Discovery of Selective RNA-Binding Small Molecules by Affinity-Selection Mass Spectrometry

Abstract

Recent advances in understanding the relevance of noncoding RNA (ncRNA) to disease have increased interest in drugging ncRNA with small molecules. The recent discovery of ribocil, a structurally distinct synthetic mimic of the natural ligand of the flavin mononucleotide (FMN) riboswitch, has revealed the potential chemical diversity of small molecules that target ncRNA. Affinity-selection mass spectrometry (AS-MS) is theoretically applicable to high-throughput screening (HTS) of small molecules binding to ncRNA. Here, we report the first application of the Automated Ligand Detection System (ALIS), an indirect AS-MS technique, for the selective detection of small molecule-ncRNA interactions, high-throughput screening against large unbiased small-molecule libraries, and identification and characterization of novel compounds (structurally distinct from both FMN and ribocil) that target the FMN riboswitch. Crystal structures reveal that different compounds induce various conformations of the FMN riboswitch, leading to different activity profiles. Our findings validate the ALIS platform for HTS screening for RNA-binding small molecules and further demonstrate that ncRNA can be broadly targeted by chemically diverse yet selective small molecules as therapeutics.

Structural Ensembles of Ribonucleic Acids From Solvent Accessibility Data: Application to the S-Adenosylmethionine (SAM)-Responsive Riboswitch

“…Indeed, the recent discovery of the antibacterial small molecule, ribocil-B, which targets the flavin mononucleotide (FMN) riboswitch supports the notion that riboswitches are druggable RNA targets (27). As a result of this and related discoveries (28), the identification and design of small molecules that target riboswitches has become an active area of research. Because individual riboswitches have evolved to bind to a specific ligand, attempts to design compounds that target riboswitches have focused on identifying compounds that are analogs of the cognate ligand or compounds that can recapitulate its interaction pattern.…”

Unveiling the druggable RNA targets and small molecule therapeutics

“…126,180 Also, ESI-MS was used by Dremann et al to identify RNA-peptide complexes specific for bacterial ribosome helix 69 (H69). 181 Fourier-transform ion cyclotron resonance (FT-ICR) MS provides high-resolution spectra, isotope-resolved precursor ion selection and accurate mass assignments. FT-ICR MS has been used to study the interaction between two closely related model RNA constructs corresponding to the decoding sites of the prokaryotic and eukaryotic rRNA and a collection of aminoglycoside antibiotics.…”

“…ALIS has been recently used to identify and characterize two RNA ligands that competitively bind to the flavin mononucleotide (FMN) riboswitch and exhibit drug-like functional capabilities, potent analog of ellipticine (WG-1) and quinolone (WG-3) class of antibacterial, respectively. 181 These MS-based screening aids in the discovery of novel chemical classes that bind RNAs that may be missed by nonbiophysical screening approaches.…”

Challenges of Connecting Chemistry to Pharmacology: Perspectives from Curating the IUPHAR/BPS Guide to PHARMACOLOGY

“…where "A" and "P" are missing) as exemplified by Nusinersen (LID 9416). We also expect to see small molecules optimized for direct binding against defined nucleic RNA sequences where we will have to replace P with a defined RNA accession number 46…”