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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.

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 associate with the selective targeting of RNA, efforts to integrate known methods of RNA binder discovery with fragment-based approaches has been fruitful, as a few bioactive ligands have been identified. Here, we review various fragment-based approaches implemented for RNA targets and provide insight 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 confers selective binding and the physicochemical properties favorable for RNA binding and bioactivity.

A solid-phase DNA-encoded library (DEL) was studied for binding the RNA repeat expansion r(

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.

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.

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.

The discovery of biofunctional natural products (NPs) has relied on the phenotypic screening of extracts and subsequent laborious work to dereplicate active NPs and define cellular targets. Herein, NPs present as crude extracts, partially purified fractions, and pure compounds were screened directly against molecular target libraries of RNA structural motifs in a library-versus-library fashion. We identified 21 hits with affinity for RNA, including one pure NP, nocathiacin I (NOC-I). The resultant data set of NOC-I-RNA fold interactions was mapped to the human transcriptome to define potential bioactive interactions. Interestingly, one of NOC-I's most preferred RNA folds is present in the nuclease processing site in the oncogenic, noncoding microRNA-18a, which NOC-I binds with low micromolar affinity. This affinity for the RNA translates into the selective inhibition of its nuclease processing in vitro and in prostate cancer cells, in which NOC-I also triggers apoptosis. In principle, adaptation of this combination of experimental and predictive approaches to dereplicate NPs from the other hits (extracts and partially purified fractions) could fundamentally transform the current paradigm and accelerate the discovery of NPs that bind RNA and their simultaneous correlation to biological targets.

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.

A hexanucleotide repeat expansion in intron 1 of the

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 (

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(G 4 C 2 ) 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(G 4 C 2 ) 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(G 4 C 2 ) 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.

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.

The ENCODE and genome-wide association projects have shown that much of the genome is transcribed into RNA and much less is translated into protein. These and other functional studies suggest that the druggable transcriptome is much larger than the druggable proteome. This review highlights approaches to define druggable RNA targets and structure-activity relationships across genomic RNA. Binding compounds can be identified and optimized into structure-specific ligands by using sequence-based design with various modes of action, for example, inhibiting translation or directing pre-mRNA splicing outcomes. In addition, strategies to direct protein activity against an RNA of interest via chemically induced proximity is a burgeoning area that has been validated both in cells and in preclinical animal models, and we describe that it may allow rapid access to new avenues to affect RNA biology. These approaches and the unique modes of action suggest that more RNAs are potentially amenable to targeting than proteins.

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, natures 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 5GAG/3CCC 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.

RNA molecules both contribute to and are causative of many human diseases. One method to perturb RNA function is to target its structure with small molecules. However, discovering bioactive ligands for RNA targets is challenging. Here, we show that the bioactivity of a linear dimeric ligand that inactivates the RNA trinucleotide repeat expansion that causes myotonic dystrophy type 1 [DM1; r(CUG)(exp)] can be improved by macrocyclization. Indeed, the macrocyclic compound is ten times more potent than the linear compound for improving DM1‐associated defects in cells, including in patient‐derived myotubes (muscle cells). This enhancement in potency is due to the macrocycle’s increased affinity and selectively for the target, which inhibit r(CUG)(exp)’s toxic interaction with muscleblind‐like 1 (MBNL1), and its superior cell permeability. Macrocyclization could prove to be an effective way to enhance the bioactivity of modularly assembled ligands targeting RNA.

