ASSAY and Drug Development TechnologiesVolume 1, Number 1-1, 2002© Mary Ann Liebert, Inc.

A FRET-Based Assay Platform for Ultra-High Density Drug Screening of Protein Kinases and Phosphatases

Steven M. Rodems*, Brian D. Hamman, Christina Lin, Jane Zhao*, Sundeep Shah, David Heidary, Lew Makings, Jeffrey H. Stack, and Brian A. Pollok*

Abstract: Protein phosphorylation is one of the major regulatory mechanisms involved in signal-induced cellular events, including cell proliferation, apoptosis, and metabolism. Because many facetsof biology are regulated by protein phosphorylation, aberrant kinase and/or phosphatase activityforms the basis for many different types of pathology. The disease relevance of protein kinases andphosphatases has led many pharmaceutical and biotechnology companies to expend significantresources in lead discovery programs for these two target classes. The existence of .500 kinases andphosphatases encoded by the human genome necessitates development of methodologies for the rapidscreening for novel and specific compound inhibitors. We describe here a fluorescence-based,molecular assay platform that is compatible with robotic, ultra-high throughput screening systemsand can be applied to virtually all tyrosine and serine/threonine protein kinases and phosphatases.The assay has a coupled-enzyme format, utilizing the differential protease sensitivity ofphosphorylated versus nonphosphorylated peptide substrates. In addition to screening individualkinases, the assay can be formatted such that kinase pathways are re-created in vitro to identifycompounds that specifically interact with inactive kinases. Miniaturization of this assay format to the1-ml scale allows for the rapid and accurate compound screening of a host of kinase and phosphatasetargets, thereby facilitating the hunt for new leads for these target classes.

9

Introduction

PROTEIN PHOSPHORYLATION has long been recognizedas one of the major regulatory mechanisms control-

ling processes such as cell growth, differentiation, andsurvival.1 Aberrant or unregulated protein phosphoryla-tion leads to uncontrolled cell growth and inhibition ofprogrammed cell death, both key determinants of onco-genesis.2–4 Protein kinases and phosphatases representgood drug targets and are a focus of small-molecule drugdiscovery efforts at almost every pharmaceutical com-pany. The most common methods for screening largechemical libraries against kinases and phosphatases in-clude traditional radiometric assays, ELISA, time-re-solved fluorescence techniques, and steady-state fluores-cent polarization methods.5–10 Although radiometricmethods can be applied to almost any kinase or phos-phatase target, radioisotopes are costly and inconvenient

to handle. Time-resolved fluorescence, ELISA, and flu-orescence polarization require specific antibody-baseddetection reagents. While generic phosphotyrosine anti-bodies are readily available, generating antibodies foreach Ser/Thr kinase substrate often requires lengthy andcostly development efforts. Currently, there is no singleassay platform that is easily and cost-effectively appliedto all classes of protein kinases and phosphatases.

With combinatorial compound libraries growing insize, genomics efforts identifying new kinase and phos-phatase drug targets, and the need to conserve preciouscompound supplies, assay miniaturization to support drugdiscovery is imperative. Addition-only, fluorescence-based assays are ideal for high-throughput, miniaturizedscreening systems due to sensitivity and lack of aspira-tion or wash steps. In an attempt to address the demandto rapidly screen the .500 potential new kinase and phos-phatase drug targets, we sought to develop a platform

Aurora Biosciences Corp., and Vertex Pharmaceuticals, Inc., San Diego, CA.*Current address: Ansata Therapeutics, La Jolla, CA.

technology that is both compatible with ultra high-throughput drug screening and applicable to all classesof protein kinases and phosphatases.

Fluorescence resonance energy transfer (FRET) is theradiationless transfer of energy from an excited donor flu-orophore to an acceptor fluorophore. Assays using aFRET-based readout take advantage of distance separa-tion of the two fluors, as FRET is a function of the sixthpower of distance. FRET-peptide assays with a donorfluor (e.g., coumarin) on one end of the peptide and apaired acceptor (e.g., fluorescein) on the other end are asensitive monitor of protease activity due to the infinitedistance separation afforded by internal protease cleav-age.11,12 As both donor and acceptor emission wavelengthscan be measured before and after protease cleavage, theemission ratio serves as a sensitive and facile way to mea-sure protease activity. FRET ratio changes of .25-fold arecommon when using coumarin and fluorescein fluors, withthe ratiometric readout (e.g., coumarin emission dividedby fluorescein emission) correcting for the sample-to-sam-ple variations of fluorescent components.

