High-throughput screening yields several small-molecule inhibitors of repeat-associated non-AUG translation

Abstract

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). 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 dosedependently 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. This work was supported by Veterans Affairs Biomedical Laboratory Research and Development Grants 1I21BX001841 and 1I01BX003231, National Institutes of Health Grants R01NS099280 and R01NS086810, and the Michigan Alzheimer's Disease Center and Protein Folding Disease Initiative (to P. K. T., M. I. I., and A. B. S.). P. K. T. served as a consultant with Denali Therapeutics, and he, K. M. G., and M. G. K. licensed technology through the University of Michigan to Denali Therapeutics that is based on the work published here. The content is solely the responsibility of the authors and does not necessarily represent the official views of the National Institutes of Health. This article contains Tables S1-S3 and Figs. S1-S7.

In silico, in vitro, and in vivo Approaches to Identify Molecular Players in Fragile X Tremor and Ataxia Syndrome

“…Three small-molecule RAN inhibitors have been recently shown to interact with CGG and GGGGCC repeat RNAs by circular dichroism and native gel analysis: BIX01294, CP-31398, and propidium iodide (Green et al, 2019). These small, bioactive compounds can selectively inhibit RAN translation across multiple disease-causing repeat expansion mutations and could therefore be employed in future treatments (Green et al, 2019).…”

Small Molecules Targeting H3K9 Methylation Prevent Silencing of Reactivated FMR1 Alleles in Fragile X Syndrome Patient Derived Cells

“…Thus, combining small molecules that reactivate FM alleles with such ASOs might provide a good strategy to restore FMRP expression while reducing the negative consequences of rCGG toxicity. Since BIX01294 has also been shown to reduce CGG RAN translation [54], related compounds with similar properties may provide added benefit. Until recently, the lack of a suitable animal model for the gene silencing seen in human FM carriers has limited preclinical evaluation of the efficacy and toxicity of FMR1-reactivating drugs.…”

A chemical screen identifies a link between lipid metabolism and mRNA translation

“…On the other hand, we here show, for the first time, that the toxicity of ISR activators such as Tunicamycin or SKI-II is alleviated by a concomitant treatment with PERKi or ISRIB, although whether this is related to translation remains unknown. To end, and despite ISR activators might be toxic for cancer cells, we should note that there are also disorders where an induction of the ISR has been proposed to be beneficial, such as Charcot-Marie-Tooth disease, opening new areas where SPHK inhibitors might be of help (11,47). In addition, mTORC1 inhibitors that lower translation are widely used in biomedical research as tools to revert age-related pathologies and some have even obtained clinical approval in the context of oncology and organ transplant.…”