Kirk Wyatt ACF Abstract FY10

“Evaluation of Non-Radioactive Luminescence Assays for Protein Kinase Activity”

Conference Name: Spring 2010/239th National Meeting of the American Chemical Society

Protein kinases (PKs) comprise approximately two percent of the human genome and play vital roles in the mediation of cellular signaling. Because of their ubiquity and importance in the cell, they have become the focus of approximately one quarter of all drug discovery efforts in the pharmaceutical industry. The development of compounds targeting PKs necessitates a reliable method for quantifying the activity of target kinases. Radioisotope-labeling assays provide high sensitivity and are often cited as the "gold-standard" for measuring kinase activity; however, the use of radioisotope-based assays, such as the [γ-32P]ATP radioisotope-labeling assay, has several disadvantages including the short shelf-life and cumbersome disposal requirements for radioisotopes. For this reason, non-radioactive assays which provide sensitivity to rival radioisotope-labeling assays are sought by protein kinase researchers. Commercially-available luminescence assays were evaluated for their suitability to be used in lieu of a radioisotope-labeling assay for the quantitation of the activity of the protein tyrosine kinases Src and FAK. Criteria for evaluation of the assays included sensitivity, reproducibility and convenience with the aim of identifying a flexible assay that can be used to identify high-affinity substrates and ATP-competitive inhibitors of the target kinases.

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