Discovery of small molecule inhibitors of Staphylococcus aureus ribonuclease P.

Swapnil Joshi¹, Wei Huang³, Loc Huynh¹, Tianshou Liu², Nidhi Kalia¹, Anna Pyle², Derek Taylor³, Michael E. Harris¹

1. Chemical Biology Division, Department of Chemistry, University of Florida, Gainesville, Florida.
2. Department of Molecular, Cellular and Developmental Biology, Yale University, New Haven, Connecticut.
3. Department of Biochemistry and Department of Pharmacology, School of Medicine, Case Western Reserve University, Cleveland, Ohio

Antimicrobial resistance continues to present a formidable medical challenge and there is an urgent need to discover and develop new antibiotics. Highly structured RNAs play essential roles in numerous biological processes and many current antibiotics target rRNA, underscoring the potential for further discovery.  Non-coding RNAs in bacteria offer a plethora of attractive but relatively unexplored targets. Bacterial RNase P contains a large (400nt) highly structured non-coding RNA (P RNA) that catalyzes an essential step in tRNA maturation. P RNA and the smaller protein unit (rnpA) are both essential genes in pathogenic bacteria by virtue of their role in supporting protein synthesis making RNase P an attractive target for developing new antimicrobials. Previous efforts to identify RNase P small molecule inhibitors have been unsuccessful or yielded leads with limited potential or targeted enzymes from non-pathogens. Using an established fluorescence polarization assay we screened three different small molecule libraries (total 27,360 compounds), including an RNA targeted collection, for inhibitors of S. aureus RNase P. Thus far secondary assays confirmed inhibition of RNase P for multiple compounds with IC₅₀ values of 20-100 μM. To establish specificity compounds were tested for inhibition of RhsP2, a tRNA modifying enzyme, and Nsp15, a viral RNA endonuclease. A subset of compounds with distinct chemical structures were discovered as apparently specific inhibitors of RNase P. An array of experimental tools available for studying RNase P assembly, substrate binding, and kinetics are being employed to establish mechanism of action and identifying binding sites. Targeting essential non-coding RNAs represents a fertile area for RNA-based drug discovery, and identification of small molecule inhibitors of S. aureus RNase P would represent an important step forward with potentially significant impact for antibiotic discovery.