Phosphorylation events on or near an amino acid thatis recognized by a site-specific protease have been shownto affect substrate cleavage.13–16 Phosphorylation of a ty-rosine residue greatly reduced the ability of chymotryspinto cleave at that tyrosine.13 In addition, phosphorylationof a serine residue immediately adjacent to the scissilebond reduced cleavage of a DEVD-containing substrateby caspase-3.16 We describe here a platform assay tech-nology that takes advantage of these observations. By us-ing FRET-peptides containing phosphorylation sites withoverlapping protease cleavage sites, we have developeda miniaturizable, fluorescence-based kinase assay that isapplicable to both tyrosine and serine/threonine kinasesand phosphatases.a The assay can also be formatted to re-create kinase signaling pathways in vitro, which may al-low for the identification of novel classes of inhibitorcompounds (e.g., not ATP-competitive). Finally, usingmicrofluidic dispensers and automation, the assay hasbeen adapted to produce a throughput of .150,000 wellsper day. This throughput combined with an automateddose–response process has provided a significant increasein the efficiency with which kinase and phosphatase drugtargets are prosecuted.

Materials and Methods

Reagents

FRET-peptides were synthesized by either MultiplePeptide Systems, Inc. (San Diego, CA), or on site. Iden-

tity and purity were verified by mass spectrometry andreverse-phase HPLC. Bovine a-chymotrypsin and stau-rosporine were purchased from Sigma (St. Louis, MO).Kinases and phosphatases were expressed and purifiedin house or purchased from New England Biolabs (Bev-erly, MA) [cyclic AMP-dependent protein kinase (PKA),CK1, protein phosphatase 1 (PP1)], Upstate Biotechnol-ogy (Charlottesville, VA) (src, PTP-1B), or Calbiochem(San Diego, CA) (abl). H-89 was purchased from Bio-mol (Plymouth Meeting, PA). Constitutively active mitogen-activated protein kinase kinase 1 (MEK1)[MEK1(DD)], nonphosphorylated MEK1, and non-phosphorylated extracellular signal-regulated kinase 2(ERK2) were expressed in baculovirus-infected insectcells and purified by Ni-NTA-agarose (Qiagen, Valen-cia, CA) chromatography. Constitutively active MKK6[MKK6(DD)] and nonphosphorylated GST-p38a wereexpressed and purified by PanVera (Madison, WI). Peptidesequences were as follows: abl FRET-peptide, EAE-AIYAAPGDK; PKA FRET-peptide, ALRRFSLGEK;ERK2 FRET-peptide, VAPFSPGGRAK; CK1 FRET-peptide, GDQDYLSLDK.

Kinase assay conditions

Assay reactions were performed in 96-, 384-, or 3,456-well plates. The reaction was scalable such that a 3,456-well assay contained 1–2 ml per well, a 384-well assaycontained 20–30 ml per well, and a 96-well assay con-tained 50–100 ml per well. Enzyme and ATP concen-trations varied depending on the application. A typicalv-abl reaction contained 5 nM v-abl, 1 mM abl FRET-peptide, and 10 mM ATP in 0.1 3 phosphate-bufferedsaline (PBS) containing 5 mM MgCl2. After room tem-perature incubation, reactions were stopped by the ad-dition of EDTA (10 mM final concentration), and chy-motrypsin (100 nM final concentration) was added toinitiate peptide cleavage. A typical PKA reaction con-tained 2 nM PKA, 1 mM PKA FRET-peptide, and 10mM ATP in 13 PKA Buffer (NEB) supplemented with0.01% Brij-35. After room temperature incubation, re-actions were stopped by the addition of EDTA (3 mMfinal concentration), and chymotrypsin (15 nM final con-centration) was added to initiate peptide cleavage. Plateswere typically read on a Cytofluor 4000 (AppliedBiosystems, Foster City, CA) with excitation using a395-nm filter [full-width half-maximum (FWHM) of 25nm] and emission using a 460-nm filter (FWHM 5 40nm) and a 530-nm filter (FWHM 5 50 nm). The Ana-lyst (Molecular Devices, Sunnyvale, CA), Acquest (Mo-lecular Devices), and the topography-compensating platereader (tcPR) (Vertex Pharmaceuticals, Inc., San Diego,CA) were also used for reading plates. Exact enzyme re-action times varied based on the concentration of en-zymes and substrates used, but were typically 5–60 min.

Rodems et al.10

aCommercialized under the PhosphoryLIGHT™ productname, by Panvera LLC (www.panvera.com).

To obtain completely phosphorylated peptide samplesfor chymotrypsin titrations and phosphatase experi-ments, kinase reactions were performed at high enzyme,high ATP (100–200 mM), and high peptide concentra-tions (100 mM). For inhibitor experiments and screens,reactions were run at ATP concentrations equivalent tothe ATP KM.

