Analytical Biochemistry 391 (2009) 31–38

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Analytical Biochemistry

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Evaluating the utility of the HTRF� TranscreenerTM ADP assay technology: Acomparison with the standard HTRF assay technology

Lin Hong, Christopher M. Quinn, Yong Jia *

Department of Kinase Biology/Oncology, Genomics Institute of the Novartis Research Foundation, San Diego, CA 92121, USA

a r t i c l e i n f o

Article history:Received 12 February 2009Available online 3 May 2009

Keywords:HTRFTranscreenerADPKinase assayATPaseInhibitor

0003-2697/$ - see front matter � 2009 Elsevier Inc. Adoi:10.1016/j.ab.2009.04.033

* Corresponding author. Fax: +1 858 812 1918.E-mail address: yjia@gnf.org (Y. Jia).

1 Abbreviations used: HTRF, homogeneous time-resopium; Tb, terbium; FRET, fluorescence resonance enepolarization; SA, streptavidin; SAXL, streptavidin-linkeput screening; RT, room temperature; BSA, bovtris-(2-carboxyethyl)phosphine; EDTA, ethylenediaminbackground; DMSO, dimethyl sulfoxide; ITK, interleuki

a b s t r a c t

The HTRF (homogeneous time-resolved fluorescence) Transcreener ADP assay is a new kinase assay tech-nology marketed by Cis-Bio International (Bagnols-Cèze, France). It measures kinase activity by detectingthe formation of ADP using a monoclonal antibody and HTRF detection principles. In this article, we com-pare this technology with a standard HTRF kinase assay using EGFR [L858R/T790M] mutant enzyme as acase study. We demonstrate that the HTRF Transcreener ADP assay generated similar kinetic constantsand inhibitor potency compared with the standard HTRF assay. However, the smaller dynamic windowand lower Z0 factor of the HTRF Transcreener ADP assay make this format less preferable for high-throughput screening. Based on the assay principle, the HTRF Transcreener ADP assay can detect bothkinase and ATPase activities simultaneously. The ability to probe ATPase activity opens up new avenuesfor assaying kinases with intrinsic ATPase activity without the need to identify substrates, and this canspeed up the drug discovery process. However, caution must be exercised because any contaminatingATPase activity will result in an invalid assay. The inability to tolerate high concentrations of ATP inthe assay will also limit the application of this technology, especially in compound mechanistic studiessuch as ATP competition. Overall, the HTRF Transcreener ADP assay provides a new alternative tool tocomplement existing assay technologies for drug discovery.

� 2009 Elsevier Inc. All rights reserved.

1

HTRF (homogeneous time-resolved fluorescence) is a pioneeringtechnology that measures biomolecular interactions. The systemuses a pair of chemically stable molecules termed as donor andacceptor. The donor is a lanthanide metal, usually europium (Eu)or terbium (Tb), encased in a cryptate molecule, whereas the accep-tor can be either a cross-linked derivative of a fluorescent allophyc-ocyanin (known as XL665) or a proprietary organic dye known as d2.When the two fluorophores are in close proximity, excitation of thedonor molecule results in a fluorescence resonance energy transfer(FRET), leading to final emission by the acceptor molecule at a dis-tinct wavelength. The spectral selectivity allows separation of donorfrom acceptor signal, whereas the fluorescence lifetime of the detec-tion complex provides the separation of free and bound acceptor sig-nals. The dual wavelength detection allows the correction of mediainterference. For these reasons, the HTRF technology is homoge-neous, sensitive, and less prone to compound interference. The prin-

ll rights reserved.

lved fluorescence; Eu, euro-rgy transfer; FP, fluorescenced XL665; HTS, high-through-ine serum albumin; TCEP,etetraacetic acid; S/B, signal/n-2 inducible T-cell kinase.

ciples and applications of the HTRF technology have been reviewedin the literature [1–6].

During recent years, kinases have emerged as one of the largestclasses of drug targets actively pursued by pharmaceutical compa-nies. This family of enzymes is involved in the regulation of manyaspects of the cell cycle, including proliferation, differentiation,secretion, and apoptosis [7,8]. The malfunction of kinases has beenimplicated in a wide variety of diseases such as cancer, inflamma-tory diseases, diabetes, atherosclerosis, and immunological disor-ders [9–12]. Consequently, the modulation of kinase activityoffers potential therapeutic value for the treatment of a wide rangeof diseases [13–15]. To meet the needs of the drug discovery com-munity, many technologies, including HTRF, have quickly foundapplications in kinase assays [16–21].

