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.

Translation of the intrinsically disordered protein α-synuclein is inhibited by a small molecule targeting its structured mRNA.

Many proteins are refractory to targeting because they lack small-molecule binding pockets. An alternative to drugging these proteins directly is to target the messenger (m)RNA that encodes them, thereby reducing protein levels. We describe such an approach for the difficult-to-target protein α-synuclein encoded by the SNCA gene. Multiplication of the SNCA gene locus causes dominantly inherited Parkinson's disease (PD), and α-synuclein protein aggregates in Lewy bodies and Lewy neurites in sporadic PD. Thus, reducing the expression of α-synuclein protein is expected to have therapeutic value. Fortuitously, the SNCA mRNA has a structured iron-responsive element (IRE) in its 5' untranslated region (5' UTR) that controls its translation. Using sequence-based design, we discovered small molecules that target the IRE structure and inhibit SNCA translation in cells, the most potent of which is named Synucleozid. Both in vitro and cellular profiling studies showed Synucleozid directly targets the α-synuclein mRNA 5' UTR at the designed site. Mechanistic studies revealed that Synucleozid reduces α-synuclein protein levels by decreasing the amount of SNCA mRNA loaded into polysomes, mechanistically providing a cytoprotective effect in cells. Proteome- and transcriptome-wide studies showed that the compound's selectivity makes Synucleozid suitable for further development. Importantly, transcriptome-wide analysis of mRNAs that encode intrinsically disordered proteins revealed that each has structured regions that could be targeted with small molecules. These findings demonstrate the potential for targeting undruggable proteins at the level of their coding mRNAs. This approach, as applied to SNCA, is a promising disease-modifying therapeutic strategy for PD and other α-synucleinopathies.

Small-molecule targeted recruitment of a nuclease to cleave an oncogenic RNA in a mouse model of metastatic cancer.

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 Designed Small Molecule Inhibitor of a Non-Coding RNA Sensitizes HER2 Negative Cancers to Herceptin.

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.

Small Molecule Targeted Recruitment of a Nuclease to RNA.

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.

Extracellular adenosine produced by ecto-5'-nucleotidase (CD73) regulates macrophage pro-inflammatory responses, nitric oxide production, and favors Salmonella persistence.

Surface enzymes CD39 (nucleoside triphosphate dephosphorylase) and CD73 (ecto-5'-nucleotidase) mediate the synthesis of extracellular adenosine that can regulate immune responses. Adenosine produced by CD39/CD73 acts via adenosine receptors (ARs). CD73 is expressed by a variety of cell types and mediates anti-inflammatory responses. Because efficient innate immune responses are required for clearance of Salmonella infection, we investigated the role of CD73 in macrophage function, including phagocytosis, intracellular killing of Salmonella, and anti-bacterial pro-inflammatory responses to Salmonella-whole cell lysate (ST-WCL) or Salmonella infection. Additionally, RAW 264.7 macrophage mRNA expression of CD39, CD73, and all ARs were measured by qPCR after ST-WCL treatment. Pro-inflammatory cytokine mRNA and nitric oxide (NO) production were quantitated in the ST-WCL treated macrophage with and without CD73-inhibitor (APCP) treatment. Phagocytosis and intracellular killing by peritoneal macrophages from CD73-deficent mice were also evaluated using E. coli BioParticles® and GFP-Salmonella infection, respectively. CD73, CD39, and A2BAR mRNA were predominantly expressed in RAW cells. ST-WCL treatment significantly reduced CD73 expression, suggesting endogenous down-regulation of CD73, and an enhanced pro-inflammatory response. ST-WCL treated and CD73-inhibited macrophages produced more NO and a higher level of pro-inflammatory cytokines than CD73-competent macrophages (e.g. IL-1ß, TNF-α). Phagocytosis of E. coli BioParticles® was significantly higher in the macrophages treated with APCP and in the peritoneal macrophages from CD73-deficent mice as compared to APCP-untreated, and CD73-competent macrophages. Internalized bacteria were more efficiently cleared from macrophages in the absence of CD73, as observed by fluorescence-microscopy and Salmonella-DNA measurement by qPCR from the infected cells. CD73 down-regulation or CD73-inhibition of macrophages during Salmonella infection can enhance the production of pro-inflammatory cytokines and NO production, improving intracellular killing and host survivability. Extracellular adenosine synthesized by CD73 suppresses antibacterial responses of macrophages, which may weaken macrophage function and impair innate immune responses to Salmonella infection.

