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Abstract：
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.
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.
摘要：
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。肌养蛋白的丧失阻止了肌肉细胞膜和细胞外基质之间关键连接的形成。DMD小鼠模型中sarcospan（SSPN）的过表达恢复了膜连接并降低了疾病的严重程度，使SSPN成为药理学上调的有希望的治疗靶标。
使用先前描述的SSPN基因表达的基于细胞的启动子报告检测（hSSPN-EGFP），我们对超过200,000个筛选的小分子文库进行了高通量筛选,以鉴定SSPN调节剂.在hSSPN-EGFP和hSSPN-荧光素酶报告细胞中验证了命中。在肌营养不良蛋白缺陷型小鼠和人肌管上进行命中选择,其中评估(1)使用定量PCR的SSPN基因表达和(2)使用免疫印迹和ELISA的SSPN蛋白表达。使用渗透压休克的膜稳定性测定用于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型mdx小鼠，和肌肉进行定量PCR以评估SSPN基因表达。
我们鉴定并验证了在缺乏肌营养不良蛋白的小鼠和人类肌肉细胞中增加SSPN基因和蛋白质表达的先导化合物。先导化合物OT-9增加了补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肌管中的膜稳定性。我们证明了膜稳定益处取决于SSPN。在DMD小鼠模型中肌内注射OT-9增加SSPN基因表达。
这项研究确定了治疗DMD的药理学方法，并为基于SSPN的治疗方法的发展奠定了基础。
使用先前描述的SSPN基因表达的基于细胞的启动子报告检测（hSSPN-EGFP），我们对超过200,000个筛选的小分子文库进行了高通量筛选,以鉴定SSPN调节剂.在hSSPN-EGFP和hSSPN-荧光素酶报告细胞中验证了命中。在肌营养不良蛋白缺陷型小鼠和人肌管上进行命中选择,其中评估(1)使用定量PCR的SSPN基因表达和(2)使用免疫印迹和ELISA的SSPN蛋白表达。使用渗透压休克的膜稳定性测定用于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型mdx小鼠，和肌肉进行定量PCR以评估SSPN基因表达。
我们鉴定并验证了在缺乏肌营养不良蛋白的小鼠和人类肌肉细胞中增加SSPN基因和蛋白质表达的先导化合物。先导化合物OT-9增加了补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肌管中的膜稳定性。我们证明了膜稳定益处取决于SSPN。在DMD小鼠模型中肌内注射OT-9增加SSPN基因表达。
这项研究确定了治疗DMD的药理学方法，并为基于SSPN的治疗方法的发展奠定了基础。