One of these technologies is the Transcreener ADP assay devel-oped by Bellbrook Labs (Madison, WI, USA). This technology de-tects the kinase reaction by measuring the formation of ADP by afluorescence polarization (FP) method [22]. It uses a monoclonalanti-ADP antibody and fluorescently labeled ADP as a tracer. Whenthe tracer binds to the anti-ADP antibody, high FP signal is gener-ated. As a kinase reaction progresses, the resulting ADP can com-pete with the tracer for binding to the anti-ADP antibody,causing a decrease in FP. This technology has been successfully ap-plied to drug discovery [23,24]. A recent collaboration betweenBellbrook Labs and Cis-Bio International (Bagnols-Cèze, France)

32 HTRF Transcreener ADP assay technology / L. Hong et al. / Anal. Biochem. 391 (2009) 31–38

led to the adaptation of the ADP Transcreener technology to theHTRF format, where the anti-ADP antibody is labeled with the Eudonor and ADP is labeled with the d2 acceptor for use as the tracermolecule. When the tracer binds to the antibody, a FRET pair formsbetween the donor and acceptor, leading to high HTRF signal.Kinase-produced ADP competes with the tracer for binding to theantibody, resulting in a loss of FRET signal (Fig. 1A). This is slightlydifferent from the standard HTRF kinase assay format, where aphospho-product is detected. In a typical standard HTRF assay,an Eu-labeled anti-phospho-antibody is used to capture the phos-phorylation site on the product, whereas the streptavidin (SA)-linked XL665 (SAXL) is used to bind to biotin (other tag/acceptorcombinations can also be used). The formation of the donor/accep-tor pair yields high HTRF signal (Fig. 1B).

Compared with the standard HTRF assay format, the HTRFTranscreener ADP assay has both advantages and drawbacks.The biggest advantage of the HTRF Transcreener ADP assay is thatit can be used as a generic assay for all kinases because the detec-tion system is based on the common reaction product ADP. TheADP-based detection should further allow any molecule—be itpeptide, protein, or lipid—to be used as a substrate. Assay devel-opment should be easy and straightforward because no customreagents are needed. This is particularly beneficial to the serine/threonine and lipid kinase families because identifying the phos-pho-specific molecules to detect the products of these reactions isoften difficult and sometimes impossible. However, because theanti-ADP antibody also has some affinity to ATP, the ATP concen-tration range that can be used in the assay is restricted and themillimolar concentrations of ATP that are believed to be physio-logically relevant are not well tolerated. Due to the competitive

Fig. 1. Principles of the HTRF Transcreener ADP and standard HTRF kinase assays.(A) HTRF Transcreener ADP kinase assay. In this assay format, the HTRF Eu donor islabeled on an anti-ADP antibody, whereas the acceptor d2 is labeled on ADP, whichserves as a tracer. In the absence of a kinase reaction, the tracer binds to theantibody, resulting in the formation of a FRET pair between the donor and acceptor,leading to high HTRF signal. Kinase-produced ADP competes with the tracer forbinding to the antibody, resulting in a loss of HTRF signal. (B) Standard HTRF kinaseassay. For a typical assay setup, the substrate is usually biotinylated, facilitatingbinding to the SAXL acceptor. The Eu donor is labeled on a phospho-specificantibody that binds to the phosphorylation site on the product. Formation of thedonor/acceptor complex results in HTRF signal. For both panels A and B, theexcitation of the donor molecule at 337 nm results in the FRET transfer to theacceptor molecule at 620 nm, leading to the emission of the acceptor molecule at665 nm. The ratio of 665/620 nm is recorded as HTRF signal.

nature of the HTRF Transcreener ADP assay detection system,the readout shows a nonlinear decrease in signal. As such, it isnecessary to generate standard curves for each ATP concentrationtested to accurately quantitate product formation, reaction kinet-ics, and ultimately compound IC50/Ki values. Generating thesestandard curves can be tedious and time-consuming, especiallywhen several ATP concentrations are being evaluated simulta-neously. In addition, the HTRF Transcreener ADP assay is verysensitive to ATPase contamination, and this may pose a frequentproblem because ATPases are often present in, and difficult to re-move from, purified kinases. The applications of standard HTRFkinase assays have been well documented and reviewed [25–38]. In contrast, the HTRF Transcreener ADP assay is relativelynew and awaits further evaluation.

