Hirose Sakura, Kobatake Yui, Tada Norihiro, Kandeel Mahmoud, Itoh Akichika, Oh-Hashi Kentaro
Graduate School of Natural Science and Technology, Gifu University, Gifu, Japan.
Faculty of Applied Biological Sciences, Gifu University, Gifu, Japan.
Cell Biochem Biophys. 2025 May 12. doi: 10.1007/s12013-025-01768-5.
Canine degenerative myelopathy (DM) is a progressive neurodegenerative disorder that shares common pathological features with amyotrophic lateral sclerosis (ALS) in humans. Both diseases are linked to mutations in the superoxide dismutase 1 (SOD1) gene. Understanding the molecular differences between wild-type (WT) and mutant SOD1 proteins is critical for developing therapeutic strategies. In this study, we employed the NanoLuc complementation (NanoBiT) reporter system to investigate the expression and functional differences between WT and E40K mutant canine SOD1 to assess the therapeutic potential of copper chaperone for SOD1 (CCS) and ebselen derivatives. E40K cSOD1 displayed significantly reduced luciferase activity compared to WT cSOD1 in all NanoBiT-tagged combinations, indicating altered homodimerization and protein stability. Co-transfection with CCS increased both WT and mutant cSOD1 protein levels and reporter activities, with a more pronounced effect on the E40K mutant. Ebselen treatment enhanced luciferase activity, particularly in E40K cSOD1-expressing cells. Two compounds (compounds 2 and 5) were stronger than the parent compound in improving mutant cSOD1-derived NanoBiT activities. Additionally, molecular docking simulations revealed stronger binding affinities of ebselen and its derivatives to E40K cSOD1, suggesting potential therapeutic benefits. In conclusion, the NanoLuc reporter system offers a valuable tool for screening potential therapeutics for SOD1-linked neurodegenerative diseases. CCS and ebselen derivatives exhibited promising effects on SOD1 activity, providing a basis for future therapeutic strategies targeting both DM and ALS.
犬退行性脊髓病（DM）是一种进行性神经退行性疾病，与人类肌萎缩侧索硬化症（ALS）具有共同的病理特征。这两种疾病都与超氧化物歧化酶1（SOD1）基因突变有关。了解野生型（WT）和突变型SOD1蛋白之间的分子差异对于制定治疗策略至关重要。在本研究中，我们采用纳米荧光素酶互补（NanoBiT）报告系统来研究WT和E40K突变型犬SOD1之间的表达和功能差异，以评估铜伴侣蛋白SOD1（CCS）和依布硒仑衍生物的治疗潜力。在所有NanoBiT标记的组合中，与WT cSOD1相比，E40K cSOD1的荧光素酶活性显著降低，表明其同源二聚化和蛋白质稳定性发生了改变。与CCS共转染可提高WT和突变型cSOD1蛋白水平及报告基因活性，对E40K突变体的影响更为显著。依布硒仑处理可增强荧光素酶活性，尤其是在表达E40K cSOD1的细胞中。两种化合物（化合物2和5）在改善突变型cSOD1衍生的NanoBiT活性方面比母体化合物更强。此外，分子对接模拟显示依布硒仑及其衍生物与E40K cSOD1的结合亲和力更强，表明具有潜在的治疗益处。总之，纳米荧光素酶报告系统为筛选SOD1相关神经退行性疾病的潜在治疗药物提供了一个有价值的工具。CCS和依布硒仑衍生物对SOD1活性表现出有前景的影响，为未来针对DM和ALS的治疗策略提供了基础。
