Targeting a Toxic RNA with Small Molecules

描述(申请人提供)：强直性肌营养不良(DM)是由重复的核苷酸扩张引起的，特别是在营养不良症肌强直蛋白激酶基因(DMPK)和锌指9基因(ZNF9)的非编码区分别发生CUG和CCUG扩张。CUG扩张型患者为I型强直性肌营养不良(DM1)，CCUG扩张型患者为II型强直性肌营养不良(DM2)。DM1和DM2的患者表现出相同的症状，这表明CUG和CCUG的扩张都通过共同的机制导致DM。假设这些非编码扩张通过RNA功能获得机制导致DM；扩展的CUG和CCUG重复RNA作为有毒RNA隔离RNA结合蛋白MBNL1，并间接增加另一种RNA结合蛋白CUGBP1的蛋白质水平，从而扰乱这些蛋白质的正常细胞功能。MBNL1和CUGBP1是前mRNA剪接因子，其活性浓度的降低和增加会导致多个转录本的选择性剪接的错误调节，对于那些含有CUG和CCUG扩增的人来说，结果是DM。导致DM1的扩展的CUG重复是一个很好的药物靶点，因为如果一个人能够识别出一个与CUG重复结合的小分子，这个结合事件可以释放隔离的MBNL1，预期的结果将是MBNL1将能够适当地调节其底物前-mRNAs的剪接，并减少或消除强直性肌营养不良的一些症状。为了实现这一目标，我们已经确定了一种小分子(五烷胺)，它可以结合CUG重复序列并释放MBNL1，在人类DM1细胞模型中逆转与DM1相关的剪接缺陷，并在DM1小鼠模型中部分逆转剪接缺陷。虽然五烷胺与CUG重复结合并逆转了与DM1相关的一些分子缺陷，但五烷胺对CUG重复的特异性似乎有限；因此，与五烷胺相比，合成五烷胺的类似物的目的是获得对CUG重复具有更高特异性和亲和力的小分子。五烷双胺破坏MNBL-CUG重复相互作用的机制正在确定中，这一知识将用于改进活性提高的类似物的设计。这项工作的长期目标是确定一种化合物，该化合物可能导致目前尚无治疗方法的强直性肌营养不良I型患者的治疗。

DESCRIPTION (provided by applicant): Myotonic dystrophy (DM) is caused by repeat nucleotide expansions, specifically CUG and CCUG expansions in the non-coding regions of the dystrophia myotonia protein kinase gene (DMPK) and the Zn finger 9 gene (ZNF9), respectively. Patients with the CUG expansions have type I myotonic dystrophy (DM1) and patients with the CCUG expansions have type II myotonic dystrophy (DM2). Patients with DM1 and DM2 display the same symptoms suggesting both CUG and CCUG expansions cause DM through a common mechanism. It is hypothesized that these non-coding expansions cause DM through an RNA gain-of-function mechanism; the expanded CUG and CCUG repeat RNAs act as toxic RNAs to sequester MBNL1, a RNA binding protein, and also indirectly increase the protein levels of CUGBP1, another RNA binding protein, which disrupts the normal cellular function of these proteins. MBNL1 and CUGBP1 are pre-mRNA splicing factors and decreasing and increasing their "active" concentration results in the mis- regulation of alternative splicing of multiple transcripts with an outcome of DM for those who harbor CUG and CCUG expansions. The expanded CUG repeats that cause DM1 are an excellent drug target because if one can identify a small molecule that binds CUG repeats, this binding event could release the sequestered MBNL1 and the expected outcome would be that MBNL1 would be able to properly regulate the splicing of its substrate pre-mRNAs and reduce or eliminate some of the symptoms of myotonic dystrophy. Towards this goal, we have identified a small molecule (pentamidine) that binds the CUG repeats and releases MBNL1, reverses the splicing defects associated with DM1 in a human DM1 cell model and partially reverses the splicing defects in a DM1 mouse model. Although pentamidine binds the CUG repeats and reverses some of the molecular defects associated with DM1, pentamidine appears to have limited specificity for the CUG repeats; therefore, analogues of pentamidine will be synthesized with the goal of obtaining small molecules with greater specificity and affinity for CUG repeats compared to pentamidine. The mechanism through which pentamidine disrupts the MNBL - CUG repeat interaction is being determined and this knowledge will be used to improve the design of analogues with improved activity. The long term goal of this work is to identify a compound that could lead to a therapy for patients with myotonic dystrophy type I for which no therapies are currently available.