Heterobifunctional small molecules to modulate RNA function.

RNA has diverse cellular functionality, including regulating gene expression, protein translation, and cellular response to stimuli, due to its intricate structures. Over the past decade, small molecules have been discovered that target functional structures within cellular RNAs and modulate their function. Simple binding, however, is often insufficient, resulting in low or even no biological activity. To overcome this challenge, heterobifunctional compounds have been developed that can covalently bind to the RNA target, alter RNA sequence, or induce its cleavage. Herein, we review the recent progress in the field of RNA-targeted heterobifunctional compounds using representative case studies. We identify critical gaps and limitations and propose a strategic pathway for future developments of RNA-targeted molecules with augmented functionalities.

Decreasing the intrinsically disordered protein α-synuclein levels by targeting its structured mRNA with a ribonuclease-targeting chimera.

α-Synuclein is an important drug target for the treatment of Parkinson's disease (PD), but it is an intrinsically disordered protein lacking typical small-molecule binding pockets. In contrast, the encoding SNCA mRNA has regions of ordered structure in its 5' untranslated region (UTR). Here, we present an integrated approach to identify small molecules that bind this structured region and inhibit α-synuclein translation. A drug-like, RNA-focused compound collection was studied for binding to the 5' UTR of SNCA mRNA, affording Synucleozid-2.0, a drug-like small molecule that decreases α-synuclein levels by inhibiting ribosomes from assembling onto SNCA mRNA. This RNA-binding small molecule was converted into a ribonuclease-targeting chimera (RiboTAC) to degrade cellular SNCA mRNA. RNA-seq and proteomics studies demonstrated that the RiboTAC (Syn-RiboTAC) selectively degraded SNCA mRNA to reduce its protein levels, affording a fivefold enhancement of cytoprotective effects as compared to Synucleozid-2.0. As observed in many diseases, transcriptome-wide changes in RNA expression are observed in PD. Syn-RiboTAC also rescued the expression of ~50% of genes that were abnormally expressed in dopaminergic neurons differentiated from PD patient-derived iPSCs. These studies demonstrate that the druggability of the proteome can be expanded greatly by targeting the encoding mRNAs with both small molecule binders and RiboTAC degraders.

Methods for the study of ribonuclease targeting chimeras (RiboTACs).

Recently, a class of heterobifunctional small molecules called ribonuclease targeting chimeras (RiboTACs) have been developed that selectively induce degradation of RNAs in cells. These molecules function by recruiting latent ribonuclease (RNase L), an endoribonuclease involved in the innate immune response, to targeted RNA structures. The RiboTACs must activate RNase L in proximity to the RNA, resulting in cleavage of the RNA and downstream degradation. To develop and validate a new RiboTAC, several steps must be taken. First, small molecule activators that bind to RNase L must be identified. Next, since RNase L is only catalytically active upon ligand-induced homodimerization, the capability of identified small molecules to activate RNase L must be assessed. RNase L-activating small molecules should then be coupled to validated RNA-binding small molecules to construct the active RiboTAC. This RiboTAC can finally be assessed in cells for RNase L-dependent degradation of target RNAs. This chapter will provide several methods that are helpful to develop and assess RiboTACs throughout this process, including recombinant RNase L expression, methods to assess RNase L engagement in vitro such as saturation transfer difference nuclear magnetic resonance (STD NMR), an in vitro assay to assess activation of RNase L, and cellular methods to demonstrate RNase L-dependent cleavage.

Altering the Cleaving Effector in Chimeric Molecules that Target RNA Enhances Cellular Selectivity.

Small molecules that target RNA and effect their cleavage are useful chemical probes and potential lead medicines. In this study, we investigate factors affecting degradation of two cancer-associated RNA targets, the mRNA that encodes the transcription factor JUN (c-Jun) and the hairpin precursor to microRNA-372 (pre-miR-372). The two RNA targets harbor the same small-molecule binding site juxtaposed with different neighboring structures. Specifically, pre-miR-372 has AU pairs and contiguous purines on one strand near the small-molecule binding site, making it an ideal substrate for oxidative cleavage via the direct degrader bleomycin A5. In contrast, while JUN mRNA has a similar number of AU pairs near the small-molecule binding site, it lacks contiguous purine nucleotides. Instead, it contains unpaired pyrimidine nucleotides, which are preferred substrates for RNase L, a ribonuclease that can be recruited to RNA with heterobifunctional ribonuclease targeting chimeras (RiboTACs). We hypothesized that structural features surrounding the binding site could be leveraged to program selective small-molecule degradation by alteration of the cleaving module. Indeed, the bleomycin degrader cleaves pre-miR-372 in gastric cancer cells but not JUN mRNA. Conversely, the RiboTAC cleaves JUN mRNA but not pre-miR-372. Thus, the selection of the appropriate cleaving effector moiety for an RNA-binding small molecule can be leveraged to cleave an RNA selectively in a predictable manner, which could have broad implications.

