Project Summary Apidaecin (Api) and Drosocin (Dro), are proline-rich antimicrobial peptides (PrAMPs) produced by honeybees and fruit flies, respectively, which share a unique mechanism of action. Our previous studies of Api showed that upon entering Gram-negative bacterial cells through the SbmA transporter, Api binds in the exit tunnel of ribosomes that have just released the newly made protein and arrests the ribosomes at stop codons by trapping the associated tRNA and release factor. As such, Api represents the first-ever described specific inhibitor of translation termination. Our subsequent whole-genome studies revealed that arresting terminating ribosomes triggers several downstream events that accentuate the inhibitory action of this PrAMP, including ribosome queuing and readthrough of stop codons. Our preliminary data indicate that Dro, despite its distinct amino acid sequence, inhibits the termination step of translation as well, by a mechanism likely resembling that of Api. Their idiosyncratic mode of binding to the target, the unique mechanism of action, and the triggering of downstream effects harmful for the bacterial cell, make these antibacterial peptides an attractive model for developing novel antibiotics. Furthermore, the biological nature of these PrAMPs opens unique opportunities for their screening and optimization by generating hundreds of thousands of peptide variants directly in bacterial cells. In the current proposal we will use the combined effort of three laboratories with expertise in biochemistry and genomics of ribosomal antibiotics, in peptide chemistry and in structural analysis of ribosome-antibiotic complexes to advance the fundamental understanding of the mechanism of action of Api- and Dro-like translation termination inhibitors and identify derivatives with superior on-target activity and expanded spectrum of antibacterial action. In order to achieve these goals we will test arrays of Api and Dro variants in bacterial cells by the tunable expression of peptide gene libraries, determine high-resolution X-ray crystal structures of ribosome-peptide complexes, and employ rational structure-based design to generate via chemical synthesis peptide variants with superior properties. 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 analyze the ribosome binding and mechanism of action of Dro-like peptides and use comparative analysis to identify the key features that define the class of antimicrobial peptides that target translation termination. The three Aims are tightly interconnected but completely independent from each other. The reagents and tools that will be generated in the course of the proposed work are aimed to serve as leads for future clinical deve...