Targeting Myotonic Dystrophy with Small Molecules
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Myotonic dystrophy (DM) is one of the most common forms of muscular dystrophy, characterized by its hallmark symptom myotonia. DM is an autosomal dominant disease caused by a toxic gain of function RNA. The toxic RNA is produced from expanded non-coding CTG/CCTG repeats, and these CUG/CCUG repeats sequester a family of RNA binding proteins. The Muscleblind-like (MBNL) family of RNA binding proteins are sequestered to the expanded CUG/CCUG repeats. The MBNL proteins are regulators of alternative splicing, and their sequestration to the toxic RNA leads to mis-splicing events, which are believed to cause the symptoms observed in DM patients. A previously reported screen for small molecules used to identify compounds that could disrupt MBNL from binding the toxic CUG repeats found that pentamidine was able to rescue splicing defects associated with DM. Herein, we present a new class of molecules (phenolsulphonphthaleins) that inhibited MBNL1/CUG repeat complex formation in a competitive electrophoretic mobility shift assay (EMSA). Additionally, one of these molecules, bromophenol blue (BPB), acted in a synergistic manner with the previously described inhibitor pentamidine. We also demonstrated that the halogenation of the phenolsulphonphthalein dyes is an important factor for activity. Moreover, we presentant analysis of a series of methylene linker variants of pentamidine that revealed heptamidine (an analog of pentamidine) could reverse splicing defects in a DM1 tissue culture model and rescue myotonia in a DM1 mouse model. Finally, we report on a new crystal structure of CUG repeats, crystallized in the context of a GAAA tetraloop/receptor which facilitated ordered packing within the crystal. This structure was consistent with previous structures showing that the repeats are essentially A-form RNA, despite having a U-U mismatch every third base pair. We also identified six types of U-U mismatch in the context of the 5'CUG/3'GUC motif, suggesting that the interactions between the uridines are dynamic. This structure also contains the highest resolution GAAA tetraloop/receptor structure (1.95 Å) reported to date. This dissertation includes previously unpublished co-authored material.