Aims:
Suppressing or eliminating pathological cytoplasmic TDP-43 has been shown to alleviate amyotrophic lateral sclerosis (ALS) symptoms in mouse models, highlighting cytoplasmic TDP-43 as a promising therapeutic target. The aim of our study was to develop a proteolysis-targeting chimera (PROTAC) and evaluate its effects on TDP-43 in cellular models as a proof of concept.
Methods:
To model cytoplasmic mislocalisation of TDP-43, we generated inducible and stable Flp-In™ T-REx™ 293 cell lines expressing either HALO-tagged TDP-43 or a cytoplasmically-localised variant (ΔNLS-HALO-TDP-43). We synthesised a PROTAC, RTK14-093p, targeting the HALO tag and recruiting the VHL E3 ligase. Western blotting and mass spectrometry evaluated levels of TDP-43 degradation, mechanisms-of-action, associated pathway alterations, and changes in protein-protein interactions following HALO-TDP-43 degradation with RTK14-093p. Additionally, flow cytometry was used to assess whether RTK14-093p improved viability in response to HALO-TDP-43 overexpression.
Results:
Our results show that the RTK14-093p PROTAC compound significantly reduced insoluble HALO-TDP-43 levels (by 3.19-fold), with a pronounced preference for degrading the endogenous insoluble HALO-TDP-43 (reduced by 3.85-fold). We characterised its interactome, finding that RTK14-093p not only degraded TDP-43 but also cleared several TDP-43 associated proteins, supporting its therapeutic potential for targeting TDP-43-positive protein aggregates.
Conclusions:
We present preliminary evidence that VHL-based PROTACs can effectively degrade TDP-43 and its associated proteins in cellular models, offering a promising therapeutic strategy for ALS. Future research will focus on evaluating the efficacy of this PROTAC in diverse ALS-relevant cellular models that recapitulate key aspects of TDP-43 pathology.