Hicham Zergzouti, Marina Zdanovskaia and Said Goueli
Promega Corporation, 2800 Woods Hollow Road, Madison, WI 53711
Because of the increasing recognition of kinases as validated drug targets, there has been an intense research interest in the development of technologies that monitor the activity of these enzymes. Although several technologies were developed, most suffer from a variety of limitations that make it difficult to address all the needs of kinase screening and profiling with one platform in an attempt to develop novel therapeutics. Towards this goal we have developed ADP-Glo™, a homogeneous luminescence-based ADP detection assay that is applicable to all types of kinases, ATPases and other ADP producing enzymes. The ADP-Glo™ Assay is universal, applicable to all kinds of kinase substrates regardless of their nature with no prior modification (peptides, proteins, alcohols, lipids, and sugars). Instead of monitoring ATP depletion (Kinase Glo®), ADP-Glo™ is a positive response assay that monitors ADP production. It detects ADP at early stages of enzyme reactions with very high signal to background (SB) ratio. It can be performed at wide range of ATP concentrations (low micromolar to millimolar), detects low ADP concentrations, and it can be carried out in high density plate formats. The assay is robust as is indicated by the high Z’ values (over 0.7) and does not require antibodies or custom synthesized substrates. Because of the high dynamic range and more importantly, the high SB values at low % ATP to ADP conversion, the assay allows the use of less amount of enzyme during high-throughput screenings. Finally, as ADP-Glo™ Assay can be used at cellular levels of ATP (mM), it is now possible to study in vitro, the mode of action of kinase inhibitors (e.g. ATP competitive vs. non competitive inhibitors) and the mechanisms of drug resistance of mutated kinases.