Project Summary Apidaecin (Api), an antimicrobial peptide produced by honeybees, has a unique mechanism of action. Our previous studies showed that after entering the cell of Gram-negative bacteria through the SbmA transporter, Api binds in the nascent peptide exit tunnel of the bacterial ribosome, traps the release factors and arrests translation at stop codons. As such, Api represents the first-ever specific inhibitor of translation termination. Our subsequent whole-genome studies have shown that arresting terminating ribosomes triggers a number of downstream events, including ribosome queuing and readthrough of stop codons, that accentuate Api's primary inhibitory action. The idiosyncratic mode of binding to the target, the unique mechanism of action, and the triggering of downstream effects harmful for the bacterial cell, make Api an attractive model for developing novel antibiotics with improved properties. In the current proposal we will use the combined effort of three laboratories with expertise in ribosomal antibiotics, peptide chemistry and structural analysis to advance the fundamental understanding of action of Api-like inhibitors and identify Api derivatives with superior on-target activity and expanded spectrum of antibacterial action. We will achieve these goals by generating and testing arrays of Api variants directly in the bacterial cell by the controlled expression of gene libraries, synthesizing peptides with specific chemical modifications, and determining X-ray crystal structures of ribosome- Api complexes. Specifically: In Aim 1, we will identify Api-derived peptides with improved activity upon ribosomes from Gram-negative and Gram-positive pathogens. In Aim 2, the spectrum of action of Api-like peptides will be expanded by bypassing the necessity for uptake by the SbmA transporter. Finally, in Aim 3, we will broaden the variety of natural antimicrobial peptides that have the ability to inhibit translation termination. All of the Aims are independent from each other, but the specific implementation of experiments planned for each aim could be influenced by the results emerging from the other aims. The reagents and tools that will be generated in the course of the proposed work will serve as leads for future clinical development. Importantly, the results obtained in the proposed studies will significantly advance the general field of ribosome-targeting antibacterial peptides, which currently is still in its infancy.