Flexible and Targeted Inhibitor Profiling using the Luminescent ADP-Glo™ Kinase Assay Platform

Part # PS138


Zegzouti Hicham, Alves Juliano, Hsiao Kevin, Vidugiriene Jolanta and Goueli Said
Promega Corporation, 2800 Woods Hollow Rd, Madison, WI 53711, USA

Profiling kinase inhibitors is a critical step during drug development. Knowing the inhibitory profile of a compound across a broad collection of kinases can be important for better understanding its biological activity, obviating any off-target activities, and in some cases identifying new targets that may lead to novel therapeutic applications. Unlike an HTS phase, where a target-specific kinase assay is used to identify hit compounds, during profiling a robust universal assay is needed to assess the selectivity and potency of the inhibitor on multiple kinases from different classes. Often, these kinases use substrates with different chemical structures and target-specific screening assays are not suitable or can be cost prohibitive. We previously introduced the luminescent ADP-Glo™ Kinase Assay that measures kinase activity by quantifying the amount of ADP produced during the enzymatic reaction. This technology addressed all the needs of kinase screening, mode of action (MOA) studies and profiling using one assay format during the drug discovery process. Here, we show that we can achieve the sensitivity and robustness required for profiling the different kinase families covering the human kinome with one platform. The ADP-Glo™ assay was previously validated with more than hundred kinases and 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 demonstrate the profiling of different kinase inhibitors using newly designed kinase strips. Each multi-well strip contains a group of kinases from the same family. The kinase stock volumes were standardized in a way that, when all the kinases were diluted into the kinase reaction, the kinases generated 5–10% ATP to ADP conversion. The substrate stocks were standardized in similar fashion and are located in a second strip at corresponding positions. We show that using the new kinase profiling strips and an optimized protocol, we could easily generate selectivity profiles using small or large kinase panels, as well as detecting 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 primary and secondary screening but also for profiling compounds in a cost-effective manner using one single platform.

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