TY - JOUR
T1 - A high-throughput screening method for small-molecule inhibitors of the aberrant mutant SOD1 and dynein complex interaction
AU - Tang, Xiaohu
AU - Seyb, Kathleen I.
AU - Huang, Mickey
AU - Schuman, Eli R.
AU - Shi, Ping
AU - Zhu, Haining
AU - Glicksman, Marcie A.
N1 - Funding Information:
The authors disclosed receipt of the following financial support for the research and/or authorship of this article: This work was supported by NIH NINDS grants U24NS049339 (to M.G.) and support from the Harvard NeuroDiscovery Center (to M.G.) and R01NS049126 (to H.Z.) and NIA grant R21AG032567 (to H.Z.).
PY - 2012/3
Y1 - 2012/3
N2 - 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.
AB - 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.
KW - CNS and PNS diseases
KW - cell-based assays
KW - fluorescence methods
KW - protein-protein interactions
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U2 - 10.1177/1087057111429595
DO - 10.1177/1087057111429595
M3 - Article
C2 - 22140121
AN - SCOPUS:84859091355
SN - 1087-0571
VL - 17
SP - 314
EP - 326
JO - Journal of Biomolecular Screening
JF - Journal of Biomolecular Screening
IS - 3
ER -