Small Molecule Inhibition of microRNA-210 Reprograms an Oncogenic Hypoxic Circuit.

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.

Comparison of small molecules and oligonucleotides that target a toxic, non-coding RNA.

Potential RNA targets for chemical probes and therapeutic modalities are pervasive in the transcriptome. Oligonucleotide-based therapeutics are commonly used to target RNA sequence. Small molecules are emerging as a modality to target RNA structures selectively, but their development is still in its infancy. In this work, we compare the activity of oligonucleotides and several classes of small molecules that target the non-coding r(CCUG) repeat expansion (r(CCUG)(exp)) that causes myotonic dystrophy type 2 (DM2), an incurable disease that is the second-most common cause of adult onset muscular dystrophy. Small molecule types investigated include monomers, dimers, and multivalent compounds synthesized on-site by using RNA-templated click chemistry. Oligonucleotides investigated include phosphorothioates that cleave their target and vivo-morpholinos that modulate target RNA activity via binding. We show that compounds assembled on-site that recognize structure have the highest potencies amongst small molecules and are similar in potency to a vivo-morpholino modified oligonucleotide that targets sequence. These studies are likely to impact the design of therapeutic modalities targeting other repeats expansions that cause fragile X syndrome and amyotrophic lateral sclerosis, for example.

How We Think about Targeting RNA with Small Molecules.

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.

Gini Coefficients as a Single Value Metric to Define Chemical Probe Selectivity.

Selectivity is a key requirement of high-quality chemical probes and lead medicines; however, methods to quantify and compare the selectivity of small molecules have not been standardized across the field. Herein, we discuss the origins and use of a comprehensive, single value term to quantify selectivity, the Gini coefficient. Case studies presented include compounds that target protein kinases, small molecules that bind RNA structures, and small molecule chimeras that bind to and degrade the target RNA. With an increasing number of transcriptome- and proteome-wide studies, we submit that reporting Gini coefficients as a quantitative descriptor of selectivity should be used broadly.

Adding Broccoli to the Biosensor Menu.

Small-molecule metabolites regulate many cellular processes but are often present at low concentrations, confounding studies of their signaling networks. In this issue of Cell Chemical Biology, You et al. (2019) describe RNA "integrators" that measure the concentrations of metabolites in live cells to enable in-depth studies of signaling pathways.

Targeted Degradation of a Hypoxia-Associated Non-coding RNA Enhances the Selectivity of a Small Molecule Interacting with RNA.

Small-molecule targeted recruitment of nucleases to RNA is a powerful method to affect RNA biology. Inforna, a sequence-based design approach to target RNA, enables the design of small molecules that bind to and cleave RNA in a selective and substoichiometric manner. Here, we investigate the ability of RNA-targeted degradation to improve the selectivity of small molecules targeting RNA. The microRNA-210 hairpin precursor (pre-miR-210) is overexpressed in hypoxic cancers. Previously, a small molecule (Targapremir-210 [TGP-210]) targeted this RNA in cells, but with a 5-fold window for DNA binding. Appendage of a nuclease recruitment module onto TGP-210 locally recruited ribonuclease L onto pre-miR-210, triggering its degradation. The chimera has enhanced selectivity compared with TGP-210 with nanomolar binding to the pre-miR-210, but no DNA binding, and is broadly selective for affecting RNA function in cells. Importantly, it cleaved pre-miR-210 substoichiometrically and induced apoptosis in breast cancer cells.

Identifying and validating small molecules interacting with RNA (SMIRNAs).

