Introduction

Dysregulation of kinase activityhas been shown to be associatedwith several human diseasesincluding cancer, diabetes andmorphological disorders. Becauseof their function, kinases arecrucial targets for drug discovery.Analysis of the human genomehas revealed the existence ofnearly 520 genes encodingkinases. The abundance of thesepotential therapeutic targetsprovides a compelling impetusfor developing efficient androbust high throughput screening(HTS) platforms for the discoveryof kinase modulators.

Time-resolved fluorescenceresonance energy transfer (TR-FRET) assays are homogeneousproximity assays where theinteraction of two labeled bind-ing partners is detected by theenergy transfer from an exciteddonor to an acceptor dye, andmeasurement of the subsequentlight emission by the acceptordye. A number of TR-FRETplatforms are currently available.They differ principally in thenature of the donor and acceptordyes used for the energy transfer.The LANCE® technology uses an europium-based chelate (Euchelate) as the donor dye. Euchelates have a high quantum

yield, large Stokes’ shift and anarrow-banded emission at ~615nm. The lifetime of their lightemission is exceptionally long,allowing for time-delayedmeasurements. These uniquefluorescence properties make Eu chelates ideal energy donorsin TR-FRET assays. In classicalLANCE assays, the acceptor dyeis allophycocyanin (APC). APCreceives the energy from irradiatedEu chelate molecules in closeproximity and in turn emits lightat 665 nm. Although APC allowsfor the efficient capture andreemission of the transferredenergy, it has some disadvan-tages. Since it is a large protein(~100 kDa), small molecules suchas peptides and oligonucleotidescannot be labeled directly and anadditional labeled component,such as APC-streptavidin (APC-SA), must be introduced in theassay set-up. Also, the bulkinessof APC can potentially createsteric hindrance in some assayconfigurations. Finally, APC islight sensitive, which requiresspecial precautions during assay set-up and incubation. To overcome these limitations,PerkinElmer has developed thenew LANCE Ultra HTS platformin which the APC acceptor dyehas been replaced by a newULight™ acceptor dye.

Development and Optimization of Kinase Assays using NewLANCE Ultra TR-FRET Reagents

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Authors Mireille Caron, Anja Rodenbrock,Philippe Roby, Nathalie Bouchard,Jaime Padrós, Valérie Paquet, ClaireNormand, Marjolaine Roy, VéroniqueBrechler and Lucille Beaudet

PerkinElmer Life and AnalyticalSciences

ULight is a small acceptor dye withspectral characteristics similar toAPC but with two distinct advan-tages. First, its low molecularweight makes it suitable for thedirect labeling of peptides and othersmall molecules; second, it is lightresistant, which simplifies thehandling of assay components andplates. LANCE Ultra reagentsinclude generic assay componentssuch as ULight-labeled Protein A,Streptavidin, anti-His tag and anti-GST tag antibodies as well as aseries of specific Eu-labeled anti-phospho-substrate antibodies withthe corresponding ULight-labeledpeptide substrates for HTS kinase assays.

A LANCE Ultra kinase assay isillustrated in Figure 1. In thepresence of kinase and ATP, theULight peptide substrate is phos-phorylated. It is then captured by aEu-anti-phospho-substrate antibody,which brings the Eu chelate donorand ULight acceptor dyes into closeproximity. Upon excitation at 320 or340 nm, the Eu chelate transfers itsenergy to the ULight dye, resultingin a fluorescent light emission at665 nm.

This application note providesdetailed guidelines for the develop-ment and validation of a LANCEUltra kinase assay. It demonstrateshow to optimize the concentrationof assay components (enzyme,ULight-labeled substrate, ATP, Eu-anti-phospho-substrate antibody) as well as the kinase assay reactionand antibody detection times. It alsoshows how to validate the perform-ance of a kinase assay by titration

of a known kinase inhibitor anddetermination of the Z'-factor. ALANCE Ultra ERK1 kinase assaydeveloped using the ULight-labeledMyelin Basic Protein peptidesubstrate (ULight-MBP) and Eu-anti-phospho-MBP antibody will be presented as an example.

Materials and Methods

MaterialsTable 1 lists the materials used forthis study, including suppliers andproduct numbers.

