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.

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.

A cross-linking approach to map small molecule-RNA binding sites in cells.

Methods to identify RNAs bound by small molecules in cells are sparse. Herein, an advance to identify the direct RNA targets of small molecules in cells is described. The approach, dubbed Chemical Cross-Linking and Isolation by Pull-down to Map Small Molecule-RNA Binding Sites (Chem-CLIP-Map-Seq), appends a cross-linker and a purification tag onto a small molecule. In cells, the compound binds to RNA and undergoes a proximity-based reaction. The cross-linked RNA is purified and then amplified using a universal reverse transcription (RT) primer and gene-specific PCR primers. At nucleotides proximal to the binding site, RT "stops" are observed. This approach has broad utility in identifying and validating the RNA targets and binding sites of small molecules in the context of a complex cellular system.

Design of a small molecule against an oncogenic noncoding RNA.

The design of precision, preclinical therapeutics from sequence is difficult, but advances in this area, particularly those focused on rational design, could quickly transform the sequence of disease-causing gene products into lead modalities. Herein, we describe the use of Inforna, a computational approach that enables the rational design of small molecules targeting RNA to quickly provide a potent modulator of oncogenic microRNA-96 (miR-96). We mined the secondary structure of primary microRNA-96 (pri-miR-96) hairpin precursor against a database of RNA motif-small molecule interactions, which identified modules that bound RNA motifs nearby and in the Drosha processing site. Precise linking of these modules together provided Targaprimir-96 (3), which selectively modulates miR-96 production in cancer cells and triggers apoptosis. Importantly, the compound is ineffective on healthy breast cells, and exogenous overexpression of pri-miR-96 reduced compound potency in breast cancer cells. Chemical Cross-Linking and Isolation by Pull-Down (Chem-CLIP), a small-molecule RNA target validation approach, shows that 3 directly engages pri-miR-96 in breast cancer cells. In vivo, 3 has a favorable pharmacokinetic profile and decreases tumor burden in a mouse model of triple-negative breast cancer. Thus, rational design can quickly produce precision, in vivo bioactive lead small molecules against hard-to-treat cancers by targeting oncogenic noncoding RNAs, advancing a disease-to-gene-to-drug paradigm.

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.

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.

Sequence-based design of bioactive small molecules that target precursor microRNAs.

Oligonucleotides are designed to target RNA using base pairing rules, but they can be hampered by poor cellular delivery and nonspecific stimulation of the immune system. Small molecules are preferred as lead drugs or probes but cannot be designed from sequence. Herein, we describe an approach termed Inforna that designs lead small molecules for RNA from solely sequence. Inforna was applied to all human microRNA hairpin precursors, and it identified bioactive small molecules that inhibit biogenesis by binding nuclease-processing sites (44% hit rate). Among 27 lead interactions, the most avid interaction is between a benzimidazole (1) and precursor microRNA-96. Compound 1 selectively inhibits biogenesis of microRNA-96, upregulating a protein target (FOXO1) and inducing apoptosis in cancer cells. Apoptosis is ablated when FOXO1 mRNA expression is knocked down by an siRNA, validating compound selectivity. Markedly, microRNA profiling shows that 1 only affects microRNA-96 biogenesis and is at least as selective as an oligonucleotide.

Inhibition of Non-ATG Translational Events in Cells via Covalent Small Molecules Targeting RNA.

One major class of disease-causing RNAs is expanded repeating transcripts. These RNAs cause diseases via multiple mechanisms, including: (i) gain-of-function, in which repeating RNAs bind and sequester proteins involved in RNA biogenesis and (ii) repeat associated non-ATG (RAN) translation, in which repeating transcripts are translated into toxic proteins without use of a canonical, AUG, start codon. Herein, we develop and study chemical probes that bind and react with an expanded r(CGG) repeat (r(CGG)(exp)) present in a 5' untranslated region that causes fragile X-associated tremor/ataxia syndrome (FXTAS). Reactive compounds bind to r(CGG)(exp) in cellulo as shown with Chem-CLIP-Map, an approach to map small molecule binding sites within RNAs in cells. Compounds also potently improve FXTAS-associated pre-mRNA splicing and RAN translational defects, while not affecting translation of the downstream open reading frame. In contrast, oligonucleotides affect both RAN and canonical translation when they bind to r(CGG)(exp), which is mechanistically traced to a decrease in polysome loading. Thus, designer small molecules that react with RNA targets can be used to profile the RNAs to which they bind in cells, including identification of binding sites, and can modulate several aspects of RNA-mediated disease pathology in a manner that may be more beneficial than oligonucleotides.

