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首页> 外文期刊>Skeletal Muscle >High-throughput screening identifies inhibitors of DUX4-induced myoblast toxicity
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High-throughput screening identifies inhibitors of DUX4-induced myoblast toxicity

机译：高通量筛选可鉴定DUX4诱导的成肌细胞毒性抑制剂

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Background Facioscapulohumeral muscular dystrophy (FSHD) is caused by epigenetic alterations at the D4Z4 macrosatellite repeat locus on chromosome 4, resulting in inappropriate expression of the DUX4 protein. The DUX4 protein is therefore the primary molecular target for therapeutic intervention. Methods We have developed a high-throughput screen based on the toxicity of DUX4 when overexpressed in C2C12 myoblasts, and identified inhibitors of DUX4-induced toxicity from within a diverse set of 44,000 small, drug-like molecules. A total of 1,280 hits were then subjected to secondary screening for activity against DUX4 expressed by 3T3 fibroblasts, for absence of activity against the tet-on system used to conditionally express DUX4, and for potential effects on cellular proliferation rate. Results This allowed us to define a panel of 52 compounds to use as probes to identify essential pathways of DUX4 activity. We tested these compounds for their ability to protect wild-type cells from other types of cell death-inducing insults. Remarkably, we found that 60% of the DUX4 toxicity inhibitors that we identified also protected cells from tert-butyl hydrogen peroxide, an oxidative stress-inducing compound. Compounds did not protect against death induced by caspase activation, DNA damage, protein misfolding, or ER stress. Encouragingly, many of these compounds are also protective against DUX4 expression in human cells. Conclusion These data suggest that oxidative stress is a dominant pathway through which DUX4-provoked toxicity is mediated in this system, and we speculate that enhancing the oxidative stress response pathway might be clinically beneficial in FSHD.
机译：背景肩肱肱型肌营养不良症（FSHD）是由4号染色体上D4Z4大卫星重复基因座的表观遗传学改变引起的，导致DUX4蛋白的不适当表达。因此，DUX4蛋白是治疗干预的主要分子靶标。方法我们基于在C2C12成肌细胞中过表达时DUX4的毒性，开发了一种高通量筛选方法，并从44,000种药物样小分子中鉴定出DUX4诱导的毒性抑制剂。然后对总共1,280个命中进行二次筛选，以筛选抗3T3成纤维细胞表达的DUX4的活性，缺乏针对有条件表达DUX4的tet-on系统的活性以及对细胞增殖速率的潜在影响。结果这使我们能够定义一组52种化合物作为探针，以鉴定DUX4活性的基本途径。我们测试了这些化合物保护野生型细胞免受其他类型的细胞死亡诱导伤害的能力。值得注意的是，我们发现我们确定的60％的DUX4毒性抑制剂还可以保护细胞免受叔丁基过氧化氢（一种氧化应激诱导化合物）的伤害。化合物不能抵抗caspase活化，DNA损伤，蛋白质错误折叠或内质网应激引起的死亡。令人鼓舞的是，这些化合物中的许多也对人类细胞中DUX4的表达具有保护作用。结论这些数据表明，氧化应激是该系统介导DUX4引起的毒性的主要途径，我们推测增强氧化应激反应途径可能对FSHD具有临床益处。

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