In this article, we describe the assays and methods developed toevaluate the utility and limitations of the HTRF Transcreener ADPassay using a potential drug target, EGFR [L858R/T790M], as a casestudy. EGFR is a receptor tyrosine kinase and a well-validated drugtarget in the clinic. Currently, small molecule inhibitors such asTarceva (Genentech/OSI) and Iressa (AstraZeneca) are used to treatpatients with non-small-cell lung cancer harboring various EGFRmutations, including a major oncogenic mutation of L858R[39–41]. However, patients can develop resistance to these treat-ments by a secondary mutation of T790M [42,43]. Thus, theL858R/T790M double mutant presents a new target opportunityfor treating patients bearing this drug-resistant mutation. We havedeveloped both HTRF Transcreener ADP and standard HTRF assaysfor this kinase and evaluated the application of the HTRF Transcre-ener ADP assay for enzyme kinetic characterization and inhibitorhigh-throughput screening (HTS). In addition, we discuss the util-ity of this assay format for kinases with coupled intrinsic ATPaseactivity.

Materials and methods

Materials

An HTRF Transcreener ADP kit (containing Eu-labeled anti-ADPantibody and ADP-d2) and PT66K were purchased from Cis-BioInternational. SAXL was purchased from Prozyme. Biotin–Lck–pep-tide was synthesized by New England Peptide. EGFR [L858R/T790M] mutant was expressed and purified as described previ-ously [44]. Plates were read on an EnVision multilabel plate reader(PerkinElmer).

ADP/ATP standard curves

For each ATP concentration used in the kinase and ATPase reac-tions, an ADP/ATP standard curve was generated (according to thegeneral methods described in the HTRF Transcreener ADP kit) toconvert the HTRF signal to the amount of ADP formed. While main-taining the combined concentration of ADP/ATP at the same con-centration of ATP used in the reaction, the ratio between ADPand ATP was varied. To mimic the kinase and ATPase reactions,10 ll of ADP/ATP solution was mixed with 5 ll of detection buffer,followed by the addition of 5 ll of detection buffer containing ADP-d2 and Eu-labeled anti-ADP antibody. After incubation at roomtemperature (RT) for 1 h, the plate was read in the EnVision platereader and a standard curve was generated using log [ADP] asthe x axis and HTRF signal as the y axis.

EGFR (L858R/T790M) mutant HTRF Transcreener ADP assays

EGFR [L858R/T790M] mutant HTRF Transcreener ADP assayswere carried out using biotin–Lck–peptide as substrate. The assay

HTRF Transcreener ADP assay technology / L. Hong et al. / Anal. Biochem. 391 (2009) 31–38 33

mixture contained 9.6 nM EGFR [L858R/T790M] mutant (MW =36.4 kDa), 1 lM biotin–Lck–peptide, and 10 lM ATP in a buffercontaining 50 mM Hepes (pH 7.1), 10 mM MgCl2, 2 mM MnCl2,0.01% bovine serum albumin (BSA), 1 mM tris-(2-carboxy-ethyl)phosphine (TCEP), and 0.1 mM Na3VO4 at a final volume of10 ll. The reactions were carried out at RT in white ProxiPlate384-Plus plates (PerkinElmer). At designated time points, the reac-tions were quenched by adding 5 ll of the detection buffer (con-taining 60 mM ethylenediaminetetraacetic acid [EDTA]) includedwith the HTRF Transcreener ADP kit, and the reaction productswere then detected by adding ADP-d2 and anti-ADP cryptateaccording to the instructions provided. The plates were incubatedat RT for 1 h and then read on an EnVision plate reader. The initialreadouts were converted to the amount of ADP formed usingGraphPad Prism software to interpolate these values from theADP/ATP standard curves.

For the ATP Km determination, the substrate concentration wasfixed at 1 lM, whereas the ATP concentration was varied from 50to 0.1 lM. For the substrate Km determination, the ATP concentra-tion was fixed at 10 lM, whereas the peptide substrate concentra-tion was varied from 500 to 0.5 lM. The linear portion of eachcurve was used to calculate the initial velocity.

EGFR [L858R/T790M] mutant standard HTRF assay

The enzymatic reactions of EGFR [L858R/T790M] mutant stan-dard HTRF assays were carried out under the same conditions asdescribed for those of EGFR [L858R/T790M] mutant HTRF Trans-creener ADP assays except that the enzyme concentration usedwas 0.82 nM. At designated time points, 5 ll of EDTA (0.2 M) wasadded to quench the reaction and the detection reagents (5 ll)were added at final concentrations of 3.4 ng/well PT66K and50 ng/well SAXL. The plates were incubated at RT for 1 h and thenread in the EnVision plate reader.

