Investigating Small Molecule Inhibitors Targeting the RNase P Enzyme from Pathogenic Bacteria

Abstract

Antimicrobial resistance (AMR) is a persistent problem in healthcare. This is particularly represented by ESKAPE pathogens—a group of highly prevalent and multidrug-resistant bacteria that pose a significant threat to public health. Targeting essential non-coding RNAs with small molecules offers an excellent avenue to develop new antibiotics because they offer a broader range of druggable sites, reduced likelihood of resistance mutations, and novel mechanisms of bacterial suppression. This study aims to develop antibiotics specifically targeting a non-coding RNA-based enzyme called Ribonuclease P (RNase P), an essential ribozyme involved in tRNA maturation. High-throughput screening efforts previously identified small molecule inhibitors of Staphylococcus aureus RNase P. Here, we evaluate those inhibitors against RNase P from Enterococcus faecium, a related ESKAPE pathogen, to determine whether similar inhibition patterns and mechanisms occur between the two RNase P enzymes. Inhibitory activity was assessed using gel electrophoresis-based assays to measure formation of a fluorescently labeled 5’ leader product generated by cleavage of the intact pre-tRNA substrate. Two small molecules exhibited promising inhibitory activity (Z118611174 and Z214790706), with the latter undergoing further analysis via IC₅₀ testing. This research lays the groundwork for future drug discovery efforts, potentially leading to a novel class of antibiotics.