Small Molecule Drugs Targeting Non-Coding RNAs as Treatments for Alzheimer's Disease and Related Dementias

Abstract

Despite the enormous burden of Alzheimer's disease and related dementias (ADRD) on patients, caregivers, and society, only a few treatments with limited efficacy are currently available. While drug development conventionally focuses on disease-associated proteins, RNA has recently been shown to be druggable for therapeutic purposes as well. Approximately 70% of the human genome is transcribed into non-protein-coding RNAs (ncRNAs) such as microRNAs, long ncRNAs, and circular RNAs, which can adopt diverse structures and cellular functions. Many ncRNAs are specifically enriched in the central nervous system, and their dysregulation is implicated in ADRD pathogenesis, making them attractive therapeutic targets. In this review, we first detail why targeting ncRNAs with small molecules is a promising therapeutic strategy for ADRD. We then outline the process from discovery to validation of small molecules targeting ncRNAs in preclinical studies, with special emphasis on primary high-throughput screens for identifying lead compounds. Screening strategies for specific ncRNAs will also be included as examples. Key challenges-including selecting appropriate ncRNA targets, lack of specificity of small molecules, and general low success rate of neurological drugs and how they may be overcome-will be discussed throughout the review.

Keywords: Alzheimer’s disease and related dementias; circRNA; drug discovery; high-throughput screens; lncRNA; miRNA; non-coding RNA; small molecules.

Figure 1

Methods for discovering small molecules modulating the expression level and activity of ncRNAs. (A) In the cell-based reporter assays, a fluorescent or luminescent reporter is used to measure ncRNA-expression level or activity: (i) if the reporter is under the control of a ncRNA promoter region, small molecules that modulate the reporter may transcriptionally modulate the ncRNA; (ii) if the reporter gene is under the control of a region interacting with an ncRNA, small molecules that modulate the reporter may regulate the expression level or activity of the ncRNA; (iii) for variants of an ncRNA produced from alternative splicing, small molecules that modulate the in-frame expression of the reporter gene may regulate alternative splicing to favor the production of a specific variant. (B) Various biochemical assays can be used to discover small molecules that bind directly to ncRNAs to modulate their configuration and function: (i) in the small molecule microarray assay, small molecules are immobilized on glass slides and then incubated with fluorophore-bound ncRNAs. After unbound ncRNAs are washed away, fluorescence signals may indicate binding between a small molecule and the ncRNA; (ii) in the fluorescent indicator displacement (FID) assay, the ncRNA is first reversibly attached to a fluorescent indicator. Small molecules that bind to and displace the indicator result in a loss of fluorescent signal; (iii) in the automated ligand identification system (ALIS), the ncRNA of interest is first incubated with various small molecules. RNA-small molecule complexes are then separated from unbound RNAs and small molecules by size-exclusion chromatography, followed by treatment under harsh conditions to release the small molecules from the complexes. The small molecules are then identified by liquid chromatography-mass spectrometry (LC-MS); (iv) computational methods can also screen millions of small molecules to predict those that are more likely to bind to a particular ncRNA. The Inforna platform utilizes known RNA motif–small molecule interactions to predict compounds for specific RNA secondary structures. RNA structures produced by X-ray crystallography or nuclear magnetic resonance (NMR) can also be used to facilitate small molecule—RNA docking. (C) Several assays can also be used to discover small molecules that modulate RNA-protein interaction: (i) in the fluorescence resonance energy transfer (FRET) assay, the protein can be tagged with a fluorophore, whereas the ncRNA is tagged with either an activator or a quencher; (ii) in the catalytic enzyme-linked click chemistry assay (cat-ELCCA), the protein is immobilized, while the RNA is tagged with a handle such as a 5′-trans-cyclooctene (TCO) that can be converted into horseradish peroxidase (HRP) using click chemistry; (iii) in the fluorescence polarization assay, small molecules bound to proteins rotate more slowly than unbound molecules, resulting in more highly polarized light emission. Small molecules that modulate the fluorescent, or luminescent, or polarized light intensity in these assays may also modulate ncRNA-protein interaction. (D) High throughput ncRNA-seq can be used to directly identify ncRNAs that are differentially expressed in cellular models treated with small molecules. This approach also enables the construction of ncRNA networks altered by a particular small molecule.

Figure 2

Preclinical discovery workflow for therapeutic small molecules targeting ncRNAs. This figure briefly outlines the workflow for discovering and validating small molecules targeting ncRNAs, with the recommended cellular and animal models at various steps on the right. The workflow starts with selecting an ADRD-relevant ncRNA target, a compound library, and a screening method. The size of the compound library, from a few hundred to millions of small molecules, will depend on various factors, including cost and estimated hit rate. For convenience and cost concerns, the primary HTS can be performed computationally, in biochemical assays with synthetic ncRNAs and small molecules, in immortalized cell lines, or, if possible, in ADRD-relevant cellular models. Once several candidates of small molecules have been identified from the primary screen, they should be validated in ADRD-relevant cellular models, including primary cells from animal models, cells differentiated from patient-derived iPSCs, organotypic brain slices, and brain organoids. An optimal dose that maximally modulates the ncRNA target should first be established. Subsequently, the mechanism of action, the specificity of action, and the functional efficacy and potential toxicity of each candidate small molecule can be investigated. Small molecules that specifically modulate the target ncRNA and its downstream targets and show neuroprotective effects with minimal toxicity can be further optimized with medicinal chemistry for improved potency, efficacy, safety, and BBB penetrance. Next, these improved small molecules can be tested for evidence of efficacy at the cellular and behavioral levels in multiple animal models of ADRD, followed by further chemistry optimization as needed. Finally, a small molecule with specific ncRNA target engagement, satisfactory brain PK/PD, good safety, and evidence of efficacy in animal models may be able to progress to human clinical trials. Abbreviations: Chem-Clip: Chemical Cross-Linking, and Isolation by Pull-down; ITC: Isothermal titration calorimetry; NMR: nuclear magnetic resonance; BBB: blood–brain barrier; PK/PD: pharmacokinetics and pharmacodynamics; iPSCs: induced pluripotent stem cells.