Targeting a Toxic RNA with Small Molecules

描述（由申请人提供）：肌强直性营养不良（DM）是由重复核苷酸扩增引起的，特别是在营养不良性肌强直蛋白激酶基因（DMPK）和锌指9基因（ZNF9）的非编码区分别出现CUG和CCUG扩增。CUG扩张的患者为I型肌强直性营养不良（DM1），而CUG扩张的患者为II型肌强直性营养不良（DM2）。DM1和DM2患者表现出相同的症状，提示CUG和CCUG扩张通过共同的机制导致DM。据推测，这些非编码扩展通过RNA功能获得机制导致糖尿病；扩增的CUG和CCUG重复RNA作为毒性RNA隔离了MBNL1（一种RNA结合蛋白），并间接增加了CUGBP1（另一种RNA结合蛋白）的蛋白水平，从而破坏了这些蛋白的正常细胞功能。MBNL1和CUGBP1是mrna前剪接因子，降低和增加它们的“活性”浓度会导致多个转录本的选择性剪接调控错误，从而导致CUG和CCUG扩增的患者发生糖尿病。引起DM1的扩展的CUG重复序列是一个很好的药物靶点，因为如果能够识别一个结合CUG重复序列的小分子，这个结合事件可以释放被隔离的MBNL1，预期的结果是MBNL1将能够适当地调节其底物前mrna的剪接，减少或消除肌强直性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（pentamidine），它结合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，并部分逆转DM1小鼠模型中的剪接缺陷。虽然pentamidine结合CUG重复序列并逆转与DM1相关的一些分子缺陷，但pentamidine似乎对CUG重复序列具有有限的特异性；因此，将合成戊脒的类似物，目的是获得比戊脒对CUG重复序列具有更大特异性和亲和力的小分子。pentamidine破坏MNBL - CUG重复相互作用的机制正在被确定，这一知识将用于改进具有更高活性的类似物的设计。这项工作的长期目标是确定一种化合物，这种化合物可以治疗目前尚无治疗方法的I型肌强直性营养不良患者。

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.