Structural dynamics influences the antibacterial activity of a cell-penetrating peptide (KFF)3K.

Given the widespread demand for novel antibacterial agents, we modified a cell-penetrating peptide (KFF)3K to transform it into an antibacterial peptide. Namely, we inserted a hydrocarbon staple into the (KFF)3K sequence to induce and stabilize its membrane-active secondary structure. The staples were introduced at two positions, (KFF)3K[5-9] and (KFF)3K[2-6], to retain the initial amphipathic character of the unstapled peptide. The stapled analogues are protease resistant contrary to (KFF)3K; 90% of the stapled (KFF)3K[5-9] peptide remained undigested after incubation in chymotrypsin solution. The stapled peptides showed antibacterial activity (with minimal inhibitory concentrations in the range of 2-16 µM) against various Gram-positive and Gram-negative strains, contrary to unmodified (KFF)3K, which had no antibacterial effect against any strain at concentrations up to 32 µM. Also, both stapled peptides adopted an α-helical structure in the buffer and micellar environment, contrary to a mostly undefined structure of the unstapled (KFF)3K in the buffer. We found that the antibacterial activity of (KFF)3K analogues is related to their disruptive effect on cell membranes and we showed that by stapling this cell-penetrating peptide, we can induce its antibacterial character.

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

The misuse and overuse of antibiotics led to the development of bacterial resistance to existing aminoglycoside (AMG) antibiotics and limited their use. Consequently, there is a growing need to develop effective antimicrobials against multidrug-resistant bacteria. To target resistant strains, we propose to combine 2-deoxystreptamine AMGs, neomycin (NEO) and amikacin (AMK), with a membrane-active antimicrobial peptide anoplin and its hydrocarbon stapled derivative. The AMG-peptide hybrids were conjugated using the click chemistry reaction in solution to obtain a non-cleavable triazole linker and by disulfide bridge formation on the resin to obtain a linker cleavable in the bacterial cytoplasm. Homo-dimers connected via disulfide bridges between the N-terminus thiol analogues of anoplin and hydrocarbon stapled anoplin were also synthesized. These hybrid compounds show a notable increase in antibacterial and bactericidal activity, as compared to the unconjugated ones or their combinations, against Gram-positive and Gram-negative bacteria, especially for the strains resistant to AMK or NEO. The conjugates and disulfide peptide dimers exhibit low hemolytic activity on sheep red blood erythrocytes.

Stapled Anoplin as an Antibacterial Agent.

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.