The Ideal Kinase Assay

Kinome_FullIf you could design the ideal kinase assay system what would it look like?

  • Would it be able to match, point for point, the results of the tried-and-true isotopic assay methods but not have any of the associated safety and waste disposal issues?
  • Would it avoid the use of specific antibodies?
  • Would it minimize false hits associated with many of the fluorescence-based assays?
  • Would it be affordable technology, adaptable to any laboratory’s throughput from 96-well to 1,536-well automated screening?
  • Would it be universal—able to assess the function of any kind of kinase (protein, lipid or sugar) that uses any kind of substrate?
  • Would it be able to detect low conversion rates (low-activity enzymes) with a high signal-to-background ratio?
  • Would you be able to use it with substrates that are multiphosphorylated?

If you answered “yes” to any of the above questions, you might want to take a look at the Promega Webinar “Enabling Kinase Research with a Luminescent ADP Detection Platform and Complete Kinase Enzyme Systems”, presented by Hicham Zegzouti, PhD, research scientist at Promega. Here he describes the ADP-Glo™ Assay platform, which meets these and several other criteria.

The precise molecular lesion that occurs with the Philadelphia Chromosome translocation—a rearrangement that creates a bcr-abl fusion in which the abl tyrosine kinase is constitutively active leading to the development of chronic myeloid leukemia is the first description of dysregulation of a kinase leading to a particular disease state. However, the human genome contains 518 protein kinases and many other atypical kinases, and one-third of all human proteins are phosphorylated. It is now estimated that over 400 human diseases are caused by dysregulation or mutation of kinases, making kinases a major target for drug discovery efforts.

The ADP-Glo™ Kinase Assay (Cat.# V9101) is a luminescent kinase assay that measures ADP formed from a kinase reaction; ADP is converted into ATP, which is converted into light by Ultra-Glo™ Luciferase. The luminescent signal positively correlates with kinase activity. The assay is well suited for measuring the effects chemical compounds have on the activity of a broad range of purified kinases, making it ideal for both primary screening as well as kinase selectivity profiling. The ADP-Glo™ Kinase Assay can be used to monitor the activity of virtually any ADP-generating enzyme (e.g., kinase or ATPase) using up to 1mM ATP. The ADP-Glo™ Max Assay (Cat.# V7001) can be used when higher concentrations (up to 5mM) are required.

The assay is performed in two steps; first, after the kinase reaction, an equal volume of ADP-Glo™ Reagent is added to terminate the kinase reaction and deplete the remaining ATP. In the second step, the Kinase Detection Reagent is added, which simultaneously converts ADP to ATP and allows the newly synthesized ATP to be measured using a coupled luciferase/luciferin reaction.
The ADP-Glo™ Assay is useful for a variety of laboratory applications including high-throughput screening, mode-of-action studies and kinase inhibitor profiling.

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A recent publication by Ahmad et al. (1) demonstrates the use of the ADP-Glo™ Assay to develop and validate a high-throughput screen for MEKK2 inhibitors. In their paper the authors describe the development of a miniaturized HTS intrinsic ATPase activity assay to identify specific inhibitors of MEKK2. In a screen of 4727 compounds, they identified one new MEKK2 inhibitor and confirmed two known inhibitors. Balazano et al. used the ADP-Glo™ Assay in “mode-of-action” studies on Haspin kinase and c-Src in their work to describe a universal mechanism of inhibitor resistance involving the hinge loop of protein kinases (2). Profiling kinase inhibitors is important to assess any side effects that may result from off-target therapeutic compound activity. Additionally, identifying new targets for a compound can lead to new applications.

In addition to the ADP-Glo™ and ADP-Glo™ Max Kinase Assays, Promega has partnered with SignalChem to create 174 Kinase Enzyme Systems that couple a specific kinase, kinase substrate, buffer and required cofactors with the ADP-Glo™ Assay to provide complete enzyme assay systems. Each enzyme assay system has been optimized to work with the ADP-Glo™ Kinase Assay for optimal results. We are also working on a “do-it-yourself” profiling system of kinase profiling strips for the ADP-Glo™ Kinase Assay and the major classes of enzyme kinases.

The ADP-Glo™ Kinase Assay technology provides a homogeneous, nonradioactive, antibody-free technology for the sensitive detection of kinase activity, including low-activity kinases. The assay is universal: suitable for any kinase and any substrate. It can be used for a variety of applications including screening, profiling and mode-of-action studies at any desired throughput.

References

  1. Ahmad, S. et al. (2012) Development and validation of a high-throughput intrinsic ATPase activity assay for the discovery of MEKK2 inhibitors. J. Biomol. Scr. 7 Nov. 2012 (epub ahead of print)
  2. Balazano, D. et al. (2011) A general framework for inhibitor resistance in protein kinases. Chemistry & Biology. 18, 966–975.

About the Webinar Series
www.promega.com/webinars provides a schedule of upcoming webinars. In addition there are links to previous webinars that allow you to either view the recording or download a pdf of the presentation. There is also a pdf of additional material available for each past webinar.
To register for a webinar, use the “Registration” link at www.promega.com/webinars This allows you to not only view the webinar, but also to participate in the live chat.
Need a reminder? You can also sign-up for monthly invitations to webinars at the webinars page (see link above). Note: Live chat is only available for live webinars, not links to recorded webinars.

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Michele Arduengo

Senior Content Developer / Social Media Lead at Promega Corporation
Michele earned her B.A. in biology at Wesleyan College in Macon, GA, and her PhD through the BCDB Program at Emory University in Atlanta, GA. Michele manages the Promega Connections blog. She enjoys leisure reading, writing creative nonfiction and knitting, and the occasional cross-country skiing jaunt.

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