Caprine Bactenecins as Promising Tools for Developing New Antimicrobial and Antitumor Drugs

Abstract

Proline-rich antimicrobial peptides (PR-AMPs) having a potent antimicrobial activity predominantly toward Gram-negative bacteria and negligible toxicity toward host cells, are attracting attention as new templates for developing antibiotic drugs. We have previously isolated and characterized several bactenecins that are promising in this respect, from the leukocytes of the domestic goat Capra hircus: ChBac5, miniChBac7.5N-α, and -β, as well as ChBac3.4. Unlike the others, ChBac3.4 shows a somewhat unusual pattern of activities for a mammalian PR-AMP: it is more active against bacterial membranes as well as tumor and, to the lesser extent, normal cells. Here we describe a SAR study of ChBac3.4 (RFRLPFRRPPIRIHPPPFYPPFRRFL-NH2) which elucidates its peculiarities and evaluates its potential as a lead for antimicrobial or anticancer drugs based on this peptide. A set of designed structural analogues of ChBac3.4 was explored for antibacterial activity toward drug-resistant clinical isolates and antitumor properties. The N-terminal region was found to be important for the antimicrobial action, but not responsible for the toxicity toward mammalian cells. A shortened variant with the best selectivity index toward bacteria demonstrated a pronounced synergy in combination with antibiotics against Gram-negative strains, albeit with a somewhat reduced ability to inhibit biofilm formation compared to native peptide. C-terminal amidation was examined for some analogues, which did not affect antimicrobial activity, but somewhat altered the cytotoxicity toward host cells. Interestingly, non-amidated peptides showed a slight delay in their impact on bacterial membrane integrity. Peptides with enhanced hydrophobicity showed increased toxicity, but in most cases their selectivity toward tumor cells also improved. While most analogues lacked hemolytic properties, a ChBac3.4 variant with two additional tryptophan residues demonstrated an appreciable activity toward human erythrocytes. The variant demonstrating the best tumor/nontumor cell selectivity was found to more actively initiate apoptosis in target cells, though its action was slower than that of the native ChBac3.4. Its antitumor effectiveness was successfully verified in vivo in a murine Ehrlich ascites carcinoma model. The obtained results demonstrate the potential of structural modification to manage caprine bactenecins' selectivity and activity spectrum and confirm that they are promising prototypes for antimicrobial and anticancer drugs design.

Keywords: antibacterial activity; antibiofilm activity; antibiotics; antitumor activity; bactenecins; proline-rich antimicrobial peptides; structure–activity relationship; synergy.

Figure 1

The roadmap of ChBac3.4 modifications’ activity testing performed in this study. The methods used were: 1) broth microdilution assay; 2) spectrophotometric membrane permeabilization assay on E. coli ML-35p; 3) crystal violet assay; 4) checkerboard titration; 5) MTT-test; 6) hemolysis assay; 7) light microscopy & cell counting with Trypan blue dye; 8) fluorescent microscopy & cell counting with Annexin V-Cy3 Apoptosis Detection kit dyes; 9) Kaplan–Meier survival curves analysis.

Figure 2

Effects of ChBac3.4 modifications on membrane permeability of E.coli ML-35p. The left panel shows changes in permeability of the outer membrane for the chromogenic marker nitrocefin; the right panel demonstrates changes in permeability of the inner membrane for the chromogenic probe molecule o-nitrophenyl-β-D-galactoside (ONPG). Concentrations of AMPs equal to 2×MIC were used to assess their membranolytic potential. Curves illustrate the accumulation of the colored products after hydrolysis by corresponding bacterial enzymes (periplasmic β-galactosidase for nitrocefin or cytoplasmic β-galactosidase for ONPG), that can only occur when membranes damaged by tested peptides allow access of the substrate to the enzyme. The lag time required for initiating the rise in optical density (OD), the rising period up to a plateau, and the slope of the curves during this period all characterize the velocity and the extent of the damaging.

Figure 3

Hemolytic action of ChBac3.4 variants toward human red blood cells. Dots with error bars represent mean values and standard deviations derived from three independent experiments performed in triplicate.

Figure 4

Dynamics of cytocidal action of ChBac3.4-1-COOH on K562 cells in comparison with the membranolytic porcine PG-1 and native ChBac3.4. (A) Time-killing curves illustrating the development of cytocidal effect for various concentrations of the peptides during the first 3 h of incubation, based on the data from the trypan blue dye exclusion assay. (B) Bar chart showing the percentage of cells dying via necrosis or apoptosis in the presence of different concentrations of the peptides.

Figure 5

Kaplan–Meier survival curves of mice treated with ChBac3.4-1-COOH in a subcutaneous Ehrlich ascites carcinoma model. Mice in experimental groups were injected with specified doses of the peptide dissolved in 0.1 ml of deionized water once per week. The control group was injected with the same amount of deionized water. A log rank test shows statistically significant difference between both 100 μg receiving group (p = 0.039) and 1 μg reseiving group (p = 0.034) and control.