Drugging the RNA World

Abstract

Although we live in the remnants of an RNA world, the world of drug discovery and chemical probes is firmly protein-centric. Developing highly selective small molecules targeting RNA is often considered to be an insurmountable challenge. Our goal is to demystify the design of such compounds. In this review, we describe various approaches to design small molecules that target RNA from sequence and the application of these compounds in RNA biology, with a focus on inhibition of human RNA-protein complexes. We have developed a library-versus-library screening approach to define selective RNA-small-molecule binding partners and applied them to disease-causing RNAs, in particular noncoding oncogenic RNAs and expanded RNA repeats, to modulate their biology in cells and animals. We also describe the design of new types of small-molecule probes that could broadly decipher the mysteries of RNA in cells.

Figure 1.

The ribosome is a well-known target of small molecules. (A) Various compounds bind to specific sites in the bacterial ribosome and protect them from chemical modification. High-resolution structures of the ribosomal subunits have been determined in the presence and absence of antibiotics. Shown here is the 30S subunit complexed with the aminoglycoside puromycin; the ribosome subunit is shown in purple and puromycin is shown in Corey, Pauling, and Kolton (CPK) colors. (B) Common secondary structural elements found in cellular RNAs.

Figure 2.

Sequence- and secondary structure-based approaches developed for small molecules targeting RNA. (A) Schematic of two-dimensional combinatorial screening (2DCS), a library-versus-library screen. A small-molecule library is immobilized onto a microarray and hybridized with an RNA motif library. The selected RNAs are sequenced and analyzed to form a chemical code for selectively targeting RNA. (B) Schematic of Inforna, a computational method to identify “druggable” RNA targets from sequence. The selected RNA motif–small-molecule partners from 2DCS are mined against the transcriptome to agnostically identify RNAs that can be targeted with these small molecules.

Figure 3.

Examples of targeting RNA with small molecules that have been designed by using Inforna. (A) Targaprimir-96 targets the Drosha site in the microRNA-96 (miR-96) hairpin precursor. The compound inhibits miR-96 biogenesis and triggers apoptosis via a miR-96-forkhead box protein O1 (FOXO1) circuit. (B) Targapremir-210 silences miR-210, induces apoptosis of hypoxic, but not normoxic, cells, and reduces tumor burden in a xenograft mouse model.

Figure 4.

Schematic of chemical cross-linking isolated by pull-down (Chem-CLIP), an approach to identify the RNA targets of small molecules in cells. (A) Small molecules targeting RNA are appended with cross-linking and biotin modules. Application of Chem-CLIP probes to cells cross-links the small molecule to its cellular RNA targets. The biotin tag allows the targets to be purified from cells, which are identified by quantitative reverse transcription polymerase chain reaction (RT-qPCR) or RNA sequencing (RNA-seq). (B) In competitive Chem-CLIP (C-Chem-CLIP), cells are cotreated with the Chem-CLIP probe and the parent compound. Bona fide targets are depleted in the pulled-down fraction.

Figure 5.

An example of on-site click chemistry to create a fluorescence resonance energy transfer (FRET) reporter to image RNA in cells without genetic modification. (A) Structures of an RNA-binding module (represented by a blue circle), 2H-K4NMeS, Aak-2H-K4NMeS-FAM (a green circle represents a fluorescein FRET donor), and TAMRA-2H-K4NMeS-N₃ (a purple circle represents TAMRA [5-arboxytetramethylrhodamine] FRET acceptor). (B) Scheme of on-site FRET pair synthesis that occurs after FRET pair precursors bind to r(CUG)^exp and undergo an RNA-catalyzed click reaction, allowing imaging of RNA in cells. (C) Images of myotonic muscular dystrophy type 1 (DM1) (CUG)₅₀₀ fibroblasts and healthy (CUG)₁₅ fibroblasts with Aak-2H-K4NMeS-FAM only (left), Aak-2H-K4NMeS-FAM with TAMRA-2H-K4NMeS-N₃ (middle), and Aak-2H-K4NMeS-FAM with TAMRA-2H-K4NMeS (lacks N³ click partner) (right). (D) On-site click chemistry generates FRET reporters to show 2H-K4NMeS selectively binds to r(CUG)₅₀₀ in DM1 fibroblasts. ***P < 0.001, as determined by a two-tailed Student’s t-test. (A and B are adapted, whereas C and D are directly reproduced, with permission, from Rzuczek et al. 2017.)