Identifying inhibitors of the bacterial peptidyl-tRNA hydrolase

Peptidyl-tRNA hydrolase (Pth) is an essential bacterial enzyme critical for protein synthesis and whose inactivation leads to cell death. Pth recycles peptidyl-tRNAs that prematurely dissociate from the translated ribosome. Additionally, Pth is involved in rescuing the large ribosomal subunit that remains associated with peptidyl-tRNA after the splitting of the stalled ribosome. It may also contribute to other ribosome rescue mechanisms. Despite its essentiality, efforts to identify Pth inhibitors have been hindered by challenges in generating suitable substrates for high-throughput screening (HTS). To address this, we designed an oligonucleotide-based Pth substrate that mimics peptidyl-tRNA and used it to develop and validate an HTS assay based on the Förster resonance energy transfer (FRET). Limited preliminary studies have identified several compounds with inhibitory activity against Pth from Escherichia coli and Klebsiella pneumoniae. This proposal aims to advance Pth inhibitor development by synthesizing and optimizing derivatives of the most potent hits with sub-micromolar IC50 values to enhance their activity against K. pneumoniae Pth and improve cellular uptake. Concurrently, we will expand the screening campaign to identify novel inhibitor scaffolds. The interaction of these compounds with Pth will be studied by SPR and X-ray crystallography, to guide medicinal chemistry optimization. In addition, we will characterize their mechanisms of action, cellular targets, effects on protein synthesis, and potential resistance mechanisms. The anticipated outcome is the identification of a lead inhibitor scaffold suitable for preclinical development as a foundation for a novel antibiotic platform.