Vascular endothelial growth factor A (VEGFA) stimulates angiogenesis in human endothelial cells, and increasing its expression is a potential treatment for heart failure. Here, we report the design of a small molecule (TGP-377) that specifically and potently enhances VEGFA expression by the targeting of a non-coding microRNA that regulates its expression. A selection-based screen, named two-dimensional combinatorial screening, revealed preferences in small-molecule chemotypes that bind RNA and preferences in the RNA motifs that bind small molecules. The screening program increased the dataset of known RNA motif–small molecule binding partners by 20-fold. Analysis of this dataset against the RNA-mediated pathways that regulate VEGFA defined that the microRNA-377 precursor, which represses Vegfa messenger RNA translation, is druggable in a selective manner. We designed TGP-377 to potently and specifically upregulate VEGFA in human umbilical vein endothelial cells. These studies illustrate the power of two-dimensional combinatorial screening to define molecular recognition events between ‘undruggable’ biomolecules and small molecules, and the ability of sequence-based design to deliver efficacious structure-specific compounds.

Expanded RNA repeats cause >30 incurable diseases. One approach to mitigate their toxicity is by using small molecules that assemble into potent, oligomeric species upon binding to the disease-causing RNA in cells. Herein, we show that the expanded repeat [r(CUG)(exp)] that causes myotonic dystrophy type 1 (DM1) catalyzes the in situ synthesis of its own inhibitor using an RNA-templated tetrazine ligation in DM1 patient-derived cells. The compound synthesized on-site improved DM1-associated defects at picomolar concentrations, enhancing potency by 10,000-fold, compared to its parent compounds that cannot undergo oligomerization. A fluorogenic reaction is also described where r(CUG)(exp) templates the synthesis of its own imaging probe to enable visualization of the repeat in its native context in live cells and muscle tissue.

Many RNAs are processed into biologically active transcripts, the aberrant expression of which can contribute to disease phenotypes. For example, the primary microRNA-17-92 (pri-miR-17-92) cluster contains six microRNAs (miRNAs) that collectively act in several disease settings. Herein, we used sequence-based design of structure-specific ligands to target a common structure in the Dicer processing sites of three miRNAs in the cluster, miR-17, miR-18a, and miR-20a, thereby inhibiting their biogenesis. The compound was optimized to afford a dimeric molecule that binds the Dicer processing site and an adjacent bulge, affording a 100-fold increase in potency. The dimer's mode of action was then extended from simple binding to direct cleavage by conjugation to bleomycin A5 in a manner that imparts RNA-selective cleavage or to indirect cleavage by recruiting an endogenous nuclease, or a ribonuclease targeting chimera (RIBOTAC). Interestingly, the dimer-bleomycin conjugate cleaves the entire pri-miR-17-92 cluster and hence functionally inhibits all six miRNAs emanating from it. The compound selectively reduced levels of the cluster in three disease models: polycystic kidney disease, prostate cancer, and breast cancer, rescuing disease-associated phenotypes in the latter two. Further, the bleomycin conjugate exerted selective effects on the miRNome and proteome in prostate cancer cells. In contrast, the RIBOTAC only depleted levels of pre- and mature miR-17, -18a, and 20a, with no effect on the primary transcript, in accordance with the cocellular localization of RNase L, the pre-miRNA targets, and the compound. These studies demonstrate a strategy to tune RNA structure-targeting compounds to the cellular localization of the target.

RNA repeat expansions cause more than 30 neurological and neuromuscular diseases with no known cures. Since repeat expansions operate via diverse pathomechanisms, one potential therapeutic strategy is to rid them from disease-affected cells, using bifunctional small molecules that cleave the aberrant RNA. Such an approach has been previously implemented for the RNA repeat that causes myotonic dystrophy type 1 [DM1, r(CUG)(exp)] with Cugamycin, which is a small molecule that selectively binds r(CUG)(exp) conjugated to a bleomycin A5 cleaving module. Herein, we demonstrate that, by replacing bleomycin A5 with deglycobleomycin, an analogue in which the carbohydrate domain of bleomycin A5 is removed, the selectivity of the resulting small-molecule conjugate (DeglycoCugamycin) was enhanced, while maintaining potent and allele-selective cleavage of r(CUG)(exp) and rescue of DM1-associated defects. In particular, DeglycoCugamycin did not induce the DNA damage that is observed with high concentrations (25 μM) of Cugamycin, while selectively cleaving the disease-causing allele and improving DM1 defects at 1 μM.