High throughput sequencing has revolutionized our ability to identify aberrant RNA expression and mutations that cause or contribute to disease. These data can be used directly to design oligonucleotide-based modalities using Watson-Crick pairing to target unstructured regions in an RNA. A complementary, although more difficult, strategy to deactivate a malfunctioning RNA is to target highly structured regions with small molecules. Indeed, RNA structures are directly causative of disease. Herein, we discuss emerging strategies to design high affinity, selective, bioactive ligands targeting RNA, or small molecules interacting with RNA (SMIRNAs), and target validation and profiling methods. An experimental foundation is required for a lead identification strategy for RNA structures, constructed from a library-vs.-library screen that probes vast libraries of small molecules for binding RNA three dimensional folds. Dubbed 2-dimensional combinatorial screening (2DCS), the resulting data can be mined against transcriptomes or the composite of RNAs that are produced in an organism to define folded RNA structures that can be targeted. By applying SMIRNAs to cells and using target validation tools such as Chemical Cross-Linking and Isolation by Pull-down (Chem-CLIP) and Small Molecule Nucleic Acid Profiling by Cleavage Applied to RNA (RiboSNAP), all targets engaged in cells can be defined, along with rules for molecular recognition to affect RNA biology. This chapter will describe lessons learned in applying these approaches in vitro, in cells, and in pre-clinical animal models of disease, enabling SMIRNAs to capture opportunities in chemical biology.

Approved Anti-cancer Drugs Target Oncogenic Non-coding RNAs.

Potential RNA drug targets for small molecules are found throughout the human transcriptome, yet small molecules known to elicit a pharmacological response by directly targeting RNA are limited to antibacterials. Herein, we describe AbsorbArray, a small molecule microarray-based approach that allows for unmodified compounds, including FDA-approved drugs, to be probed for binding to RNA motif libraries in a massively parallel format. Several drug classes bind RNA including kinase and topoisomerase inhibitors. The latter avidly bound the motif found in the Dicer site of oncogenic microRNA (miR)-21 and inhibited its processing both in vitro and in cells. The most potent compound de-repressed a downstream protein target and inhibited a miR-21-mediated invasive phenotype. The compound's activity was ablated upon overexpression of pre-miR-21. Target validation via chemical crosslinking and isolation by pull-down showed direct engagement of pre-miR-21 by the small molecule in cells, demonstrating that RNAs should indeed be considered druggable.

Extracellular adenosine generation in the regulation of pro-inflammatory responses and pathogen colonization.

Adenosine, an immunomodulatory biomolecule, is produced by the ecto-enzymes CD39 (nucleoside triphosphate dephosphorylase) and CD73 (ecto-5'-nucleotidase) by dephosphorylation of extracellular ATP. CD73 is expressed by many cell types during injury, infection and during steady-state conditions. Besides host cells, many bacteria also have CD39-CD73-like machinery, which helps the pathogen subvert the host inflammatory response. The major function for adenosine is anti-inflammatory, and most recent research has focused on adenosine's control of inflammatory mechanisms underlying various autoimmune diseases (e.g., colitis, arthritis). Although adenosine generated through CD73 provides a feedback to control tissue damage mediated by a host immune response, it can also contribute to immunosuppression. Thus, inflammation can be a double-edged sword: it may harm the host but eventually helps by killing the invading pathogen. The role of adenosine in dampening inflammation has been an area of active research, but the relevance of the CD39/CD73-axis and adenosine receptor signaling in host defense against infection has received less attention. Here, we review our recent knowledge regarding CD73 expression during murine Salmonellosis and Helicobacter-induced gastric infection and its role in disease pathogenesis and bacterial persistence. We also explored a possible role for the CD73/adenosine pathway in regulating innate host defense function during infection.

A clipped [3]rotaxane derived from bis-nor-seco-cucurbit[10]uril.

This paper describes the synthesis of host 1 by the double bridging reaction of bis-ns-CB[10] with 2 under acidic conditions. Host 1 functions as a double cavity host for aliphatic and aromatic ammonium ions (3-17) in water. Conducting the bridging reaction in the presence of guest 4 delivers [3]rotaxane 1·4(2) by a clipping process.