Larson B, Banks P, Pierre N, Graham S, Jacquemart L, Degorce F
Biotek instruments, Inc., Winooski, Vermont, USA. Cisbio Bioassays, Codolet, France.
MipTec 2012, Basel, Switzerland / SLAS 2013, Orlando USA
Cellular kinases play an important role in relaying signals from activated receptors residing at the cell membrane to the interior of the cell, through signal transduction. The cellular processes in which they are associated include angiogenesis, cell growth, cell migration, and apoptosis. Overexpression of the kinase, or constitutive activity, has also been linked to a number of disease states, including vascular disease, bone disorders, and multiple forms of cancer. Therefore, cellular kinases continue to be an important target for small molecule and large molecule drug development. The ability to screen compounds or antibodies for their antagonistic effects on these signaling molecules, in a rapid and robust way, is critical to current efforts in this area. Here we describe a method using HTRF® technology to quantify cellular kinase activity without overexpression of kinase or its substrate. Two specifi c kinases were included in the study: 1. Vascular Endothelial Growth Factor Receptor 2 (VEGFR2), one of multiple subtypes of the receptor tyrosine kinase VEGFR, is involved in mediation of almost all cellular responses to VEGF. 2. Signal Transducer and Activator of Transcription 3 (STAT3), acts as a transcription activator, and is phosphorylated by receptor-associated kinases in response to cytokines and growth factors. Detection of phosphorylated kinase involves a sandwich immunoassay which includes an anti-phospho specifi c antibody coupled with an anti-total antibody labeled with either donor or acceptor fl uors. Activation of the appropriate signaling cascade causes an increase in HTRF signal, whereas inhibition will demonstrate the opposite effect. Automation of all assay component dispense steps were performed in a high throughput format using a non-contact dispenser. Simultaneous, rapid detection of the fl uorescent signal from the donor and acceptor molecules, in LV384-well format, was accomplished through the combination of a high performance Xenon fl ash lamp and dual matched PMTs incorporated into a novel HTS microplate reader. Optimization, validation, and pharmacology data demonstrate how the combination of assay chemistry, automation, and detection technologies create a robust method for high throughput screening of potential modulators of these important cell signaling pathways.