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Figure 1. Principle of the LANCE Ultra assay

Method

ERK1 kinase LANCE Ultra assayAll assay components were titratedindividually (see Results andDiscussion). Optimized concentra-tions of reagents for the LANCEUltra ERK1 kinase assay were 1 nMERK1 kinase, 50 nM ULight-MBPand 4 µM ATP. Kinase Assay Bufferconsisted of 50 mM Tris-HCl pH7.5, 10 mM MgCl2, 1 mM EGTA, 2 mM DTT and 0.01% Tween-20.Kinase assay components wereprepared as concentrated pre-mixesin Kinase Assay Buffer and added tothe wells of a 384-well OptiPlate™.The total volume of the kinasereaction was 10 µL. In the opti-mized assay, kinase reactions wereincubated for 60 min at 23 °C andstopped by the addition of 10 mMEDTA. The Eu-anti-phospho-MBPantibody diluted in Detection Buffer was then added to a finalconcentration of 2 nM. Detectionreactions were incubated for 1 h at 23 °C. The LANCE signal wasmeasured on an EnVision™

Multilabel Microplate Reader.Excitation wavelength was set at 320 nm and emission recorded at 665 nm.

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Table 1. Reagents and Consumables

Item Supplier Product No.

ULight-MBP peptide PerkinElmer TRF0109

Eu-anti-phospho-MBP PerkinElmer TRF0201

LANCE Detection Buffer 10X PerkinElmer CR97-100

White OptiPlates-384 PerkinElmer 6007290

MAP kinase 1/ERK1, active Millipore Corp. 14-439

Staurosporine Sigma-Aldrich S4400

ATP Sigma-Aldrich A2383

TopSeal-A™ PerkinElmer 6005250

EnVision Multilabel Reader PerkinElmer 2103-0010

Mirror: LANCE/DELFIA™ Dual PerkinElmer 2100-4160

Excitation Filter: UV2(TRF) 320 nm PerkinElmer 2100-5060

Emission Filter: Eu 615 nm PerkinElmer 2100-5090

Emission Filter: LANCE 665 nm PerkinElmer 2100-5110

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Results and Discussion

Cross-titration of ERK1 kinaseand ULight labeled MBP substrate A cross-titration of ERK1 kinaseand ULight-MBP substrate wasperformed at a non-limiting ATPconcentration (20 µM) in order todetermine optimal concentrationsof enzyme and substrate for theassay. Figure 2 shows that increas-ing amounts of enzyme leads to anincreased LANCE signal, whichreflects the higher level of phospho-rylation of the ULight-MBP sub-strate. A signal to background (S/B)ratio of 8.5 was obtained using aslittle as 0.25 nM of ERK1 enzymeand 50 nM of ULight-MBP.However, since a significantlyhigher S/B ratio was obtained using 1 nM of ERK1 and 50 nM of ULight-MBP (S/B ratio of 31),these enzyme and substrate concentrations were selected for further experiments.

ATP Titration In order to identify competitiveinhibitors for ATP, the concentra-tion of ATP in the assay should benear the Km value of the enzyme forATP. In LANCE Ultra assays, theEC50 value for ATP can be used asan apparent Km of the enzyme forATP. ATP titration was performedusing the optimized ERK1 enzymeand ULight-MBP substrate concen-trations (Figure 3). An apparent Km value of 4.2 µM was obtained.Consequently, all subsequentexperiments were performed using4 µM ATP in the kinase reaction.

Figure 2. Optimization of ERK1 enzyme and ULight-MBP substrate concentrations. ERK1 enzymewas titrated from 0.25 to 4 nM and ULight-MBP substrate from 25 to 500 nM in Kinase AssayBuffer supplemented with 20 µM ATP (final concentrations in kinase reactions). Reactions wereterminated after 90 min by the addition of 10 mM EDTA. ULight-MBP peptide phosphorylationwas detected by the addition of 2 nM Eu-anti-phospho-MBP and measured after 60 min on theEnVision Multilabel Reader.

Figure 3. ATP titration. ATP was serially diluted in assay reactions containing 1 nM ERK1 kinaseand 50 nM ULight-MBP substrate. Reactions were terminated after 90 min by the addition of 10 mM EDTA. ULight-MBP peptide phosphorylation was detected by the addition of 2 nM Eu-anti-phospho-MBP and measured after 60 min incubation at RT on the EnVision Multilabel Reader.