In vitro kinase pathway reactions

The MEK1–ERK2 kinase pathway reactions contained0.5 ng of constitutively active MEK1 and 2.5 ng of in-active ERK2 in a 1-ml reaction containing 100 mM ATPand 1 mM ERK2 FRET-peptide in a buffer consisting of10 mM HEPES, pH 7.4, 10 mM MgCl2, 25 mM NaCl, 1mM dithiothreitol, and 0.01% Brij-35. The MKK6–p38kinase pathway reactions contained 11 ng of constitu-tively active MKK6 and 330 ng of inactive p38a in thebuffer conditions described above, except 300 mM ATPwas used in a 20-ml reaction volume.

3,456-well assays

The PKA screen in 3,456-well plates was performedin 1.5-ml (per well) kinase reaction volumes containing2 nM PKA (NEB), 1 mM PKA FRET-peptide, and 10mM ATP in 13 PKA Buffer (NEB) supplemented with0.01% Brij-35. Compounds (10 nl of a 2 mM stock in75% dimethyl sulfoxide) were preprinted into 3,456-wellplates (one compound per well) by using the piezo sam-ple distribution robot (Vertex Pharmaceuticals).17 Eachcompound was represented once in the screen. Com-pound plates were stored until use at 4°C in sealed bagsto minimize evaporation. Compounds were preincubatedin a 1-ml volume with kinase and buffer for 15 min atroom temperature before the addition of peptide and ATPin 0.5 ml. Assay reagents were dispensed with the flyingreagent dispenser (FRD; Vertex Pharmaceuticals). Thereaction was allowed to proceed for 60 min at room tem-perature during which time the plates were read on thetcPR (Vertex Pharmaceuticals) to determine possible in-terference from compound fluorescence. Chymotrypsin(15 nM final concentration), EDTA (3 mM final concen-tration), and Brij-35 (0.01% final concentration) wereadded in a 0.5-ml volume, and peptide cleavage pro-gressed for 45 min at room temperature before a finalreading on the tcPR.

For the HEK/CRE cell-based assay, HEK293 cellscontaining the b-lactamase reporter gene under controlof a promoter consisting of multimerized (43) cyclicAMP response element binding protein (CREB) responseelements were dispensed by the FRD (5,000 cells/well)into 3,456-well plates containing test compound. Cellswere stimulated with 1 mM forskolin (final concentra-tion) for 4 h, followed by incubation with the fluorescent

b-lactamase substrate, CCF4-AM (an improved form ofCCF2-AM18). After 1 h at room temperature, the plateswere read on the tcPR.

Phosphatase assays

FRET-peptides used in phosphatase assays were phos-phorylated before use as described. PTP-1B (agarose con-jugate; Upstate Biotechnology) reactions were performedin 0.13 PBS in microfuge tubes so that the agarose con-jugate could be removed prior to transfer of the reactionto 96-well plates. For inhibitor experiments, orthovana-date was incubated with PTP-1B for 10 min prior to ini-tiation of the reaction by addition of the prephosphory-lated abl FRET-peptide substrate. PP1 assays wereperformed in 50-ml reaction volumes containing 0.1 U ofPP1 (NEB), 13 PP1 Buffer (NEB), 1 mM MnCl2, andphosphorylated CK1 FRET-peptide (1 mM) for 60 minat room temperature. For inhibitor experiments, 100 nMmicrocystin-LR was preincubated with PP1 for 10 minat room temperature before the addition of peptide. Chy-motrypsin (500 ng) was added, and peptide cleavage oc-curred for 60 min before the plate was read.

Results

FRET-peptide kinase assay format

The FRET-based kinase assay platform described hereutilizes a differential rate of chymotrypsin cleavage of thepeptide substrate that is dependent on the phosphorylationstate of the peptide (Fig. 1A). An 11-amino acid peptide(abl FRET-peptide), containing the consensus phospho-rylation sequence for v-abl kinase,19 was synthesized withfluorescein on the N-terminus and coumarin on the C-ter-minus. The intact peptide exhibits FRET; excitation ofthe coumarin at 405 nm results in low-level emission at460 nm and high-level emission at 530 nm due to effi-cient energy transfer from the donor fluor (coumarin) tothe acceptor (fluorescein). When the peptide is cleavedby an endoprotease such as chymotrypsin, FRET is lostand excitation at 405 nm results in increased emission at460 nm and reduced emission at 530 nm. The 460 nm/530nm ratio is used to quantitate the FRET change andamount of peptide cleavage.

Phosphorylation of the abl FRET-peptide by v-abl re-sulted in greatly reduced cleavage efficiency of the pep-tide by chymotrypsin (Fig. 1B). This differential proteasesensitivity provided a window where a specific concen-tration of chymotrypsin could be chosen such that 100%of the nonphosphorylated peptide was cleaved, yet nearlyall of the phosphorylated peptide remained intact withinthe time frame of the assay.

