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Trinucleotide repeat expansions fold into long, stable hairpins and cause a variety of incurable RNA gain-of-function diseases such as Huntington's disease, the myotonic dystrophies, and spinocerebellar ataxias. One approach for treating these diseases is to bind small molecules to the structured RNAs. Both Huntington's disease-like 2 (HDL2) and myotonic dystrophy type 1 (DM1) are caused by a r(CUG) repeat expansion, or r(CUG)exp. The RNA folds into a hairpin structure with a periodic array of 1×1 nucleotide UU loops (5'CUG/3'GUC; where the underlined nucleotides indicate the Us in the internal loop) that sequester various RNA-binding proteins (RBP) and hence the source of its gain-of-function. Here, we report NMR-refined structures of single 5'CUG/3'GUC motifs in complex with three different small molecules, a di-guandinobenzoate (1), a derivative of 1 where the guanidino groups have been exchanged for imidazole (2), and a quinoline with improved drug-like properties (3). These structures were determined using nuclear magnetic resonance (NMR) spectroscopy and simulated annealing with restrained molecular dynamics (MD). Compounds 1, 2, and 3 formed stacking and hydrogen bonding interactions with the 5'CUG/3'GUC motif. Compound 3 also formed van der Waals interactions with the internal loop. The global structure of each RNA-small molecule complexes retains an A-form conformation, while the internal loops are still dynamic but to a lesser extent compared to the unbound form. These results aid our understanding of ligand-RNA interactions and enable structure-based design of small molecules with improved binding affinity for and biological activity against r(CUG)exp. As the first ever reported structures of RNA r(CUG) repeats bound to ligands, these structures can enable virtual screening campaigns combined with machine learning assisted de novo design.
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Various approaches have been developed to target RNA and modulate its function with modes of action including binding and cleavage. Herein, we explored how small molecule binding is correlated with cleavage induced by heterobifunctional ribonuclease targeting chimeras (RiboTACs), where RNase L is recruited to cleave the bound RNA target, in a transcriptome-wide, unbiased fashion. Only a fraction of bound targets was cleaved by RNase L, induced by RiboTAC binding. Global analysis suggested that (i) cleaved targets generally form a region of stable structure that encompasses the small molecule binding site; (ii) cleaved targets have preferred RNase L cleavage sites nearby small molecule binding sites; (iii) RiboTACs facilitate a cellular interaction between cleaved targets and RNase L; and (iv) the expression level of the target influences the extent of cleavage observed. In one example, we converted a binder of LGALS1 (galectin-1) mRNA into a RiboTAC. In MDA-MB-231 cells, the binder had no effect on galectin-1 protein levels, while the RiboTAC cleaved LGALS1 mRNA, reduced galectin-1 protein abundance, and affected galectin-1-associated oncogenic cellular phenotypes. Using LGALS1, we further assessed additional factors including the length of the linker that tethers the two components of the RiboTAC, cellular uptake, and the RNase L-recruiting module on RiboTAC potency. Collectively, these studies may facilitate triangulation of factors to enable the design of RiboTACs.
Assuntos
Transcriptoma , Humanos , Linhagem Celular Tumoral , Endorribonucleases/metabolismo , Endorribonucleases/química , RNA Mensageiro/metabolismo , RNA Mensageiro/genética , Ribonucleases/metabolismo , Ribonucleases/químicaRESUMO
Trinucleotide repeat expansions fold into long, stable hairpins and cause a variety of incurable RNA gain-of-function diseases such as Huntington's disease, the myotonic dystrophies, and spinocerebellar ataxias. One approach for treating these diseases is to bind small molecules to these structured RNAs. Both Huntington's disease-like 2 (HDL2) and myotonic dystrophy type 1 (DM1) are caused by a r(CUG) repeat expansion, or r(CUG)exp. The RNA folds into a hairpin structure with a periodic array of 1 × 1 nucleotide UU loops (5'CUG/3'GUC; where the underlined nucleotides indicate the Us in the internal loop) that sequester various RNA-binding proteins (RBPs) and hence the source of its gain-of-function. Here, we report nuclear magnetic resonance (NMR)-refined structures of single 5'CUG/3'GUC motifs in complex with three different small molecules, a di-guandinobenzoate (1), a derivative of 1 where the guanidino groups have been exchanged for imidazole (2), and a quinoline with improved drug-like properties (3). These structures were determined using NMR spectroscopy and simulated annealing with restrained molecular dynamics (MD). Compounds 1, 2, and 3 formed stacking and hydrogen bonding interactions with the 5'CUG/3'GUC motif. Compound 3 also formed van der Waals interactions with the internal loop. The global structure of each RNA-small molecule complexes retains an A-form conformation, while the internal loops are still dynamic but to a lesser extent compared to the unbound form. These results aid our understanding of ligand-RNA interactions and enable structure-based design of small molecules with improved binding affinity for and biological activity against r(CUG)exp. As the first ever reported structures of a r(CUG) repeat bound to ligands, these structures can enable virtual screening campaigns combined with machine learning assisted de novo design.
