Targeting MicroRNAs with Small Molecules.

MicroRNAs (miRs) have been implicated in numerous diseases, presenting an attractive target for the development of novel therapeutics. The various regulatory roles of miRs in cellular processes underscore the need for precise strategies. Recent advances in RNA research offer hope by enabling the identification of small molecules capable of selectively targeting specific disease-associated miRs. This understanding paves the way for developing small molecules that can modulate the activity of disease-associated miRs. Herein, we discuss the progress made in the field of drug discovery processes, transforming the landscape of miR-targeted therapeutics by small molecules. By leveraging various approaches, researchers can systematically identify compounds to modulate miR function, providing a more potent intervention either by inhibiting or degrading miRs. The implementation of these multidisciplinary approaches bears the potential to revolutionize treatments for diverse diseases, signifying a significant stride towards the targeting of miRs by precision medicine.

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

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

Targeted Degradation of Structured RNAs via Ribonuclease-Targeting Chimeras (RiboTacs).

INTRODUCTION: RNA structural motifs can serve as recognition sites for proteins or regulatory elements. Notably, these specific RNA shapes are directly related to many diseases. Targeting specific RNA motifs using small molecules is an emerging domain of study within the area of drug discovery. Targeted degradation strategies are a relatively modern technology in drug discovery, offering important clinical and therapeutic outcomes. These approaches involve using small molecules to selectively degrade specific biomacromolecules associated with a disease. "Ribonuclease-Targeting Chimeras" (RiboTaCs) represent a promising type of targeted degradation strategy due to their ability to selectively degrade structured RNA targets. AREAS COVERED: In this review, the authors present the evolution of RiboTaCs, their underlying mechanism, and their in-vitro validation. The authors summarize several disease-associated RNAs that have been previously targeted for degradation using the RiboTaC strategy and discuss how their degradation led to alleviating disease-associated phenotypes in-vitro and in-vivo. EXPERT OPINION: There are several future challenges that still need to be adressed for RiboTaC technology to fully realize its potential. Despite these challenges, the authors are optimistic about its prospects, which have the potential to fundamentally transform the treatment of a wide range of diseases.

Programming inactive RNA-binding small molecules into bioactive degraders.

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

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

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

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

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

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

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

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

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

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

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

A Druglike Small Molecule that Targets r(CCUG) Repeats in Myotonic Dystrophy Type 2 Facilitates Degradation by RNA Quality Control Pathways.

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.

Combining Colistin and Fluconazole Synergistically Increases Fungal Membrane Permeability and Antifungal Cidality.

The increasing emergence of drug-resistant fungal pathogens, together with the limited number of available antifungal drugs, presents serious clinical challenges to treating systemic, life-threatening infections. Repurposing existing drugs to augment the antifungal activity of well-tolerated antifungals is a promising antifungal strategy with the potential to be implemented rapidly. Here, we explored the mechanism by which colistin, a positively charged lipopeptide antibiotic, enhances the antifungal activity of fluconazole, the most widely used orally available antifungal. In a range of susceptible and drug-resistant isolates and species, colistin was primarily effective at reducing fluconazole tolerance, a property of subpopulations of cells that grow slowly in the presence of a drug and may promote the emergence of persistent infections and resistance. Clinically relevant concentrations of colistin synergized with fluconazole, reducing fluconazole minimum inhibitory concentration 4-fold. Combining fluconazole and colistin also increased survival in a C. albicans Galleria mellonella infection, especially for a highly fluconazole-tolerant isolate. Mechanistically, colistin increased permeability to fluorescent antifungal azole probes and to intracellular dyes, accompanied by an increase in cell death that was dependent upon pharmacological or genetic inhibition of the ergosterol biosynthesis pathway. The positive charge of colistin is critical to its antifungal, and antibacterial, activity: colistin directly binds to several eukaryotic membrane lipids (i.e., l-α-phosphatidylinositol, l-α-phosphatidyl-l-serine, and l-α-phosphatidylethanolamine) that are enriched in the membranes of ergosterol-depleted cells. These results support the idea that colistin binds to fungal membrane lipids and permeabilizes fungal cells in a manner that depends upon the degree of ergosterol depletion.