Figure 4. Time course of ERK1 phosphorylation of ULight-MBP substrate. ERK1 enzyme (1 nM)was incubated with ULight-MBP substrate (50 nM) in Kinase Assay Buffer supplemented with 4 µM ATP. Reactions were terminated at specific time points by the addition of 10 mM EDTA.ULight-MBP peptide phosphorylation was detected by the addition of 2 nM Eu-anti-phospho-MBP and measured after 60 min incubation at RT on the EnVision Multilabel Reader.

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Time course of ULight-MBPsubstrate phosphorylation A time course of substrate phospho-rylation was performed to ensurethat the assay incubation time wasin the linear range of the kinasereaction. As shown in Figure 4,signal generated by the LANCEUltra ERK1 assay was linear for upto 90 min (r2 = 0.99) with 53,000counts measured at 60 min, corre-sponding to an S/B ratio of 8.4.Based on these results, incubationwas reduced from 90 to 60 min,which permits a significant reduction of the LANCE Ultraassay time and obtaining results in just two hours.

Titration of the Eu-anti-phospho-MBP antibodyThe Eu-anti-phospho-MBP used forthe capture of the phosphorylatedULight-MBP substrate was titratedto optimize its concentration in thedetection reaction. As shown inFigure 5, S/B ratios (indicatedabove bars) reached a maximum of 8.7 using 2 nM of Eu-labeledantibody. At higher antibodyconcentration, both specific (+ATP)and non-specific (-ATP) signalsincreased, which led to a signifi-cant decrease of S/B ratios.Therefore, 2 nM was kept as the Eu-anti-phospho-substrateantibody concentration in thedetection reaction.

Antibody incubation time and signal stabilityThe Eu-anti-phospho-MBP anti-body is used for the detection ofphosphorylated products after thetermination of the enzymaticreaction by EDTA. In order todetermine 1) the equilibrium timefor the detection step and 2) thestability of the LANCE Ultra signal

over time, a time course of detec-tion was conducted over a 20-hourperiod. As shown in Figure 6,equilibrium of the detectionreaction was reached after 30 min,with an S/B ratio of 8.3. Signal was stable over at least 6 h. Afterovernight incubation, the signaldecreased but the S/B ratioremained relatively stable (S/B ratioof 7.4). This time course experi-ment confirmed that the LANCEUltra ERK1 assay is suitable forscreening campaigns, where platesare often read offline.

DMSO toleranceAs DMSO is routinely used as acarrier solvent for compoundlibraries, it is essential that itspresence in the assay reaction doesnot impact significantly the assayperformance. Figure 7 shows thataddition of DMSO to ERK1 kinasereactions results in a concentration-dependent decrease of the LANCEsignal. This effect is not due to adecrease in the performance of theLANCE Ultra reagents since thedetection of an ULight phosphory-lated MBP peptide by the Eu-anti-Phospho-MBP antibody was not

Figure 5. Titration of Eu-anti-phospho-MBP. ERK1 enzyme (1 nM) was incubated with ULight-MBPsubstrate (50 nM) in Kinase Assay Buffer supplemented with 4 µM ATP (final concentrations inkinase reactions). Reactions were terminated after 60 min by the addition of 10 mM EDTA. ULight-MBP peptide phosphorylation was detected by the addition of Eu-anti-phospho-MBP (dilutionsfrom 0.2 to 20 nM in the detection reactions) and measured after 60 min incubation at RT on theEnVision Multilabel Reader.

Figure 6. Time course of detection. ERK1 enzyme (1 nM) was incubated with ULight-MBPsubstrate (50 nM) in Kinase Assay Buffer supplemented with 4 µM ATP (final concentrations in the kinase reaction). Reactions were terminated after 60 min by the addition of 10 mM EDTA.ULight-MB peptide phosphorylation was detected by the addition of 2 nM Eu-anti-phospho-MBPand measured after the indicated incubation times at RT on the EnVision Multilabel Reader.

affected by DMSO concentrationsup to 10% (data not shown). Thisresult rather indicates that DMSOcauses a reduction in the ERK1enzyme activity. Since HTS assaysmight require the addition of DMSOat concentrations up to 2%, wedecided to use 2% DMSO for thevalidation of the ERK1 assay in thepresence of staurosporine.