The design and discovery of small molecule medicines has largely been focused on a small number of druggable protein families. A new paradigm is emerging, however, in which small molecules exert a biological effect by interacting with RNA, both to study human disease biology and provide lead therapeutic modalities. Due to this potential for expanding target pipelines and treating a larger number of human diseases, robust platforms for the rational design and optimization of small molecules interacting with RNAs (SMIRNAs) are in high demand. This review highlights three major pillars in this area. First, the transcriptome-wide identification and validation of structured RNA elements, or motifs, within disease-causing RNAs directly from sequence is presented. Second, we provide an overview of high-throughput screening approaches to identify SMIRNAs as well as discuss the lead identification strategy, Inforna, which decodes the three-dimensional (3D) conformation of RNA motifs with small molecule binding partners, directly from sequence. An emphasis is placed on target validation methods to study the causality between modulating the RNA motif in vitro and the phenotypic outcome in cells. Third, emergent modalities that convert occupancy-driven mode of action SMIRNAs into event-driven small molecule chemical probes, such as RNA cleavers and degraders, are presented. Finally, the future of the small molecule RNA therapeutics field is discussed, as well as hurdles to overcome to develop potent and selective RNA-centric chemical probes.

The most common cause of amyotrophic lateral sclerosis and frontotemporal dementia (c9ALS/FTD) is an expanded G(4)C(2) RNA repeat [r(G(4)C(2))(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(G(4)C(2))(exp). The compound was able to selectively bind r(G(4)C(2))(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 patient–derived 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(G(4)C(2))(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(G(4)C(2))(exp) is a strategy for mitigating c9ALS/FTD-associated pathologies and studying disease-associated pathways in preclinical models.

The hexanucleotide repeat expansion GGGGCC [r(G(4)C(2))(exp)] within intron 1 of C9orf72 causes genetically defined amyotrophic lateral sclerosis and frontotemporal dementia, collectively named c9ALS/FTD. , the repeat expansion causes neurodegeneration via deleterious phenotypes stemming from r(G(4)C(2))(exp) RNA gain- and loss-of-function mechanisms. The r(G(4)C(2))(exp) RNA folds into both a hairpin structure with repeating 1 × 1 nucleotide GG internal loops and a G-quadruplex structure. Here, we report the identification of a small molecule (CB253) that selectively binds the hairpin form of r(G(4)C(2))(exp). Interestingly, the small molecule binds to a previously unobserved conformation in which the RNA forms 2 × 2 nucleotide GG internal loops, as revealed by a series of binding and structural studies. NMR and molecular dynamics simulations suggest that the r(G(4)C(2))(exp) hairpin interconverts between 1 × 1 and 2 × 2 internal loops through the process of strand slippage. We provide experimental evidence that CB253 binding indeed shifts the equilibrium toward the 2 × 2 GG internal loop conformation, inhibiting mechanisms that drive c9ALS/FTD pathobiology, such as repeat-associated non-ATG translation formation of stress granules and defective nucleocytoplasmic transport in various cellular models of c9ALS/FTD.

