As mediators of eukaryotic signal transduction, controlling multiple cellular processes such as gene transcription, cell cycling, migration, apoptosis, and differentiation, protein kinases are considered important targets in drug discovery. Kinase HTS screens ideally require kinase assay platforms with broad applicability in the inhibitor drug discovery process and are thus generically applicable to both the kinase target and substrate. Most non-radioactive kinase assay formats are limited in this respect in that they require either speciﬁc antibodies or afﬁnity capture reagents, to detect generation of the phosphorylated substrate. Often speciﬁc antibodies to the phosphorylated substrate are unavailable while afﬁnity capture methods have limitations in the kinase assay conditions that can be used. During substrate phosphorylation, all kinases consume ATP and assays have been developed to measure ATP depletion occurring during the kinase reaction. However, this approach is limited by the ATP concentration required by the kinase, resulting in a high assay background. Consequently, small signal decreases, as occurs with weakly active kinases are difﬁcult to detect.
An optimal approach is to measure the accumulation of a generic product of the kinase reaction i.e. ADP. This technique results in an increase in assay signal directly proportional to kinase activity and has the marked advantage that the assay is performed at a range of ATP concentrations, including those at the ATP Km value. Consequently, the inhibitory potency of novel compounds, when evaluated at the ATP Km, is a robust measure of activity at the ATP binding site and is easily compared to similar measurements at other kinases. Moreover, measurement of ADP accumulation allows ﬂexibility in choice of the kinase substrate, so that both peptides and endogenous substrates (including during auto-phosphorylation) the kinase may be utilized.
DiscoverX has developed a homogeneous ﬂuorescence based assay to measure the generation of ADP, a universal product of kinase activity. The assay uses an enzyme-coupled reaction that produces a red-shifted ﬂuorescence signal that is directly proportional to the amount of ADP in the solution. ADP Hunter is a biochemical assay to measure the accumulation of ADP (ﬁgure 1), a universal product of kinase enzyme activity.
ADP Hunter is speciﬁcally designed for high-throughput screening of kinase inhibitors. The assay has been designed for use in full-volume 384-well microplates, but can also be run in additional microplate formats. To allow for automation, a Stop Solution is also provided for added signal and background stabilization. Unlike alternative generic approaches that monitor the depletion of ATP from a kinase reaction, this method follows the product of the reaction, and offers a convenient gain-of-signal assay format. This application note describes the use of the ADP Hunter assay measured on BMG LABTECH’s PHERAstar FS multi-mode microplate reader.
Materials & Methods
The PHERAstar FS is desinged to read all leading HTS detection modes such as ﬂuorescence intensity, time-resolved ﬂuorescence, ﬂuorescence polarization, luminescence and absorption in all formats up to 1536. The PHERAstar FS was run in ﬂuorescence mode for the monitoring of the ADP Hunter demo kit (DiscoverX Corporation) containing the following reagents:
Table 1: ADP Hunter demo kit components
|1||ADP Hunter Reagent A||8 mL|
|2||ADP Hunter Reagent B||15 mL|
|3||ADP Hunter Stop Solution||4 mL|
|4||ADP Hunter Standard (360 μM)||2 mL|
|5||ADP Hunter Assay Buffer||20 mL|
The standard curve for DiscoverX’s ADP Hunter kit was run according to the package insert protocol (table 2) in black walled 384-well plates (non-binding poly-propylene plates; Greiner).
Table 2: ADP Hunter protocol for a standard curve
|Full Volume 384-well Plate||ADP Standard|
|Step 1: Standard Dilutions||8 μL ADP Standard dilutions|
|Step 2: ADP Detection||a) Add 8 μL Reagent A
b) Add 16 μL Reagent B
c) Incubate 60 minutes
|Step 3: Stop Solution||Add 4 μL Stop Solution|
Read fluorescence intensity signal:
|Note: The signal may be measured up to 2 hours after
addition of Stop Solution
The ﬂuorescence signal (Ex: 530-10 nm/Em: 590-20) was read 1 hour after the addition of the last reagent on the PHERAstar FS using the following protocol for plate reader setup (ﬁgure 2).
Results & Discussion
DiscoverX ADP Hunter assay was prepared in 384-well format and standard curves were run on BMG LABTECH’s PHERAstar FS in ﬂuorescence mode. ADP standard detection reagents were added according to the assay protocol and the ﬂuorescence signal was read 1 hour after addition of the last reagent. The ADP standard curve is illustrated in the ﬁgure below (ﬁgure 3).
The ADP Hunter assay format was designed to produce a positive signal in direct proportion to the amount of ADP generated. The dynamic range of the assay is 1.5 μM to 120 μM ADP. The simple homogeneous assay format provides robust and reproducible results with signal to background ratios > 10 and typical Z’ values > 0.7. ADP Hunter is based on enzyme couples reaction that produces a positive red-shifted ﬂuorescence signal, minimizing interference from ﬂuorescent compounds. In addition, ADP Hunter incorporates a stop solution, which stabilizes the detection signal for screening plates in batch mode. Here we have described the value of a generic kinase screening assay when analyzed on a PHERAstar FS instrument. Together, they offer a powerful solution for kinase screening needs.
This assay is a useful tool for those customers who do not have access to a modiﬁed substrate, a phosphorylation-state speciﬁc antibody, or the ability or desire to use radioactivity. These reagents are designed to be robust and applicable to high throughput ﬂuid dispensing systems using simple plate readers. The PHERAstar FS with its innovative and user-friendly, intuitive software allows for the greatest ﬂexibility and ease of use.
For more information on DiscoverX assays please refer to the website: www.discoverx.com
ADP Hunter, DiscoverX and the DiscoverX logo are registered trademarks of the DiscoverX Corporation.