For the ATP Km determination, the substrate concentration wasfixed at 1 lM, whereas the ATP concentration was varied from 100to 0.1 lM. For the substrate Km determination, the ATP concentra-tion was fixed at 10 lM, whereas the peptide substrate concentra-tion was varied from 400 to 1 lM. Each reaction product was thendiluted to a final substrate/product concentration of 1 lM fordetection using a general method described previously [34]. TheHTRF signal was corrected for the dilution factor where applicable,and the linear portion of the curve was used to calculate the initialvelocity.

Fig. 2. Time courses of the EGFR [L858R/T790M] mutant catalyzed reactions. (A) HTRF sigpeptide reaction measured by the HTRF Transcreener ADP assay. An ADP/ATP standard cubiotin–Lck–peptide reactions measured by the HTRF Transcreener ADP assay (�) and theATPase reaction (e). The y axis on the left represents the HTRF signal measured by theconcentration measured by the HTRF Transcreener ADP assay (�) and the ATPase assay (ewas interpolated using the experimental HTRF signal readout based on the standard ADPMaterials and Methods.

EGFR [L858R/T790M] mutant ATPase assays

Both the reaction and detection steps of EGFR [L858R/T790M]mutant ATPase assays were carried out under similar conditionsas those of EGFR [L858R/T790M] mutant HTRF Transcreener ADPassays except that the peptide substrate was not present in thereaction. The initial readouts were converted to the amount ofADP formed using GraphPad Prism software to interpolate thesevalues from the ADP/ATP standard curves.

For the ATP Km determination, the ATP concentration was var-ied from 100 to 0.1 lM. The linear portion of the curve was usedto calculate the initial velocity.

Z0 factor, apparent Km, and compound IC50 determinations

The Z0 factor was determined by using the positive and negativecontrols of an enzymatic reaction as described previously [45]. Theapparent ATP Km and substrate Km values were determined accord-ing to the methods described previously [33]. The compound IC50

values were determined from 12-point inhibition curves coveringa concentration range from 50 to 0.000283 lM according to themethods described previously [33].

Results and discussion

Assay development

The HTRF Transcreener ADP assay for EGFR [L858R/T790M] mu-tant was developed at 10 lM ATP and 1 lM biotin–Lck–peptide,and the detection was performed according to the instructionsfrom the HTRF Transcreener ADP kit. A standard curve was gener-ated (Fig. 2A, inset) to convert the HTRF signal (Fig. 2A) to theamount of ADP formed so as to show the kinetics of the timecourse (Fig. 2B, �). The signal/background (S/B) ratio for this assayis approximately 3-fold (Fig. 2A). One major drawback of the HTRFTranscreener ADP assay is that the detection range is rather nar-row, and is defined by each ADP/ATP standard curve. If the HTRFsignal reading is above or below the dynamic range defined bythe standard curve, it cannot be accurately converted to theamount of ADP formed using that standard curve. In comparison,a standard HTRF assay was also developed for EGFR [L858R/T790M] mutant at the same ATP and substrate concentrations withan S/B ratio of approximately 14-fold (Fig. 2B, �). The higher dy-namic window of the standard HTRF assay, together with the fact

nal versus time generated from EGFR [L858R/T790M] mutant catalyzed biotin–Lck–rve is shown in the inset. (B) Time courses of EGFR [L858R/T790M] mutant catalyzedstandard HTRF kinase assay (�) and time course of the EGFR [L858R/T790M] mutantstandard HTRF kinase assay (�), whereas the y axis on the right represents the ADP). The amount of ADP formed for both the HTRF Transcreener ADP and ATPase assays/ATP curve shown in the inset in panel A. The assays were conducted as described in

34 HTRF Transcreener ADP assay technology / L. Hong et al. / Anal. Biochem. 391 (2009) 31–38

that the amount of enzyme used in the standard HTRF assay is lessthan 10% of the amount used in the HTRF Transcreener ADP assay,indicates that the standard HTRF assay is much more robust thanthe HTRF Transcreener ADP assay. The signal-increasing nature ofthe standard HTRF assay makes data analysis easy and straightfor-ward because HTRF signal can be plotted directly to analyze thetime course and standard curves are not needed for further datamanipulation.

