Oncocin

The proline-rich antimicrobial peptide oncocin is remarkably active in vitro against a number of important Gram-negative bacteria of concern to humans owing to their increasing resistance to antibiotics, i.e. Enterobacteriaceae (Escherichia coli, Klebsiella pneumoniae and Enterobacter cloacae) and non-fermenting species (Acinetobacter baumannii and Pseudomonas aeruginosa). Degradation of oncocin in mouse serum was investigated in this study. Several approaches to stabilise the main cleavage sites (C-terminal to Arg-15 and N-terminal to Arg-19) by substituting either or both arginine (Arg) residues with non-proteinogenic amino acids, i.e. α-amino-3-guanidino-propionic acid, homoarginine, nitro-arginine, N-methyl-arginine, β-homoarginine, D-arginine (D-Arg) or ornithine (Orn), were tested. These modifications were found to increase the half-life of oncocin in full mouse serum. For oncocin with two Orn residues in positions 15 and 19, the half-life in full serum increased from 25 min to 3 h. An increase of >8 h was observed for oncocin with two D-Arg residues at these same positions. The antibacterial activities of these modified sequences were slightly better than the original oncocin sequence. Moreover, the three most stable analogues were found to be bactericidal against E. coli and were not toxic to HeLa cells or haemolytic to human erythrocytes.

Oncocin is a proline-rich antimicrobial peptide that inhibits protein synthesis by binding to the bacterial ribosome. In this work, the antimicrobial activity of oncocin was improved by systematic peptide mutagenesis and activity evaluation. We found that a pair of cationic substitutions (P4K and L7K/R) improves the activity by 2-4 fold (p<0.05) against multiple Gram-negative bacteria. An in vitro transcription / translation assay indicated that the increased activity was not because of stronger ribosome binding. Rather a cellular internalization assay revealed a higher internalization rate for the optimized analogs thereby suggesting a mechanism to increase potency. In addition, we found that the optimized peptides' benefit is dependent upon nutrient-depleted media conditions. The molecular design and characterization strategies have broad potential for development of antimicrobial peptides.

Design and synthesis of conjugates consisting of the macrolide antibiotic desmycosin and fragments of the antibacterial peptide oncocin were performed in attempt to develop new antimicrobial compounds. New compounds were shown to bind to the E. coli 70S ribosomes, to inhibit bacterial protein synthesis in vitro, as well as to suppress bacterial growth. The conjugates of N-terminal hexa- and tripeptide fragments of oncocin and 3,2',4''-triacetyldesmycosin were found to be active against some strains of macrolide-resistant bacteria. By simulating molecular dynamics of the complexes of these compounds with the wild-type bacterial ribosomes and with ribosomes, containing A2059G 23S RNA mutation, the specific structural features of their interactions were revealed.