kinase screening:layout 1

Several years ago in DDW we reviewed kinaseprofiling1 and highlighted the increasingcompetition between fee-for-service providers

and the marked expansion in the size of kinasespanels offered. With the publication of its latestmarket report on kinases in September 2006HTStec has re-examined current practices andpreferences in kinase screening and profiling, par-ticularly with respect to the need for outsourcedservices. We now discuss some of this report’s keyfindings and review assay vendor and fee-for-serv-ice provider updates on the latest kinase screeningand profiling offerings.

Therapeutic focus and mostinvestigated kinases The therapeutic area making the greatest use ofkinase assays today was oncology (Figure 1).

The second biggest use of kinase assays wasmade by inflammatory disease. In comparison,all other therapeutic areas (eg diabetes and obe-sity, autoimmune disease, neurology, cardiovas-cular and metabolic disease) made less use ofkinase assays.

Tyrosine kinases remain the class of kinasesmost investigated in-house, closely followed by ser-ine and threonine kinases (Figure 2). More thanthree quarters of all respondents were investigatingkinases of these classes in-house. In comparison,lipid kinases and other (eg inactive kinases) wereonly investigated by minority of respondents in-house. The percentage of respondents that investi-gated these kinase classes at a fee-for serviceprovider (outsourced) followed the same trend butwas in all cases a reduced percentage relative to in-house testing.

By Dr John Comley

Drug Discovery World Winter 2006/7 27

Screening

Whereas wider coverage of the kinome was the principal focus of kinaseprofiling a few years ago, today’s vendors of kinase screening and profilingproducts are: 1) increasingly applying automated and industrialised approachesto their fee-for-service offerings; 2) making greater use of time-resolvedfluorescence assay formats; 3) pursuing more universal assay approaches; 4) making wider use of assays that measure the accumulation of ADP; 5) placing greater emphasis on screening inhibitors that bind to inactive andlow activity kinases; 6) extending the diversity of cellular kinase approaches; 7) developing bench-top turnkey profiling instrument solutions; and 8) becoming increasingly aware of the potential of label-free approaches.Overall, the end user is spoilt for choice with the range of alternative offerings, methodologies and approaches currently available for kinasescreening and profiling.

– spoilt for choice

KINASE SCREENINGand PROFILING

Kinase screening:Layout 1 19/1/07 18:45 Page 27

Kinase profiling approachThe preferred approach to kinase panel profilingby around half of the respondents surveyed was touse a combination of in-house and outsourced

panels for selected lead compounds (Figure 3).Less than 10% of respondents preferred to useonly in-house or only outsourced profiling forselected lead compounds. Profiling of focusedlibraries against a kinase panel (in-house or out-sourced) was also much less used compared toselected lead compounds.

The number of kinases survey respondents usedtoday in their in-house profiling panels or accessin outsourced profiling panels is shown in Figure4. The mean size of the kinase profiling panelsbeing used was 34 kinases in-house, versus 103kinases outsourced.

Assay technology preferencesAssay technology preferences for kinase panel profil-ing in-house and outsourced are presented in Figure5. When outsourcing, the majority (41%) of respon-dents did not have a preferred kinase panel profilingassay technology and were happy to go with CRO’srecommendation. Of the named technologies radio-metric filter (eg P33 incorporation) was preferred foroutsourcing (24% share), followed by TR-FRET (egLANCE, HTRF, TR-IMAP) (6% share). TR-FRETwas the preferred assay technology for in-house pro-filing, although only around one in four (23%) ofrespondents choose it. The next preferred in-housetechnology was FP (Fluorescent Polarisation) withligand (ie no antibody) (11% share), followed by anumber of different technologies each with between10% and 5% share (radiometric filter, glow lumines-cence, FlashPlate/Image FlashPlate (radiometric),AlphaScreen and FI (fluorescent intensity) withmicrofluidic (on chip) separation). In total, surveyrespondents chose 17 different kinase assay tech-nologies as their preferred technologies and sourcedthese from 16 different technology suppliers/vendorsand 11 outsourced fee-for-service providers. Theseobservations highlight the broad range of technolo-gies that are now available for kinases and largediversity of opinion among end users in this highlycompetitive marketplace.

Respondent satisfaction with data quality fromcurrent kinase screening methods is presented inFigure 6. Only a small minority (5% in-house, 11%outsourced) were dissatisfied with the data qualitygenerated by their current kinase screening methods.The remainder are currently satisfied with existingkinase screening methods, although most (56% in-house, 81% outsourced) would be open to betterapproaches if they were available. On the basis of thisresponse we can conclude that most of the currentkinase offerings are adequate for end users needs.

