Duchenne muscular dystrophy (DMD) is a degenerative muscle disease caused by mutations in the dystrophin gene. Loss of dystrophin prevents the formation of a critical connection between the muscle cell membrane and the extracellular matrix. Overexpression of sarcospan (SSPN) in the mouse model of DMD restores the membrane connection and reduces disease severity, making SSPN a promising therapeutic target for pharmacological upregulation. Using a previously described cell-based promoter reporter assay of SSPN gene expression (hSSPN-EGFP), we conducted high-throughput screening on libraries of over 200,000 curated small molecules to identify SSPN modulators. The hits were validated in both hSSPN-EGFP and hSSPN-luciferase reporter cells. Hit selection was conducted on dystrophin-deficient mouse and human myotubes with assessments of (1) SSPN gene expression using quantitative PCR and (2) SSPN protein expression using immunoblotting and an ELISA. A membrane stability assay using osmotic shock was used to validate the functional effects of treatment followed by cell surface biotinylation to label cell surface proteins. Dystrophin-deficient mdx mice were treated with compound, and muscle was subjected to quantitative PCR to assess SSPN gene expression. We identified and validated lead compounds that increased SSPN gene and protein expression in dystrophin-deficient mouse and human muscle cells. The lead compound OT-9 increased cell membrane localization of compensatory laminin-binding adhesion complexes and improved membrane stability in DMD myotubes. We demonstrated that the membrane stabilizing benefit is dependent on SSPN. Intramuscular injection of OT-9 in the mouse model of DMD increased SSPN gene expression. This study identifies a pharmacological approach to treat DMD and sets the path for the development of SSPN-based therapies.

中文翻译：

---

高通量筛选鉴定了肌跨调节剂，其稳定肌肉细胞并在杜氏肌营养不良症小鼠模型中表现出活性。


杜氏肌营养不良症 (DMD) 是一种由肌营养不良蛋白基因突变引起的退行性肌肉疾病。抗肌营养不良蛋白的丧失会阻碍肌细胞膜和细胞外基质之间关键连接的形成。在 DMD 小鼠模型中过度表达 sarcospan (SSPN) 可恢复膜连接并降低疾病严重程度，使 SSPN 成为药理学上调的有希望的治疗靶点。使用先前描述的基于细胞的 SSPN 基因表达启动子报告基因测定 (hSSPN-EGFP)，我们对超过 200,000 个精心策划的小分子文库进行了高通量筛选，以鉴定 SSPN 调节剂。命中结果在 hSSPN-EGFP 和 hSSPN-荧光素酶报告细胞中得到验证。对抗肌营养不良蛋白缺陷小鼠和人肌管进行命中选择，并使用定量 PCR 评估 SSPN 基因表达和 (2) 使用免疫印迹和 ELISA 评估 SSPN 蛋白表达。使用渗透休克的膜稳定性测定来验证处理的功能效果，然后进行细胞表面生物素化以标记细胞表面蛋白。用化合物治疗肌营养不良蛋白缺陷的 mdx 小鼠，并对肌肉进行定量 PCR 以评估 SSPN 基因表达。我们鉴定并验证了可增加抗肌营养不良蛋白缺陷小鼠和人类肌肉细胞中 SSPN 基因和蛋白质表达的先导化合物。先导化合物 OT-9 增加了代偿性层粘连蛋白结合粘附复合物的细胞膜定位，并改善了 DMD 肌管的膜稳定性。我们证明了膜稳定性的好处取决于 SSPN。在 DMD 小鼠模型中肌内注射 OT-9 会增加 SSPN 基因的表达。 这项研究确定了治疗 DMD 的药理学方法，并为基于 SSPN 的疗法的开发奠定了道路。