High-Content Screening to identify small molecules for refolding SOD1 mutants

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Amyotrophic Lateral Sclerosis (ALS) is a fatal neurodegenerative disease characterized by progressive degeneration of motor neurons, leading to muscular atrophy, paralysis and death in 3-5 years from the onset of symptoms. The only FDA-approved drug for the treatment of ALS, Riluzole, has only moderate effects, prolonging survival by three months on average. Therefore, new strategies are urgently needed to develop drugs that can significantly improve survival and quality of life of ALS patients. Compelling evidence indicates that the misfolding of copper and zinc superoxide dismutase (SOD1) into toxic conformation underlies the pathogenesis of about 20% of familiar ALS and maybe, also some sporadic ALS. Therefore, stabilization of SOD1 structure and restoration of its native conformation should be a desirable approach to cure SOD1-related ALS. Small molecule chemical chaperones have been approved by FDA for treating other diseases caused by misfolded proteins, such as cystic fibrosis and transthyretin amyloidoses. However, no such chemical chaperone has been reported for refolding of misfolded SOD1, even at experimental stage. In this proposal, we will develop a high-content screening (HCS) approach to identify small molecules that promote refolding of SOD1 mutants to their wild type (wt) conformation. Successful development of such a screening strategy is strongly supported by our preliminary studies. We have identified a peptide sequence that is normally concealed in native wt SOD1 but exposed in ALS-linked SOD1 mutants and a misfolded form of wt SOD1. Importantly the exposed peptide sequence resembles nuclear export signal (NES) and indeed, has nuclear export activity. Consequently, misfolded wt SOD1 and SOD1 mutants are cleared from the nucleus due to the NES-like peptide(NLP)-mediated nuclear export, while native wt SOD1 is localized in both the nucleus and the cytoplasm. We propose an imaging-based assay to identify small molecules that restore nuclear localization of SOD1 mutants as an indicator of refolding to native wt SOD1 conformation. We have generated an antibody against NLP that specifically recognizes SOD1 mutants and misfolded wt SOD1, but not native wt SOD1 in cells. Accordingly, the NLP antibody will be used to distinguish the misfolded conformation from the native form of SOD1, and thereby can validate the folding status of SOD1 mutants after compound treatment. These preliminary studies lead us to pursue three specific aims: 1) develop a cell-based biplex platform for primary and counterscreens to identify small molecule chaperones that refold mutant SOD1 to its native wt conformation, 2) perform an automated pilot screening of NCGC Pharmaceutical Collection compound library to identify mutant SOD1 refolding chaperones, and 3) hit validation and characterization. Upon completion, the proposed research will establish the first HCS assay and hit validation pipeline for chemical chaperones that refold SOD1. Future studies will include large-scale screen in collaboration with Dr. Mark Henderson at NCATS. Furthermore, candidate chaperones identified in this study will undergo preclinical assessment and optimization in collaboration with experts in related fields, with the ultimate goal being a clinically relevant class of drugs to treat SOD1-linked ALS through direct correction of SOD1 misfolding.