Use of a small molecule microarray screen to identify inhibitors of the catalytic RNA subunit of Methanobrevibacter smithii RNase P.

Abstract

Despite interest in developing therapeutics that leverage binding pockets in structured RNAs-whose dysregulation leads to diseases-such drug discovery efforts are limited. Here, we have used a small molecule microarray (SMM) screen to find inhibitors of a large ribozyme: the Methanobrevibacter smithii RNase P RNA (Msm RPR, ∼300 nt). The ribonucleoprotein form of RNase P, which catalyzes the 5'-maturation of precursor tRNAs, is a suitable drug target as it is essential, structurally diverse across life domains, and present in low copy. From an SMM screen of 7,300 compounds followed by selectivity profiling, we identified 48 hits that bound specifically to the Msm RPR-the catalytic subunit in Msm (archaeal) RNase P. When we tested these hits in precursor-tRNA cleavage assays, we discovered that the drug-like M1, a diaryl-piperidine, inhibits Msm RPR (KI, 17 ± 1 μM) but not a structurally related archaeal RPR, and binds to Msm RPR with a KD(app) of 8 ± 3 μM. Structure-activity relationship analyses performed with synthesized analogs pinpointed groups in M1 that are important for its ability to inhibit Msm RPR. Overall, the SMM method offers prospects for advancing RNA druggability by identifying new privileged scaffolds/chemotypes that bind large, structured RNAs.