DMSO effect

We investigated the effect of dimethyl sulfoxide (DMSO) onboth the HTRF Transcreener ADP assay and the standard HTRF as-say, and the results are shown in Fig. S1 in the supplementarymaterial. Both assays can tolerate DMSO concentrations up to 2%,but at 5% DMSO the signal window was reduced in both cases.These data indicate that both assay formats are suitable for HTSapplications because most screening assays contain 62% DMSO.In all studies described in this article, the DMSO concentrationused was 60.5%.

Z0 factor determination

The Z0 factor is a standard parameter for evaluating the robust-ness of an assay and its suitability for HTS [45]. In theory, the Z0 fac-tor can be any value between 0 and 1, with a higher Z0 factorindicating a more robust assay. We determined the Z0 factor ofthe EGFR [L858R/T790M] reaction in both the HTRF TranscreenerADP assay and standard HTRF assay formats according to themethod described by Zhang and coworkers [45]. The Z0 factor forthe HTRF Transcreener ADP assay turned out to be 0.54 (Fig. 3A),which is much lower than the 0.84 value for the standard HTRF as-say (Fig. 3B). This is not surprising because a larger dynamic win-dow and lower variability in an assay contribute directly to ahigher Z0 factor. An assay with a Z0 factor of P0.5 is consideredas suitable for HTS. Thus, both the EGFR [L858R/T790M] HTRFTranscreener ADP and standard HTRF assays are robust enoughto be used for HTS applications, with the standard HTRF assaybeing preferred due to the higher Z0 factor.

Steady-state enzyme kinetic constant characterization

HTRF is a fluorescence-based technology with a complicateddetection scheme, making the measurement of the kinetic con-stants of the enzyme less straightforward. In our previous publica-tion, we did a detailed analysis of the HTRF reaction and detectionsystems and described methods to determine enzyme kinetic con-stants such as ATP Km, substrate Km, and kcat for standard HTRF

Fig. 3. Z0 factor determination for EGFR [L858R/T790M] mutant catalyzed biotin–Lck–peHTRF kinase assay (B). The Z0 factor was obtained according to the published methods [45open symbols represent negative controls. The Z0 factor for the HTRF Transcreener ADP asdetermined to be 0.84.

kinase assays [34]. The methods for the HTRF Transcreener ADPassay share many similarities with those of a standard HTRF assay,yet there are obvious differences. In the standard HTRF assay, theATP Km determination is rather straightforward because the con-centration of the detectable substrate is fixed, whereas the ATPconcentration varies. The initial velocity from each reaction canbe compared directly, and the ATP Km can be obtained by fittingthe data to the Michaelis–Menten equation. On the other hand,the substrate Km determination is more challenging because theexperiment requires that the concentration of the detectable sub-strate be changed, so one detection condition does not apply toall reaction conditions. The approach we took to circumvent thisproblem was to adjust the final reaction product to the same sub-strate concentration prior to detection. The initial rate of each reac-tion is corrected by the dilution factor and then fitted to theMichaelis–Menten equation to obtain the substrate Km [34]. How-ever, for the HTRF Transcreener ADP assay, the situation is a bitdifferent.

In the HTRF Transcreener ADP assay, determining the substrateKm is more straightforward because the varying substrate (from500 to 0.5 lM) is not the detectable product. The assay was carriedout with the ATP concentration fixed at 10 lM, so only one ADP/ATP standard curve needed to be generated. The initial rate of eachreaction was determined from the experimental data using theADP/ATP standard curve and plotted versus substrate concentra-tion to obtain the apparent substrate Km (Fig. 4A). In contrast,determining the ATP Km in this assay format is more tedious andchallenging. Because the ATP concentration varies throughout theexperiment (from 50 to 0.1 lM), multiple ADP/ATP standard curvesneed to be generated for each ATP concentration tested so as toconvert the HTRF signal readout to the amount of ADP formed.Each reaction rate was then plotted versus ATP concentration toobtain the apparent ATP Km (Fig. 4B). One inherent problem ofthe HTRF Transcreener ADP assay is its low tolerance to high con-centrations of ATP due to the anti-ADP antibody also having someaffinity toward ATP. High concentrations of ATP can reduce the dy-namic window of the standard curve dramatically, leading to alowered Z0 factor and a loss of confidence in the assay. The conse-quence is that for enzymes with a high ATP Km, it will be challeng-ing to determine this kinetic parameter using the HTRFTranscreener ADP assay. This is very different from the standardHTRF assay, which can tolerate a wide range of ATP concentrationswithout an upper limit.