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

Myotonic dystrophy type 1 (DM1) is caused by a highly structured RNA repeat expansion, r(CUG)exp, harbored in the 3' untranslated region (3' UTR) of dystrophia myotonica protein kinase (DMPK) 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)expin vitro, affording perimidin-2-amine diazirine (1) that bound to r(CUG)exp. The interactions between the small molecule and RNA were further studied by nuclear magnetic resonance (NMR) spectroscopy and molecular modeling. Binding of 1 in DM1 myotubes was profiled transcriptome-wide, identifying 12 transcripts including DMPK that were bound by 1. Augmenting the functionality of 1 with cleaving capability created a chimeric degrader that specifically targets r(CUG)exp for elimination. The degrader broadly improved DM1-associated defects as assessed by RNA-seq, while having limited effects on healthy myotubes. This study (i) provides a platform to investigate molecular recognition of ligands directly in disease-affected cells; (ii) illustrates that RNA degraders can be more specific than the binders from which they are derived; and (iii) suggests that repeating transcripts can be selectively degraded due to the presence of multiple ligand binding sites.

Programming inactive RNA-binding small molecules into bioactive degraders.

Target occupancy is often insufficient to elicit biological activity, particularly for RNA, compounded by the longstanding challenges surrounding the molecular recognition of RNA structures by small molecules. Here we studied molecular recognition patterns between a natural-product-inspired small-molecule collection and three-dimensionally folded RNA structures. Mapping these interaction landscapes across the human transcriptome defined structure-activity relationships. Although RNA-binding compounds that bind to functional sites were expected to elicit a biological response, most identified interactions were predicted to be biologically inert as they bind elsewhere. We reasoned that, for such cases, an alternative strategy to modulate RNA biology is to cleave the target through a ribonuclease-targeting chimera, where an RNA-binding molecule is appended to a heterocycle that binds to and locally activates RNase L1. Overlay of the substrate specificity for RNase L with the binding landscape of small molecules revealed many favourable candidate binders that might be bioactive when converted into degraders. We provide a proof of concept, designing selective degraders for the precursor to the disease-associated microRNA-155 (pre-miR-155), JUN mRNA and MYC mRNA. Thus, small-molecule RNA-targeted degradation can be leveraged to convert strong, yet inactive, binding interactions into potent and specific modulators of RNA function.

A blood-brain penetrant RNA-targeted small molecule triggers elimination of r(G4C2)exp in c9ALS/FTD via the nuclear RNA exosome.

A hexanucleotide repeat expansion in intron 1 of the C9orf72 gene is the most common genetic cause of amyotrophic lateral sclerosis and frontotemporal dementia, or c9ALS/FTD. The RNA transcribed from the expansion, r(G4C2)exp, causes various pathologies, including intron retention, aberrant translation that produces toxic dipeptide repeat proteins (DPRs), and sequestration of RNA-binding proteins (RBPs) in RNA foci. Here, we describe a small molecule that potently and selectively interacts with r(G4C2)exp and mitigates disease pathologies in spinal neurons differentiated from c9ALS patient-derived induced pluripotent stem cells (iPSCs) and in two c9ALS/FTD mouse models. These studies reveal a mode of action whereby a small molecule diminishes intron retention caused by the r(G4C2)exp and allows the liberated intron to be eliminated by the nuclear RNA exosome, a multi-subunit degradation complex. Our findings highlight the complexity of mechanisms available to RNA-binding small molecules to alleviate disease pathologies and establishes a pipeline for the design of brain penetrant small molecules targeting RNA with novel modes of action in vivo.

Targeting RNA structures with small molecules.

RNA adopts 3D structures that confer varied functional roles in human biology and dysfunction in disease. Approaches to therapeutically target RNA structures with small molecules are being actively pursued, aided by key advances in the field including the development of computational tools that predict evolutionarily conserved RNA structures, as well as strategies that expand mode of action and facilitate interactions with cellular machinery. Existing RNA-targeted small molecules use a range of mechanisms including directing splicing - by acting as molecular glues with cellular proteins (such as branaplam and the FDA-approved risdiplam), inhibition of translation of undruggable proteins and deactivation of functional structures in noncoding RNAs. Here, we describe strategies to identify, validate and optimize small molecules that target the functional transcriptome, laying out a roadmap to advance these agents into the next decade.

