Furamidine rescues myotonic dystrophy type I associated mis-splicing through multiple mechanisms.

Myotonic dystrophy type 1 (DM1) is an autosomal dominant, CTG•CAG microsatellite expansion disease. Expanded CUG repeat RNA sequester the muscleblind-like (MBNL) family of RNA-binding proteins, thereby disrupting their normal cellular function leading 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 rescue mis-splicing in the HSALR 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 HSALR DM1 mouse model to investigate furamidine's mechanism of action. Mis-splicing rescue was observed in DM1 myotubes and the HSALR DM1 mouse with furamidine treatment. Interestingly, while furamidine was found to bind CTG•CAG repeat DNA with nanomolar affinity, a reduction in expanded CUG repeat transcript levels was observed in the HSALR 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 HSALR DM1 mouse model. Importantly, over 20% of the differentially expressed genes were shown to be returned, to varying degrees, to wild type expression levels.