A Small Molecule that Binds an RNA Repeat Expansion Stimulates Its Decay via the Exosome Complex.

Many diseases are caused by toxic RNA repeats. Herein, we designed a lead small molecule that binds the structure of the r(CUG) repeat expansion [r(CUG)exp] that causes myotonic dystrophy type 1 (DM1) and Fuchs endothelial corneal dystrophy (FECD) and rescues disease biology in patient-derived cells and in vivo. Interestingly, the compound's downstream effects are different in the two diseases, owing to the location of the repeat expansion. In DM1, r(CUG)exp is harbored in the 3' untranslated region, and the compound has no effect on the mRNA's abundance. In FECD, however, r(CUG)exp is located in an intron, and the small molecule facilitates excision of the intron, which is then degraded by the RNA exosome complex. Thus, structure-specific, RNA-targeting small molecules can act disease specifically to affect biology, either by disabling the gain-of-function mechanism (DM1) or by stimulating quality control pathways to rid a disease-affected cell of a toxic RNA (FECD).

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

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

Macrocyclization of a Ligand Targeting a Toxic RNA Dramatically Improves Potency.

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.

Optimization of the Linker Domain in a Dimeric Compound that Degrades an r(CUG) Repeat Expansion in Cells.

RNA repeat expansions are responsible for more than 30 incurable diseases. Among them is myotonic dystrophy type 1 (DM1), the most common form of adult on-set muscular dystrophy. DM1 is caused by an r(CUG) repeat expansion [r(CUG)exp] located in the 3' untranslated region (UTR) of the dystrophia myotonica protein kinase gene. This repeat expansion is highly structured, forming a periodic array of 5'CUG/3'GUC internal loop motifs. We therefore designed dimeric compounds that simultaneously bind two of these motifs by connecting two RNA-binding modules with peptoid linkers of different geometries and lengths. The optimal linker contains two proline residues and enhances compound affinity. Equipping this molecule with a bleomycin A5 cleaving module converts the simple binding compound into a potent allele-selective cleaver of r(CUG)exp. This study shows that the linker in modularly assembled ligands targeting RNA can be optimized to afford potent biological activity.

Progress toward the development of the small molecule equivalent of small interfering RNA.

Given that many small molecules could bind to structured regions at sites that will not affect function, approaches that trigger degradation of RNA could provide a general way to affect biology. Indeed, targeted RNA degradation is an effective strategy to selectively and potently modulate biology. We describe several approaches to endow small molecules with the power to cleave RNAs. Central to these strategies is Inforna, which designs small molecules targeting RNA from human genome sequence. Inforna deduces the uniqueness of a druggable pocket, enables generation of hypotheses about functionality of the pocket, and defines on- and off-targets to drive compound optimization. RNA-binding compounds are then converted into cleavers that degrade the target directly or recruit an endogenous nuclease to do so. Cleaving compounds have significantly contributed to understanding and manipulating biological functions. Yet, there is much to be learned about how to affect human RNA biology with small molecules.

A Toxic RNA Catalyzes the Cellular Synthesis of Its Own Inhibitor, Shunting It to Endogenous Decay Pathways.

Myotonic dystrophy type 2 (DM2) is a genetically defined disease caused by a toxic expanded repeat of r(CCUG) [r(CCUG)exp], harbored in intron 1 of CCHC-type zinc-finger nucleic acid binding protein (CNBP) pre-mRNA. This r(CCUG)exp causes toxicity via a gain-of-function mechanism, resulting in three pathological hallmarks: aggregation into nuclear foci; sequestration of muscleblind-like-1 (MBNL1) protein, leading to splicing defects; and retention of CNBP intron 1. We studied two types of small molecules with different modes of action, ones that simply bind and ones that are templated by r(CCUG)exp in cells, i.e., the RNA synthesizes its own drug. Indeed, our studies completed in DM2 patient-derived fibroblasts showed that the compounds disrupt the r(CCUG)exp-MBNL1 complex, reduce intron retention, subjecting the liberated intronic r(CCUG)exp to native decay pathways, and rescue other DM2-associated cellular defects. Importantly, this study shows that small molecules can modulate RNA biology by shunting toxic transcripts toward native decay pathways.

