Application Note
Screen and profile kinases and ATPases with ease using a new bioluminescent ADP monitoring assay
B
ecause kinases and ATPases are val- idated drug targets, there is intense interest in robust technologies to monitor the activities of these enzymes in drug discovery programmes to develop novel therapeutics. Although several tech- nologies exist, most of them are limited in their ability to address all the needs of kinase screening and profiling using a sin- gle platform. We recently developed a uni- versal and homogeneous luminescence- based ADP detection assay, ADP-Glo™ Kinase Assay, that is applicable to all types of kinases, ATPases and other ADP pro- ducing enzymes.
The ADP-Glo Kinase Assay is universal and applicable to all classes of kinase sub- strates regardless of their nature with no prior modification (peptides, proteins, alcohols, lipids and sugars). The assay monitors ADP production and is directly proportional to light output via a coupled luciferase reaction. Sensitivity and linearity are hallmarks of the ADP assay. As demon-
By Dr Hicham Zegzouti and Dr Said Goueli
strated in panel A of Figure 1, there is a lin- ear relationship between the luminescent signal and the amount of ADP in the reac- tion buffer at all ATP+ADP concentration series tested. In panel B, the high signal-to- background ratios at low ATP conversions illustrate the high sensitivity of the assay. Most kinase researchers adopt radiomet- ric assays as the most sensitive format, and such assays are often referred to as the ‘gold standard’ for kinase studies. The ADP-Glo Assay correlates with radioactive assays in terms of kinase inhibitor potency determination (data not shown). The assay produces high signal-to- background ratios for all kinase enzymes tested to date. As evident in Table 1, only small amounts of enzyme per reaction are required to generate a signal-to-back-
Table 1: ADP-Glo Kinase Assay displays high signal strength at low ATP conversion ENZYME
SIGNAL-TO-
BACKGROUND RATIO OF 5 (SB5)
EGFR FAK DNA-PK IKK PI3K PI3 Kinase Sphingosine Kinase 1 Hexokinase 0.3 ng 0.6 ng 0.4 ng 4.5 ng 0.9 ng 0.15 ng 0.8 ng 0.1 ng Drug Discovery World Summer 2010 2.2 2.5 2.7 2.9 1.6 2.0 2.9 4.6 B. Signal-to-Background Ratios Percent ADP in an ATP + ADP Mixture 100 80 60 40 20 10 5 4 3 2 1 0
1µM 80 54 41 28 15 8 4 4 3 2 2 1 10µM 135 110 84 57 31 16 8 7 6 4 2 1 100µM 125 97 77 56 31 17 9 8 6 5 3 1 1mM 117 91 74 51 28 14 8 7 6 4 2 1
ground ratio of 5 (SB5). In this data set, the percentage of ATP converted into ADP ranges between 2-5% for the enzymes listed.
Conclusion
The ADP-Glo Kinase Assay offers many advantages as a platform technology for the screening and profiling of kinases, ATPases and other ADP producing enzymes. The lack of fluorescence interfer- ence, signal stability and ability to distin- guish competitive from non-competitive ATP inhibitors render the assay ideal for primary and secondary screening; the uni- versality and sensitivity render the assay suitable for profiling of lead compounds.
For more information on the new ADP-Glo Kinase Assay please see:
www.promega. com/adpglo
A.
0.5 × 105 1.0 × 105 1.5 × 105 2.0 × 105 2.5 × 105 3.0 × 105
0
% ATP TO ADP CONVERSION AT SB5
1µM R² = 0.99
0 20 40 60 80 100 120 Percent ATP-to-ADP Conversion
0.4 × 107 0.8 × 107 1.2 × 107 1.6 × 107 2.0 × 107
0 100µM
0.5 × 105 1.0 × 105 1.5 × 105 2.0 × 105 2.5 × 105
0 10µM
R² = 0.99
0 20 40 60 80 100 120 Percent ATP-to-ADP Conversion
R² = 0.99
0 20 40 60 80 100 120 Percent ATP-to-ADP Conversion
0.3 × 108 0.6 × 108 0.9 × 108 1.2 × 108 1.5 × 108 1.8 × 108
0 1mM
R² = 0.99
0 20406080 100 120 Percent ATP-to-ADP Conversion
Figure 1
Sensitivity and linearity of the ADP-Glo Kinase Assay. Panel A: ADP-Glo Kinase Assay was performed on ATP-to-ADP conversion reactions for four different combinations of ATP+ADP. Panel B: Signal to-background ratios for varying amounts of ADP in a series of ATP+ADP mixtures
49
[ATP + ADP]
Luminescence (RLU)
Luminescence (RLU)
Luminescence (RLU)
Luminescence (RLU)
8052MA
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