High-Throughput Biochemical Kinase Selectivity Assays: Panel Development and Screening Applications

Abstract

Kinases represent attractive targets for drug discovery. Eight small-molecule kinase inhibitors are currently marketed in the area of oncology, and numerous others are in clinical trials. Characterization of the selectivity profiles of these compounds is important to target appropriate patient populations and to reduce the potential of toxicity due to off-target effects. The authors describe the development, validation, and utilization of a biochemical kinase assay panel for the selectivity profiling of inhibitors. The panel was developed as 29 radiometric Flashplate assays, and then an initial 13 were transitioned to a nonradiometric Caliper mobility shift assay format. Generation of high-quality data from the panel is detailed along with a comparison of the assay formats. Both assay technologies were found to be suitable for panel screening, but mobility shift assays yielded higher data quality. The selectivity data generated here should be useful in computational modeling and help facilitate, in conjunction with sequence and structural information, the rational design of inhibitors with well-defined selectivity profiles.

A Comparison of Protein Kinases Inhibitor Screening Methods Using Both Enzymatic Activity and Binding Affinity Determination

“…Utilizing fluorescent-labeled peptides, real-time detection and quantification of both substrate and product is possible, and the reaction turnover can be determined [22]. The sensitivity of the mobility shift assay is comparable to that of radiometric assays [23]. A concern with activity-based screens is that most inhibitors compete with ATP for binding, for which reason the results depend on the ATP concentration [24].…”

Measuring and interpreting the selectivity of protein kinase inhibitors

“…These assays can be carried out in 384-well plate format and have been used in high-throughput to screen 32,000 compounds [115] as well as for screening inhibitors against kinase panels. Electrophoretic separation assays have been compared with a radiometric assay format and found to be more reliable [29]. In our own research, we have used this technology to profile both potent kinase inhibitors and also to investigate the selectivity patterns of low molecular weight fragments (MW < 200) tested at high compound concentrations ( [131]; Smyth et al unpublished results).…”

A Benzothiophene Inhibitor of Mitogen-Activated Protein Kinase-Activated Protein Kinase 2 Inhibits Tumor Necrosis Factor α Production and Has Oral Anti-Inflammatory Efficacy in Acute and Chronic Models of Inflammation

“…PF-3644022 was evaluated for inhibition of 200 human kinases by using an in-house 30 kinase selectivity panel (Card et al, 2009) and 170 kinases from the Upstate Kinase Profiler service (Millipore Corporation, Billerica, MA). The kinase selectivity experiments were performed with the MgATP concentration fixed at the K m(app) determined for each enzyme.…”