Identifying and Studying RNA Loop-Small Molecule Interactions

描述（由申请人提供）：1型肌强直性肌营养不良（DM1）和2型（DM2）是由扩展重复rna的毒性功能获得引起的。这两个扩展的重复序列都位于非编码区，并与剪接调节因子肌盲（MBNL1）结合；rna -蛋白复合物的形成通过使MBNL1失活导致糖尿病。我们的团队已经开发了一系列模块化组装的多价配体，它们以纳米摩尔亲和力特异性地结合DM rna，并在体外以纳米摩尔IC50有效地抑制DM RNA-MBNL1复合物的形成。当简单地加入培养基时，这些化合物也能渗透到小鼠成肌细胞中；配体的一个子集定位于细胞核，这是RNA-MBNL1相互作用在体内发生的地方。由于这些化合物具有高亲和力、特异性和细胞渗透性，因此具有潜在的生物活性。我们的目标是双重的：确定模块化组装的多价化合物在细胞培养中靶向DM1重复序列的功效，并使用x射线晶体学了解rna -配体复合物的分子识别，以便设计更有效和选择性的抑制剂。如果成功，这些研究可能会导致设计针对有毒三联体重复rna的化合物的一般策略的发展。具体而言，我们建议：1。优化先前鉴定的多价配体a)破坏DM1 RNA- MBNL1相互作用的形成，并在体外特异性结合DM1 RNA； b)改善小鼠成肌细胞系和受DM1影响的成肌细胞的摄取、细胞定位和毒性谱。确定结合DM1 RNA的配体在肽样主干上多价显示。在拟议的研究中，将改变配体之间的间距模块以优化抑制效力并改变细胞摄取和定位特性。2. 使用荧光素酶报告系统确定一系列符合Specific Aim 1标准的先导化合物是否可以增加含有rcug重复rna的翻译，使用荧光原位杂交（FISH）检测破坏DM1影响的成肌细胞的核病灶，并使用RT-PCR纠正与DM1相关的剪接缺陷（胰岛素受体）。3. 测定配体存在下dm1和DM2 rna的结构。这样的研究将确定对结合RNA很重要的小分子的特征。然后可以制造衍生物来提高亲和力和特异性。我们期望拟议的工作将通过雇用额外的科学人员来刺激经济，与通知号no -09-058的目标一致，NIH宣布为竞争性修订申请提供恢复法案资金。

DESCRIPTION (provided by applicant): 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. PUBLIC HEALTH RELEVANCE: 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.