A Rational Approach to Targeting Unstable RNA Repeats

项目总结 一些遗传性疾病，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，在美国也被称为Lou Gehrig病)是不稳定重复的结果 扩张。对于许多这样的重复序列，一旦被转录，它们就会采用一种特殊的发夹结构， 与RNA剪接因子肌样蛋白1(MBNL1)的结合部位非常相似。自动减支 MBNL1的缺失不仅会导致其功能的丧失，而且还会使基因转录本无法输出到 并将其转化为一种功能蛋白质。几个实验室已经证明， RNAexp-MBNL1复合体的破坏会导致疾病表型的逆转，特别是DM1。而当 MBNL1长期以来一直被认为是DM1的真正治疗靶点，但它的设计仍然是一个挑战 能够识别和结合引起DM1的CUGexp的分子，具有高亲和力、序列特异性和 选择性，并取代MBNL1。这项拟议的研究旨在开发一种新的分子平台 以CUGexp为靶点，作为治疗与重复发作相关的神经退行性疾病的概念验证 扩张。 目的1.化学构筑块及其相应配体的合成。在初步研究中，我们有 进行了分子动力学模拟，并进行了化学反应，验证了设计理念的有效性 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 为结合研究准备相应的配体。 目的2.配体结合性质的测定。我们将采用一系列生物物理方法， 包括UV-Vis、CD、ITC、SPR和电泳迁移率改变分析，以确定其结合特性 新设计的配体。充分了解结合动力学和热力学是一种 朝着开发治疗上述遗传病的分子疗法迈出的重要第一步。 所提出的分子设计概念是通用的，不仅适用于靶向CUGexp，而且还适用于许多 其他重复的扩展序列。如果开发成功，拟议中的研究将产生深远的影响 对与重复扩张相关的神经退行性疾病的治疗具有指导意义。

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.