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.
scite is a Brooklyn-based organization that helps researchers better discover and understand research articles through Smart Citations–citations that display the context of the citation and describe whether the article provides supporting or contrasting evidence. scite is used by students and researchers from around the world and is funded in part by the National Science Foundation and the National Institute on Drug Abuse of the National Institutes of Health.
hi@scite.ai
10624 S. Eastern Ave., Ste. A-614
Henderson, NV 89052, USA
Copyright © 2024 scite LLC. All rights reserved.
Made with 💙 for researchers
Part of the Research Solutions Family.