Fragile X-associated tremor and ataxia syndrome (FXTAS) is a late-onset, progressive neurodegenerative disorder characterized by tremors, ataxia and neuropsychological problems. This disease is quite common in the general population with approximately 20 million carriers worldwide. The risk of developing FXTAS increases dramatically with age, with about 45% of male carriers over the age of 50 being affected. FXTAS is caused by a CGG-repeat expansion (CGGexp) in the fragile X mental retardation 1 (FMR1) gene. CGGexp RNA is translated into the FMRpolyG protein by a mechanism called RAN translation. Although both gene and pathogenic trigger are known, no therapeutic interventions are available at this moment. Here, we present, for the first time, primary hippocampal neurons derived from the ubiquitous inducible mouse model which is used as a screening tool for targeted interventions. A promising candidate is the repeat binding, RAN translation blocking, small molecule 1a. Small molecule 1a shields the disease-causing CGGexp from being translated into the toxic FMRpolyG protein. Primary hippocampal neurons formed FMRpolyG-positive inclusions, and upon treatment with 1a, the numbers of FMRpolyG-positive inclusions are reduced. We also describe for the first time the formation of FMRpolyG-positive inclusions in the liver of this mouse model. Treatment with 1a reduced the insoluble FMRpolyG protein fraction in the liver but not the number of inclusions. Moreover, 1a treatment had a reducing effect on the number of Rad23b-positive inclusions and insoluble Rad23b protein levels. These data suggest that targeted small molecule therapy is effective in an FXTAS mouse model and has the potential to treat CGGexp-mediated diseases, including FXTAS.

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.

Myotonic dystrophy type 2 (DM2) is one of >40 microsatellite disorders caused by RNA repeat expansions. The DM2 repeat expansion, r(CCUG)(exp) (where “exp” denotes expanded repeating nucleotides), is harbored in intron 1 of the CCHC-type zinc finger nucleic acid binding protein (CNBP). The expanded RNA repeat causes disease by a gain-of-function mechanism, sequestering various RNA-binding proteins including the pre-mRNA splicing regulator MBNL1. Sequestration of MBNL1 results in its loss-of-function and concomitant deregulation of the alternative splicing of its native substrates. Notably, this r(CCUG)(exp) causes retention of intron 1 in the mature CNBP mRNA. Herein, we report druglike small molecules that bind the structure adopted by r(CCUG)(exp) and improve DM2-associated defects. These small molecules were optimized from screening hits from an RNA-focused small-molecule library to afford a compound that binds r(CCUG)(exp) specifically and with nanomolar affinity, facilitates endogenous degradation of the aberrantly retained intron in which it is harbored, and rescues alternative splicing defects.

RNAs, particularly noncoding RNAs (ncRNAs), are becoming increasingly important therapeutic targets, because they are causative and antagonists of human disease. Indeed, aberrant RNA structural elements and expression deregulate biological processes. In this review, we describe methodologies to discover and optimize small molecules interacting with RNA (SMIRNAs), including the evaluation of direct target engagement and the rescue of RNA-mediated phenotypes in vitro and in vivo. Such studies are essential to fully characterize the mode of action of SMIRNAs and advance our understanding of rationally and efficiently drugging RNAs for therapeutic benefit.

Aberrant RNA structure and function operate in neurological disease progression and severity. As RNA contributes to disease pathology in a complex fashion, that is, via various mechanisms, it has become an attractive therapeutic target for small molecules and oligonucleotides. In this review, we discuss the identification of RNA structures that cause or contribute to neurological diseases as well as recent progress toward the development of small molecules that target them, including small molecule modulators of pre-mRNA splicing and RNA repeat expansions that cause microsatellite disorders such as Huntington’s disease and amyotrophic lateral sclerosis. The use of oligonucleotide-based modalities is also discussed. There are key differences between small molecule and oligonucleotide targeting of RNA. The former targets RNA structure, while the latter prefers unstructured regions. Thus, some targets will be preferentially targeted by oligonucleotides and others by small molecules.