Assuntos
RNA , Bibliotecas de Moléculas Pequenas , Expansão das Repetições de Trinucleotídeos , RNA/química , RNA/metabolismo , Bibliotecas de Moléculas Pequenas/química , Bibliotecas de Moléculas Pequenas/farmacologia , Bibliotecas de Moléculas Pequenas/síntese química , Humanos , Conformação de Ácido Nucleico , Estrutura Molecular , Espectroscopia de Ressonância Magnética , Quinolinas/química , Modelos MolecularesRESUMO
Small molecules targeting RNA can be valuable chemical probes and potential therapeutics. The interactions between small molecules, particularly fragments, and RNA, however, can be difficult to detect due to their modest affinities and short residence times. Here, we describe the procedures for mapping the molecular fingerprints of small molecules in vitro and throughout the human transcriptome in live cells, identifying both the targets bound by the small molecule and the sites of binding therein. For complete details on the use and execution of this protocol, please refer to 1.
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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.
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RNA , Bibliotecas de Moléculas Pequenas , Humanos , RNA/metabolismo , RNA/química , Bibliotecas de Moléculas Pequenas/farmacologia , Bibliotecas de Moléculas Pequenas/química , AnimaisRESUMO
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.
Assuntos
Proteínas Intrinsicamente Desordenadas , Doença de Parkinson , Humanos , alfa-Sinucleína/genética , RNA Mensageiro/genética , Proteínas Intrinsicamente Desordenadas/genética , Doença de Parkinson/tratamento farmacológico , Doença de Parkinson/genética , Regiões 5' não Traduzidas , RibonucleasesRESUMO
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.
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Endorribonucleases , Ribonucleases , Endorribonucleases/genética , RNA/química , Imunidade InataRESUMO
Previously, we have identified a novel human metastasis-inducing lncRNA (named SKAI1BC), that suppresses the KAI1/CD82 metastasis-suppressing gene and is upregulated in triple negative breast cancer and melanoma derived cell lines. Modeling of the SKAI1BC lncRNA secondary structure and its potential interaction with Inforna compounds, led us to identify several compounds that might bind the SKAI1BC lncRNA. We found that these compounds inhibit metastasis invasion and cell migration in culture, in all eight types of solid human cancers tested: several of which are the most lethal and/or frequent human malignancies. Moreover, in most cases, the mechanism of action of several of our compounds involves enhancement of KAI1/CD82 RNA level depending on the specific compound and the human tumor type. With the epigenetic inactivation of KAI1/CD82 in at least ten additional solid human cancers, this implies a very good chance to broaden the spectrum of human cancers affected by our compounds. This is the first time that modeling of a large lncRNA (> 700 bp) secondary structure followed by its potential interaction with Inforna like compounds database has led to the identification of potential biologically active small molecule drugs.
