Systematically Studying the Effect of Small Molecules Interacting with RNA in Cellular and Preclinical Models

Abstract

The interrogation and manipulation of biological systems by small molecules is a powerful approach in chemical biology. Ideal compounds selectively engage a target and mediate a downstream phenotypic response. Although historically small molecule drug discovery has focused on proteins and enzymes, targeting RNA is an attractive therapeutic alternative, as many disease-causing or -associated RNAs have been identified through genome-wide association studies. As the field of RNA chemical biology emerges, the systematic evaluation of target validation and modulation of target-associated pathways is of paramount importance. In this Review, through an examination of case studies, we outline the experimental characterization, including methods and tools, to evaluate comprehensively the impact of small molecules that target RNA on cellular phenotype.

Figure 1.

Phenotypic screening of small molecules and summary of target validation strategies. (a) Schematic representation of benchmarks for small molecule identification through phenotypic screening. (b) Tools to validate target engagement of small molecules identified by phenotypic screening.

Figure 2.

Didehydro-cortistatin A inhibits transcription of the HIV provirus. (a) The viral protein Tat mediates transcription of HIV by binding to the transactivation response (TAR) element in the 5′ UTR of HIV mRNA and recruiting a host protein complex that recruits positive transcription elongation factor (pTEFb). (b) Structure of didehydro-cortistatin A (dCA). (c) Binding of dCA to Tat inhibits the protein’s interaction with the TAR element, thus inhibiting initiation of transcription.

Figure 3.

Targeting of the FMN riboswitch by ribocil. (a) Chemical structure of the natural riboswitch ligand, flavin mononucleotide (FMN) and the synthetic mimic ribocil. (b) Schematic representation of the FMN riboswitch. Key mutations indicated in red. Key residues in X-ray crystallography studies indicated in black. (c) Phenotypic screening and validation techniques used in the identification of ribocil.

Figure 4.

Small molecule targeting of the FMN riboswitch. (a) Structures of FMN, riboflavin, and roseoflavin and principles of the in-line binding assay. (b) Sequence and predicted structure of the 165 nt ribD 5′ UTR from Bacillus subtilis. (c) Phenotypic screening and validation techniques used in the identification of roseoflavin, including mutational analysis, both in cells and in vitro.

Figure 5.

Phenotypic screening and validation techniques of small molecules that modulate Tau splicing. (a) Small molecule binding to the splicing regulatory element (SRE) stabilizes the hairpin structure and promotes exclusion of MAPT exon 10, leading to translation of 3R Tau. (b) Phenotypic screening and target validation techniques used in the identification of the lead compound.

Figure 6.

Targeting of SMN2 by Risdiplam. (a) Schematic representation of the mechanism of alternative SMN2 gene splicing. (b) Sequence and structure of SMN2 exon 7 and adjacent features. (c) Chemical structure of risdiplam. (d) Phenotypic screening and validation techniques used in the identification of risdiplam.

Figure 7.

Targeting of the oncogenic miRNA cluster miR-17–92 with small molecules. (a) The miR-17–92 cluster encodes six different miRNAs, including miR-17, -18a, and -20a that share the same structure at their Dicer processing sites (blue); miR-17 and -20a have an identical G bulge (yellow) adjacent to the Dicer site, and miR-18a has a similarly structured A bulge at same location (purple). (b) Structure of the hit monomeric compound, the optimized dimeric compound (1), its derivatives used for Chem-CLIP (2 and 3), and a bleomycin conjugated small molecule (4) used for proteomics studies. (c) Mode of action of 1, inducing apoptosis in DU-145 cells.

Figure 8.

Small molecules that reduce miR-21 abundance by two different mechanisms and pathogenic phenotype. (a) Structures of the miR-21-targeting dimeric small molecule binder, ribonuclease targeting chimera (RIBOTAC), and a control RIBOTAC compound with a less efficient RNase L recruiting module. (b) Secondary structure of pre-miR-21 (mature miR-21 shown in red). (c) Effect of dimeric binder, RIBOTAC, and control RIBOTAC on expression levels of miR-21. RNA fluorescence in situ hybridization (FISH) histology for the detection of miR-21 in lungs of mice xenografted with a metastatic breast cancer cell line. Breast cancer metastasis to lungs, as evidenced by lung nodules, is inhibited by treatment with 6.