Although the mechanism whereby phosphorylation in-

FRET-Based Assay for Ultra-High Throughput Screening 11

hibits chymotrypsin cleavage of the peptide is not wellestablished, it is likely that the phosphate on the tyrosineeither sterically or electrostatically hinders recognitionand/or positioning of the tyrosine in the active site ofchymotrypsin. We hypothesized that phosphorylation ofneighboring amino acids might also cause inhibition ofchymotrypsin cleavage. As there are no known proteasesthat specifically recognize either a serine or threonine,we tested whether a peptide with a phenylalanine (whichis also recognized by chymotrypsin) immediately adja-cent to a serine would be cleaved by chymotrypsin if theserine was phosphorylated. An 11-amino acid peptide

(PKA FRET-peptide), containing a consensus phospho-rylation sequence for PKA, was synthesized with fluo-rescein on the N-terminus and coumarin on the C-termi-nus. Phosphorylation of the PKA FRET-peptide by PKAresulted in reduced cleavage by chymotrypsin (Fig. 1C).Although the differential protease sensitivity of the PKAFRET-peptide (phosphorylated versus nonphosphory-lated) was less pronounced than for the abl FRET-pep-tide, a chymotrypsin concentration was readily deter-mined that could discriminate between phosphorylatedand nonphosphorylated peptides. These data indicatedthat an addition-only, high-throughput, FRET-peptide ki-

Rodems et al.12

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FIG. 1. The FRET-peptide kinase assay. (A) Tyrosine kinase FRET-peptides contain fluorescein (Fl) attached to the N-termi-nus and coumarin (Cou) conjugated to a C-terminal lysine via linkage to the «-NH2 side chain. The tyrosine (Y) serves as boththe phosphorylation site and the chymotrypsin recognition determinant. Chymotrypsin cleaves the amide bond C-terminal to thetyrosine in the nonphosphorylated peptide, but not the phosphorylated peptide. An intact peptide exhibits FRET, whereas noFRET is observed with a cleaved peptide. (B) Completely phosphorylated (open circles) and nonphosphorylated (filled circles)abl FRET-peptides were incubated with varying concentrations of chymotrypsin. Nonphosphorylated peptides are cleaved at lowerchymotrypsin concentrations as indicated by an increase in 460/530 ratio (i.e., loss of FRET). (C) Completely phosphorylated(open circles) and nonphosphorylated (filled circles) PKA FRET-peptides were incubated with varying concentrations of chy-motrypsin. Nonphosphorylated peptides are cleaved at lower chymotrypsin concentrations as indicated by an increase in 460/530ratio (loss of FRET).

A

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nase assay could be developed based on differential pro-tease sensitivity of phosphorylated peptides. In all cases,it is the rate of chymotrypsin cleavage that differs forphosphorylated and unmodified peptides, as phosphory-lated peptides can eventually be cleaved by increasingthe chymotrypsin concentration and/or the time of pro-tease digestion.

The FRET-peptide kinase assay can be used todetermine standard kinetic parameters

In developing the FRET-peptide kinase assay for indi-vidual kinases, standard reaction conditions were opti-mized, including incubation time, enzyme concentration,and ATP concentration. As expected, increasing amountsof v-abl (Fig. 2A) or increasing reaction time (data notshown) resulted in increasing levels of phosphorylation ofthe abl FRET-peptide. The enzyme dependence curve ob-tained using FRET was identical to that observed when thepeptide was labeled with [g-32P]ATP and substrate phos-phorylation detected radiometrically, suggesting that theFRET detection did not affect the enzyme kinetics (datanot shown). By titrating ATP in the reaction, the ATP KM

for v-abl was determined to be 7.7 mM (Fig. 2B), which

is consistent with literature values of 12–18 mM.20,21 Thesedata indicate that the FRET-peptide kinase assay can beused to determine standard kinetic parameters.

To show that the FRET-peptide kinase assay could ac-curately detect inhibitors of kinase activity, we incubatedv-abl in the presence of varying amounts of the general ki-nase inhibitor staurosporine. A dose-dependent decrease inv-abl activity was detected (Fig. 2C), and the IC50 deter-mined by using the FRET-peptide kinase assay was 76 nM.Similarly, PKA inhibition by the specific inhibitor H-89was determined to be 18 nM (Fig. 2D), which is in agree-ment with reported values of 48 nM.22 It should be notedthat in order to generate sufficient dynamic range to resultin an acceptable Z9 value23 under typical screening condi-tions, 30–50% of the peptide substrate will be phosphory-lated. Under such substrate turnover conditions, the rate ofreaction may no longer be in the linear range. However,kinetic simulations of various reaction times and substrateturnover (assuming simple first-order kinetics and fittingthe data to the hyperbolic equation for IC50) suggested thatallowing the reaction to proceed to 50% substrate conver-sion should theoretically result in no greater than a 40%increase in the apparent inhibitor IC50 (Dr. Paul England,personal communication).

