Myotonic muscular dystrophy type 1 (DM1) and type 2 (DM2) are caused by a toxic gain of function by expanded repeating RNAs. Both expanded repeats reside in noncoding regions and bind to the splicing regulator muscleblind (MBNL1);the formation of the RNA-protein complexes causes DM by inactivating MBNL1. Our group has developed a series of modularly assembled, multivalent ligands that specifically bind the DM RNAs with nanomolar affinities and that potently inhibit the formation of the DM RNA-MBNL1 complex in vitro with nanomolar IC50's. These compounds are also permeable to mouse myoblasts when simply added to the culture medium;a subset of the ligands localize to the nucleus, which is where the RNA-MBNL1 interaction occurs in vivo. Because these compounds are high affinity, specific, and cell permeable, they have the potential to be biologically active.
Our aims are two-fold: determine the efficacy of modularly assembled, multivalent compounds that target the DM1 repeats in cell culture and to understand the molecular recognition of the RNA-ligand complexes using X-ray crystallography in order to design more potent and selective inhibitors. If successful, these investigations may result in the development of a general strategy to design compounds that target toxic triplet repeating RNAs. Specifically, we propose to: 1. Optimize previously identified, multivalent ligands a.) to disrupt the formation of the DM1 RNA- MBNL1 interaction and to specifically bind the DM1 RNA in vitro and b.) for improved uptake, cellular localization, and toxicity profiles in a mouse myoblast cell line and DM1-affected myoblasts. Ligands identified to bind the DM1 RNA were multivalently displayed on a peptoid backbone. In the proposed studies, the spacing modules between ligands will be changed to optimize inhibition potency and to alter cellular uptake and localization properties. 2. Determine if a series of lead compounds that meet the criteria in Specific Aim 1 can increase translation of rCUG-repeat containing RNAs using a luciferase reporter system, disrupt nuclear foci in DM1-affected myoblasts using a fluorescence in situ hybridization (FISH) assay, and correct splicing defects (insulin receptor) that are associated with DM1 using RT-PCR. 3. Determine the structure of DM 1 and DM2 RNAs in the presence of ligand. Such studies will identify the features of the small molecule that are important for binding the RNA. Derivatives can then be made to improve affinity and specificity. We expect that the proposed work will stimulate the economy by enabling hiring of additional scientific staff, consistent with the goals of Notice Number NOT-09-058, NIH Announces the Availability of Recovery Act Funds for Competitive Revision Applications.
Myotonic dystrophy (DM) is a genetic disease characterized by wasting of muscle function including organ wasting that leads to cardiac disease, respiratory impairment, cataracts, and a host of other significant problems. At present, there are no therapeutics that treat the cause of DM, the formation of an RNA-protein complex. In this proposal, we describe the determining the biological efficacy of previously developed small molecules to move towards the development of DM therapies and the structural determination of RNA-ligand complexes to design more potent and specific compounds.
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