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Kinase Profiling using a universal luminescent ADP detection platform with complete Kinase Enzyme Systems

Zegzouti Hicham*; Worzella Tracy, Bessetti Joseph, Cameron Gregg, and Goueli Said
Promega Corporation, 2800 Woods hollow Road, Madison, WI 53711, USA

Because the selectivity of kinase inhibitor towards the target of interest is critical for its advancement through the various phases of drug development, profiling of the inhibitor against other kinases is a common practice in drug discovery. Thus, a robust and universal Kinase assay is desired in order to assess the selectivity and potency of the inhibitor on multiple kinases from different classes that often use substrates with different chemical structures. Radiometric assays have been widely accepted as the most reliable platform for profiling kinases. 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 screening, mode of action (MOA) studies and profiling using a single platform during the drug discovery process. Here, we describe the use of luminescence to detect ADP, the universal kinase reaction product; we can achieve the sensitivity and robustness required for profiling the different kinase families covering the human kinome. ADP-Glo™ technology which measures kinase activity by quantifying the amount of ADP produced is applicable to all kinds of kinase substrates regardless of their nature and with no prior modification, making it easier to generate inhibitor profile of different kinase families using one platform. The assay is as sensitive as a radiometric method but without the use of radioactivity and the cost of disposing of radioactive waste. Because of the high signal to background (S/B) values even at low % ATP to ADP conversion, the assay allows significant savings of enzyme usage in kinase assays. As the ADP-Glo™ technology was used and validated with hundreds of kinases; the assay is now optimized for use with a large panel of complete Kinase Enzyme Systems (KES) that span different families of the human kinome. We also show the results of an optimized automation protocol for profiling different Kinase inhibitors using ADP-Glo™ and 70 KES. The fact that ADP-Glo™ assay offers so many positive attributes makes it an ideal assay not only for primary and secondary screening but also for profiling of lead compounds.
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