FRET-Based Assay for Ultra-High Throughput Screening 13

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FIG. 2. Measurement of kinetic parameters using the FRET-peptide kinase assay. (A) Titration of v-abl enzyme in the reac-tion predictably resulted in increased phosphorylation of the abl-FRET peptide. (B) To determine the ATP KM for v-abl, the v-abl FRET-peptide was incubated with v-abl and increasing concentrations of ATP. Increasing the amount of ATP resulted in in-creased v-abl FRET-peptide phosphorylation with a calculated ATP KM of 7.7 mM. (C) The v-abl FRET-peptide was incubatedwith v-abl, ATP, and increasing concentrations of staurosporine. Staurosporine inhibited the phosphorylation of the v-abl FRET-peptide with a calculated IC50 of 76 nM. (D) The PKA FRET-peptide was incubated with PKA, ATP, and increasing concen-trations of H-89. H-89 inhibited the phosphorylation of the PKA FRET-peptide with a calculated IC50 of 18 nM.

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Multi-kinase pathways can be assayed in vitro usingthe FRET-peptide kinase assay

Protein kinases involved in signal transduction oftenfunction within a multi-enzyme cascade in which active up-stream kinases phosphorylate and activate downstream ki-nases. Although most kinase inhibitors bind to the ATPbinding pocket of active kinases, there is at least one ex-ample of a compound (PD 98059) that binds noncompeti-tively for ATP to the inactive form of MEK1 and preventsits activation by Raf.24 Inactive (nonphosphorylated) pro-tein kinases adopt a different three-dimensional structurethan active (phosphorylated) kinases, particularly within theactivation loop, presenting the possibility that novel classesof inhibitors could be found by screening for compoundsthat selectively interact with inactive kinases.25–27 As in-active kinases cannot efficiently phosphorylate their sub-strates, the assay has to be configured such that the inac-tive kinase of interest is phosphorylated by its activatingkinase in the presence of test compound. The FRET-pep-tide kinase assay is well suited for this approach becausethe FRET-peptide is specific for the kinase of interest.

Using the FRET-peptide kinase assay format, in vitropathway assays were developed in which a constitutivelyactive MKK phosphorylates an inactive mitogen-acti-vated protein kinase (MAPK). Two examples were cho-sen to demonstrate this case, the ERK2 and p38 path-ways. In both cases, the assay was dependent on theconstitutively active upstream kinase (MEK1 or MKK6,respectively). Figure 3A shows that neither inactiveERK2 alone, constitutively active MEK1 [MEK(DD)]alone, nor inactive MEK1 with inactive ERK2 was suf-ficient to phosphorylate the ERK2 FRET-peptide. Kinaseactivity in the assay was only detected when MEK1(DD)was combined with the inactive ERK2. Similar resultswere seen for the MKK6/p38 pathway (Fig. 3B). By re-creating an MKK/MAPK pathway in the in vitro assay,there are three different potential targets: the active MKK,the inactive MAPK, and the activated MAPK. Deconvo-lution of compound hits using individual FRET-peptidesubstrates with the respective kinase is readily achievedusing a single kinase in this assay format (data notshown). Taken together, these data demonstrate that theFRET-peptide assay can be configured to screen for po-tentially novel inhibitors that recognize the inactive formof protein kinases.

The FRET-peptide kinase assay can be miniaturized tothe 3,456-well format

As the FRET-peptide kinase assay is an addition-onlyformat with a robust, ratiometric fluorescence readout, itis ideally suited for miniaturization. The FRET-peptidekinase assay has been adapted to a 3,456-well/1 ml for-mat using the FRD (Vertex Pharmaceuticals), which is amicrofluidic device capable of dispensing as little as 200

nl into the assay plates. To determine whether there iscomparable assay performance in the miniaturized for-mat, we performed a head-to-head comparison of the as-say in a 384-well format using identical assay reagents.

