SLF peptides and SFTI-1. Secondary shifts were obtained by subtracting experimental ¹H NMR Hα chemical shifts from random coil shifts for the corresponding residue (43). The ORB/ORB2k open and cyclic peptides have similar shifts in loop 1, whereas the Rana-E/T and pYR peptides have similar secondary shifts in loop 1. Loop 1 contains a highly conserved BBI reactive loop (dotted gray box).

FIG 2

NMR solution structures of open and cyclic pYR. (A and B) Structural ensembles of open (A) and cyclic (B) pYR. Disulfide bonds are highlighted in gray. (C) Three-dimensional structural comparison of pYR peptides with truncated ORB2K (PDB accession number 2O9Q) and SFTI-1 (PDB accession number 1JBL).

FIG 3

Relative trypsin inhibitory activities of SLF peptides, compared with SFTI-1. SFTI-1 has an equilibrium dissociation constant K_i of 17 pM (29). The statistical significance was calculated with respect to SFTI-1 by one-way analysis of variance (ANOVA) and the post hoc Bonferroni test. ****, P < 0.0001.

FIG 4

Cytotoxic activity of selected ORB and pYR peptides against noncancerous (HFF-1) (A) and cancerous (MM96L) (B) cells. Both open and cyclic forms of the peptides were tested, and melittin was used as a positive control (0% viability corresponds to 100% cell death).

FIG 5

In vivo efficacy of SLF peptides against S. aureus in the murine thigh infection model. Both open (A) and cyclic (B) forms were tested at 1 and 3 mg · kg⁻¹ (5 animals tested per compound). PBS and neomycin sulfate (3 mg · kg⁻¹) were used as negative (C−) and positive (C+) controls, respectively. The statistical significance was calculated with respect to the untreated group (C−) by one-way ANOVA and the post hoc Bonferroni test. **, P < 0.01; ***, P < 0.001; ****, P < 0.0001.

FIG 6

Serum stability of open and cyclic forms of pYR and SFTI-1. Backbone-cyclized pYR and SFTI-1 had better stability in human serum than did their open forms.