Synergy with rifampin and kanamycin enhances potency, kill kinetics, and selectivity of de novo-designed antimicrobial peptides

Abstract

By choosing membranes as targets of action, antibacterial peptides offer the promise of providing antibiotics to which bacteria would not become resistant. However, there is a need to increase their potency against bacteria along with achieving a reduction in toxicity to host cells. Here, we report that three de novo-designed antibacterial peptides (DeltaFm, DeltaFmscr, and Ud) with poor to moderate antibacterial potencies and kill kinetics improved significantly in all of these aspects when synergized with rifampin and kanamycin against Escherichia coli. (DeltaFm and DeltaFmscr [a scrambled-sequence version of DeltaFm] are isomeric, monomeric decapeptides containing the nonproteinogenic amino acid alpha,beta-didehydrophenylalanine [DeltaF] in their sequences. Ud is a lysine-branched dimeric peptide containing the helicogenic amino acid alpha-aminoisobutyric acid [Aib].) In synergy with rifampin, the MIC of DeltaFmscr showed a 34-fold decrease (67.9 microg/ml alone, compared to 2 microg/ml in combination). A 20-fold improvement in the minimum bactericidal concentration of Ud was observed when the peptide was used in combination with rifampin (369.9 microg/ml alone, compared to 18.5 microg/ml in combination). Synergy with kanamycin resulted in an enhancement in kill kinetics for DeltaFmscr (no killing until 60 min for DeltaFmscr alone, versus 50% and 90% killing within 20 min and 60 min, respectively, in combination with kanamycin). Combination of the dendrimeric peptide DeltaFq (a K-K2 dendrimer for which the sequence of DeltaFm constitutes each of the four branches) (MIC, 21.3 microg/ml) with kanamycin (MIC, 2.1 microg/ml) not only lowered the MIC of each by 4-fold but also improved the therapeutic potential of this highly hemolytic (37% hemolysis alone, compared to 4% hemolysis in combination) and cytotoxic (70% toxicity at 10x MIC alone, versus 30% toxicity in combination) peptide. Thus, synergy between peptide and nonpeptide antibiotics has the potential to enhance the potency and target selectivity of antibacterial peptides, providing regimens which are more potent, faster acting, and safer for clinical use.

FIG. 1.

Chemical structures of kanamycin (22) (A) and rifampin (KEGG drug database) (B).

FIG. 2.

Synergy in bactericidal action. Histogram showing results for untreated and antibiotic-treated samples of E. coli ML35p. The antibiotic concentrations used are indicated below the bars. Cells were treated with or without drug for 18 h and plated on MH agar plates. After 20 h of incubation at 37°C, the colonies were counted. Synergy in bactericidal activity was observed for Ud and rifampin at an FBC index of 0.16. The log₁₀ CFU/ml observed at the end point is indicated above each bar. Standard deviations from triplicate experiments are plotted. RIF, rifampin.

FIG. 3.

Synergy enhances kill kinetics of antimicrobial peptides. Kill kinetics of ΔFmscr and the combination of ΔFmscr and kanamycin (KAN) at an FIC index of 0.5 against E. coli ML35p at the concentrations indicated.

FIG. 4.

Hemolytic activities of peptide and nonpeptide antibiotics alone (A) and in combination (B) at MIC and 10× MIC. The values above the bars indicate the concentrations (MIC [lightface] and 10× MIC [boldface]) at which the antibiotics alone or in combination were studied. For the combinations, the concentrations are given as x/y, where x and y represent the concentrations (μg/ml) of peptide and nonpeptide antibiotics, respectively. Peptide and nonpeptide antibiotics were incubated with 0.4% RBCs in PBS. The results are expressed as percent hemolysis. RBCs incubated with 0.1% Triton X-100 were considered to be 100% lysed. The percent hemolysis was calculated as follows: [OD₄₁₄ (antibiotic + RBCs) − OD₄₁₄ (RBCs in PBS)]/[OD₄₁₄ (RBCs in Triton X-100) − OD₄₁₄ (RBCs in PBS)] × 100. RIF, rifampin; KAN, kanamycin. Standard deviations from three observations are plotted.

FIG. 5.

Mammalian-cell cytotoxicity (MTT assay) of peptide and nonpeptide antibiotics at MIC and 10× MIC alone and in combination against L929 (A) and HeLa (B) cell lines. Test samples (individual antibiotics or antibiotic mixtures) were incubated with cells for 24 h in RPMI 1640. Untreated cells served as the negative control. The values above the bars indicate the concentrations (MIC [lightface] and 10× MIC [boldface]) at which the antibiotics alone or in combination were studied. For the combinations, the concentrations are given as x/y, where x and y represent the concentrations of peptide and nonpeptide antibiotics, respectively. The ratio of OD₅₇₀ for peptide-treated cells to OD₅₇₀ for untreated cells was used to calculate the percent viability of cells. RIF, rifampin; KAN, kanamycin. Standard deviations from three observations are plotted.