Identifying and Studying RNA Loop-Small Molecule Interactions

描述(由申请人提供)：强直性肌营养不良1型(DM1)和2型(DM2)是由扩展的重复RNA获得的有毒功能引起的。这两个扩展的重复序列都位于非编码区，并与剪接调节蛋白(MBNL1)结合；RNA-蛋白质复合体的形成通过使MBNL1失活而导致DM。我们的团队已经开发出一系列模块化组装的多价配体，这些配体可以将DM RNA与纳米分子亲和力特异地结合在一起，并在体外有效地抑制DMRNA-MBNL1与纳米分子IC50‘S复合体的形成。当这些化合物简单地添加到培养液中时，这些化合物也对小鼠成肌细胞具有渗透性；部分配体定位于细胞核，也就是体内发生RNA-MBNL1相互作用的地方。由于这些化合物具有高亲和力、特异性和细胞渗透性，它们具有潜在的生物活性。我们的目标有两个：确定在细胞培养中针对DM1重复序列的模块化组装的多价化合物的有效性，以及利用X射线结晶学了解RNA配体复合体的分子识别，以便设计更有效和更具选择性的抑制剂。如果成功，这些研究可能导致开发出一种通用策略，以设计针对有毒的三联体重复RNA的化合物。具体地说，我们建议：1.优化先前确定的多价配体a。)破坏DM1RNA-MBNL1相互作用的形成，并在体外与DM1RNA特异性结合。用于改善小鼠成肌细胞系和DM1影响的成肌细胞的摄取、细胞定位和毒性分布。与DM1RNA结合的配体以多价形式显示在类肽骨架上。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满足特定目的1标准的先导化合物是否能够增加含有rCUG重复序列的RNA的翻译，使用荧光原位杂交(FISH)方法破坏DM1影响的成肌细胞中的核焦点，并使用RT-PCR纠正与DM1相关的剪接缺陷(胰岛素受体)。3.在配体存在的情况下确定DM1和DM2 RNA的结构。这样的研究将确定小分子的特征，这些特征对于结合RNA是重要的。然后，可以制作衍生品来提高亲和力和特异性。我们预计拟议的工作将通过雇佣更多的科学人员来刺激经济，与通知编号NOT-09-058的目标一致，NIH宣布可为竞争性修订申请提供恢复法案资金。 公共卫生相关性：强直性肌营养不良(DM)是一种遗传性疾病，其特征是肌肉功能衰竭，包括导致心脏疾病、呼吸功能障碍、白内障和许多其他重大问题的器官萎缩。目前，还没有治疗糖尿病的疗法，糖尿病是一种RNA-蛋白质复合体的形成。在这项建议中，我们描述了先前开发的小分子的生物学有效性的测定，以走向DM治疗的发展，以及RNA-配体复合体的结构测定，以设计更有效和特定的化合物。

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.