Rodems et al.14

MEK1-ERK2

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FIG. 3. Assay of multi-kinase pathways using FRET-peptides. Kinase pathway assays were re-created in vitro by us-ing an active upstream kinase, an inactive target kinase, and aFRET-peptide substrate of the target kinase. The upstream ki-nase phosphorylates and activates the target kinase, which inturn phosphorylates the FRET-peptide. (A) Constitutively ac-tive MEK1 [MEK1(DD)] in combination with an inactiveERK2 was required to phosphorylate the ERK2 FRET-peptide;inactive ERK2 was not sufficient. In addition, inactive MEK1was not able to phosphorylate ERK2 or the ERK2 FRET-pep-tide. Importantly, MEK1(DD) alone could not phosphorylatethe ERK2 FRET-peptide. (B) The combination of constitutivelyactive MKK6 [MKK6(DD)] and inactive p38a was required tophosphorylate the ERK2 FRET-peptide because neither inac-tive p38a alone nor MKK6(DD) alone resulted in significantpeptide phosphorylation.

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In the experiment, assay reagents were prepared and thenaliquoted so that the same reagents were dispensed into384-well plates by hand-held pipettors, or into 3,456-wellplates by the FRD. Fluorescence detection was performedusing appropriate plate readers for each format; 384-wellplates were read in an LJL Analyst and 3,456-well plateswere read using Vertex’s tcPR. In both 384-well and3,456-well plates, ,40% of the PKA FRET-peptide wasphosphorylated by PKA under identical assay conditions(data not shown). In addition, the IC50 for the PKA in-hibitor, H-89, was nearly identical between the formats:14.3 nM in 384-well plates and 19.0 nM in 3,456-wellplates (Fig. 4). The dynamic ranges (1.7-fold in 384-well;1.6-fold in 3,456-well) and the Z9 factors (0.49 in 384-well; 0.55 in 3,456-well) were nearly identical betweenthe formats, indicating that the standard deviations of thereplicate samples were very similar between the 384-welland 3,456-well formats. Taken together, these data indi-cate that there is no loss in assay performance when theFRET-peptide kinase assay is miniaturized to the 3,456-well scale.

Use of the FRET-peptide kinase assay to screencompound libraries in 3,456-well assay plates

After it was determined that there was no loss in as-say performance when miniaturized to the 3,456-wellplate scale, the FRET-peptide kinase assay was used toscreen ,110,000 compounds against PKA using the PKAFRET-peptide as described in Materials and Methods.The 110,000-compound PKA screen was performed in asingle day (,8 h) using 44 3,456-well plates for a totalof 152,064 wells screened (including controls and blankwells). Due to the miniaturization (1.5 ml per well), theentire screen required only 20 mg of PKA. The screenyielded a hit rate of 1.4% (using a hit “cutoff” of 50%inhibition of phosphorylation), with an average Z9 valueof 0.8 (Fig. 5A and B). From 1,423 compounds with.50% inhibition in the primary screen, 591 (41%)showed .50% inhibition on retesting. Of the confirmedcompound hits, .300 were chosen for dose–responseanalysis in 3,456-well plates. In one such plate, 96 com-pounds can be tested in dose–response mode with 11 dif-ferent concentrations in triplicate per compound.

A duplicate copy of each 3,456-well plate containingactive compounds was prepared so that parallel dose–re-sponse experiments could be performed using the FRET-peptide kinase assay and a corresponding cell-based as-say to determine which compounds also had activity incell models of PKA signaling. The “PKA cellular assay”consisted of a HEK293T cell clone engineered with theb-lactamase reporter gene under control of a CREB-recognition site (HEK/CRE). HEK/CRE cells were addedto individual wells of the 3,456-well plate (5,000 cellsper well) and then stimulated with 1 mM forskolin (final

concentration) for 4 h at 37°C. After a 1-h room-tem-perature incubation in the presence of the fluorescent b-lactamase substrate, CCF4, the fluorescence values weredetermined by using the tcPR. We found that ,10% ofthe confirmed hits from the biochemical FRET peptideassay had IC50 values in the range of 1–20 mM in thecell-based assay. Figure 5C shows the data from sevenrepresentative compounds analyzed in both the PKAFRET-peptide and PKA cellular assays. As can be ex-pected, compounds generally had higher IC50 values inthe cell assay. A lack of cell permeability, rapid removalfrom the cell, or an inability to compete with the high in-tracellular ATP concentration could all be factors as towhy the majority of compounds failed to show cellularactivity. Thus, by performing biochemical and cellularassays in parallel in miniaturized formats, data can be ob-tained rapidly and with minimal compound usage.

Adaptation of the FRET-peptide kinase assay platformto measure protein phosphatase activity

The FRET-peptide kinase assay technology can beused to measure protein phosphatase activity by using aprephosphorylated FRET-peptide as a substrate for thephosphatase. Under this scenario, the phosphorylated

FRET-Based Assay for Ultra-High Throughput Screening 15

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FIG. 4. Comparison of 384-well and 3,456–well assay for-mats. An H-89 titration was performed to compare assay per-formance in 384-well and 3,456-well formats. Solutions wereprepared and then split such that identical preparations ofreagents were used for each plate type in the experiment.Reagents were added to 384-well plates with hand-held pipet-tors, and to 3,456-well plates with microfluidic dispensers. Af-ter identical reaction times, the plates were read on fluorescenceplate readers (LJL Analyst for 384-well plates, Vertex’s tcPRfor 3,456-well plates). Nearly identical inhibitor IC50 values in-dicated that there was no loss in assay performance when minia-turized to the 3,456-well scale.

