Abstract
Repeat-associated non-AUG (RAN) translation is a noncanonical translation initiation event that occurs at nucleotide-repeat expansion mutations that are associated with several neurodegenerative diseases, including fragile X-associated tremor ataxia syndrome (FXTAS), ALS, and frontotemporal dementia (FTD). Translation of expanded repeats produces toxic proteins that accumulate in human brains and contribute to disease pathogenesis. Consequently, RAN translation constitutes a potentially important therapeutic target for managing multiple neurodegenerative disorders. Here, we adapted a previously developed RAN translation assay to a high-throughput format to screen 3,253 bioactive compounds for inhibition of RAN translation of expanded CGG repeats associated with FXTAS. We identified five diverse small molecules that dose-dependently inhibited CGG RAN translation, while relatively sparing canonical translation. All five compounds also inhibited RAN translation of expanded GGGGCC repeats associated with ALS and FTD. Using CD and native gel analyses, we found evidence that three of these compounds, BIX01294, CP-31398, and propidium iodide, bind directly to the repeat RNAs. These findings provide proof-of-principle supporting the development of selective small-molecule RAN translation inhibitors that act across multiple disease-causing repeats.
Highlights
Repeat-associated non-AUG (RAN) translation is a noncanonical translation initiation event that occurs at nucleotiderepeat expansion mutations that are associated with several neurodegenerative diseases, including fragile X–associated tremor ataxia syndrome (FXTAS), ALS, and frontotemporal dementia (FTD)
To identify small-molecule inhibitors of RAN translation of FXTAS-associated CGG repeats, we adapted a previously developed in vitro RAN translation assay [26] to a 384-well format (Fig. 1A). This assay utilized a ϩ1CGGx100 RAN translation nanoluciferase (NLuc) reporter mRNA, which was added to wells containing rabbit reticulocyte lysate (RRL) treated with one of 3,253 bioactive compounds at 20 M (Fig. 1A)
As a positive control for translation inhibition, the last two columns of each plate were treated with 3 M cycloheximide, and as an internal negative control, the first two columns were treated with DMSO (Fig. 1A)
Summary
To better define the mechanism of RAN translation and identify potential inhibitors of this process, we developed an in vitro, reporter-based small molecule screen for bioactive compounds that selectively and dose-dependently inhibit RAN translation of CGG repeats. From this screen, we identified five novel CGG RAN translation inhibitors and found that each compound inhibits RAN translation of C9ALS/FTD-associated GGGGCC repeats, in multiple sense-strand reading frames. These studies establish that RAN translation from multiple repeat expansions and across multiple reading frames can be selectively targeted by small-molecule inhibitors, providing insights into the mechanisms by which RAN translation occurs and establishing a framework for future therapeutic development in nucleotide repeat expansion disorders
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