High-throughput screening for inhibitors of the e3 ubiquitin ligase APC
Jianing Huang1, Julie Sheung, Guoqiang Dong
1Rigel Pharmaceuticals, Inc., South San Francisco, California, USA.
Abstract:
The anaphase-promoting complex (APC) is an E3 ubiquitin ligase that mediates the ubiquitination and degradation of the securin protein and mitotic cyclins, resulting in the regulation of the onset of sister-chromatid separation and mitotic exit. In an effort to identify novel therapeutic compounds that modulate cell proliferation and, therefore, have potential applications in oncology, a plate-based in vitro ubiquitination assay that uses recombinant purified E1, E2 (UbcH5c), E3 (APC11/APC2), and Flag-ubiquitin has been established and used to screen for small molecule inhibitors of APC E3 ligase activity. In this assay, APC2/APC11 is immobilized on the plate, and its E3 ligase activity (i.e., the incorporation of Flag-tagged polyubiquitin chain onto APC2/APC11 as a result of auto-ubiquitination) is detected with anti-Flag-horseradish peroxidase-conjugated antibody by monitoring the luminescence signal from the plate. Here we describe in detail the protocol for high-throughput screening of APC, including expression and purification of the individual proteins, assay development, and optimization. This assay has been validated in a 96-well plate format and successfully implemented to identify novel small molecule compounds that potently inhibit APC2/APC11 ligase activity.
Insights
Researchers developed a high-throughput screening assay to identify novel small molecule inhibitors of the anaphase-promoting complex (APC), a key regulator of cell division with potential applications in oncology.
Area of Science:
- Cell Biology
- Biochemistry
- Drug Discovery
Background:
- The anaphase-promoting complex (APC) is a crucial E3 ubiquitin ligase regulating cell cycle progression by targeting securin and mitotic cyclins for degradation.
- Dysregulation of APC activity is implicated in various cancers, making it a promising target for anti-cancer drug development.
Purpose of the Study:
- To establish and detail a robust, high-throughput screening assay for identifying small molecule inhibitors of APC E3 ligase activity.
- To validate the assay's utility in discovering novel compounds with potential therapeutic applications in oncology.
Main Methods:
- Development of a plate-based in vitro ubiquitination assay utilizing purified recombinant proteins (E1, E2 [UbcH5c], E3 [APC11/APC2], and Flag-ubiquitin).
- Immobilization of APC2/APC11 on a plate for detecting auto-ubiquitination via an anti-Flag-horseradish peroxidase-conjugated antibody and luminescence signal.
- Optimization and validation of the assay in a 96-well plate format for high-throughput screening.
Main Results:
- A reliable high-throughput screening assay for APC E3 ligase activity was successfully established and optimized.
- The assay was validated and demonstrated to be effective in identifying potent small molecule inhibitors of APC2/APC11.
Conclusions:
- The developed assay provides a valuable tool for the discovery of novel APC inhibitors.
- This screening platform holds significant potential for identifying new anti-cancer therapeutics targeting cell proliferation.
