title: A real-time fluorescence polarization activity assay to screen for inhibitors of bacterial ribonuclease P.
abstract: Ribonuclease P (RNase P) is an essential endonuclease that catalyzes the 5' end maturation of precursor tRNA (pre-tRNA). Bacterial RNase P is an attractive potential antibacterial target because it is essential for cell survival and has a distinct subunit composition compared to the eukaryal counterparts. To accelerate both structure-function studies and discovery of inhibitors of RNase P, we developed the first real-time RNase P activity assay using fluorescence polarization/anisotropy (FP/FA) with a 5' end fluorescein-labeled pre-tRNAAsp substrate. This FP/FA assay also detects binding of small molecules to pre-tRNA. Neomycin B and kanamycin B bind to pre-tRNAAsp with a Kd value that is comparable to their IC50 value for inhibition of RNase P, suggesting that binding of these antibiotics to the pre-tRNA substrate contributes to the inhibitory activity. This assay was optimized for high-throughput screening (HTS) to identify specific inhibitors of RNase P from a 2880 compound library. A natural product derivative, iriginol hexaacetate, was identified as a new inhibitor of Bacillus subtilis RNase P. The FP/FA methodology and inhibitors reported here will further our understanding of RNase P molecular recognition and facilitate discovery of antibacterial compounds that target RNase P.
authors.affiliation: Department of Chemistry, University of Michigan, Ann Arbor, MI 48109, USA.
authors.forename: Xin
authors.initials: X
authors.lastname: Liu
authors.affiliation: Department of Chemistry, University of Michigan, Ann Arbor, MI 48109, USA.
authors.forename: Yu
authors.initials: Y
authors.lastname: Chen
authors.affiliation: Department of Chemistry, University of Michigan, Ann Arbor, MI 48109, USA Department of Biological Chemistry, University of Michigan Medical School, Ann Arbor, MI 48109, USA fierke@umich.edu.
authors.forename: Carol A
authors.initials: CA
authors.lastname: Fierke
mesh_terms: D000900:Anti-Bacterial Agents / Q000494:pharmacology*; D001412:Bacillus subtilis / Q000201:enzymology; D004791:Enzyme Inhibitors / Q000494:pharmacology*; D005454:Fluorescence Polarization / Q000379:methods*; D005601:Framycetin / Q000494:pharmacology; D057166:High-Throughput Screening Assays / Q000379:methods; D007612:Kanamycin / Q000031:analogs & derivatives / Q000494:pharmacology; D059367:RNA Cleavage; D012343:RNA, Transfer / Q000378:metabolism; D043262:Ribonuclease P / Q000037:antagonists & inhibitors*
chemical_list: D000900:Anti-Bacterial Agents; D004791:Enzyme Inhibitors; C001495:bekanamycin; D005601:Framycetin; D007612:Kanamycin; D012343:RNA, Transfer; D043262:Ribonuclease P