Stapled Anoplin as an Antibacterial Agent

Abstract

Anoplin is a linear 10-amino acid amphipathic peptide (Gly-Leu-Leu-Lys-Arg-Ile-Lys-Thr-Leu-Leu-NH2) derived from the venom sac of the solitary wasp. It has broad antimicrobial activity, including an antibacterial one. However, the inhibition of bacterial growth requires several dozen micromolar concentrations of this peptide. Anoplin is positively charged and directly interacts with anionic biological membranes forming an α-helix that disrupts the lipid bilayer. To improve the bactericidal properties of anoplin by stabilizing its helical structure, we designed and synthesized its analogs with hydrocarbon staples. The staple was introduced at two locations resulting in different charges and amphipathicity of the analogs. Circular dichroism studies showed that all modified anoplins adopted an α-helical conformation, both in the buffer and in the presence of membrane mimics. As the helicity of the stapled anoplins increased, their stability in trypsin solution improved. Using the propidium iodide uptake assay in Escherichia coli and Staphylococcus aureus, we confirmed the bacterial membrane disruption by the stapled anoplins. Next, we tested the antimicrobial activity of peptides on a range of Gram-negative and Gram-positive bacteria. Finally, we evaluated peptide hemolytic activity on sheep erythrocytes and cytotoxicity on human embryonic kidney 293 cells. All analogs showed higher antimicrobial activity than unmodified anoplin. Depending on the position of the staple, the peptides were more effective either against Gram-negative or Gram-positive bacteria. Anoplin[5-9], with a lower positive charge and increased hydrophobicity, had higher activity against Gram-positive bacteria but also showed hemolytic and destructive effects on eukaryotic cells. Contrary, anoplin[2-6] with a similar charge and amphipathicity as natural anoplin effectively killed Gram-negative bacteria, also pathogenic drug-resistant strains, without being hemolytic and toxic to eukaryotic cells. Our results showed that anoplin charge, amphipathicity, and location of hydrophobic residues affect the peptide destructive activity on the cell wall, and thus, its antibacterial activity. This means that by manipulating the charge and position of the staple in the sequence, one can manipulate the antimicrobial activity.

γ-Core Guided Antibiotic Design Based on Human Enteric Defensin 5

“…Considering that the hydrophobic residues contribute to the membrane interactions of RC18, and because hydrophobic residues may account for the virulence of certain AMPs [ 40 , 41 ], we next asked if RC18 was toxic to host cells. Actually, the important cytotoxicity dose often hampers the development of many potent AMPs, such as melittin and latarcin [ 42 , 43 ], into antibiotic candidates.…”

Conjugates of Aminoglycosides with Stapled Peptides as a Way to Target Antibiotic-Resistant Bacteria

“…Peptide Synthesis. Anoplin and anoplin [2][3][4][5][6] were synthesized according to the procedures described by Wojciechowska et al 49 Alkyne-anoplin, alkyne-anoplin [2][3][4][5][6], Cys-anoplin, Cys-anoplin [2][3][4][5][6] were obtained by coupling of 10-undecynoic acid or Fmoc-Cys(Mmt)-OH at the Nterminus of anoplin and anoplin [2][3][4][5][6]. These residues, as well as all preceding amino acids, were manually added during solid phase peptide synthesis using 9-fluorenylmethoxycarbonyl (Fmoc) strategies.…”

“…Anoplin [2][3][4][5][6] proved a helical peptide that effectively penetrated the membrane and cell wall mimics of the E. coli K12 strain and showed 4−16 μM MIC against Gram-negative strains. 49 Therefore, we suppose that the better activity of AMG-anoplin [2][3][4][5][6] conjugates is related to stronger affinity of the stapled peptide to the bacterial cell membrane. Anoplin [2][3][4][5][6] as a permeabilizing agent affects bacterial cell membrane integrity.…”

“…48 We also showed that hydrocarbon stapling of anoplin stabilizes its helical structure and increases its proteolytic stability and antibacterial activity (up to 8−16 fold as compared to unmodified anoplin). 49 In addition, anoplin stapled between the second and sixth amino acid is neither hemolytic nor cytotoxic. 49 Several strategies to develop new antimicrobial agents were based on conjugation of two compounds in order to increase their uptake and activity.…”

“…We focus on the anoplin peptide whose modifications and antibacterial potential have been recently studied. − Anoplin is a naturally found amphipathic peptide (N ter -Gly-Leu-Leu-Lys-Arg-Ile-Lys-Thr-Leu-Leu-C ter ) derived from the venom sac of the solitary wasp, with rather low antibacterial activity. However, we and others demonstrated the antibacterial potential of anoplin derivatives. , Typically, amphipathic and stable secondary structures are key for cytoplasmic membrane disruption and effective antibacterial activity of peptides . We showed that anoplin adopts a helical structure in the presence of membrane mimics and lipopolysaccharides .…”