title: Development of a high throughput time-resolved fluorescence resonance energy transfer assay for TRAF6 ubiquitin polymerization.
abstract: Interleukin 1 receptor activation innervates a cascade of signal transduction events that ultimately lead to the activation of inflammatory and immune response genes. TRAF6 is a Ub ligase (E3) involved in this pathway, and inhibition of this critical enzyme may provide a means for treating inflammatory and immune diseases. A TR-FRET assay has been developed and evaluated for HTS for TRAF6 inhibitors. Bio-Ub and Eu-Ub were polymerized in the presence of Ub activating enzyme E1, conjugating enzyme E2, and TRAF6. Following a 2-h incubation, the reaction was stopped with a buffer containing 10 m M EDTA and the fluorescence donor SA-APC. Fluorescence energy transfer from Eu to APC was measured as a ratio of fluorescence intensity at 655 nm to that at 615 nm (excitation at 340 nm). This homogeneous assay has been optimized and validated in a 384-well format. A window of five- to eightfold and Z' factor of 0.6-0.8 suggests that this assay can be applied to screen for inhibitors of the polyubiquitination activity of TRAF6.
authors.affiliation: Tularik Inc., South San Francisco, CA, USA.
authors.forename: Catherine A
authors.initials: CA
authors.lastname: Hong
authors.forename: Elissa
authors.initials: E
authors.lastname: Swearingen
authors.forename: Rommel
authors.initials: R
authors.lastname: Mallari
authors.forename: Xiong
authors.initials: X
authors.lastname: Gao
authors.forename: Zhaodan
authors.initials: Z
authors.lastname: Cao
authors.forename: Anne
authors.initials: A
authors.lastname: North
authors.forename: Stephen W
authors.initials: SW
authors.lastname: Young
authors.forename: Shu-Gui
authors.initials: SG
authors.lastname: Huang
mesh_terms: D004492:Edetic Acid / Q000737:chemistry; D031541:Fluorescence Resonance Energy Transfer / Q000295:instrumentation / Q000379:methods*; D011108:Polymers; D015203:Reproducibility of Results; D025801:Ubiquitin / Q000737:chemistry*; D044764:Ubiquitin-Activating Enzymes / Q000737:chemistry*; D044763:Ubiquitin-Conjugating Enzymes / Q000737:chemistry*; D044767:Ubiquitin-Protein Ligases / Q000037:antagonists & inhibitors / Q000737:chemistry*
chemical_list: D011108:Polymers; D025801:Ubiquitin; D004492:Edetic Acid; D044763:Ubiquitin-Conjugating Enzymes; D044767:Ubiquitin-Protein Ligases; D044764:Ubiquitin-Activating Enzymes