Unstructured proteins as therapeutic targets for neuromuscular diseases

Dr. Leonidas Emmanouilidis, ETH Zürich



Neuromuscular diseases constitute a complex challenge for healthcare and society, characterized by the progressive degeneration of neurons. Amyotrophic Lateral Sclerosis (ALS) exemplifies the intricate interplay between genetic susceptibility, cellular dysfunction, and clinical manifestation. This devastating disorder leads to motor neuron degeneration, paralysis, and fatality, highlighting the need for innovative therapies. Genetic predisposition and environmental factors contribute to ALS etiology, with mutations in genes like SOD1, TDP- 43, C9orf72, and FUS being implicated in familial cases. ALS-associated mutations induce the formation of cytoplasmic condensates through liquid-liquid phase separation (LLPS). Recent advances highlight the significance of phase transitions in neuromuscular diseases. LLPS involves molecular assembly into distinct phases within cells, playing pivotal role in compartmentalization and function of the molecules involved. In ALS, LLPS drives the formation of pathological inclusions. Familial ALS-linked mutations, particularly in FUS and TDP-43, accelerate liquid-to-solid transition, leading to potentially toxic protein network formation. This insight unveils new opportunities for intervention, suggesting small peptides to disrupt aberrant phase separation events. Our project objectives encompass identifying peptides targeting FUS and TDP-43, key proteins in ALS pathogenesis, to prevent aberrant LLPS or to dissolve preexisting condensates. Preliminary experiments indicate that our peptides indeed inhibit FUS droplet formation. Further studies will optimize binding sequences, exploring systematically the chemical space of the peptide chemistry. Small molecules will be designed to mimic effective peptide binding, addressing limitations of peptides such as cell permeability and stability. In addition, selected libraries of small molecules will be selected and tested for their in vitro effect on LLPS of FUS and TDP-43. The relevance of the in vitro results will be cross-validated with in vivo assays where cellular condensates of wild type and mutant FUS/TDP-43 will be monitored to assess the molecules' ability to prevent or dissolve these structures, serving as a cellular model for ALS pathology. In conclusion, our study bridges the gap between neurodegenerative diseases and LLPS-driven protein aggregation in ALS. By designing peptides and small molecules targeting critical aromatic motifs, we aim to interfere with aberrant LLPS, offering a potential therapeutic avenue to disrupt the formation of toxic protein networks, ultimately contributing to the development of novel ALS treatments.