To validate the kinetic constants obtained by the HTRF Trans-creener ADP assay, we also carried out the same experiments usingthe standard HTRF assay. The apparent ATP Km and substrate Km

were determined under the same conditions (see Figs. S2A andS2B in supplementary material) and according to the methods

ptide reactions measured by the HTRF Transcreener ADP assay (A) and the standard]. In both panels A and B, the solid symbols represent positive controls, whereas the

say was determined to be 0.54, whereas that for the standard HTRF kinase assay was

Fig. 4. Apparent ATP Km and substrate Km of EGFR [L858R/T790M] mutant catalyzed biotin–Lck–peptide reaction measured by the HTRF Transcreener ADP assay. (A)Substrate (biotin–Lck–peptide) Km. Assays were carried out at a fixed ATP concentration (10 lM) while varying the substrate peptide concentration from 500 to 0.5 lM. TheHTRF signal readout was converted to the amount of ADP formed using the ADP/ATP standard curve and then plotted versus reaction time, as shown in the inset. The initialrates of each reaction were determined for the different substrate concentrations indicated in the legend. The initial velocities were then plotted versus the substrateconcentrations, and the biotin–Lck–peptide Km was obtained by curve fitting based on the Michaelis–Menten equation. (B) ATP Km. Assays were carried out at a fixed biotin–Lck–peptide concentration (1 lM) while varying the ATP concentration from 50 to 0.1 lM. The HTRF signal readout was converted to the amount of ADP formed using theADP/ATP standard curve and then plotted versus reaction time, as shown in the inset. The initial rates of each reaction were determined for the different ATP concentrationsindicated in the legend. The initial velocities were then plotted versus the ATP concentrations, and the ATP Km was obtained by curve fitting based on the Michaelis–Mentenequation.

HTRF Transcreener ADP assay technology / L. Hong et al. / Anal. Biochem. 391 (2009) 31–38 35

described previously [34]. The kinetic constants obtained fromboth assay formats are comparable (Table 1), validating the HTRFTranscreener ADP assay format.

In terms of determining kinetic parameters, the HTRF Transcre-ener ADP and standard HTRF assays exhibit different characteris-tics. With the standard HTRF assay, it is easy to determine theATP Km but more complicated to obtain the substrate Km. Espe-cially in cases where the substrate Km is high, the approach we de-scribed previously might not be technically feasible [34]. Incontrast, it is straightforward to measure the substrate Km withthe HTRF Transcreener ADP assay but is more tedious and difficultto obtain the ATP Km. In situations where the ATP Km is high, thelimitation of this technology in assaying ATP concentrations inthe millimolar range will create a challenge for determining thiskinetic constant. These two HTRF technologies do complement eachother nicely, presenting researchers with more tools and opportu-nities to achieve their goals. For example, in some instances thedifferent technologies may be used to determine different kineticconstants based on which assay poses less technical challenges(i.e., using standard HTRF to determine the ATP Km while usingHTRF Transcreener ADP to determine the substrate Km).

Compound inhibition

The ultimate goal of assay development in drug discovery is tocharacterize compound inhibition of the target. We selected agroup of 26 compounds that are ATP competitive and have varyingpotencies on the EGFR [L858R/T790M] enzyme, and we testedthem in both the HTRF Transcreener ADP and standard HTRF as-says. The experiments were carried out under the same ATP andsubstrate concentrations so as to compare the results. IC50 valuesobtained from both experiments are graphed and compared inFig. 5A. IC50 values from the HTRF Transcreener ADP assay are plot-ted on the x axis, whereas those from the standard HTRF assay areplotted on the y axis. Each symbol represents one compound. As

Table 1Apparent ATP Km and substrate Km of EGFR [L858R/T790M] catalyzed biotin–Lck–peptide reaction determined by the HTRF Transcreener ADP and standard HTRF assayformats.

Assay format Apparent ATP Km (lM) Apparent substrate Km (lM)

HTRF Transcreener ADP 4.4 ± 0.2 54.0 ± 5.5Standard HTRF 2.2 ± 0.1 47.7 ± 4.1

shown in Fig. 5A, we observed a good general correlation betweenthe HTRF Transcreener ADP and standard HTRF assays, further val-idating the HTRF Transcreener ADP assay method. However, high-potency compounds (IC50 < 30 nM) appeared to be more active inthe standard HTRF assay. This observation indicates greater sensi-tivity in the standard HTRF assay, probably due to a larger dynamicrange and a much lower amount of enzyme in the assay.

