Optimization of a Protein-Targeted Medicine into an RNA-Specific Small Molecule.

Protein-targeted small molecule medicines often bind RNAs and affect RNA-mediated pathways in cells. Historically, small molecule engagement and modulation of RNA have not been considered in medicine development; however, RNA should be considered both a potential on- and off-target. Kinase inhibitors have emecrged as common RNA binders with dovitinib, a classic receptor tyrosine kinase (RTK) inhibitor, inhibiting RTKs and the biogenesis of oncogenic microRNA-21 through direct engagement.

Transcriptome-Wide Studies of RNA-Targeted Small Molecules Provide a Simple and Selective r(CUG) Degrader in Myotonic Dystrophy.

Myotonic dystrophy type 1 (DM1) is caused by a highly structured RNA repeat expansion, r(CUG), harbored in the 3' untranslated region (3' UTR) of dystrophia myotonica protein kinase () mRNA and drives disease through a gain-of-function mechanism. A panel of low-molecular-weight fragments capable of reacting with RNA upon UV irradiation was studied for cross-linking to r(CUG), affording perimidin-2-amine diazirine () that bound to r(CUG). The interactions between the small molecule and RNA were further studied by nuclear magnetic resonance (NMR) spectroscopy and molecular modeling.

Conformational dynamics of RNA G4C2 and G2C4 repeat expansions causing ALS/FTD using NMR and molecular dynamics studies.

G4C2 and G2C4 repeat expansions in chromosome 9 open reading frame 72 (C9orf72) are the most common cause of genetically defined amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD), or c9ALS/FTD. The gene is bidirectionally transcribed, producing G4C2 repeats [r(G4C2)exp] and G2C4 repeats [r(G2C4)exp]. The c9ALS/FTD repeat expansions are highly structured, and structural studies showed that r(G4C2)exp predominantly folds into a hairpin with a periodic array of 1 × 1 G/G internal loops and a G-quadruplex.

DNA-Encoded Library Screening To Inform Design of a Ribonuclease Targeting Chimera (RiboTAC).

Ribonuclease targeting chimeras (RiboTACs) induce degradation of an RNA target by facilitating an interaction between an RNA and a ribonuclease (RNase). We describe the screening of a DNA-encoded library (DEL) to identify binders of monomeric RNase L to provide a compound that induced dimerization of RNase L, activating its ribonuclease activity. This compound was incorporated into the design of a next-generation RiboTAC that targeted the microRNA-21 (miR-21) precursor and alleviated a miR-21-associated cellular phenotype in triple-negative breast cancer cells.

A Small Molecule Exploits Hidden Structural Features within the RNA Repeat Expansion That Causes c9ALS/FTD and Rescues Pathological Hallmarks.

The hexanucleotide repeat expansion GGGGCC [r(GC)] within intron 1 of causes genetically defined amyotrophic lateral sclerosis and frontotemporal dementia, collectively named c9ALS/FTD. , the repeat expansion causes neurodegeneration via deleterious phenotypes stemming from r(GC) RNA gain- and loss-of-function mechanisms. The r(GC) RNA folds into both a hairpin structure with repeating 1 × 1 nucleotide GG internal loops and a G-quadruplex structure.

Affecting RNA biology genome-wide by binding small molecules and chemically induced proximity.

The ENCODE and genome-wide association projects have shown that much of the genome is transcribed into RNA and much less is translated into protein. These and other functional studies suggest that the druggable transcriptome is much larger than the druggable proteome. This review highlights approaches to define druggable RNA targets and structure-activity relationships across genomic RNA.

Structural Features of Small Molecules Targeting the RNA Repeat Expansion That Causes Genetically Defined ALS/FTD.

Genetically defined amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD), collectively named c9ALS/FTD, are triggered by hexanucleotide GGGGCC repeat expansions [r(GC)] within the gene. In these diseases, neuronal loss occurs through an interplay of deleterious phenotypes, including r(GC) RNA gain-of-function mechanisms. Herein, we identified a benzimidazole derivative, CB096, that specifically binds to a repeating 1 × 1 GG internal loop structure, 5'CG/3'GC, that is formed when r(GC) folds.

Visualizing a protonated RNA state that modulates microRNA-21 maturation.

MicroRNAs are evolutionarily conserved small, noncoding RNAs that regulate diverse biological processes. Due to their essential regulatory roles, microRNA biogenesis is tightly regulated, where protein factors are often found to interact with specific primary and precursor microRNAs for regulation. Here, using NMR relaxation dispersion spectroscopy and mutagenesis, we reveal that the precursor of oncogenic microRNA-21 exists as a pH-dependent ensemble that spontaneously reshuffles the secondary structure of the entire apical stem-loop region, including the Dicer cleavage site.

Probing excited conformational states of nucleic acids by nitrogen CEST NMR spectroscopy.

Characterizing low-populated and short-lived excited conformational states has become increasingly important for understanding mechanisms of RNA function. Interconversion between RNA ground and excited conformational states often involves base pairing rearrangements that lead to changes in the hydrogen-bond network. Here, we present two N chemical exchange saturation transfer (CEST) NMR experiments that utilize protonated and non-protonated nitrogens, which are key hydrogen-bond donors and acceptors, for characterizing excited conformational states in RNA.

NMR characterization of RNA small molecule interactions.

Exciting discoveries of naturally occurring ligand-sensing and disease-linked noncoding RNAs have promoted significant interests in understanding RNA-small molecule interactions. NMR spectroscopy is a powerful tool for characterizing intermolecular interactions. In this review, we describe protocols and approaches for applying NMR spectroscopy to investigate interactions between RNA and small molecules.