Defining RNA motif-aminoglycoside interactions via two-dimensional combinatorial screening and structure-activity relationships through sequencing.

RNA is an extremely important target for the development of chemical probes of function or small molecule therapeutics. Aminoglycosides are the most well studied class of small molecules to target RNA. However, the RNA motifs outside of the bacterial rRNA A-site that are likely to be bound by these compounds in biological systems is largely unknown. If such information were known, it could allow for aminoglycosides to be exploited to target other RNAs and, in addition, could provide invaluable insights into potential bystander targets of these clinically used drugs. We utilized two-dimensional combinatorial screening (2DCS), a library-versus-library screening approach, to select the motifs displayed in a 3×3 nucleotide internal loop library and in a 6-nucleotide hairpin library that bind with high affinity and selectivity to six aminoglycoside derivatives. The selected RNA motifs were then analyzed using structure-activity relationships through sequencing (StARTS), a statistical approach that defines the privileged RNA motif space that binds a small molecule. StARTS allowed for the facile annotation of the selected RNA motif-aminoglycoside interactions in terms of affinity and selectivity. The interactions selected by 2DCS generally have nanomolar affinities, which is higher affinity than the binding of aminoglycosides to a mimic of their therapeutic target, the bacterial rRNA A-site.

Defining the RNA internal loops preferred by benzimidazole derivatives via 2D combinatorial screening and computational analysis.

RNA is an important therapeutic target; however, RNA targets are generally underexploited due to a lack of understanding of the small molecules that bind RNA and the RNA motifs that bind small molecules. Herein, we describe the identification of the RNA internal loops derived from a 4096 member 3 × 3 nucleotide loop library that are the most specific and highest affinity binders to a series of four designer, druglike benzimidazoles. These studies establish a potentially general protocol to define the highest affinity and most specific RNA motif targets for heterocyclic small molecules. Such information could be used to target functionally important RNAs in genomic sequence.

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.

A Massively Parallel Selection of Small Molecule-RNA Motif Binding Partners Informs Design of an Antiviral from Sequence.

Many RNAs cause disease; however, RNA is rarely exploited as a small-molecule drug target. Our programmatic focus is to define privileged RNA motif small-molecule interactions to enable the rational design of compounds that modulate RNA biology starting from only sequence. We completed a massive, library-versus-library screen that probed over 50 million binding events between RNA motifs and small molecules. The resulting data provide a rich encyclopedia of small-molecule RNA recognition patterns, defining chemotypes and RNA motifs that confer selective, avid binding. The resulting interaction maps were mined against the entire viral genome of hepatitis C virus (HCV). A small molecule was identified that avidly bound RNA motifs present in the HCV 30 UTR and inhibited viral replication while having no effect on host cells. Collectively, this study represents the first whole-genome pattern recognition between small molecules and RNA folds.

Rapid Generation of miRNA Inhibitor Leads by Bioinformatics and Efficient High-Throughput Screening Methods.

The discovery of microRNAs (miRNAs) has opened an entire new avenue for drug development. These short (15-22 nucleotides) noncoding RNAs, which function in RNA silencing and posttranscriptional regulation of gene expression, have been shown to critically affect numerous pathways in both development and disease progression. Current miRNA drug development focuses on either reintroducing the miRNA into cells through the use of a miRNA mimic or inhibiting its function via use of a synthetic antagomir. Although these methods have shown some success as therapeutics, they face challenges particularly with regard to cellular uptake and for use as systemic reagents. We recently presented a novel mechanism of inhibiting miR-544 by directed inhibition of miRNA biogenesis. We found that inhibition of DICER processing of miR-544 through the use of a small molecule abolished miR-544 function in regulating adaptation of breast cancer cells to hypoxic stress. Herein, we describe a protocol that utilizes bioinformatics to first identify lead small molecules that bind to DICER cleavage sites in pre-miRNAs and then employ an efficient, high-throughput fluorescent-based screening system to determine the inhibitory potential of the lead compounds and their derivatives.

Defining RNA-Small Molecule Affinity Landscapes Enables Design of a Small Molecule Inhibitor of an Oncogenic Noncoding RNA.

