Development and Validation of a High-Throughput Cell-Based AlphaLISA Assay to Identify Small Molecule 20S Proteasome Modulators

Abstract

<b>Abstract ID 54242</b> <b>Poster Board 318</b> Intrinsically Disordered Proteins (IDPs) comprise a large fraction of the human proteome, and the regulation of IDPs is exquisitely modulated through protein synthesis and degradation mechanisms. Alterations in IDP homeostasis have been implicated in many diseases, including cancer, neurodegenerative diseases and diabetes. IDPs inherently lack a folded domain and can be degraded by the 20S proteasome core without previous ubiquitination. However, aggregation of IDPs due to oligomerization is a major contributor to neurodegenerative diseases, in part due to a decline of 20S proteasome activity as age increases. Therefore, enhancing 20S proteasome activity by small molecules is an attractive therapeutic target. Although several <i>in&nbsp;vitro</i> screens have been performed for 20S proteasome activators, no high-throughput cell-based assay was available to screen for 20S proteasome activators using an IDP as a target protein. In collaboration with the Assay Development and Drug Repurposing Core (ADDRC) at Michigan State University, we took advantage of the AlphaLISA bead technology to determine the protein levels of a GFP:ODC fusion protein in stably transfected HEK293T cells as a readout for 20S proteasome activity. We optimized this assay to a 384 well format and a Z’&gt;0.6 and then validated this assay with our current 20S proteasome activator, TCH-165. This assay was used to screen the FDA Prestwick Library and NIH Clinical Library to identify compounds that could increase 20S proteasome activity in cells. After cell-based validation, these compounds were also tested <i>in&nbsp;vitro</i> against both the 20S and 26S proteasomes using standard fluorogenic small peptide assays to select for 20S proteasome specific small molecules. Finally, we validated these compounds in western blots against another IDP, a-synuclein. Overall, our results show that this assay is robust, high-throughput and useful for identifying proteasome modulators in a cellular environment. This work was supported by the National Institutes of Aging, NS111347