Poster abstracts

Poster number 128 submitted by Vaishnavi Sidharthan

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

Vaishnavi Sidharthan (Department of Chemistry & Biochemistry, and Center for RNA Biology, The Ohio State University), Christopher D. Sibley, Kara Dunne-Dombrink, Mo Yang (Chemical Biology Laboratory, National Cancer Institute), Walter J. Zahurancik (Department of Chemistry & Biochemistry, and Center for RNA Biology, The Ohio State University), Christian Heryakusuma, Biswarup Mukhopadhyay (Department of Biochemistry, Virginia Tech University), Damien B. Wilburn (Department of Chemistry & Biochemistry, and Center for RNA Biology, The Ohio State University), John S. Schneekloth Jr, Venkat Gopalan (Chemical Biology Laboratory, National Cancer Institute; Department of Chemistry & Biochemistry, and Center for RNA Biology, The Ohio State University)

Abstract:
There is great interest in leveraging binding pockets in structured long non-coding (lnc) RNAs to develop therapeutics that allow metabolic fine-tuning. For example, regulating methanogens in intestinal tracts may offer a modality to mitigate methane emission from livestock and to treat type 2 diabetes mellitus in human. In this context, drug targets in Methanobrevibacter smithii (Msm) warrant consideration as Msm and close relatives constitute a major part of cattle rumen and human intestine methanogen population. To this end, we have used a small molecule microarray (SMM) screen to find inhibitors of a lncRNA ribozyme: the Msm 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. Moreover, M1 retards the growth of Msm in culture. Overall, the SMM method offers prospects for advancing RNA druggability by identifying new privileged scaffolds/chemotypes that bind lncRNAs.

References:
1.Gopalan et al. (2018) RNA 24, 1–5
2.Connelly et al. (2017) Methods Mol Biol 1518, 157−175
3.Danielson et al. (2017) Front Microbiol 8, 226

Keywords: RNase P, drug discovery, small molecule microarray, Methanobrevibacter smithii