- 作者: Yu-Ling Tseng, Po-Chao Lu, Chi-Chang Lee, Ruei-Yu He, Yung-An Huang, Yin-Chen Tseng, Ting-Jen Rachel Cheng, Joseph Jen-Tse Huang & Jim-Min Fang
- 作者服務機構: 1.Chemical Biology and Molecular Biophysics, Taiwan International Graduate Program, Academia Sinica, Taipei, 115, Taiwan 2.Department and Graduate Institute of Pharmacology, National Taiwan University, Taipei, 100, Taiwan 3.Department of Applied Chemistry, National Chiayi University, Chiayi City, 600, Taiwan 4.Department of Chemistry, National Taiwan University, Taipei, 106, Taiwan 5.Institute of Chemistry, Academia Sinica, Taipei, 115, Taiwan 6.Neuroscience Program of Academia Sinica, Academia Sinica, Taipei, 115, Taiwan 7.Sustainable Chemical Science and Technology, Taiwan International Graduate Program, Academia Sinica, Taipei, 115, Taiwan 8.The Genomics Research Center, Academia Sinica, Taipei, 115, Taiwan
- 中文摘要:
- 英文摘要:
Background
Amyotrophic lateral sclerosis (ALS) associated with TAR DNA-binding protein 43 (TDP-43) aggregation has been considered as a lethal and progressive motor neuron disease. Recent studies have shown that both C-terminal TDP-43 (C-TDP-43) aggregates and oligomers were neurotoxic and pathologic agents in ALS and frontotemporal lobar degeneration (FTLD). However, misfolding protein has long been considered as an undruggable target by applying conventional inhibitors, agonists, or antagonists. To provide this unmet medical need, we aim to degrade these misfolding proteins by designing a series of proteolysis targeting chimeras (PROTACs) against C-TDP-43.
Methods
By applying filter trap assay, western blotting, and microscopy imaging, the degradation efficiency of C-TDP-43 aggregates was studied in Neuro-2a cells overexpressing eGFP-C-TDP-43 or mCherry-C-TDP-43. The cell viability was characterized by alarmarBlue assay. The beneficial and disaggregating effects of TDP-43 PROTAC were examined with the YFP-C-TDP-43 transgenic C. elegans by motility assay and confocal microscopy. The impact of TDP-43 PROTAC on C-TDP-43 oligomeric intermediates was monitored by fluorescence lifetime imaging microscopy and size exclusion chromatography in the Neuro-2a cells co-expressing eGFP-C-TDP-43 and mCherry-C-TDP-43.
Results
Four PROTACs with different linker lengths were synthesized and characterized. Among these chimeras, PROTAC 2 decreased C-TDP-43 aggregates and relieved C-TDP-43-induced cytotoxicity in Neuro-2a cells without affecting endogenous TDP-43. We showed that PROTAC 2 bound to C-TDP-43 aggregates and E3 ligase to initiate ubiquitination and proteolytic degradation. By applying advanced microscopy, it was further shown that PROTAC 2 decreased the compactness and population of C-TDP-43 oligomers. In addition to cellular model, PROTAC 2 also improved the motility of transgenic C. elegans by reducing the C-TDP-43 aggregates in the nervous system.
Conclusions
Our study demonstrated the dual-targeting capacity of the newly-designed PROTAC 2 against both C-TDP-43 aggregates and oligomers to reduce their neurotoxicity, which shed light on the potential drug development for ALS as well as other neurodegenerative diseases. - 中文關鍵字:
- 英文關鍵字: Neurodegenerative diseases, Amyotrophic lateral sclerosis, TDP-43 cytotoxicity, Aggregate and oligomer, PROTACs, Protein degradation, Transgenic C. elegans