Furamidine Rescues Myotonic Dystrophy Type I Associated Mis-Splicing through Multiple Mechanisms

Myotonic dystrophy type 1 (DMI) is an autosomal dominant, CTG center dot CAG microsatellite expansion disease. Expanded CUG repeat RNA sequester the muscleblind-like (MBNL) family of RNA-binding proteins, thereby disrupting their normal cellular function which leads to global mis-regulation of RNA processing. Previously, the small molecule furamidine was shown to reduce CUG foci and rescue mis-splicing in a DM1 HeLa cell model and to rescue mis-splicing in the HSA(LR) DM1 mouse model, but furamidine's mechanism of action was not explored. Here we use a combination of biochemical, cell toxicity, and genomic studies in DM1 patient-derived myotubes and the HSA(LR) DM1 mouse model to investigate furamidine's mechanism of action. Mis-splicing rescue was observed in DM1 myotubes and the HSA(LR) DM1 mouse with furamidine treatment. Interestingly, while furamidine was found to bind CTG center dot CAG repeat DNA with nanomolar affinity, a reduction in expanded CUG repeat transcript levels was observed in the HSA(LR) DM1 mouse but not DM1 patient-derived myotubes. Further investigation in these cells revealed that furamidine treatment at nanomolar concentrations led to up regulation of MBNL1 and MBNL2 protein levels and a reduction of ribonuclear foci. Additionally, furamidine was shown to bind CUG RNA with nanomolar affinity and disrupted the MBNL1-CUG RNA complex in vitro at micromolar concentrations. Furamidine's likely promiscuous interactions in vitro and in vivo appear to affect multiple pathways in the DM1 mechanism to rescue mis-splicing, yet surprisingly furamidine was shown globally to rescue many mis-splicing events with only modest off target effects on gene expression in the HSA(LR) DM1 mouse model. Importantly, over 20% of the differentially expressed genes were shown to be returned, to varying degrees, to wild-type expression levels.