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Melanoma , RNA Longo não Codificante , Humanos , RNA Longo não Codificante/genética , Proteína Kangai-1/genética , Proteína Kangai-1/metabolismo , Genes Supressores de Tumor , Linhagem Celular , Melanoma/tratamento farmacológico , Melanoma/genética , Metástase NeoplásicaRESUMO
A summit held March 2023 in Scottsdale, Arizona (USA) focused on the intronic hexanucleotide expansion in the C9ORF72 gene and its relevance in frontotemporal dementia (FTD) and amyotrophic lateral sclerosis (ALS; C9ORF72-FTD/ALS). The goal of this summit was to connect basic scientists, clinical researchers, drug developers, and individuals affected by C9ORF72-FTD/ALS to evaluate how collaborative efforts across the FTD-ALS disease spectrum might break down existing disease silos. Presentations and discussions covered recent discoveries in C9ORF72-FTD/ALS disease mechanisms, availability of disease biomarkers and recent advances in therapeutic development, and clinical trial design for prevention and treatment for individuals affected by C9ORF72-FTD/ALS and asymptomatic pathological expansion carriers. The C9ORF72-associated hexanucleotide repeat expansion is an important locus for both ALS and FTD. C9ORF72-FTD/ALS may be characterized by loss of function of the C9ORF72 protein and toxic gain of functions caused by both dipeptide repeat (DPR) proteins and hexanucleotide repeat RNA. C9ORF72-FTD/ALS therapeutic strategies discussed at the summit included the use of antisense oligonucleotides, adeno-associated virus (AAV)-mediated gene silencing and gene delivery, and engineered small molecules targeting RNA structures associated with the C9ORF72 expansion. Neurofilament light chain, DPR proteins, and transactive response (TAR) DNA-binding protein 43 (TDP-43)-associated molecular changes were presented as biomarker candidates. Similarly, brain imaging modalities (i.e., magnetic resonance imaging [MRI] and positron emission tomography [PET]) measuring structural, functional, and metabolic changes were discussed as important tools to monitor individuals affected with C9ORF72-FTD/ALS, at both pre-symptomatic and symptomatic disease stages. Finally, summit attendees evaluated current clinical trial designs available for FTD or ALS patients and concluded that therapeutics relevant to FTD/ALS patients, such as those specifically targeting C9ORF72, may need to be tested with composite endpoints covering clinical symptoms of both FTD and ALS. The latter will require novel clinical trial designs to be inclusive of all patient subgroups spanning the FTD/ALS spectrum.
The C9ORF72 Summit was held in March 2023 in Scottsdale, Arizona (USA). Some people who have the disease frontotemporal dementia or the disease amyotrophic lateral sclerosis have a change in one of their genes; the name of the gene is C9ORF72. People who carry this genetic difference usually inherited it from a parent. Researchers are improving their understanding of how the change in the C9ORF72 gene affects people, and efforts are being made to use this knowledge to develop treatments for amyotrophic lateral sclerosis and frontotemporal dementia. In addition to studying the cellular and molecular mechanisms of how the C9ORF72 mutation leads to cellular dysfunction and frontotemporal dementia and amyotrophic lateral sclerosis clinical symptoms, a large effort of the research community is aimed at developing measurements, called biomarkers, that could enhance therapy development efforts in multiple ways. Examples include monitoring of disease activity, identifying those at risk of developing amyotrophic lateral sclerosis or frontotemporal dementia, predicting which people might benefit from a particular treatment, and showing that a drug has had a biological effect. Markers that identify healthy people who are at risk of developing amyotrophic lateral sclerosis or frontotemporal dementia could be used to test treatments that would start before a person shows any symptoms and hopefully would delay or even prevent their onset.
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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. In this study, we use knowledge of the molecular recognition of both protein and RNA targets by dovitinib to design molecules that specifically inhibit the RNA target but lack activity against canonical protein targets in cells. As it is now becoming apparent that RNA can be both an on- and off-target for small molecule medicines, this study lays a foundation to use design principles to maximize desired compound activity while minimizing off-target effects.
Assuntos
MicroRNAs , MicroRNAs/metabolismo , Receptores Proteína Tirosina Quinases , Inibidores de Proteínas Quinases/farmacologiaRESUMO
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.
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MicroRNAs , RNA , RNA/metabolismo , Sítios de Ligação , Ribonucleases/metabolismo , MicroRNAs/metabolismo , RNA Mensageiro/genéticaRESUMO
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.
