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A Small Molecule that Targets an RNA Repeat Expansion

Toxic structured RNAs cause many different diseases.  For example, myotonic dystrophy type 1 (DM1) is caused by r(CUG) repeat expansion [r(CUG)exp] harboured in the 3’ untranslated region (UTR) of the dystrophia myotonica protein kinase (DMPK) mRNA. DM1 is one of the most common forms of adult-onset muscular dystrophy, affecting approximately 1 in 8,000 people.  r(CUG)exp binds to and sequesters various proteins, particularly the pre-mRNA splicing regulator muscleblind-like 1 (MBNL1), which limits the number of MBNL1 available to regulate pre-mRNA splicing and causes system-wide defects. Recently, r(CUG)exp was confirmed to cause another disease called Fuchs endothelial corneal dystrophy (FECD), in which the repeat expansion resides in intron 3 of the transcription factor 4 (TCF4) pre-mRNA. FECD is a dominantly inherited corneal disease that affects as many as 5% of Caucasian males and results in vision impairment. Similar to DM1, r(CUG)exp also sequesters MBNL1 in FECD, causing pre-mRNA splicing defects (Figure1).

Figure1. DM1 and FECD are caused by r(CUG)exp found in the 3’ UTR of DMPK mRNA or in intron 3 of TCF4 mRNA, respectively

A common way to rescue the microsatellite diseases is by the binding of antisense oligonucleotides (ASOs) that target unstructured regions in the coding mRNA. However, ASOs are limited by their poor specificity as tandem repeats are ubiquitous in the genome. Besides, repeat-targeting ASOs also promote the risk of parallel knockdown of transcripts from the wild-type allele, which may give rise to loss-of-function phenotypes. Therefore, ASOs are required to customize for each disease, even if different diseases are caused by the same repeat expansion (Figure2).

Figure2. Using antisense oligonucleotides (ASOs) to target disease-causing RNAs requires customization for each transcript

To overcome the limitations of ASOs, Alicia J. Angelbello et al. designed a series of small molecules that bind r(CUG)exp, compound 2b was selected out with nanomolar affinity and is broadly selective (Figure3). This small molecule directly engages the target, and as a result, it improves r(CUG)exp-mediated defects in both DM1 and FECD. Specifically, the small molecule promotes the excision of intron retained in TCF4 in FECD-affected cells, the excised intron is then degraded by the nuclear exosome complex (Figure4).

Figure3. Design of compounds that bind r(CUG)exp. (A) Structures of small molecules interacting with RNA (SMIRNAs) that target r(CUG)exp. (B) Studying compounds in vitro via a previously reported TR-FRET assay to identify compounds that potently inhibit the r(CUG)exp-MBNL1 complex. 

Figure 4. Mechanism of r(CUG)exp-containing intron 3 decay in FECD cells

Obviously, RNA structure-specific ligands can be applied across diseases that are mediated by the same toxic structure, although targeting RNA structures with small molecules are still at an early stage and the clinical development of such compounds is just beginning to emerge. Several other diseases are also mediated by repeat expansions in retained introns, including myotonic dystrophy type 2 (DM2) and C90rf72-mediated amyotrophic lateral sclerosis/frontotemporal dementia (c9ALS/FTD). It will be interesting to check if exosomal decay can also be activated by ligands that target these repeat expansions. If so, these targets may be more druggable than they immediately appear. 

 References: 
bioRxiv preprint doi: https://doi.org/10.1101/2020.05.11.088427.

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