Target Identification of a Class of Pyrazolone Protein Aggregation Inhibitor Therapeutics for Amyotrophic Lateral Sclerosis.

Amyotrophic lateral sclerosis (ALS) is a fatal neurodegenerative disease with no cure, and current treatment options are very limited. Previously, we performed a high-throughput screen to identify small molecules that inhibit protein aggregation caused by a mutation in the gene that encodes superoxide dismutase 1 (SOD1), which is responsible for about 25% of familial ALS. This resulted in three hit series of compounds that were optimized over several years to give three compounds that were highly active in a mutant SOD1 ALS model. Here we identify the target of two of the active compounds ( and ) with the use of photoaffinity labeling, chemical biology reporters, affinity purification, proteomic analysis, and fluorescent/cellular thermal shift assays. Evidence is provided to demonstrate that these two pyrazolone compounds directly interact with 14-3-3-E and 14-3-3-Q isoforms, which have chaperone activity and are known to interact with mutant SOD1 aggregates and become insoluble in the subcellular JUNQ compartment, leading to apoptosis. Because protein aggregation is the hallmark of all neurodegenerative diseases, knowledge of the target compounds that inhibit protein aggregation allows for the design of more effective molecules for the treatment of ALS and possibly other neurodegenerative diseases.