Kinase-coupled intrinsic ATPase activity

When we looked carefully at the time course of EGFR [L858R/T790M] in the HTRF Transcreener ADP assay (Fig. 2B, �), we no-ticed that the amount of ADP formed (�2.5 lM ADP at 60 min) isgreater than the amount of peptide substrate used in the reaction(1 lM). This was initially puzzling because the maximal ADP for-mation should be 1 lM at 100% substrate turnover, so what couldlead to the extra ADP formed in our assay? Because it is known thatmany purified kinases have either intrinsic or contaminating ATP-ase activity [24,46–49], we speculated that this additional ADP wasproduced by an ATPase. We then ran our enzyme assay in the ab-sence of peptide substrate, and indeed a clear ADP formation andcatalytic turnover were observed (�2 lM ADP at 60 min [Fig. 2B,e]), confirming the presence of ATPase activity in our enzymepreparation.

A key question is whether this ATPase activity is intrinsic or dueto contamination. In other words, is the assay detecting the activityof our target or the activity of some other contaminating ATPase?EGFR [L858R/T790M] was purified through multiple columns witha final purity P99%, making contaminating ATPase activity veryunlikely, so we hypothesized that the ATPase activity is intrinsicto the enzyme. One approach to test this hypothesis is to deter-mine the potency of some ATP competitive inhibitors in both thekinase and ATPase assays. For kinases displaying intrinsic ATPaseactivity, the kinase and ATPase activity is generally propagated atthe same binding site for ATP. Therefore, if the ATPase activity isintrinsic for the EGFR [L858R/T790M] mutant, the IC50 for theATP competitive inhibitors should agree in two assays [24,46–48]. If the IC50 agreement is poor, the ATPase activity is likelydue to a contaminant. We carried out this experiment with thesame group of compounds in the ATPase assay and compared theIC50 values with those obtained from the HTRF Transcreener ADPand standard HTRF assays. As shown in Figs. 5B and C, IC50 valuesgenerated by all three assays showed excellent correlation, con-firming our hypothesis that that EGFR [L858R/T790M] mutant

Fig. 5. IC50 correlations of the EGFR [L858R/T790M] HTRF Transcreener ADP assay with the standard HTRF assay (A), the HTRF Transcreener ADP assay with the ATPase assay(B), and the standard HTRF assay with the ATPase assay (C). Each symbol represents one compound. The solid line represents a perfect agreement where the IC50 values fromthe two assays are identical, and the broken lines represent a ±5-fold variation. The closer the symbol is to the solid line, the better the correlation between the two IC50

values.

36 HTRF Transcreener ADP assay technology / L. Hong et al. / Anal. Biochem. 391 (2009) 31–38

has both kinase and ATPase activity and that the ATPase activity isintrinsic and coupled to that of kinase.

We then determined the ATP Km of this ATPase reaction, obtain-ing a value of 13.7 lM (see Fig. S3 in supplementary material),which is higher than the apparent ATP Km of 4.4 lM from the HTRFTranscreener ADP assay. The lower apparent ATP Km of the HTRFTranscreener ADP assay can be explained by the mechanism ofsubstrate-stimulated ATPase activity that has been reported inthe literature [47]. In the case of EGFR [L858R/T790M], this ATPasestimulation by peptide substrate is at least partially due to the Km

effect.It has been shown that a fair amount of kinases possesses

intrinsic ATPase activity [24,46–49], and this feature can actuallyprovide added benefits for researchers in drug discovery. Kinasesare engaged in complicated signaling networks that are not wellunderstood, and the substrates for many kinases are unknown,and this often poses a problem for assay development. If a kinasehas intrinsic ATPase activity coupled to its kinase activity, it wouldenable researchers to develop enzyme assays using the ATPaseactivity without the need to find the substrate for the kinase, andthis in turn would allow HTS and further drug discovery activitiesto progress. In fact, a publication by Kashem and coworkers de-scribed the HTS of interleukin-2 inducible T-cell kinase (ITK) bythree mechanistically different assay technologies: two that mea-sured the kinase activity of the enzyme and one that measuredthe intrinsic ATPase activity [49]. Their results showed that a gen-erally good correlation was obtained with these three assay for-mats, with the ATPase activity assay identifying the mostcomprehensive hit set, the largest number of unique hits, and thefewest unique misses. The authors further argued that measuringintrinsic ATPase activity was a convenient, reliable, and inexpen-sive way to identify inhibitors of certain kinases by HTS [49]. TheHTRF Transcreener ADP assay can be advantageous over the stan-dard HTRF assay in that it enables fast assay development for ATP-

