Kinases have become one of the most important and widely studied families of enzymes in biochemical and medical research. Kinases are a class of phosphotransferases that use ATP to catalyze the transfer of phosphate to a wide variety of cellular substrates. Many cellular processes such as cell signaling, cell division and growth, development, differentiation, and cell death are orchestrated by kinases (1) (2). Under normal physiological conditions, the regulation of kinases is tightly regulated. However, under pathological conditions, kinase activities can be dysregulated, and the disruption of the intracellular signaling networks governed by these enzymes leads to many diseases including cancer and inflammation (3) (4) (5) (6). Consequently, kinases have become an important target for drug discovery, resulting in successful kinase inhibitor-based drugs, such as Gleevec® (imatinib) and Tarceva® (erlotinib) (7) (8).
Given the importance of kinase inhibitors as potential therapeutics, researchers have focused on developing technologies that monitor changes in kinase activities and their modulation by small-molecule inhibitors (9). Isotopic-based methods for measuring kinase activity have long been considered the gold standard. However, due to safety concerns and waste management issues, alternative approaches have been sought (9) (10) . The ideal kinase assay platform should be able to: 1) detect activity of many enzyme classes 2) use universal substrates 3) detect activity at low substrate conversion rates 4) have a large dynamic range and low data variability 5) be amenable to high-throughput applications, and 6) reduce false-positive results. With these goals in mind, we have developed a luminescence-based ADP detection assay, ADP-Glo™ Kinase Assay, that measures kinase activity by quantifying the amount of ADP produced in the kinase reaction (11). This assay can be used to monitor the activity of virtually any ADP-generating enzyme (e.g., kinase or ATPase). Since the ADP-Glo™ Kinase Assay is universal and can be used with any kinase/substrate combination (11) (12) (13), we applied it to screening compounds for kinase inhibitor activity and creating inhibitor selectivity profiles for several kinase families spanning the human kinome.