A small molecule (1) with overlapping affinity for two microRNA (miRNA) precursors was used to inform design of a dimeric compound (2) selective for one of the miRNAs. In particular, 2 selectively targets the microRNA(miR)-515 hairpin precursor to inhibit production of miR-515 that represses sphingosine kinase 1 (SK1), a key enzyme in the biosynthesis of sphingosine 1-phosphate (S1P). Application of 2 to breast cancer cells enhanced SK1 and S1P levels, triggering a migratory phenotype. Knockout of SK1, forced overexpression of miR-515, and application of a small molecule SK1 inhibitor all ablated 2's effect on phenotype, consistent with its designed mode of action. Target profiling studies via Chem-CLIP showed that 2 bound selectively to the miR-515 hairpin precursor in cells. Global neoprotein synthesis upon addition of 2 to MCF-7 breast cancer cells demonstrated 2's selectivity and upregulation of cancer-associated proteins regulated by S1P. The most upregulated protein was human epidermal growth factor receptor 2 (ERBB2/HER2), which is regulated by the SK1/S1P pathway and is normally not expressed in MCF-7 cells. Like triple negative breast cancer (TNBC) cells, the lack of HER2 renders them insusceptible to Herceptin and its antibody-drug conjugate Kadcyla. In addition to proteomics, an RNA-seq study supports that 2 has limited off target effects and other studies support that 2 is more selective than an oligonucleotide. We therefore hypothesized that 2 could sensitize MCF-7 cells to anti-HER2 therapies. Indeed, application of 2 sensitized cells to Herceptin. These results were confirmed in two other cell lines that express miR-515 and are HER2-, the hepatocellular carcinoma cell line HepG2 and the TNBC line MDA-MB-231. Importantly, normal breast epithelial cells (MCF-10A) that do not express miR-515 are not affected by 2. These observations suggest a precision medicine approach to sensitize HER2- cancers to approved anticancer medicines. This study has implications for broadening the therapeutic utility of known targeted cancer therapeutics by using a secondary targeted approach to render otherwise insensitive cells, sensitive to a targeted therapeutic.

Mutations in the human tau gene result in alternative splicing of the tau protein, which causes frontotemporal dementia and Parkinsonism. One disease mechanism is linked to the stability of a hairpin within the microtubule-associated protein tau (MAPT) mRNA, which contains an A-bulge. Here we employ computational methods to investigate the structural and thermodynamic properties of several A-bulge RNAs with different closing base-pairs. We find that the current amber RNA force field has a preference to overstabilize base-triple over stacked states, even though some of the A-bulges are known to prefer stacked states according to NMR studies. We further determined that if the neighboring base-pairs of A-bulges are AU, this situation can lead to base slippage. However, when the 3′-side of the A-bulge has an UA base-pair, the stacked state is stabilized by an extra interaction that is not observed in the other sequences. We suggest that these A-bulge RNA systems could be used as benchmarks to improve the current RNA force fields.

SARS-CoV-2 has exploded throughout the human population. To facilitate efforts to gain insights into SARS-CoV-2 biology and to target the virus therapeutically, it is essential to have a roadmap of likely functional regions embedded in its RNA genome. In this report, we used a bioinformatics approach, ScanFold, to deduce the local RNA structural landscape of the SARS-CoV-2 genome with the highest likelihood of being functional. We recapitulate previously-known elements of RNA structure and provide a model for the folding of an essential frameshift signal. Our results find that SARS-CoV-2 is greatly enriched in unusually stable and likely evolutionarily ordered RNA structure, which provides a large reservoir of potential drug targets for RNA-binding small molecules. Results are enhanced via the re-analyses of publicly-available genome-wide biochemical structure probing datasets that are broadly in agreement with our models. Additionally, ScanFold was updated to incorporate experimental data as constraints in the analysis to facilitate comparisons between ScanFold and other RNA modelling approaches. Ultimately, ScanFold was able to identify eight highly structured/conserved motifs in SARS-CoV-2 that agree with experimental data, without explicitly using these data. All results are made available via a public database (the RNAStructuromeDB: https://structurome.bb.iastate.edu/sars-cov-2) and model comparisons are readily viewable at https://structurome.bb.iastate.edu/sars-cov-2-global-model-comparisons.