ase activity without the need to identify kinase substrates. In thecase where multiple HTSs are needed for a target, using mechanis-tically distinct assay formats, including both kinase and ATPaseactivity assays, can provide more comprehensive and unique hitsets with fewer false negatives, thereby increasing the chances ofsuccess of a drug discovery program. However, intrinsic ATPaseactivity assay cannot be used as a universal screening format be-cause not all kinases have this activity.

Conclusions

The HTRF Transcreener ADP assay is a relatively new technologyon the market that shares the FRET detection principle of HTRFtechnology but measures the accumulation of ADP instead of aphospho-product. Compared with the standard HTRF assay, onemajor advantage of the HTRF Transcreener ADP assay is its genericformat, with no requirement for phospho-specific antibodies. Thisis particularly beneficial for some serine/threonine and lipid ki-nases because identifying phospho-specific antibodies for theseclasses of kinases is not trivial and sometimes even proves to beimpossible. Like the standard HTRF assay, the HTRF TranscreenerADP assay is a fluorescence-based technology. Because the fluores-cence signal is strictly proportional to the fluorophore concentra-tion, the reaction conditions are scalable for both larger andsmaller reaction volumes. Based on the detection principle, dNTPs,dNDPs, dNMPs, and other phosphate-containing reagents mayinterfere with the HTRF Transcreener ADP assay due to the poten-tial for cross-reactivity with the anti-ADP antibody.

The HTRF Transcreener ADP assay is less robust than the stan-dard HTRF assay, with a smaller dynamic window and a lower Z0

factor, making it a less optimal choice for HTS. The fact that thestandard HTRF assay uses approximately 12-fold less enzyme com-pared with the Transcreener ADP assay not only makes the stan-

HTRF Transcreener ADP assay technology / L. Hong et al. / Anal. Biochem. 391 (2009) 31–38 37

dard HTRF assay more robust but also can have a practical impacton the cost and feasibility of an HTS campaign in the case of ki-nases that are difficult to express. In addition, Figs. 5A and C showthat the lower sensitivity of the Transcreener ADP assay does notallow differentiation of highly active inhibitors. The HTRF Trans-creener ADP assay is also particularly prone to the activity of con-taminating ATPases that are commonly present in purified kinases.Caution must be exercised when using this technology to avoiddeveloping an invalid assay.

For kinases with intrinsic ATPase activity, there are two reac-tions progressing simultaneously. Because the phospho-productis unique to the kinase reaction, the standard HTRF assay, whichdetects only phospho-product formation, specifically measures ki-nase activity regardless of the presence of ATPase activity. How-ever, because ADP is the common product of both kinase andATPase reactions, the HTRF Transcreener ADP assay, which detectsthe formation of ADP, simultaneously measures both kinase andATPase activities. Data analysis and deconvolution for the situa-tions involving multiple reactions can be difficult due to the com-plicated kinetics. On the other hand, the ability to measureintrinsic ATPase activity by the HTRF Transcreener ADP assayopens up new avenues to researchers, allowing faster assay devel-opment and the possibility of running an HTS without identifyingkinase substrates. This is particularly valuable for kinases withoutknown substrates. In addition, assaying the intrinsic ATPase activ-ity of a kinase provides more drug discovery tools and can be syn-ergistic with kinase activity assays. Our studies have demonstratedthat the HTRF Transcreener ADP assay is a valid assay technologythat yields similar enzyme kinetic constants and inhibitor poten-cies compared with the standard HTRF assay. However, the restric-tions in testing high ATP concentrations in this assay will certainlylimit the applications of this technology, especially for compoundmechanistic studies such as ATP competition. Nonetheless, theHTRF Transcreener ADP assay technology provides a new alterna-tive tool for researchers and can be complementary to, and syner-gistic with, other assay technologies in kinase drug discovery.

Acknowledgment

The authors thank C.-H. Yun and M. Eck (Dana Farber CancerInstitute) for providing the purified EGFR [L858R/T790M] enzyme.

Appendix A. Supplementary data

Supplementary data associated with this article can be found, inthe online version, at doi:10.1016/j.ab.2009.04.033.

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