By Laurence Jacquemart, Marion De Decker, and Bastien Caumes  Fig.1: (a) Assay principle: HTRF Transcreener ADP assay principle: during the enzymatic step, the kinase catalyses the transfer of a phosphate group from ATP to a substrate. For ATPase, ATP is converted into ADP and inorganic phosphate. Then in the detection step, native ADP and d2 labeled ADP compete for the anti-ADP antibody labeled with Eu3+ Cryptate. The signal is inversely proportional to the concentration of ADP in the sample. (b) Standard curve: The standard curve is based on a mixture of ATP and ADP. ATP solutions are serially diluted in ADP solutions in order to mimic the conversion of ATP into ADP (from 0 to 100%): ADP concentration ranges from 0 to 100 µM final. Click here to enlarge. |
Enzymes represent on average 45% of all targets screened by the pharmaceutical industry. Some of the most investigated and well-known are Tyrosine (Tyr), Serine/Threonine (Ser/Thr), lipid kinases and ATPases. (Hightech Business Decisions, 2007)When studying kinase and ATPase activity, finding the right assay is important to researchers using high-throughput screening, specifically an assay with homogeneous and non-radioactive properties that can be used for the study of different kinds of substrates for kinases, that has a format flexible enough for different enzymatic conditions, and that is sensitive enough for low enzyme activities.
To date, assays have been developed to address the above challenges in two ways: by measuring phosphorylated substrate for kinases or either ADP (adenosine 5’-diphosphate) generation or ATP (adenosine triphosphate) depletion for both kinases and ATPases. ADP is the universal product from kinase and ATPase activity. Traditional methods of measuring the accumulation of ADP from kinases and ADP producing enzymes can result in a number of limitations. Examples include the use of radioactivity (i.e. 33P) or the reliance upon a secondary enzyme (i.e. luciferase, peroxidase, pyruvate kinase) for detection. With radioactive methods, particular attention must be paid to safety and the proper disposal of materials, which can be costly. Further, assay sensitivity can be compromised as a result of high background levels. With enzyme coupled detection methods, the resulting data can be subject to high false-positive rates and sensitivity limitations can arise if high turnover by the target enzyme is required for the product’s detection by the secondary enzyme.
A homogeneous, non-radioactive screening method, HTRF Transcreener ADP, has been developed by Cisbio Bioassays for identifying and characterizing the phosphotransferase activity induced by ATPases and kinases. Compared to existing methods, enzyme-free detection enables a low false positive rate and, for kinases, this kit represents a highly flexible solution as it is compatible with any substrate. This competitive immunoassay is performed in two steps, the enzymatic step followed by the detection step (Fig. 1a). Standard curves are drawn up to mimic ADP generation during an enzymatic reaction; total adenosine concentration (ATP + ADP) remains constant for each sample, while the percentage of ADP within each sample varies (Fig. 1b).
The following describes a typical four-step-development with the Eg5 ATPase using the HTRF Transcreener ADP assay.
 Fig.2: (a) Standard curves ATP/ADP (b) Km calculation. The concentration of ADP produced for each concentration of ATP is then calculated on the corresponding standard curve, i.e. the ratio obtained for Eg5 with ATP 5 µM final is read on the standard curve obtained with ATP/ADP 5 µM final in order to obtain a concentration of ADP. Km is calculated using the Michaelis-Menten equation from the resulting plot of the ADP concentration produced versus ATP concentration. Click here to enlarge. |
Human Eg5, a member of the kinesin super family, plays a key role in mitosis, and is responsible for the formation and maintenance of the bipolar spindle. Eg5 has garnered substantial interest as a potential chemotherapeutic target in cancer treatment.
Materials
Eg5 was from Cytoskeleton; S-Trityl-L-cysteine (Eg5 inhibitor), purchased from Calbiochem (Merck); ATP and MgCl2 were obtained from SIGMA. All other reagents are kit components. Eg5, ATP, and compounds were prepared in the enzymatic buffer provided with the kit, supplemented with 10 mM MgCl2. HTRF detection reagents were prepared in the detection buffer, which contains 60 mM EDTA and 400 mM KF. For each step, an ATP/ADP titration curve was realized with the amount of ATP used in the assay.
