A Rational Approach to Targeting Unstable RNA Repeats

靶向不稳定 RNA 重复序列的合理方法

基本信息

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
项目类别：
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

项目摘要

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.

项目概要许多遗传性疾病，包括强直性肌营养不良（DM1 和 DM2）、脆性 X 震颤/共济失调、亨廷顿病、马查多-约瑟夫病、脊髓小脑共济失调以及最近的肌萎缩侧索硬化症硬化症（ALS，在美国也称为卢伽雷氏病），由于不稳定的重复而发生扩张。对于许多这样的重复序列，一旦转录，它们就会采用一种特殊的发夹结构，与 RNA 剪接因子肌肉盲样 1 (MBNL1) 蛋白的结合位点非常相似。封存 MBNL1的缺失并不会导致其功能丧失，还会阻止基因转录本被输出到细胞质并被翻译成功能性蛋白质。多个实验室已经证明 RNAexp-MBNL1 复合物的破坏会导致疾病表型的逆转，特别是 DM1。尽管 MBNL1长期以来被认为是DM1的真正治疗靶点，但设计仍然是一个挑战能够识别并结合 CUGexp（DM1 的原因）的分子，具有高亲和力、序列特异性和选择性，并取代MBNL1。拟议的研究旨在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验证扩张。目标 1. 化学结构单元和相应配体的合成。在初步研究中我们有进行MD模拟并进行化学反应，证明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，我们将扩大单体生产和制备相应的配体用于结合研究。目标 2. 测定配体的结合特性。我们将采用一系列生物物理方法，包括 UV-vis、CD、ITC、SPR 和电泳迁移率变化测定，以确定新设计的配体。充分了解结合动力学和热力学是一个开发治疗上述遗传疾病的分子疗法的重要第一步。所提出的分子设计概念是通用的，不仅适用于靶向 CUGexp，还适用于一系列其他重复的扩展序列。如果开发成功，所提出的研究将具有深远的影响对治疗与重复扩张相关的神经退行性疾病的意义。