title: High-throughput screening for inhibitors of the e3 ubiquitin ligase APC.
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.
authors.affiliation: Rigel Pharmaceuticals, Inc., South San Francisco, California, USA.
authors.forename: Jianing
authors.initials: J
authors.lastname: Huang
authors.forename: Julie
authors.initials: J
authors.lastname: Sheung
authors.forename: Guoqiang
authors.initials: G
authors.lastname: Dong
authors.forename: Christina
authors.initials: C
authors.lastname: Coquilla
authors.forename: Sarkiz
authors.initials: S
authors.lastname: Daniel-Issakani
authors.forename: Donald G
authors.initials: DG
authors.lastname: Payan
mesh_terms: D004353:Drug Evaluation, Preclinical; D004591:Electrophoresis, Polyacrylamide Gel; D004791:Enzyme Inhibitors / Q000494:pharmacology*; D005982:Glutathione Transferase / Q000378:metabolism; D011994:Recombinant Proteins / Q000037:antagonists & inhibitors / Q000378:metabolism; D044767:Ubiquitin-Protein Ligases / Q000037:antagonists & inhibitors* / Q000378:metabolism
chemical_list: D004791:Enzyme Inhibitors; D011994:Recombinant Proteins; D044767:Ubiquitin-Protein Ligases; D005982:Glutathione Transferase