NanoBiT-based Analysis of Canine SOD1 Protein Dynamics: Understanding the Role of CCS and Ebselen Derivatives as Potential Therapeutics for Canine Degenerative Myelopathy.

Abstract

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.