Staurosporine inhibitionStaurosporine is a broad spectrumkinase inhibitor that interferes withATP binding. To evaluate stau-rosporine inhibition of the LANCEUltra ERK1 assay, the activity of theenzyme was measured in thepresence of increasing concentra-tions of staurosporine (Figure 8). An IC50 value of 2.8 µM wasobtained, consistent with valuesreported in the literature usingother technologies1 and showingthat ERK1 is relatively resistant tostaurosporine inhibition.

Assay robustness and performanceThe robustness and performance ofthe LANCE Ultra ERK1 kinase assaywas evaluated by manually con-ducting a Z' analysis2 in 384-wellformat. Two series of 48 replicateswere analyzed: in the absence (totalsignal) or presence (minimal signal)of 30 µM staurosporine (Figure 9).DMSO was added to all reactions to a final concentration of 2% tosimulate screening conditions.Percent coefficient of variation(%CV), S/B ratio, and Z'-factor werecalculated. A %CV of 3.2 and anS/B ratio of 4 were obtained for thetotal signal. These values, combinedwith a calculated Z'-factor of 0.83,clearly demonstrate the robustnessof the optimized ERK1 LANCEUltra assay and its suitability forHTS applications.

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Figure 7. DMSO Tolerance of the LANCE Ultra ERK1 assay. ERK1 enzyme (1 nM) was incubatedwith ULight-MBP substrate (50 nM) in Kinase Assay Buffer supplemented with 4 µM ATP andincreasing DMSO concentrations. Reactions were terminated after 60 min by the addition of 10mM EDTA. ULight-MBP peptide phosphorylation was detected by the addition of 2 nM Eu-anti-phospho-MBP and measured after 60 min incubation at RT on the EnVision Multilabel Reader.

Figure 8. Staurosporine inhibition. ERK1 enzyme (1 nM) was incubated with ULight-MBPsubstrate (50 nM) in the presence of serial dilutions of staurosporine (1 nM to 30 µM) in KinaseAssay Buffer supplemented with 4 µM ATP, in the presence of 2% DMSO (final concentrations in the kinase reaction). Reactions were terminated after 60 min by the addition of 10 mM EDTA.ULight-MBP peptide phosphorylation was detected by the addition of 2 nM Eu-anti-phospho-MBP and measured after 60 min incubation at RT on the EnVision Multilabel Reader.

Figure 9. Z' analysis for a 384-well manual ERK1 assay. ERK1 enzyme (1 nM) was incubated with ULight-MBP substrate (50 nM) in Kinase Assay Buffer supplemented with 4 µM ATP, in the absence (total signal) or presence of 30 µM staurosporine (final concentration in the kinase reaction). Reactions were terminated after 60 min by the addition of 10 mM EDTA.Phosphorylation was detected by the addition of 2 nM Eu-anti-phospho-MBP and measured after 60 min incubation at RT on the EnVision Multilabel Reader.

Total Signal Staurosporine-Inhibited Total/StaurosporineSignal

Average %CV Average %CV S/B Z'

52,566 3.2 13,189 4.4 4.0 0.83

Conclusion

The new LANCE Ultra reagents,ULight-MBP and Eu-anti-phospho-MBP were used for the developmentof a sensitive and robust ERK1kinase assay that allowed:

• Minimizing enzyme consumptionwhile maintaining a high S/Bratio by careful optimization ofthe concentration of each assay component.

• Working at a concentration nearthe apparent Km value for ATP (4 µM), which provides the mostsensitive screen for detecting ATP competitive inhibitors.

• Developing a robust assay,suitable for HTS applications, as demonstrated by (1) the assay’s signal stability over 6 h,(2) the accuracy of evaluatingstaurosporine potency and (3) a Z'-factor of 0.83.

References

1. Meggio, et al. (1995) Eur. J.Biochem. 234: 317–322.

2. Zhang, et al. (1999) J. Biomol.Screen. 4(2): 67–73.

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©2006 PerkinElmer, Inc. All rights reserved. The PerkinElmer logo and design are registered trademarks of PerkinElmer, Inc. LANCE is a registered trademark and DELFIA, EnVision,OptiPlate, TopSeal-A and ULight are trademarks of PerkinElmer, Inc. or its subsidiaries, in the United States and other countries. ULight is covered under patent numbers US20050202565and US20040166515. All other trademarks not owned by PerkinElmer, Inc. or its subsidiaries that are depicted herein are the property of their respective owners. PerkinElmer reserves theright to change this document at any time without notice and disclaims liability for editorial, pictorial or typographical errors.

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