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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. A small molecule probe revealed that r(G4C2)exp also adopts a hairpin structure with 2 × 2 GG/GG internal loops. We studied the conformational dynamics adopted by 2 × 2 GG/GG loops using temperature replica exchange molecular dynamics (T-REMD) and further characterized the structure and underlying dynamics using traditional 2D NMR techniques. These studies showed that the loop's closing base pairs influence both structure and dynamics, particularly the configuration adopted around the glycosidic bond. Interestingly, r(G2C4) repeats, which fold into an array of 2 × 2 CC/CC internal loops, are not as dynamic. Collectively, these studies emphasize the unique sensitivity of r(G4C2)exp to small changes in stacking interactions, which is not observed in r(G2C4)exp, providing important considerations for further principles in structure-based drug design.
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Esclerose Lateral Amiotrófica , Proteína C9orf72 , Demência Frontotemporal , Humanos , Esclerose Lateral Amiotrófica/genética , Proteína C9orf72/genética , Expansão das Repetições de DNA , Demência Frontotemporal/genética , Espectroscopia de Ressonância Magnética , Simulação de Dinâmica Molecular , RNARESUMO
Although fragment-based drug discovery (FBDD) has been successfully implemented and well-explored for protein targets, its feasibility for RNA targets is emerging. Despite the challenges associated with the selective targeting of RNA, efforts to integrate known methods of RNA binder discovery with fragment-based approaches have been fruitful, as a few bioactive ligands have been identified. Here, we review various fragment-based approaches implemented for RNA targets and provide insights into experimental design and outcomes to guide future work in the area. Indeed, investigations surrounding the molecular recognition of RNA by fragments address rather important questions such as the limits of molecular weight that confer selective binding and the physicochemical properties favorable for RNA binding and bioactivity.
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Desenho de Fármacos , Descoberta de Drogas , LigantesRESUMO
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.
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Endorribonucleases , MicroRNAs , RNA Mensageiro , Humanos , Genes jun/genética , Genes myc/genética , MicroRNAs/antagonistas & inibidores , MicroRNAs/química , MicroRNAs/genética , MicroRNAs/metabolismo , Conformação de Ácido Nucleico , RNA Mensageiro/antagonistas & inibidores , RNA Mensageiro/química , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Relação Estrutura-Atividade , Especificidade por Substrato , Endorribonucleases/química , Endorribonucleases/metabolismo , TranscriptomaRESUMO
RNA is challenging to target with bioactive small molecules, particularly those of low molecular weight that bind with sufficient affinity and specificity. In this report, we developed a platform to address this challenge, affording a novel bioactive interaction. An RNA-focused small-molecule fragment collection (n = 2500) was constructed by analyzing features in all publicly reported compounds that bind RNA, the largest collection of RNA-focused fragments to date. The RNA-binding landscape for each fragment was studied by using a library-versus-library selection with an RNA library displaying a discrete structural element, probing over 12.8 million interactions, the greatest number of interactions between fragments and biomolecules probed experimentally. Mining of this dataset across the human transcriptome defined a drug-like fragment that potently and specifically targeted the microRNA-372 hairpin precursor, inhibiting its processing into the mature, functional microRNA and alleviating invasive and proliferative oncogenic phenotypes in gastric cancer cells. Importantly, this fragment has favorable properties, including an affinity for the RNA target of 300 ± 130 nM, a molecular weight of 273 Da, and quantitative estimate of drug-likeness (QED) score of 0.8. (For comparison, the mean QED of oral medicines is 0.6 ± 0.2). Thus, these studies demonstrate that a low-molecular weight, fragment-like compound can specifically and potently modulate RNA targets.
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MicroRNAs , Bibliotecas de Moléculas Pequenas , Humanos , Bibliotecas de Moléculas Pequenas/farmacologia , Bibliotecas de Moléculas Pequenas/química , Peso Molecular , MicroRNAs/metabolismo , CarcinogêneseRESUMO
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. The RNA-binding module in the RiboTAC is Dovitinib, a known receptor tyrosine kinase (RTK) inhibitor, which was previously identified to bind miR-21 as an off-target. Conversion of Dovitinib into this RiboTAC reprograms the known drug to selectively affect the RNA target. This work demonstrates that DEL can be used to identify compounds that bind and recruit proteins with effector functions in heterobifunctional compounds.
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MicroRNAs , Neoplasias de Mama Triplo Negativas , Humanos , Ribonucleases , DNARESUMO
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.