RNase P Inhibitors Identified as Aggregators

Abstract

RNase P is an essential enzyme responsible for tRNA 5'-end maturation. In most bacteria, the enzyme is a ribonucleoprotein consisting of a catalytic RNA subunit and a small protein cofactor termed RnpA. Several studies reported small molecule inhibitors directed against bacterial RNase P that were identified by high-throughput screenings. Using the bacterial RNase P enzymes from Thermotoga maritima, Bacillus subtilis and Staphylococcus aureus as model systems, we found that such compounds, including RNPA2000 and derivatives, iriginol hexaacetate and purpurin, induce the formation of insoluble aggregates of RnpA rather than acting as specific inhibitors. In the case of RNPA2000, aggregation was induced by Mg2+ ions. These findings were deduced from solubility analyses by microscopy and HPLC, RnpA-inhibitor co-pulldown experiments, detergent addition and RnpA titrations in enzyme activity assays. Finally, we used a B. subtilis RNase P depletion strain, whose lethal phenotype could be rescued by a protein-only RNase P of plant origin, for inhibition zone analyses on agar plates. These cell-based experiments argued against RNase P-specific inhibition of bacterial growth by RNPA2000. We were also unable to confirm the previously reported non-specific RNase activity of S. aureus RnpA itself. Our results indicate that high-throughput screenings searching for bacterial RNase P inhibitors are prone to the identification of “false positives” that are also termed Pan-assay interference compound s (PAINS).

The specificity landscape of bacterial ribonuclease P

“…Synthetic substrates derived from minihelices or bipartite pre-tRNAs severed at the anticodon loop have also been employed with end-attached fluorophore/quencher pairs. However, promising RNase P targeting drugs were recently identified as protein aggregators that do not inhibit RNase P with specificity, which calls into question their mode of action in vivo ( 109 ).…”

Targeting Ribonucleases with Small Molecules and Bifunctional Molecules

“…Additionally, RNPA2000 was reported to show weak RNase P inhibitory activity (IC 50 : 125 μM, Figure ). A concern for the identified RNase P inhibitors, including RNPA2000, iriginol hexaacetate, and purpurin, was that they acted as aggregators and are likely unspecific RNase P inhibitors. , In addition to small molecules, a FRET assay was employed to evaluate rationally designed and modified oligonucleotides as inhibitors of RNase P. Antisense oligonucleotides targeting the RNA component of RNase P were coupled to cell-penetrating peptides to yield conjugates that inhibited bacterial growth. The best-performed conjugates showed IC 50 values of ∼100 nM, being the most potent RNase P inhibitors reported to date …”