Kinases are a large and diverse group of enzymes which are the focus of roughly 1/3 of all drug development efforts due to their association with multiple diseases. Consequently, there is demand for assays that can detect kinase activity and subsequently their modulation by drug compounds. This has led to multiple commercial offerings that employ a variety of different detection technologies. Many of these assays employ microplates to allow for the measurement of 96 up to 1536 samples in parallel for which a multi-mode microplate reader is employed to read the experiment.
Protein kinase assays have taken advantage of this fact to develop assays that, monitor interactions with the kinase active site, evaluate conversion of ATP to ADP and attachment of a phosphate group onto the substrate. Choice of which approach you should use depends on the question you wish to answer, the type of sample to which you have access and the detection capability you prefer.
Eutheria Bio utilizes the LanthaScreen Kinase Binding assay platform and NanoBRET™ Target Engagement Intracellular Kinase Assay.
LanthaScreen was designed to identify any compound that alters binding to the active site (aka ATP-site) on a kinase. The platform employs a fluorescently labeled tracer of which there are now six. The tracers are based on known inhibitors that bind to kinases. Specificity for a kinase is achieved using a tagged version of the kinase. This tag can bind a Europium-labeled antibody such that when antibody and tracer are both bound to the kinase a high TR-FRET signal is observed. Potential inhibitors are discovered based on their ability to displace the tracer resulting in a decreased TR-FRET (fig. 3). It has been shown that this assay platform can be used to study both active and inactive kinases and to assess the role of phosphorylation on the interaction with the tracer.
The NanoBRET Intracellular Kinase Assay measures the compound binding of select target kinases in intact cells. The platform employs an energy transfer technique designed to measure molecular proximity in living cells. The assay measures the apparent affinity of test compounds by competitive displacement of the NanoBRET tracer, reversibly bound to a NanoLuc luciferase-kinase fusion construct in cells. The intracellular binding affinity and selectivity are physiologically relevant and fundamental to the pharmacological mechanism of the compounds. While biochemical and biophysical assays identify the kinase inhibitors in vitro, the NanoBRET target engagement assay serves as a great tool to determine the direct interaction of the compounds binding to target kinases in cells. The NanoBRET assay provides real-time data of the binding affinity of a compound to kinase target including occupancy time Intact cells provide a relevant environment for the compound-kinase interaction in regards of ion and ATP concentration, pH, or co-factors. In addition, to function inside the cell, compounds must overcome the cellular membrane.
A few different assays seek to assess kinase activity based on the amount of ATP that has been consumed. Unlike the kinase binding assay these are truly kinase activity assays as they measure conversion of ATP to ADP as a part of the phosphorylation event. These types of assays can be applied to any kinase, or really any enzyme that converts ATP to ADP as a part of their activity.
We offer Kinase-Glo® and ADP-Glo™
Both use the fact that firefly luciferase/D-luciferin require ATP to generate light. Thus, light production can be linked to the amount of ATP that was consumed by a kinase under a particular condition.
Kinase-Glo which measure residual ATP after a kinase reaction was carried out. So, a highly active kinase will deplete ATP and have little light production. In a kinase inhibition assay the presence of inhibition would be observed as higher light signal due to more ATP left because of less kinase activity.
ADP-Glo measures the amount of ADP generated by first adding a reagent that stops kinase activity and depletes residual ATP. Then a second reagent is added that stops ATP degradation, converts ADP to ATP and has the luciferase/luciferin reagents to generate light. The result is a signal that tracks in the opposite direction of Kinase-Glo. High light production for active kinases which is reduced by inhibitors.
An additional approach to measure kinase activity utilises the monitoring of substrate conversion. An example is the pyruvate kinase activity assay. This coupled enzyme assay comprises two different enzymatic reactions. The first one is the transfer of a phosphate group from phosphoenolpyruvate to ADP by pyruvate kinase, yielding pyruvate and ATP. The second enzymatic reaction then uses pyruvate and a fluorescent peroxidase substrate to generate a fluorescence signal. Since it is dependent on the generation of pyruvate, this fluorescence signal can be used as readout for the kinetic measurement of pyruvate kinase activity. Alternatively, the product can be detected with an absorbance measurement
The Cellular Phosphorylation Assay quantifies changes in the phosphorylation status of a substrate as a result of treatment with your inhibitor in intact cells. The assay was designed to address compound activity in a physiological environment on a physiological substrate. The assay is available with either an endogenously expressed target kinase or with transduced cells for overexpression of the kinase of interest. Cells are preincubated with the compound to allow thorough target binding. To perform substrate phosphorylation some kinases are constitutively active and some kinases need to be activated with incubation of a ligand. After stopping the reaction by cell lysis, the substrate phosphorylation is quantified.
See our list of possible assays