title: A high-throughput screening method for small-molecule inhibitors of the aberrant mutant SOD1 and dynein complex interaction.
abstract: Aberrant protein-protein interactions are attractive drug targets in a variety of neurodegenerative diseases due to the common pathology of accumulation of protein aggregates. In amyotrophic lateral sclerosis, mutations in SOD1 cause the formation of aggregates and inclusions that may sequester other proteins and disrupt cellular processes. It has been demonstrated that mutant SOD1, but not wild-type SOD1, interacts with the axonal transport motor dynein and that this interaction contributes to motor neuron cell death, suggesting that disrupting this interaction may be a potential therapeutic target. However, it can be challenging to configure a high-throughput screening (HTS)-compatible assay to detect inhibitors of a protein-protein interaction. Here we describe the development and challenges of an HTS for small-molecule inhibitors of the mutant SOD1-dynein interaction. We demonstrate that the interaction can be formed by coexpressing the A4V mutant SOD1 and dynein intermediate complex in cells and that this interaction can be disrupted by compounds added to the cell lysates. Finally, we show that some of the compounds identified from a pilot screen to inhibit the protein-protein interaction with this method specifically disrupt the interaction between the dynein complex and mtSOD1 but not the dynein complex itself when applied to live cells.
authors.affiliation: Department of Molecular and Cellular Biochemistry, College of Medicine, University of Kentucky, Lexington, KY, USA.
authors.forename: Xiaohu
authors.initials: X
authors.lastname: Tang
authors.forename: Kathleen I
authors.initials: KI
authors.lastname: Seyb
authors.forename: Mickey
authors.initials: M
authors.lastname: Huang
authors.forename: Eli R
authors.initials: ER
authors.lastname: Schuman
authors.forename: Ping
authors.initials: P
authors.lastname: Shi
authors.forename: Haining
authors.initials: H
authors.lastname: Zhu
authors.forename: Marcie A
authors.initials: MA
authors.lastname: Glicksman
mesh_terms: D000690:Amyotrophic Lateral Sclerosis / Q000235:genetics / Q000378:metabolism / Q000473:pathology; D002460:Cell Line; D004398:Dyneins / Q000378:metabolism*; D004791:Enzyme Inhibitors; D005982:Glutathione Transferase; D057809:HEK293 Cells; D057166:High-Throughput Screening Assays / Q000379:methods*; D006801:Humans; D020836:Protein Structure, Quaternary; D013482:Superoxide Dismutase / Q000037:antagonists & inhibitors / Q000235:genetics* / Q000378:metabolism*; D000072105:Superoxide Dismutase-1
chemical_list: D004791:Enzyme Inhibitors; C000606290:SOD1 protein, human; D013482:Superoxide Dismutase; D000072105:Superoxide Dismutase-1; D005982:Glutathione Transferase; D004398:Dyneins