Because EDTA contained in the detection buffer does not stop Eg5 activity, all experiments were performed in one step: 2 µL of Eg5 enzyme, 4 µL of ATP, and 4 µL of enzymatic buffer were mixed with 5 µL of ADP-d2 and 5 µL of anti-ADP labeled with Eu3+ Cryptate.
Methodology
 Fig.3: S-Trityl-L-cysteine inhibition. Click here to enlarge. |
The first step of the development consisted in the enzyme titration performed in order to obtain the optimal enzyme concentration. A two-fold dilution series of Eg5 was run, ranging from 250 nM to 7.8 nM final reaction concentrations (20 µL), and incubated at 100 µM final concentration of ATP (non limiting ATP concentration) (20 µL). HTRF detection reagents were added at the same time, and all the reagents were incubated at room temperature for 30 minutes. Eg5 optimal concentration was found at 60 nM.
For the second step, related to enzyme kinetics, three different concentrations were used around the optimal, from 30 nM to 90 nM and 100 µM ATP. The plate was then read at different end points: 3’, 6’, 10’, 15’, 20’, 25’, 35’, 45 minutes, 1h, 2h, 3h, 4h, 5h, and 22 hours. The optimal incubation period for the 3 concentrations of Eg5 to achieve maximum signal and a linear time course was found to be 25 minutes (R2: 0.955 for Eg5 60 nM).
The third step was the ATP titration: a standard curve was generated for each ATP concentration used in the assay. Assays were run with a fixed concentration of enzyme (60 nM) and different concentrations of ATP from 5 µM to 200 µM final (Fig. 2a) for 25 minutes. The amount of ADP produced was plotted against ATP concentrations using the Michaelis-Menten equation. The apparent Km was obtained at 75 µM (Fig. 2b).
For the inhibition experiment, Eg5 enzyme at 60 nM was first pre-incubated at 37°C for 90 minutes in presence of various concentrations of S-Trityl-L-cysteine from 500 µM to 30 nM, with a three-fold dilution series between each concentration. Then ATP (100 µM) and HTRF detection reagents were added and the plate incubated at room temperature for 30 minutes. The IC50 was calculated using the inhibition curve (Fig. 3) and found at 0.87 µM.
In Salvatore DeBonis’ paper1, S-Trityl-L-cysteine is defined as a reference inhibitor of Eg5 activity and IC50 they obtained was 1.0 µM. Using HTRF Transcreener ADP assay the IC50 value (0.87 µM) is in the same range as that published.
Conclusion
Ideal kinase target investigation involves tools enabling researchers to study all kinases and ATPases, not ones limited to the study of Tyr and Ser/Thr kinases only. With reliable tools, researchers are able to study a broader number of targets. This article has outlined the guidelines for an assay development relating to a specific ATPase target. As HTRF Transcreener ADP is universal and non-radioactive, it is well-suited to a broad range of targets such as lipid kinases, all ATPases, and HSP. The homogeneous format and enzyme-free detection enable high throughput applications.
Reference
1. in vitro screening for inhibitors of the human mitotic kinesin Eg5 with antimitotic and antitumor activities Salvatore DeBonis, Dimitrios A. Skoufias, Luc Lebeau, Roman Lopez, Gautier Robin, Robert L. Margolis, Richard H. Wade, and Frank Kozielski, Mol Cancer Ther. 2004;3:1079-1090
About the authors
Laurence Jacquemart is manager of technical marketing in Cisbio Bioassays’ services group. She earned a degree in life sciences from the University of Orsay in France.
Marion De Decker is HTRF Product Manager at Cisbio Bioassays, where she works with products related to kinases, biomarkers, and customer services. She has a degree in science and business from the Ecole Supérieure Privée d’Application des Sciences, part of the Catholic University in Lille, France and earned a master’s degree in applied marketing from the Institut d’Administration des Entreprises in Aix en Provence, France.
Bastien Caumes is part of Cisbio Bioassays’ team focused on kinases, kinasines, ATPases and assay development. He is currently finishing his master’s degree in biotechnology at the University of Montpellier.