The development of new therapeutics targeting protein kinases is crucial in the fight against diseases like cancer. Kinase drug discovery programs rely on the precise characterization and screening of potent kinase inhibitors. A clinically proven and validated assay platform is essential for integrating into these active discovery and development pipelines.
Traditional methods for measuring kinase activity, such as those using radioisotopes or simple binding studies, can be challenging, unsafe, and often fail to correlate binding data with functional enzymatic activity strongly. Furthermore, due to the presence of conserved active sites and high structural homology among kinases, developing selective kinase inhibitors is a complex process that requires validation at every step.
HitHunter Kinase Activity Kits: A Reliable Solution
The Eurofins DiscoverX HitHunter® Kinase Activity Assay Kits are developed using the well-established ADP Hunter Plus™ technology to measure kinase activity. These kits have undergone extensive activity testing, including optimizations for ATP and kinase concentrations, which confirms their suitability in assay development. They are innovative, ready-to-use, and validated for screening kinase inhibitors with key characteristics.
- Ready-to-use and Convenient: Kits come with all necessary reagents (including the target kinase) and are rigorously optimized for performing screening kinase activity
- High-throughput Compatibility: Applicable for both small-scale (96-well) and high-throughput screening (HTS) formats
- Non-radioactive and Antibody-free: The simple, homogeneous (no wash) assay format is easy to run, requires no antibodies, and is safer than traditional radioactive methods
How does the ADP Hunter Assay work?
The kits utilize the ADP Hunter Plus technology to directly measure kinase activity (Figure 1). This technology focuses on the universal byproduct of kinase activity, ADP.
- The ADP Hunter Plus detection reagents use a coupled enzyme system to generate hydrogen peroxide (H2O2) from the produced ADP (typical of a kinase reaction wherein enzyme transfers a phosphate from ATP to a substrate, producing ADP).
- The H2O2 then reacts with a fluorescent dye precursor (ADHP) in the presence of peroxidase to generate a highly fluorescent dye called resorufin.
- The resorufin dye provides a stable, bright fluorescence that is red-shifted (~590 nm), thereby reducing interference from biological autofluorescence and improving signal-to-noise ratio and assay sensitivity.
- This convenient gain-of-signal assay format provides results in about 90 minutes using a fluorometric readout.
Figure 1. Kinase Enzyme Activity Kit Assay Principle. In Step 1, the kinase transfers a phosphate group from ATP to its substrate, generating ADP as a byproduct. In Step 2, the generated ADP is converted to hydrogen peroxide through a coupled enzyme reaction. The H2O2 then reacts with ADHP in the presence of peroxidase to produce resorufin, a bright, red-shifted fluorescent dye (Ex 530 nm / Em 590 nm) proportional to kinase activity.
Validation for Kinase Inhibitor Testing (AMPK Example)
Eurofins DiscoverX has released 50 assays (as of Nov. 2025) and continues to expand its portfolio. Each assay has been rigorously optimized to enable researchers to save time and screen kinases with confidence.
As an example, assay optimization and validation for kinase inhibitor testing were performed for the AMPKα1β1γ1 kinase complex using the ADP Hunter technology.
Key steps in the assay development process include:
- Determining Assay Suitability: An enzyme titration confirmed a robust signal at a low kinase concentration (1 ng/well).
- Optimizing ATP and Substrate Concentrations: The concentrations of ATP and the substrate were determined based on their calculated Km values to ensure that they were not rate-limiting. For example, the final assay ATP concentration was set at 3 x Km ATP (87 μM) — a critical step for screening ATP-competitive inhibitors.
- Inhibitor Testing: The assay was confirmed as suitable for inhibitor screening by testing both the reference inhibitor, staurosporine (an ATP competitive inhibitor), and the specific inhibitor, BAY-3827 (Figure 2).
Reference and Specific Inhibitors Testing
To confirm suitability for inhibitor testing, the assay measured kinase activity at 1 ng/well (50 ng/mL) kinase against a range of reference inhibitor concentrations. After demonstrating suitability (Figure 2. A.), the AMPK specific inhibitor BAY-3827 was tested in the assay to provide additional validation data. Kinase activity was measured at a range of specific inhibitor concentrations, as shown in Figure 2. B.
Figure 2: Inhibitor testing to measure kinase activity. A. Reference inhibitor using a range of staurosporine concentrations reveals a low nanomolar IC50 (0.45 nM) and a robust assay with a signal-to-noise ratio of 9 between controls containing no inhibitor or enzyme. Since staurosporine is an ATP competitive inhibitor, the low nM IC50 indicates that the ATP concentration in the assay is suitable for measuring responses to this type of inhibitor. B. Specific inhibitor testing using a range of BAY-3827 concentrations reveals an IC50 of 8.2 nM, which is in good agreement with published values (1.4 nM to 15 nM).
Conclusion
HitHunter Kinase Enzyme Activity Kits are rigorously tested with a variety of kinases and substrates and serve as a readily available choice to use for screening kinase inhibitors and accelerating new kinase inhibitor drug discovery. The assays enable a better understanding of structure/activity relationships (SAR), avoid toxic liabilities, and thereby support the selection of superior drug candidates.
Explore targets, assay principles, applications, and resources under Eurofins DiscoverX’s kinase activity assay kits portfolio, and accelerate kinase drug discovery.
Resources
Assay Development & Application of Optimized & Validated HitHunter Kinase Activity Kits for Inhibitor Screening view application note
Reliable PI3K Inhibitor Screening Using Validated HitHunter Kinase Enzyme Activity Assay Kits view application note