Hoskins Jason W, Ofori Leslie O, Chen Catherine Z, Kumar Amit, Sobczak Krzysztof, Nakamori Masayuki, Southall Noel, Patnaik Samarjit, Marugan Juan J, Zheng Wei, Austin Christopher P, Disney Matthew D, Miller Benjamin L, Thornton Charles A
Department of Neurology, University of Rochester, Rochester, NY 14642, USA.
Department of Chemistry, University of Rochester, Rochester, NY 14642, USA.
Nucleic Acids Res. 2014 Jun;42(10):6591-602. doi: 10.1093/nar/gku275. Epub 2014 May 5.
Myotonic dystrophy type 1 (DM1) is a dominantly inherited neuromuscular disorder resulting from expression of RNA containing an expanded CUG repeat (CUG(exp)). The pathogenic RNA is retained in nuclear foci. Poly-(CUG) binding proteins in the Muscleblind-like (MBNL) family are sequestered in foci, causing misregulated alternative splicing of specific pre-mRNAs. Inhibitors of MBNL1-CUG(exp) binding have been shown to restore splicing regulation and correct phenotypes in DM1 models. We therefore conducted a high-throughput screen to identify novel inhibitors of MBNL1-(CUG)12 binding. The most active compound was lomofungin, a natural antimicrobial agent. We found that lomofungin undergoes spontaneous dimerization in DMSO, producing dilomofungin, whose inhibition of MBNL1-(CUG)12 binding was 17-fold more potent than lomofungin itself. However, while dilomofungin displayed the desired binding characteristics in vitro, when applied to cells it produced a large increase of CUG(exp) RNA in nuclear foci, owing to reduced turnover of the CUG(exp) transcript. By comparison, the monomer did not induce CUG(exp) accumulation in cells and was more effective at rescuing a CUG(exp)-induced splicing defect. These results support the feasibility of high-throughput screens to identify compounds targeting toxic RNA, but also demonstrate that ligands for repetitive sequences may have unexpected effects on RNA decay.
1型强直性肌营养不良症（DM1）是一种常染色体显性遗传的神经肌肉疾病，由含有扩展CUG重复序列（CUG(exp)）的RNA表达所致。致病性RNA保留在核仁中。肌肉盲样（MBNL）家族中的多聚（CUG）结合蛋白被隔离在核仁中，导致特定前体mRNA的可变剪接失调。在DM1模型中，MBNL1与CUG(exp)结合的抑制剂已被证明可恢复剪接调控并纠正表型。因此，我们进行了一项高通量筛选，以鉴定MBNL1与（CUG）12结合的新型抑制剂。活性最强的化合物是洛莫真菌素，一种天然抗菌剂。我们发现洛莫真菌素在二甲基亚砜（DMSO）中会自发二聚化，生成双洛莫真菌素，其对MBNL1与（CUG）12结合的抑制作用比洛莫真菌素本身强17倍。然而，虽然双洛莫真菌素在体外表现出所需的结合特性，但应用于细胞时，由于CUG(exp)转录本的周转减少，它会使核仁中的CUG(exp) RNA大量增加。相比之下，单体不会在细胞中诱导CUG(exp)积累，并且在挽救CUG(exp)诱导的剪接缺陷方面更有效。这些结果支持了通过高通量筛选来鉴定靶向毒性RNA的化合物的可行性，但也表明针对重复序列的配体可能会对RNA衰变产生意想不到的影响。
