Enabling Kinase Research with a Luminescent ADP Detection Platform and Complete Kinase Enzyme Systems

Many cellular processes are orchestrated by kinases and the disruption of the intracellular signaling networks governed by these enzymes leads to many diseases like cancer. Because of the importance of studying kinases and their increasing recognition as validated drug targets, there have been intense research interests in the development of technologies that monitor the activity of these enzymes. Although several other technologies were developed in the last few years, most suffer from a variety of limitations that makes it difficult to address all the needs of kinase research with one platform. Towards this goal we have developed a luminescence-based ADP detection assay (ADP-Glo) that measures kinase activity by quantifying the amount of ADP produced during a kinase reaction. This assay can be used to monitor the activity of virtually any ADP-generating enzyme (e.g., kinase or ATPase) using a broad range of ATP concentrations in the reaction. This assay is universal, applicable to all kinds of kinase substrates regardless of their nature with no prior modification (peptides, proteins, alcohols, lipids, and sugars). It is shown to be as sensitive as a radioisotope based method, and thus significantly reduces the amount of enzyme needed in kinase assays. The ADP-Glo™ assay has been validated for more than hundred seventy kinases including disease relevant mutant kinases and is optimized for use with a large panel of complete Kinase Enzyme Systems (KES) that span different families of the human kinome. We show that using the new kinase systems we could easily generate inhibitor selectivity profiles using small or large kinase panels, as well as identifying compound promiscuity towards members of a single kinase subfamily or different subfamilies of the kinome. The fact that ADP-Glo™ platform offers so many positive attributes makes it an ideal assay not only for basic kinase characterization but also for primary and secondary inhibitor screening, mode of action (MOA) studies and profiling compounds in a cost-effective manner using one single platform.