Structure-Specific Cleavage of an RNA Repeat Expansion with a Dimeric Small Molecule Is Advantageous over Sequence-Specific Recognition by an Oligonucleotide.

Myotonic dystrophy type 2 (DM2) is a genetically defined muscular dystrophy that is caused by an expanded repeat of r(CCUG) [r(CCUG)exp] in intron 1 of a CHC-type zinc finger nucleic acid binding protein (CNBP) pre-mRNA. Various mechanisms contribute to DM2 pathology including pre-mRNA splicing defects caused by sequestration of the RNA splicing regulator muscleblind-like-1 (MBNL1) by r(CCUG)exp. Herein, we study the biological impacts of the molecular recognition of r(CCUG)exp's structure by a designer dimeric small molecule that directly cleaves the RNA in patient-derived cells. The compound is comprised of two RNA-binding modules conjugated to a derivative of the natural product bleomycin. Careful design of the chimera affords RNA-specific cleavage, as attachment of the bleomycin cleaving module was done in a manner that disables DNA cleavage. The chimeric cleaver is more potent than the parent binding compound for alleviating DM2-associated defects. Importantly, oligonucleotides targeting the r(CCUG)exp sequence for cleavage exacerbate DM2 defects due to recognition of a short r(CCUG) sequence that is embedded in CNBP, argonaute-1 (AGO1), and MBNL1, reducing their levels. The latter event causes a greater depletion of functional MBNL1 than the amount already sequestered by r(CCUG)exp. Thus, compounds targeting RNA structures can have functional advantages over oligonucleotides that target the sequence in some disease settings, particularly in DM2.

Antifungal activity, mode of action variability, and subcellular distribution of coumarin-based antifungal azoles.

Azole antifungals inhibit the biosynthesis of ergosterol, the fungal equivalent of cholesterol in mammalian cells. Here we report an investigation of the activity of coumarin-substituted azole antifungals. Screening against a panel of Candida pathogens, including a mutant lacking CYP51, the target of antifungal azoles, revealed that this enzyme is inhibited by triazole-based antifungals, whereas imidazole-based derivatives have more than one mode of action. The imidazole-bearing antifungals more effectively reduced trailing growth associated with persistence and/or recurrence of fungal infections than triazole-based derivatives. The imidazole derivatives were more toxic to mammalian cells and more potently inhibited the activity of CYP3A4, which is one of the main causes of azole toxicity. Using live cell imaging, we showed that regardless of the type of azole ring fluorescent 7-diethylaminocoumarin-based azoles localized to the endoplasmic reticulum, the organelle that harbors CYP51. This study suggests that the coumarin is a promising scaffold for development of novel azole-based antifungals that effectively localize to the fungal cell endoplasmic reticulum.

Fluorescent Tracking of the Endoplasmic Reticulum in Live Pathogenic Fungal Cells.

In fungal cells, the endoplasmic reticulum (ER) harbors several of the enzymes involved in the biosynthesis of ergosterol, an essential membrane component, making this organelle the site of action of antifungal azole drugs, used as a first-line treatment for fungal infections. This highlights the need for specific fluorescent labeling of this organelle in cells of pathogenic fungi. Here we report on the development and evaluation of a collection of fluorescent ER trackers in a panel of Candida, considered the most frequently encountered pathogen in fungal infections. These trackers enabled imaging of the ER in live fungal cells. Organelle specificity was associated with the expression of the target enzyme of antifungal azoles that resides in the ER; specific ER labeling was not observed in mutant cells lacking this enzyme. Labeling of live Candida cells with a combination of a mitotracker and one of the novel fungal ER trackers revealed sites of contact between the ER and mitochondria. These fungal ER trackers therefore offer unique molecular tools for the study of the ER and its interactions with other organelles in live cells of pathogenic fungi.