Improving the Activity of Trp-Rich Antimicrobial Peptides by Arg/Lys Substitutions and Changing the Length of Cationic Residues

Abstract

Antimicrobial peptides (AMPs) constitute a promising alternative for the development of new antibiotics that could potentially counteract the growing number of antibiotic-resistant bacteria. However, the AMP structure⁻function relationships remain unclear and detailed studies are still necessary. The positively charged amino acid residues (Arg and Lys) play a crucial role in the activity of most AMPs due to the promotion of electrostatic interactions between the peptides and bacterial membranes. In this work we have analyzed the antimicrobial and structural properties of several Trp-rich AMPs containing exclusively either Arg or Lys as the positively charged residues. Their antimicrobial activity and mechanism of action were investigated, showing that Lys residues give rise to a reduced antimicrobial potency for most peptides, which was correlated, in turn, with a decrease in their ability to permeabilize the cytoplasmic membrane of Escherichia coli. Additionally, the presence of Arg and Lys renders the peptides susceptible to degradation by proteases, such as trypsin, limiting their therapeutic use. Therefore, modifications of the side chain length of Arg and Lys were investigated in an attempt to improve the protease resistance of AMPs. This approach resulted in enhanced stability to trypsin digestion, and in several cases, shorter sidechains conserved or even improved the antimicrobial activity. All together, these results suggest that Arg-to-Lys substitutions, coupled with side chain length modifications, can be extremely useful for improving the activity and stability of AMPs.

Keywords: antimicrobial peptides; arginine; lysine; protease degradation; tritrpticin; trypsin.

Figure 1

Chemical structures of the amino acids arginine (Arg) and lysine (Lys) as well as their analogs with different side chain lengths. Abbreviations: Agb, (S)-2-amino-4-guanidinobutyric acid; hArg, homo-arginine; Dap, 2,3-diaminopropionic acid; Dab, 2,4-diaminobutyric acid; Orn, ornithine.

Figure 2

Permeabilization of the inner membrane of Eschericia coli ML35p by the action of several Tritrp analogs with different Arg and Lys side chain lengths. Bacterial cells (optical density (OD)₆₀₀ = 0.3) were incubated at 37 °C for 1 h in the presence of peptides at concentrations (µM) corresponding to the values indicated by the legends on the right. Results are average ± standard error of the mean (S.E.M.) (n = 4). Melittin was used as a control peptide.

Figure 3

Permeabilization of the inner membrane of E. coli ML35p by the action of several Tritrp analogs with Pro-to-Ala and Arg-to-Lys substitutions. Bacterial cells (OD₆₀₀ = 0.3) were incubated at 37 °C for 1 h in the presence of peptides at concentrations (µM) corresponding to the values indicated by the legends on the right. Results are average ± S.E.M. (n = 4).

Figure 4

Permeabilization of the inner membrane of E. coli ML35p by the action of several Trp-rich AMPs and magainin2-F5W with Arg-to-Lys mutations. Bacterial cells (OD₆₀₀ = 0.3) were incubated at 37 °C for 1 h in the presence of peptides at concentrations (µM) corresponding to the values indicated by the legends on the right. Results are average ± S.E.M. (n = 3).

Figure 5

Calcein leakage induced by analogs of the AMP Tritrp and melittin against large unilamellar vesicles (LUVs) composed of egg-derived phosphatidyl-choline (ePC)/egg-derived phosphatidyl-glycerol (ePG) (1:1) and ePC/cholesterol (Chol) (2.5:1). Peptides (0.2 µM) containing different numbers of aliphatic carbons in the Arg or Lys side chain where incubated in the presence of LUVs (2 µM) for 15 min at 25 °C. Results are average ± standard deviation (S.D.) (n = 3).

Figure 6

Far-ultraviolet (UV) circular dichroism spectra of several Tritrp-derived peptides in the presence (black) or absence (gray) of SDS micelles. The peptides (50 µM) were incubated in Tris buffer (10 mM) pH 7.5 in the presence or absence of 30 mM SDS.

Figure 7

Far-UV circular dichroism spectra of several Trp-rich AMPs in the presence (black) or absence (gray) of SDS micelles. The peptides (50 µM) were incubated in Tris buffer (10 mM) pH 7.5 in the presence or absence of 30 mM SDS. Magainin2-F5W was used as a helical control peptide.

Figure 8

Reverse-phase chromatography of trypsin digestion of AMPs. The peptides (10 µM) were incubated in the presence of trypsin (30 units) at 37 °C. Samples collected at times 0 (gray) and 60 min (black) were analyzed by reversed-phase high-performance liquid chromatography (RP-HPLC). The originally discovered and nonamidated tritrpticin peptide was used as control and analyzed at time intervals of 0, 15, 30, and 60 min (gray to black color scale, respectively). Abbreviation: Abs, absorbance.