[Image: see text] RNA contributes to disease pathobiology and is an important therapeutic target. The downstream biology of disease-causing RNAs can be short-circuited with small molecules that recognize structured regions. The discovery and optimization of small molecules interacting with RNA is, however, challenging. Herein, we demonstrate a massively parallel one-bead-one-compound methodology, employed to optimize the linker region of a dimeric compound that binds the toxic r(CUG) repeat expansion [r(CUG)(exp)] causative of myotonic dystrophy type 1 (DM1). Indeed, affinity selection on a 331,776-member library allowed the discovery of a compound with enhanced potency both in vitro (10-fold) and in DM1-patient-derived myotubes (5-fold). Molecular dynamics simulations revealed additional interactions between the optimized linker and the RNA, resulting in ca. 10 kcal/mol lower binding free energy. The compound was conjugated to a cleavage module, which directly cleaved the transcript harboring the r(CUG)(exp) and alleviated disease-associated defects.

The NIH Virtual SARS-CoV-2 Antiviral Summit, held on 6 November 2020, was organized to provide an overview on the status and challenges in developing antiviral therapeutics for coronavirus disease 2019 (COVID-19), including combinations of antivirals. Scientific experts from the public and private sectors convened virtually during a live videocast to discuss severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) targets for drug discovery as well as the preclinical tools needed to develop and evaluate effective small-molecule antivirals. The goals of the Summit were to review the current state of the science, identify unmet research needs, share insights and lessons learned from treating other infectious diseases, identify opportunities for public-private partnerships, and assist the research community in designing and developing antiviral therapeutics. This report includes an overview of therapeutic approaches, individual panel summaries, and a summary of the discussions and perspectives on the challenges ahead for antiviral development.

Significance The development of selective bioactive compounds that target RNA structures is difficult. Here we report an approach to identify low molecular weight fragments that bind to a cancer-causing RNA. By determining where the fragments bind within an RNA target, they can be assembled to provide a high-affinity and potent inhibitor. This approach could be a general way to develop bioactive small molecules that target RNA, which previously was considered “undruggable.” It was applied to provide a precision lead medicine against an RNA that contributes to cancer metastasis., RNAs have important functions that are dictated by their structure. Indeed, small molecules that interact with RNA structures can perturb function, serving as chemical probes and lead medicines. Here we describe the development of a fragment-based approach to discover and optimize bioactive small molecules targeting RNA. We extended the target validation method chemical cross-linking and isolation by pull-down (Chem-CLIP) to identify and map the binding sites of low molecular weight fragments that engage RNA or Chem-CLIP fragment mapping (Chem-CLIP-Frag-Map). Using Chem-CLIP-Frag-Map, we identified several fragments that bind the precursor to oncogenic microRNA-21 (pre-miR-21). Assembly of these fragments provided a specific bioactive compound with improved potency that inhibits pre-miR-21 processing, reducing mature miR-21 levels. The compound exerted selective effects on the transcriptome and selectively mitigated a miR-21–associated invasive phenotype in triple-negative breast cancer cells. The Chem-CLIP-Frag-Map approach should prove general to expedite the identification and optimization of small molecules that bind RNA targets.

Genetically defined amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD), collectively named c9ALS/FTD, are triggered by hexanucleotide GGGGCC repeat expansions [r(G(4)C(2))(exp)] within the C9orf 72 gene. In these diseases, neuronal loss occurs through an interplay of deleterious phenotypes, including r(G(4)C(2))(exp) RNA gain-of-function mechanisms. Herein, we identified a benzimidazole derivative, CB096, that specifically binds to a repeating 1×1 GG internal loop structure, 5′CGG/3′GGC, that is formed when r(G(4)C(2))(exp) folds. Structure−activity relationship (SAR) studies and molecular dynamics (MD) simulations were used to define the molecular interactions formed between CB096 and r(G(4)C(2))(exp) that results in the rescue of disease-associated pathways. Overall, this study reveals a unique structural feature within r(G(4)C(2))(exp) that can be exploited for the development of lead medicines and chemical probes.

Summary 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 β 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.