Truncated and Disubstituted (P4K and L7R) Oncocin Peptides Target the Ribosome with Improved Antibacterial Activity.

Development of therapeutics with alternative mechanisms of action is essential to combat bacteria with rapidly emerging drug resistance. The proline-rich antimicrobial peptide (PrAMP) oncocin (Onc) targets bacterial ribosomes but differs from conventional antibiotics in its mechanism of action. A cell-based plasmid expression system was deployed in Escherichia coli (E. coli) to gain direct knowledge of structure-activity relationships with truncated and/or disubstituted (P4K and L7R) oncocin variants. We identified a shortened oncocin sequence that maintained antibiotic activity and favorable cationic amino acid substitutions that led to a 2-fold decrease in the minimum inhibitory concentration (MIC) compared to the parent 19mer oncocin sequence. In cellulo dimethyl sulfate probing and in silico molecular modeling were performed to reveal the key oncocin variant interactions with the peptidyl transferase center and P site of E. coli 23S rRNA. Multiple sequence alignment of over 1000 bacterial 23S rRNAs and 3D modeling highlight the interactions between the oncocin variant and rRNA nucleotides that are highly conserved across a broad range of bacterial species. The integration of structural and sequence analyses provides a useful framework for the future structure-guided development of PrAMPs. Overall, our multipronged approach has led to the characterization of the inhibitory activity of PrAMPs under cellular conditions with potential implications for further development of peptide antibiotics.