Transcriptome-Wide Mapping of Small-Molecule RNA-Binding Sites in Cells Informs an Isoform-Specific Degrader of QSOX1 mRNA.

The interactions between cellular RNAs in MDA-MB-231 triple negative breast cancer cells and a panel of small molecules appended with a diazirine cross-linking moiety and an alkyne tag were probed transcriptome-wide in live cells. The alkyne tag allows for facile pull-down of cellular RNAs bound by each small molecule, and the enrichment of each RNA target defines the compound's molecular footprint. Among the 34 chemically diverse small molecules studied, six bound and enriched cellular RNAs. The most highly enriched interaction occurs between the novel RNA-binding compound F1 and a structured region in the 5' untranslated region of quiescin sulfhydryl oxidase 1 isoform a (QSOX1-a), not present in isoform b. Additional studies show that F1 specifically bound RNA over DNA and protein; that is, we studied the entire DNA, RNA, and protein interactome. This interaction was used to design a ribonuclease targeting chimera (RIBOTAC) to locally recruit Ribonuclease L to degrade QSOX1 mRNA in an isoform-specific manner, as QSOX1-a, but not QSOX1-b, mRNA and protein levels were reduced. The RIBOTAC alleviated QSOX1-mediated phenotypes in cancer cells. This approach can be broadly applied to discover ligands that bind RNA in cells, which could be bioactive themselves or augmented with functionality such as targeted degradation.

DNA-encoded library versus RNA-encoded library selection enables design of an oncogenic noncoding RNA inhibitor.

Nature evolves molecular interaction networks through persistent perturbation and selection, in stark contrast to drug discovery, which evaluates candidates one at a time by screening. Here, nature's highly parallel ligand-target search paradigm is recapitulated in a screen of a DNA-encoded library (DEL; 73,728 ligands) against a library of RNA structures (4,096 targets). In total, the screen evaluated ∼300 million interactions and identified numerous bona fide ligand-RNA three-dimensional fold target pairs. One of the discovered ligands bound a 5'GAG/3'CCC internal loop that is present in primary microRNA-27a (pri-miR-27a), the oncogenic precursor of microRNA-27a. The DEL-derived pri-miR-27a ligand was cell active, potently and selectively inhibiting pri-miR-27a processing to reprogram gene expression and halt an otherwise invasive phenotype in triple-negative breast cancer cells. By exploiting evolutionary principles at the earliest stages of drug discovery, it is possible to identify high-affinity and selective target-ligand interactions and predict engagements in cells that short circuit disease pathways in preclinical disease models.

A structure-specific small molecule inhibits a miRNA-200 family member precursor and reverses a type 2 diabetes phenotype.

MicroRNA families are ubiquitous in the human transcriptome, yet targeting of individual members is challenging because of sequence homology. Many secondary structures of the precursors to these miRNAs (pri- and pre-miRNAs), however, are quite different. Here, we demonstrate both in vitro and in cellulis that design of structure-specific small molecules can inhibit a particular miRNA family member to modulate a disease pathway. The miR-200 family consists of five miRNAs, miR-200a, -200b, -200c, -141, and -429, and is associated with type 2 diabetes (T2D). We designed a small molecule that potently and selectively targets pre-miR-200c's structure and reverses a pro-apoptotic effect in a pancreatic ß cell model. In contrast, an oligonucleotide targeting the RNA's sequence inhibited all family members. Global proteomics and RNA sequencing analyses further demonstrate selectivity for miR-200c. Collectively, these studies establish that miR-200c plays an important role in T2D, and small molecules targeting RNA structure can be an important complement to oligonucleotides.

Bioinformatic Searching for Optimal RNA Targets of Dimeric Compounds Informs Design of a MicroRNA-27a Inhibitor.

Various studies have shown that selective molecular recognition of RNA targets by small molecules in cells, although challenging, is indeed possible. One facile strategy to enhance selectivity and potency is binding two or more sites within an RNA simultaneously with a single molecule. To simplify the identification of targets amenable to such a strategy, we informatically mined all human microRNA (miRNA) precursors to identify those with two proximal noncanonically paired sites. We selected oncogenic microRNA-27a (miR-27a) for further study as a lead molecule binds its Drosha site and a nearby internal loop, affording a homodimer that potently and specifically inhibits miR-27a processing in both breast cancer and prostate cancer cells. This reduction of mature miR-27a ameliorates an oncogenic cellular phenotype with nanomolar activity. Collectively, these studies demonstrate that synergistic bioinformatic and experimental approaches can define targets that may be more amenable to small molecule targeting than others.

