Abstract

Identification of Compounds that Restore FMR1 Gene Expression in Fragile X Syndrome

Daman Kumari, Staff Scientist, Laboratory of Cell and Molecular Biology, National Institute of Diabetes and Digestive and Kidney Diseases, National Institutes of Health (NIH)

Fragile X syndrome (FXS) is the leading cause of inherited cognitive disability and autism spectrum disorder. The most common mutation in FXS patients is the expansion of a CGG-repeat sequence in the 5’-untranslated region of the Fragile X Mental Retardation-1 (FMR1) gene to >200 repeats that causes transcriptional silencing and loss of its protein product, FMRP. Most of the available therapeutic options for FXS target behavioral symptoms and currently there is no cure. Identification of FMRP targets and the elucidation of molecular mechanisms involved in the disease pathology have lead to the development of drugs that target the altered signaling pathways in the brains of FXS patients. However, these strategies are limited by the fact that loss of FMRP affects multiple pathways. Given that the FMR1 gene is silenced in FXS by aberrant epigenetic modifications which can be reversed, and the fact that expanded CGG-repeats are not a part of the open reading frame which is otherwise normal, strategies aimed at restoring FMRP production may be worthwhile. We have used two parallel approaches to identify small molecule compounds that are capable of restoring FMR1 expression in FXS patient cells. These include an unbiased high throughput screening (HTS) approach for identifying compounds that restore FMRP expression, and elucidating the mechanism of FMR1 gene silencing to target specific pathways with known small molecule inhibitors for gene reactivation. We developed a sensitive and quantitative assay for FMRP detection and optimized it for HTS. Using this assay in pilot screens with FXS neural stem cells, we identified a few compounds that were able to increase FMRP expression. This provides proof of principle that screening large compound libraries might yield better hits that could be developed into lead compounds. We have also identified a role for FMR1 mRNA in its own gene silencing. Our data suggest that the ability to interfere with the recruitment of repressive chromatin modifiers by FMR1 mRNA to the gene will allow the development of strategies that specifically target the FMR1 locus for restoring gene expression.