Poster abstracts

Poster number 42 submitted by Vaishnavi Sidharthan

Identification of inhibitors of Methanobrevibacter smithii RNase P using small molecule microarrays

Vaishnavi Sidharthan (Department of Chemistry & Biochemistry, and Center for RNA Biology, The Ohio State University), Kara Dunne-Dombrink (Chemical Biology Laboratory, National Cancer Institute), Walter J. Zahurancik, Ila A. Marathe, Lien B. Lai (Department of Chemistry & Biochemistry, and Center for RNA Biology, The Ohio State University), John S. Schneekloth Jr. (Chemical Biology Laboratory, National Cancer Institute), Venkat Gopalan (Department of Chemistry & Biochemistry, and Center for RNA Biology, The Ohio State University)

Abstract:
RNase P is an essential and ubiquitous endoribonuclease that catalyzes the Mg(II)-dependent 5' maturation of precursor tRNAs (pre-tRNAs). The ribonucleoprotein form of this enzyme comprises a catalytic RNase P RNA (RPR) and a variable number of RNase P protein (RPP) cofactors: one in bacteria, up to five in archaea, and up to ten in eukaryotes1. Since the five archaeal RPPs have eukaryotic homologs, the simpler composition and biochemical tractability of archaeal RNase P have rendered it an experimental surrogate for the larger eukaryotic relative1. This idea inspired advances including the characterization of different archaeal RNase P variants, which we plan to leverage for our drug discovery initiative that seeks to exploit the structural diversity of RNase P across the three domains of life. Here, motivated by two reasons, we sought to identify inhibitors of RNase P from Methanobrevibacter smithii (Msm), a methanogenic archaeon found in the animal gut. First, Msm RPR (~350 nt) is functional in vitro without protein cofactors. Second, the presence of Msm in the mouse gut is associated with increased energy harvest and host obesity2. As selective targeting of Msm RNase P (without drastic alterations to the gut microbial landscape) may allow a new anti-obesity therapeutic approach, we set out to identify binders of Msm RPR using a small molecule microarray (SMM) 3 library of ~7000 compounds. Selected hits were then tested in pre-tRNA cleavage assays with Msm RPR to identify bona fide inhibitors. Surprisingly, Michaelis-Menten analyses revealed that one of the hits is an uncompetitive inhibitor and that it is completely ineffective against another structurally related archaeal RPR. Ongoing structure-activity relationship analyses with derivatives of this compound will help uncover and improve the basis for its inhibition and selectivity. Since a pre-tRNA was not included in the SMM screen, the identification of an uncompetitive inhibitor suggests that the Msm RPR conformational ensemble must include structures pre-organized for substrate binding and cleavage. While our results heighten the promise of identifying inhibitors of Msm RNase P, they also showcase the value of SMM-centered screens to find functional modulators of large RNAs.

References:
1. Gopalan et al. (2018) RNA 24, 1–5
2. Samuel et al. (2006) Proc Natl Acad Sci USA 103, 10011–10016
3. Connelly et al. (2017) Methods Mol Biol 1518, 157−175

Keywords: Msm RNase P, small molecule microarray, drug targeting