Targeting a Toxic RNA with Small Molecules

描述（由申请人提供）：强直性肌营养不良（DM）是由重复核苷酸扩增引起的，特别是分别在肌营养不良性肌强直蛋白激酶基因（DMPK）和锌指9基因（ZNF 9）的非编码区中的CUG和CCUG扩增。具有CUG扩增的患者患有I型肌强直性营养不良（DM1），并且具有CCUG扩增的患者患有II型肌强直性营养不良（DM2）。DM 1和DM 2患者表现出相同的症状，表明CUG和CCUG扩张通过共同的机制引起DM。据推测，这些非编码扩增通过RNA功能获得机制引起DM;扩增的CUG和CCUG重复RNA作为毒性RNA来隔离MBNL1（一种RNA结合蛋白），并间接增加CUGBP1（另一种RNA结合蛋白）的蛋白水平，从而破坏这些蛋白的正常细胞功能。MBNL1和CUGBP1是前体mRNA剪接因子，降低和增加它们的"活性"浓度会导致多种转录物的可变剪接的失调，对于携带CUG和CCUG扩增的那些人会导致DM。导致DM 1的扩增的CUG重复序列是一个极好的药物靶标，因为如果可以鉴定出结合CUG重复序列的小分子，则该结合事件可以释放隔离的MBNL1，并且预期的结果将是MBNL1将能够适当地调节其底物前mRNA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，我们已经确定了一个小分子（喷他脒），结合CUG重复和释放MBNL1，逆转剪接缺陷与DM1在人DM1细胞模型和部分逆转的DM1小鼠模型中的剪接缺陷。尽管喷他脒结合CUG重复序列并逆转与DM1相关的一些分子缺陷，但喷他脒似乎对CUG重复序列具有有限的特异性;因此，将合成喷他脒的类似物，目的是获得与喷他脒相比对CUG重复序列具有更大特异性和亲和力的小分子。正在确定喷他脒破坏MNBL-CUG重复相互作用的机制，这一知识将用于改进具有改进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，可以导致治疗强直性肌营养不良I型患者，目前没有治疗方法。

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.