FRET-peptide is resistant to protease cleavage, and de-phosphorylation of the FRET-peptide by the phosphataseresults in increased protease sensitivity and loss of FRET.To demonstrate this feature, the phosphorylated ablFRET-peptide was treated with leukocyte antigen-related(LAR) protein tyrosine phosphatase. Incubation withLAR resulted in complete dephosphorylation of the abl

FRET-peptide, as indicated by the high 460/530 ratiopresent at the low end of the dose–response curve usingthe general phosphatase inhibitor orthovanadate (Fig.6A). Orthovanadate was able to inhibit LAR activity withan IC50 of 45 nM, indicating that the FRET-peptide as-say can be used to detect inhibitors of protein tyrosinephosphatases. The FRET-peptide assay platform has also

Rodems et al.16

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3.9 m M12 m M20 m M7.6 m M12 m M11 m M

13.7 m M

FIG. 5. Ultra high-throughput PKA screen using the FRET-peptide technology. A PKA screen was performed using 44 3,456-well plates (,110,000 compounds, plus controls). (A) Scatter plot of assay results from one 3,456-well plate. The “1 ATP” con-trol defines 0% inhibition (open green triangles), the “No ATP” control defines 100% inhibition (filled red squares). Compoundsare represented by filled blue circles. (B) Histogram of entire screen. The hit rate (compounds resulting in .50% inhibition ofPKA activity) was 1.4%. (C) Comparative activity of confirmed hits (by IC50) between the FRET-peptide PKA assay and a cell-based reporter gene assay for forskolin-induced PKA activation (HEK-CRE b-lactamase cell assay). Duplicate 3,456-well platescontaining compound dilutions were prepared and assayed in the FRET-peptide kinase assay and in the reporter gene assay. Sev-eral primary screen hits were retested in a dose–response format; representative data from seven compounds are shown.

A

B

C

been used to measure activities of other tyrosine phos-phatases, including PTP-1B and YOP (yersinia entero-colitica outer protein phosphatase; data not shown).

Serine phosphatase activity can be assayed by theFRET-peptide technology, as shown using PP1. PP1 de-phosphorylation of the prephosphorylated CK1 FRET-peptide gave a fourfold dynamic range in the assay, anddetected inhibition of PP1 activity by 0.1 mM micro-cystin-LR, a general phosphatase inhibitor (Fig. 6B). Thisassay technology was also successfully used to measurePP2A activity on different Ser/Thr kinase FRET-peptidesubstrates (data not shown).

Discussion

With .500 kinases and phosphatases encoded by thehuman genome, there exists a need to rapidly profile thesekey signaling enzymes against the growing number ofcompounds. The FRET-peptide assay platform whenused in combination with miniaturization technologies

has allowed us to greatly reduce the time required toprogress from assay development to completed screen.The assay format is readily adaptable to all classes of pro-tein kinases and phosphatases. Although the assay is de-pendent on engineering a chymotrypsin cleavage sitewithin the substrate peptide, the single amino acid sub-stitution (tyrosine or phenylalanine) within the consen-sus kinase recognition sequence has been tolerated in allpeptides tested to date. FRET-peptide kinase assays havebeen developed for multiple tyrosine kinases, as well asfor basophilic (e.g. PKA), acidophilic (e.g., CK1), andproline-directed (e.g., ERK2) serine/threonine kinases.To date, assays for .20 protein kinases have been de-veloped with the FRET-peptide system. By using a com-mon assay format for the kinase target class, assay de-velopment has proven to be extremely efficient.

Although developing an assay to a new protein kinaseoften requires the synthesis of a new FRET-peptide, wehave found that [similar to the use of poly(EY) peptidesfor tyrosine kinases] a single peptide sequence can beused for several different protein kinases. For example,

FRET-Based Assay for Ultra-High Throughput Screening 17

Inhibition of LAR byorthovanadate

10-9 10-8 10-7 10-6 10-5 10-40.0

2.5

5.0

7.5

10.0

12.5

15.0

17.5

[OV], M

Rat

io (

460/

530)