Ribonuclease recruitment using a small molecule reduced c9ALS/FTD r(G4C2) repeat expansion in vitro and in vivo ALS models.

The most common cause of amyotrophic lateral sclerosis and frontotemporal dementia (c9ALS/FTD) is an expanded G4C2 RNA repeat [r(G4C2)exp] in chromosome 9 open reading frame 72 (C9orf72), which elicits pathology through several mechanisms. Here, we developed and characterized a small molecule for targeted degradation of r(G4C2)exp. The compound was able to selectively bind r(G4C2)exp's structure and to assemble an endogenous nuclease onto the target, provoking removal of the transcript by native RNA quality control mechanisms. In c9ALS patient­derived spinal neurons, the compound selectively degraded the mutant C9orf72 allele with limited off-targets and reduced quantities of toxic dipeptide repeat proteins (DPRs) translated from r(G4C2)exp. In vivo work in a rodent model showed that abundance of both the mutant allele harboring the repeat expansion and DPRs were selectively reduced by this compound. These results demonstrate that targeted small-molecule degradation of r(G4C2)exp is a strategy for mitigating c9ALS/FTD-associated pathologies and studying disease-associated pathways in preclinical models.

Reprogramming of Protein-Targeted Small-Molecule Medicines to RNA by Ribonuclease Recruitment.

Reprogramming known medicines for a novel target with activity and selectivity over the canonical target is challenging. By studying the binding interactions between RNA folds and known small-molecule medicines and mining the resultant dataset across human RNAs, we identified that Dovitinib, a receptor tyrosine kinase (RTK) inhibitor, binds the precursor to microRNA-21 (pre-miR-21). Dovitinib was rationally reprogrammed for pre-miR-21 by using it as an RNA recognition element in a chimeric compound that also recruits RNase L to induce the RNA's catalytic degradation. By enhancing the inherent RNA-targeting activity and decreasing potency against canonical RTK protein targets in cells, the chimera shifted selectivity for pre-miR-21 by 2500-fold, alleviating disease progression in mouse models of triple-negative breast cancer and Alport Syndrome, both caused by miR-21 overexpression. Thus, targeted degradation can dramatically improve selectivity even across different biomolecules, i.e., protein versus RNA.

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

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.

Targeting the SARS-CoV-2 RNA Genome with Small Molecule Binders and Ribonuclease Targeting Chimera (RIBOTAC) Degraders.

COVID-19 is a global pandemic, thus requiring multiple strategies to develop modalities against it. Herein, we designed multiple bioactive small molecules that target a functional structure within the SARS-CoV-2's RNA genome, the causative agent of COVID-19. An analysis to characterize the structure of the RNA genome provided a revised model of the SARS-CoV-2 frameshifting element, in particular its attenuator hairpin. By studying an RNA-focused small molecule collection, we identified a drug-like small molecule (C5) that avidly binds to the revised attenuator hairpin structure with a K d of 11 nM. The compound stabilizes the hairpin's folded state and impairs frameshifting in cells. The ligand was further elaborated into a ribonuclease targeting chimera (RIBOTAC) to recruit a cellular ribonuclease to destroy the viral genome (C5-RIBOTAC) and into a covalent molecule (C5-Chem-CLIP) that validated direct target engagement and demonstrated its specificity for the viral RNA, as compared to highly expressed host mRNAs. The RIBOTAC lead optimization strategy improved the bioactivity of the compound at least 10-fold. Collectively, these studies demonstrate that the SARS-CoV-2 RNA genome should be considered druggable.

Small molecule recognition of disease-relevant RNA structures.

Targeting RNAs with small molecules represents a new frontier in drug discovery and development. The rich structural diversity of folded RNAs offers a nearly unlimited reservoir of targets for small molecules to bind, similar to small molecule occupancy of protein binding pockets, thus creating the potential to modulate human biology. Although the bacterial ribosome has historically been the most well exploited RNA target, advances in RNA sequencing technologies and a growing understanding of RNA structure have led to an explosion of interest in the direct targeting of human pathological RNAs. This review highlights recent advances in this area, with a focus on the design of small molecule probes that selectively engage structures within disease-causing RNAs, with micromolar to nanomolar affinity. Additionally, we explore emerging RNA-target strategies, such as bleomycin A5 conjugates and ribonuclease targeting chimeras (RIBOTACs), that allow for the targeted degradation of RNAs with impressive potency and selectivity. The compounds discussed in this review have proven efficacious in human cell lines, patient-derived cells, and pre-clinical animal models, with one compound currently undergoing a Phase II clinical trial and another that recently garnerd FDA-approval, indicating a bright future for targeted small molecule therapeutics that affect RNA function.