ATP is one of the substrates of luciferase. ATP concentrations can be measured by quantitating the light output from a luciferase reaction. As kinases also use ATP, it is possible to assay kinase activity through the loss of luminescence in a coupled luciferase reaction. We have applied this luminescence-based ATP depletion approach to a model serine/threonine kinase. We find that the method may be run as an endpoint assay, in which ATP detection reagents (containing luciferase and luciferin) are added at the end of the reaction, or in a kinetic mode, where the ATP detection reagents are present throughout the reaction. The ATP depletion approach is capable of detecting kinase inhibitors. Six inhibitors of the model kinase, previously identified using other screening methods, are also active in the luminescence-based approach and display a similar rank order of potency. An advantage of the method is that kinase inhibitors, because they increase luminescence (by reversing the enzyme-dependent loss of signal), are immediately distinguishable from compounds such as luciferase inhibitors and luminescence quenchers, which further reduce the luminescence. The compound collections that we screened were rich in compounds that reduced luminescence. Compounds that have dual kinase and luciferase inhibitory activity, or kinase inhibitory activity combined with luminescence quenching, might be missed by being classified as false negatives. We show that the kinetic form of the assay can be used to minimize this possibility.

IMAP is a fluorescence polarisation-based assay method which can be applied to the measurement of protein kinase activity. Using a model serine/threonine kinase we found that IMAP generated a good assay window (Z' > 0.8), was very tolerant of DMSO, and was flexible with respect to sample processing (stopped reactions were stable over a period of several days). Using a set of six low molecular weight inhibitors of the kinase, we found a good correlation between IMAP and scintillation proximity assay (SPA) potency data. IMAP, which measures product accumulation, was compared in an HTS setting with a substrate depletion method (luminescence-based measurement of ATP concentration). There was a reasonable (approximately 50%) overlap in primary hits from a 17,000 compound set, but more apparent false positives were generated from the IMAP method. We followed up the compounds that showed activity in the IMAP method but not in the luminescence assay. Approximately 10% of these compounds displayed intrinsic fluorescence, suggesting that they were false actives by virtue of intrinsic spectroscopic properties. Compound activity by competition of phosphopeptide binding to IMAP beads can occur with high concentrations of chelating compounds, but did not occur with any of the false actives, suggesting that this form of interference is rare.

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Future kinase assays will be designed to detect diverse inhibitors that use a wider, or different, range of binding sites.