IC50 = 45 nM

FIG. 6. Detection of phosphatase activity using FRET-peptides. The FRET-peptide assay design can be used to measure phos-phatase activity by starting with a prephosphorylated FRET-peptide. Dephosphorylation of the peptide renders it susceptible tochymotrypsin cleavage, resulting in an increased 460/530 ratio (loss of FRET). (A) Phosphorylated v-abl FRET-peptide was in-cubated with LAR protein tyrosine phosphatase in the presence of different concentrations of the phosphatase inhibitor ortho-vanadate (OV). Increasing OV caused inhibition of phosphatase activity with an IC50 of 45 nM. (B) Phosphorylated CK1 FRET-peptide was incubated with 0.1 U of PP1 in the presence or absence of 0.1 mM microcystin-LR (M-LR). M-LR (at 0.1 mMcompletely inhibited PP1 activity, as indicated by the CK1 FRET-peptide’s resistance to chymotrypsin cleavage.

Inhibition of PP1 bymicrocystin-LR

0

5

10

15

Rat

io (

460/

530)

p-C

K1

p-C

K1

p-C

K1

CK

1FRETpeptide

PP1

M-LR

- - 0.1 U 0.1 U

- - - 0.1 m M

A B

the same proline-directed kinase sequence (ERK2 FRET-peptide) was used to develop assays for both ERK2 andp38. A peptide derived from the Kemptide (PKA FRET-peptide) has been used for several different Ser/Thr ki-nases in addition to PKA. This feature of the assay plat-form improves the efficiency with which kinases can bescreened in addition to reducing the cost. Our experiencehas shown that establishing a panel of FRET-peptidescorresponding to consensus recognition motifs for vari-ous protein kinases greatly facilitates the assay develop-ment process.

Addition-only assay formats are optimal for high-throughput screening systems, and are required as screen-ing formats go to higher densities. We have routinely per-formed the FRET-peptide kinase assay in a 3,456-wellformat. At this density, a 100,000-compound screen iseasily performed in a single working day. This level ofminiaturization and speed allows the accelerated prose-cution of kinase inhibitor screens. Cost savings are alsoachieved in the amount of enzyme used per screen. Asthe assays are performed in 1–1.5-ml volumes, kinase en-zyme requirements for the screen are in the microgramto single milligram range instead of the 10–100 mg re-quired for lower density formats. Finally, dose–responseanalysis can be performed on up to 96 compounds per3,456-well plate, greatly improving the efficiency withwhich high-quality information on compound potencycan be obtained. The efficiencies derived from miniatur-ized, automation-compatible platform technologies suchas the FRET peptide kinase technology permit imple-mentation of a broad screening/rapid counterscreeningstrategy, which leads to more complete profiling of com-pound selectivity and action.

Most kinase inhibitors to date are ATP-competitive;this may derive, in part, from screening assays havingbeen run at relatively low concentrations of ATP (typi-cally at Km for ATP). One drawback to targeting the ATPbinding pocket of active kinases is that the overall struc-ture of the pocket in the active state is similar betweenmany different kinases, as they all have to bind the samesubstrate molecule, ATP.26 Structure-based drug designefforts have dealt with this fact by taking advantage ofthe limited amino acid diversity within the pocket. How-ever, generating highly specific ATP-competitive kinaseinhibitors has been extremely challenging. In contrast, thestructures of inactive kinases are very diverse.26 An in-triguing approach is to identify inhibitors that bind to in-active kinases and prevent their activation; such inhibi-tors may provide greater specificity and have less toxicity.Indeed, the binding of Gleevec to the bcr-abl oncoproteinseems to lock it in an inactive conformation.28 In addi-tion, Alessi et al. have identified a novel class of inhibi-tors that appear to prevent the activation of MEK1 byRaf.24 This class of compounds was identified by re-cre-ating the Raf-MEK-ERK pathway in vitro. One advan-

tage of the present FRET-peptide assay technology is thatit can be configured to screen multi-kinase pathways. Inthis format, compounds are preincubated in the presenceof an inactive target kinase. Upon addition of ATP and aspecific FRET-peptide substrate, the upstream activatingkinase will phosphorylate and activate the target kinase,which then phosphorylates the FRET-peptide. Com-pounds that interact with the target kinase and precludethe activation reaction from occurring should be identifi-able. Current efforts are focused on evaluating internalscreening results to discern whether our pathway-basedassay approach will generate novel types of kinase in-hibitors.

Acknowledgments

We are grateful to Paul England and Gregor Zlokarnikfor key input and helpful discussions. We thank AlbertGriffin and Dennis Hurley for peptide design and syn-thesis. We also thank Minh Vuong and his team for tcPRand FRD development.

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Address reprint requests to:Jeffrey H. Stack

Vertex Pharmaceuticals, Inc.11010 Torreyana Road

San Diego, CA 92121

FRET-Based Assay for Ultra-High Throughput Screening 19