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P2016
Profiling the Activity of Proteasome Inhibitors Using Promega’s Proteasome-Glo™ Cell-Based Assay
Presenter Khristofer Garcia, Millennium Pharmaceuticals Inc., USA
Additional Authors: Khristofer Garcia, Jonathan Blank, Jane Liu, Paul Hales, Chris Tsu, and Larry Dick
A homogeneous, robust, antibody-free fluorescence lifetime assay was developed for Lck tyrosine kinase. PURETIME14, a long lifetime acridone dye derivative was used to label the N-terminus of substrate and product peptides. Fluorescence lifetimes of the substrate (11.5ns +/-0.1ns) and phospho-tyrosine product (13.7ns +/-0.1ns) were sufficiently different to permit precise quantification of conversion rates. Enzyme time courses and inhibition measurements were made. A Lck activity assay with 30% conversion was configured for detection of the inhibitor PP2 and was found to have a Z’ value of 0.82. The assay was validated with a mini-screen of 80 drug-like compounds drawn at random from the Maybridge Hitfinder library. One highly fluorescent compound in the set was used to show that fluorescence lifetime successfully detected inhibition that was a false negative with fluorescence intensity measurement.