As the area of small molecules interacting with RNA advances, general routes to provide bioactive compounds are needed as ligands can bind RNA avidly to sites that will not affect function. Small-molecule targeted RNA degradation will thus provide a general route to affect RNA biology. A non–oligonucleotide-containing compound was designed from sequence to target the precursor to oncogenic microRNA-21 (pre–miR-21) for enzymatic destruction with selectivity that can exceed that for protein-targeted medicines. The compound specifically binds the target and contains a heterocycle that recruits and activates a ribonuclease to pre–miR-21 to substoichiometrically effect its cleavage and subsequently impede metastasis of breast cancer to lung in a mouse model. Transcriptomic and proteomic analyses demonstrate that the compound is potent and selective, specifically modulating oncogenic pathways. Thus, small molecules can be designed from sequence to have all of the functional repertoire of oligonucleotides, including inducing enzymatic degradation, and to selectively and potently modulate RNA function in vivo.
A hypoxic state is critical to the metastatic and invasive characteristics of cancer. Numerous pathways play critical roles in cancer maintenance, many of which include noncoding RNAs such as microRNA (miR)-210 that regulates hypoxia inducible factors (HIFs). Herein, we describe the identification of a small molecule named Targapremir-210 that binds to the Dicer site of the miR-210 hairpin precursor. This interaction inhibits production of the mature miRNA, derepresses glycerol-3-phosphate dehydrogenase 1-like enzyme (GPD1L), a hypoxia-associated protein negatively regulated by miR-210, decreases HIF-1α, and triggers apoptosis of triple negative breast cancer cells only under hypoxic conditions. Further, Targapremir-210 inhibits tumorigenesis in a mouse xenograft model of hypoxic triple negative breast cancer. Many factors govern molecular recognition of biological targets by small molecules. For protein, chemoproteomics and activity-based protein profiling are invaluable tools to study small molecule target engagement and selectivity in cells. Such approaches are lacking for RNA, leaving a void in the understanding of its druggability. We applied Chemical Cross-Linking and Isolation by Pull Down (Chem-CLIP) to study the cellular selectivity and the on- and off-targets of Targapremir-210. Targapremir-210 selectively recognizes the miR-210 precursor and can differentially recognize RNAs in cells that have the same target motif but have different expression levels, revealing this important feature for selectively drugging RNAs for the first time. These studies show that small molecules can be rapidly designed to selectively target RNAs and affect cellular responses to environmental conditions, resulting in favorable benefits against cancer. Further, they help define rules for identifying druggable targets in the transcriptome.
The choreography between RNA synthesis and degradation is a key determinant in biology. Engineered systems such as CRISPR have been developed to rid a cell of RNAs. Here, we show that a small molecule can recruit a nuclease to a specific transcript, triggering its destruction. A small molecule that selectively binds the oncogenic microRNA(miR)-96 hairpin precursor was appended with a short 2'-5' poly(A) oligonucleotide. The conjugate locally activated endogenous, latent ribonuclease (RNase L), which selectively cleaved the miR-96 precursor in cancer cells in a catalytic and sub-stoichiometric fashion. Silencing miR-96 derepressed pro-apoptotic FOXO1 transcription factor, triggering apoptosis in breast cancer, but not healthy breast, cells. These results demonstrate that small molecules can be programmed to selectively cleave RNA via nuclease recruitment and has broad implications.
RNA offers nearly unlimited potential as a target for small molecule chemical probes and lead medicines. Many RNAs fold into structures that can be selectively targeted with small molecules. This Perspective discusses molecular recognition of RNA by small molecules and highlights key enabling technologies and properties of bioactive interactions. Sequence-based design of ligands targeting RNA has established rules for affecting RNA targets and provided a potentially general platform for the discovery of bioactive small molecules. The RNA targets that contain preferred small molecule binding sites can be identified from sequence, allowing identification of off-targets and prediction of bioactive interactions by nature of ligand recognition of functional sites. Small molecule targeted degradation of RNA targets (ribonuclease-targeted chimeras, RIBOTACs) and direct cleavage by small molecules have also been developed. These growing technologies suggest that the time is right to provide small molecule chemical probes to target functionally relevant RNAs throughout the human transcriptome.
Highlights d A small molecule locally recruits ribonuclease to degrade a hypoxia-associated RNA d Appendage of a ribonuclease recruitment module increases selectivity and potency d Degradation of target RNA induces apoptosis in hypoxic breast cancer cells d Targeted small-molecule degradation of RNA sequence is broadly applicable
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