A Rational Approach to Targeting Unstable RNA Repeats

靶向不稳定 RNA 重复序列的合理方法

• 批准号: 9316040
• 负责人: DANITH H LY
• 金额: $ 22.36万
• 依托单位: CARNEGIE-MELLON UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2017
• 资助国家: 美国
• 起止时间: 2017-04-01 至 2019-03-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/9316040
• 关键词: Acids; Adopted; Affect; Affinity; Alleles; Amino Acid Sequence; Amyotrophic Lateral Sclerosis; Ataxia; Atrophic; Back; Base Pairing; Binding; Binding Sites; Biophysics; Chemical Structure; Code; Complex; Cytoplasm; DNA; Development; Disease; Drug Kinetics; Electrophoretic Mobility Shift Assay; Fragile X Syndrome; Genes; Goals; Grant; Hand; Hereditary Disease; Huntington gene; Hydrogen Bonding; Individual; Intervention; Kinetics; Lateral; Lead; Length; Ligand Binding; Ligands; Machado-Joseph Disease; Methods; Molecular; Mus; Muscle; Mutation; Myotonic Dystrophy; Neurodegenerative Disorders; Oligonucleotides; Pathogenicity; Peptides; Pharmaceutical Preparations; Physiological; Production; Property; Proteins; Publishing; RNA; RNA Splice Sites; RNA Splicing; Research; Research Project Grants; Roentgen Rays; Route; Scheme; Sclerosis; Series; Specificity; Spinobulbar Muscular Atrophy; Structure; TNFSF5 gene; Testing; Therapeutic; Thermodynamics; Transcript; Translating; Tremor; Triplet Multiple Birth; Ursidae Family; Vertebral column; X-Ray Crystallography; base; biophysical techniques; blind; chemical reaction; chemical synthesis; citral B; design; disease phenotype; indium arsenide; monomer; neuromuscular; novel; nucleobase; protein function; prototype; scale up; simulation; small molecule; therapeutic development; therapeutic target; therapy development

PROJECT SUMMARY A number of genetic disorders, including myotonic dystrophy (DM1 and DM2), fragile X tremor/ataxia, Huntingtin's disease, Machado-Joseph disease, spinocerebella ataxia, and more recently, amyotrophic lateral sclerosis (ALS, also known in the U.S. as Lou Gehrig's disease), occur as the result of unstable repeat expansion. For many of these repeat sequences, once transcribed they adopt a peculiar hairpin structure that closely resembles the binding site of an RNA splicing factor muscleblind-like 1 (MBNL1) protein. Sequestration of MBNL1 not results in the loss of its function, but also traps the gene transcript from being exported to the cytoplasm and from being translated into a functional protein. It has been demonstrated by several labs that disruption of the RNAexp-MBNL1 complex leads to reversion in the disease phenotype, in particular DM1. While MBNL1 has long been recognized as a bona fide therapeutic target of DM1, it remains a challenge to design molecules that can recognize and bind CUGexp, the cause of DM1, with high affinity, sequence-specificity, and selectivity, and displace MBNL1. The proposed research aims at developing a novel molecular platform for targeting CUGexp, as a proof-of-concept for treating neurodegenerative diseases associated with repeat expansion. Aim 1. Synthesis of chemical building blocks and the corresponding ligands. In the preliminary study we have performed MD simulations and carried out chemical reactions demonstrating the validity of the design concept and the feasibility of the synthetic routes. In the proposed study, we will scale up the monomer production and prepare the corresponding ligands for binding study. Aim 2. Determination of the binding properties of ligands. We will employ an array of biophysical methods, including UV-vis, CD, ITC, SPR, and electrophoretic mobility-shift assays to determine the binding properties of the newly designed ligands. Gaining a full understanding of the binding kinetics and thermodynamics is an important first step toward developing molecular therapies for treating the aforementioned genetic diseases. The proposed molecular design concept is general, applicable not only to targeting CUGexp, but also a slew of other repeated expansion sequences. If successfully developed, the proposed research will have far-reaching implication for the treatment of neurodegenerative diseases associated with repeat expansion.

项目摘要 许多遗传疾病，包括肌发育症（DM1和DM2），脆弱的X震颤/共济失调， 亨廷顿氏病，马查多 - 约瑟夫病，脊髓疾病，以及最近的肌萎缩性侧向 硬化症（ALS，在美国也被称为Lou Gehrig氏病），由于重复不稳定而发生 扩张。对于许多这些重复序列的许多 与RNA剪接因子肌肉闪光1（MBNL1）蛋白的结合位点非常相似。隔离 MBNL1的效果并不导致其功能的丧失，但也将基因转录本从出口到该基因转录本 细胞质和转化为功能蛋白。几个实验室已经证明了 RNAExp-MBNL1复合物的破坏会导致疾病表型的恢复，特别是DM1。尽管 MBNL1长期以来一直被认为是DM1的真正治疗靶标，设计仍然是一个挑战 可以识别和结合CugeXP的分子，DM1的原因，具有高亲和力，序列特异性和 选择性和移位MBNL1。拟议的研究旨在为开发一个新颖的分子平台 针对CugeXP，作为治疗与重复相关的神经退行性疾病的概念证明 扩张。 目标1。化学构建块和相应配体的合成。在初步研究中，我们有 进行了MD模拟并进行了化学反应，证明了设计概念的有效性 以及合成路线的可行性。在拟议的研究中，我们将扩展单体生产，并 准备相应的配体进行结合研究。 目标2。确定配体的结合特性。我们将采用一系列生物物理方法， 包括UV-VIS，CD，ITC，SPR和电泳迁移率迁移测定，以确定结合特性 新设计的配体。完全了解结合动力学和热力学是一种 开发用于治疗上述遗传疾病的分子疗法的重要第一步。 提出的分子设计概念是一般的，不仅适用于靶向CugeXP，还适用 其他重复的扩展序列。如果成功开发，拟议的研究将具有深远的影响 对与重复扩张相关的神经退行性疾病的治疗的影响。