RNA drug targets are pervasive in cells, but methods to design small molecules that target them are sparse. Herein, we report a general approach to score the affinity and selectivity of RNA motif-small molecule interactions identified via selection. Named High Throughput Structure-Activity Relationships Through Sequencing (HiT-StARTS), HiT-StARTS is statistical in nature and compares input nucleic acid sequences to selected library members that bind a ligand via high throughput sequencing. The approach allowed facile definition of the fitness landscape of hundreds of thousands of RNA motif-small molecule binding partners. These results were mined against folded RNAs in the human transcriptome and identified an avid interaction between a small molecule and the Dicer nuclease-processing site in the oncogenic microRNA (miR)-18a hairpin precursor, which is a member of the miR-17-92 cluster. Application of the small molecule, Targapremir-18a, to prostate cancer cells inhibited production of miR-18a from the cluster, de-repressed serine/threonine protein kinase 4 protein (STK4), and triggered apoptosis. Profiling the cellular targets of Targapremir-18a via Chemical Cross-Linking and Isolation by Pull Down (Chem-CLIP), a covalent small molecule-RNA cellular profiling approach, and other studies showed specific binding of the compound to the miR-18a precursor, revealing broadly applicable factors that govern small molecule drugging of noncoding RNAs.

Inforna 2.0: A Platform for the Sequence-Based Design of Small Molecules Targeting Structured RNAs.

The development of small molecules that target RNA is challenging yet, if successful, could advance the development of chemical probes to study RNA function or precision therapeutics to treat RNA-mediated disease. Previously, we described Inforna, an approach that can mine motifs (secondary structures) within target RNAs, which is deduced from the RNA sequence, and compare them to a database of known RNA motif-small molecule binding partners. Output generated by Inforna includes the motif found in both the database and the desired RNA target, lead small molecules for that target, and other related meta-data. Lead small molecules can then be tested for binding and affecting cellular (dys)function. Herein, we describe Inforna 2.0, which incorporates all known RNA motif-small molecule binding partners reported in the scientific literature, a chemical similarity searching feature, and an improved user interface and is freely available via an online web server. By incorporation of interactions identified by other laboratories, the database has been doubled, containing 1936 RNA motif-small molecule interactions, including 244 unique small molecules and 1331 motifs. Interestingly, chemotype analysis of the compounds that bind RNA in the database reveals features in small molecule chemotypes that are privileged for binding. Further, this updated database expanded the number of cellular RNAs to which lead compounds can be identified.

Small molecule chemical probes of microRNA function.

MicroRNAs (miRNAs) are small, non-coding RNAs that control protein expression. Aberrant miRNA expression has been linked to various human diseases, and thus miRNAs have been explored as diagnostic markers and therapeutic targets. Although it is challenging to target RNA with small molecules in general, there have been successful campaigns that have identified small molecule modulators of miRNA function by targeting various pathways. For example, small molecules that modulate transcription and target nuclease processing sites in miRNA precursors have been identified. Herein, we describe challenges in developing chemical probes that target miRNAs and highlight aspects of miRNA cellular biology elucidated by using small molecule chemical probes. We expect that this area will expand dramatically in the near future as progress is made in understanding small molecule recognition of RNA.

Small Molecule Inhibition of miR-544 Biogenesis Disrupts Adaptive Responses to Hypoxia by Modulating ATM-mTOR Signaling.

Hypoxia induces a complex circuit of gene expression that drives tumor progression and increases drug resistance. Defining these changes allows for an understanding of how hypoxia alters tumor biology and informs design of lead therapeutics. We probed the role of microRNA-544 (miR-544), which silences mammalian target of rapamycin (mTOR), in a hypoxic breast cancer model by using a small molecule (1) that selectively impedes the microRNA's biogenesis. Application of 1 to hypoxic tumor cells selectively inhibited production of the mature microRNA, sensitized cells to 5-fluorouracil, and derepressed mRNAs affected by miR-544 in cellulo and in vivo, including boosting mTOR expression. Thus, small molecule inhibition of miR-544 reverses a tumor cell's physiological response to hypoxia. Importantly, 1 sensitized tumor cells to hypoxia-associated apoptosis at a 25-fold lower concentration than a 2'-O-methyl RNA antagomir and was as selective. Further, the apoptotic effect of 1 was suppressed by treatment of cell with rapamycin, a well-known inhibitor of the mTOR signaling pathway, illustrating the selectivity of the compound. Thus, RNA-directed chemical probes, which could also serve as lead therapeutics, enable interrogation of complex cellular networks in cells and animals.

Two-dimensional combinatorial screening enables the bottom-up design of a microRNA-10b inhibitor.

The RNA motifs that bind guanidinylated kanamycin A (G Kan A) and guanidinylated neomycin B (G Neo B) were identified via two-dimensional combinatorial screening (2DCS). The results of these studies enabled the "bottom-up" design of a small molecule inhibitor of oncogenic microRNA-10b.