Screening for Inhibitors of Kinase Autophosphorylation
Autophosphorylation of kinases influences their conformational state and can also regulate enzymatic activity. Recently, this has become an area of interest for drug discovery. Using Alk2 as an example, we present two protocols — one based on phosphate-binding Alphascreen beads, the other on coupled luminescence measurements of ADP formation — that can be used to screen for inhibitors of autophosphorylation.
Key wordsKinase Autoactivation Autophosphorylation Alphascreen Luminescence ADP-Glo Alk2 Assay development High-throughput screening
We highly appreciate the supply of recombinant Alk2 and its substrate Smad1-MH2 by Sonia Faut and Myriam Duckely as well as protein characterization through LC-MS by Peggy Lefeuvre and Francis Bitsch. The support from Geoffrey Cutler in implementing the miniaturized ADP formation assay on an automated screening system was also very helpful. The whole Alk2 project team is acknowledged for input and stimulating discussions. Last but not least, we would like to thank Joerg Trappe for suggesting and encouraging the drafting of this manuscript.
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