Identifying Therapeutic Inhibitors of TDP43 Phase-Separation.

Amyotrophic Lateral Sclerosis (ALS) is a devastating neurodegenerative disease for which there has been little improvement in treatment over the past 25 years. ALS is linked to mutations in dozens of genes, but despite its genetic complexity, 97% of patients share a distinct molecular pathology: cytoplasmic inclusions of TAR DNA-Binding protein 43 (TDP-43). Further, TDP-43 aggregates are toxic to healthy neurons, suggesting that TDP-43 aggregates contribute to neurodegeneration in ALS. Due to the near-ubiquity of TDP-43 inclusions in ALS patients and their contribution to ALS pathology, prevention or dissolution of these TDP-43 inclusions presents a promising therapeutic strategy. In cells, TDP43 can form both solid aggregates or liquid droplets, through liquid-liquid phase separation (LLPS). Here, we screened a library of small molecules and RNAs to identify inhibitors of TDP-43 LLPS using an in vitro assay which quantifies TDP-43 liquid droplet formation. Hits from this screen were further characterized based on their ability to reverse and inhibit both TDP-43 droplets and aggregates to obtain functional profiles for each promising inhibitor. Pairing functional data of inhibitors with structural information regarding their binding to TDP-43 enables a close-dissection of the interactions that drive TDP-43 self-assembly. Thus, small molecule screens which target protein self-assembly present a powerful approach for both developing novel therapeutics and identifying the interactions that govern self-assembly; these inform each other and may lead to the discovery of novel therapeutic agents which target protein self-assembly to treat severe diseases, like ALS.