Herein we seek to advance antibiotic development of the new target Peptidyl-tRNA hydrolase (Pth). Pth enzymes are essential in all living organisms, recovering limited tRNA molecules from peptidyl-tRNA produced primarily by ribosome stalling. Bacteria have one essential Pth enzyme, Pth1. It is highly conserved across species and very different from the redundant, multi-component Pth system in eukaryotes. Thus Pth1 is a promising target for novel antibiotic development. Moreover, interrupting protein biosynthesis is a proven antibiotic strategy. Targeting an ancillary essential enzyme, Pth1 not the ribosome, holds considerable potential for synergy with existing antibiotics that block protein synthesis. Since there are 10 to 100 times less Pth1 than ribosomes in bacterial cells, small molecule inhibition of Pth1 has a considerable stoichiometric advantage as well. Our past work reveals the potential for both broad and narrow spectrum Pth1 inhibition, further elevating potential for Pth1 as an effective antibiotic target. Taken together, Pth1 is an outstanding candidate for new antibiotic development. The objectives of this proposal are to determine the effects removing Pth1 from human cells, search largely unexplored aquatic fungal extracts for Pth1 inhibitors, and map the evolution of resistance when bacteria are faced with Pth1 inhibition. In Aim 1, we delete Pth1 from the genome to determine the effects on human cells. In yeast, knockout of Pth1 had no effect, thus minimal impacts are expected. This aim will characterize what effects Pth1 inhibition has in human cells. In Aim 2, we search an aquatic fungal library for Pth1 inhibitors. I addition to identifying small molecules that inhibit Pth1, this aim evaluates the prospect of and establishes expectations for future larger scale efforts. Aim 3 takes advantage of a newly discovered Pth1 inhibitor to understand evolution of resistance in bacteria. The time required for resistance to develop and mechanisms by which it develops will be the first information of its kind. Together these aims provide new insight into understudied Pth enzymes and Pth1 inhibition. They also set a strong foundation for furthering Pth1 as an antibiotic target. This project is highly relevant to human health addressing the continuous emergence of pathogenic bacteria resistant to currently used antibiotics. Innovation is evident from providing fundamental insight into the cellular effects of compromised Pth1 function in human cells, searching the largely unexplored realm of aquatic fungi for inhibitors, and for the first time mapping mechanisms by which bacteria evolve resistance to Pth1 inhibition.