The features of a new kinase screening platformof greatest interest to respondents are ranked in

28 Drug Discovery World Winter 2006/7

Screening

Figure 1: Therapeutic areas using kinase assays today

OncologyInflammatory diseaseDiabetes and obesityAutoimmune disease

NeurologyCardiovascular

Metabolic diseaseOther

0% 10% 20% 30% 40% 50% 60% 70% 80%

% Using© HTStec 2006

Figure 2: Relative interest in different classes of kinase

Tyrosine kinases

Serine kinases

Threonine kinases

Lipid kinases

Others

0% 10% 20% 30% 40% 50% 60% 70% 80% 90%

% Investigating

In-houseOutsourced

© HTStec 2006

Figure 3: Preferred approach to kinase panel profiling today

In-house and outsourced –Selected lead compounds

We don’t have oneIn-house – Selected lead

compoundsOutsourced – Selected lead

compoundsIn-house – Focused libraries

In-house and outsourced –Focused libraries

Outsourced – Focused libraries

Other

0% 5% 10% 15% 20% 25% 30% 35% 40% 45% 50%

% Preferring approach© HTStec 2006

Kinase screening:Layout 1 19/1/07 18:45 8

Figure 7. A non-radioactive method was ranked asthe feature of most interest, closely followed bylower cost per data point than radioactive assays;cell-based assay format and then label-free tech-nology. A microfluidic (on chip) format wasranked the least important feature, possibly reflect-ing the fact that chip formats (eg CaliperLSLabchips) have gained greatest acceptance formobility shift separations of product from sub-strate, for kinase assays performed off chip.Interestingly, with respect to label-free assays 18%of respondents said they have implemented themfor kinase profiling and a further 30% stated theyare actively investigating them. Currently duringthe lead optimisation of kinase inhibitors, low

throughput fluidic chip-based label-free methods(eg Biacore) are widely used in kinetic analysis (ieto determine on and off-rates). However, it seemsprobable that higher throughput plate-based label-free systems (eg Corning EPIC) will have a futurerole in screening low activity or inactive kinaseforms in an area where current catalytic assay for-mats offer very little. Another application area forlabel-free is the detection of cell interactionsbetween antibodies or ligands and screening poten-tial therapeutic Abs to surface markers and rank-ing them in the context of kinases receptor TKs.Label-free methods may also facilitate to screeningpotential structural constructs in proof of foldingand crystallography trials, if an interaction with a


Screening

Figure 5: Preferred assay technology for kinase panel profiling

We Don't Have One – go with CRO’s recommendationRadiometric Filter (eg P33 incorporation)

TR-FRET (Time Resolved FRET eg LANCE, HTRF, TR-IMAP)Other Technology

FI (Fluorescent Intensity) with Microfluidic (on chip) SeparationFP (Fluorescent Polarisation)/Antibody

Other Radiometric AssaysFP (Fluorescent Polarisation)/Ligand (ie NO antibody)

FI (Fluorescent Intensity) including fluorescent quench assaysECL (ElectroChemiLuminescence)

FRET (Fluorescent Resonance Energy Transfer)Glow Luminescence

FI (Fluorescent Intensity) based ELISA assaysAlphaScreen

FlashPlate/Image FlashPlate (Radiometric)SPA/LEADseeker (Radiometric)

Chemiluminescent based ELISA AssaysTRF (Time Resolved Fluorescence eg DELFIA)

FLT (Fluorescence Lifetime)

0% 5% 10% 15% 20% 25% 30% 35% 40% 45%

% Preferring technology

In-house

Outsourced

© HTStec 2006

Figure 4: Size of kinase panel used in 2006

35%

30%

25%

20%

15%

10%

5%

0%N/A 0 1-10 11-25 26-50 51-100 101-150 151-200 201-250 >250

No of kinases in panel

% R

espo

ndin

g

In-houseOutsourced

© HTStec 2006

Figure 6: Satisfaction with data quality from current kinase screening methods

In-house assays

Outsourced assays

0% 10% 20% 30% 40% 50% 60% 70% 80% 90% 100%

% Response

YES, very happy with our current setup YES, but open to better approaches NO, needs to be improved

© HTStec 2006


compound is observed. Clearly label-free is an areaof growing importance and market share in kinasescreening and profiling.

Changing dynamic of kinase profilingmarketplaceIn Table 1 we compare some of the kinase profil-ing statistics which were estimated in HTStec’s2004 and 2006 market surveys. In-house profilinghas shown a decline in the total number of datapoints generated and the number of FTEs (full timeequivalents) that support it, with a shift from %singlet data points to more full dose-response andduplicate data points. In addition, the average costpaid per single data point generated has increasedfour fold. Over the same period the total numberof data points profiled using outsourcing has morethan doubled, with increases in the % singlet and% full dose-response data points. The average costpaid per single data point outsourced appears tohave changed little. In 2004 around 10X more datapoints were profiled in-house than outsourced, in2006 this has decreased to less than 3X more in-house. As a consequence of this shifting dynamic,the 2006 market for outsourced profiling has dou-bled to $37.8 million since 2004. In contrast, the


Screening

Figure 7: Most important features of a new kinase screening platform

Non-radioactive method

Lower cost per data point than radioactive assays

Cell-based assay format

Label-free technology

Antibody-free

Ability to measure large protein substrates

Miniaturised microplate format

Cell-lysate assay format

Multiplexed format

Array format

Microfluidic (on chip) format

1.00 1.50 2.00 2.50 3.00 3.50 4.00 4.50 5.00

Ranking score 1 to 5, where 1 = no interest (least importance) and 5 = greatest interest

© HTStec 2006

1 all data from HTStec’s published Trends Reports2 refers to total pharma/biotech spend in house on reagents for profiling3 refers to total pharma/biotech spend on fee-for-service profiling

IN HOUSE PROFILING 2004 2006

Estimated Market Size2 $12.2M $16.6M

Average Cost/DP $0.84 $4.09

No. DP Generated/Lab 262,000 151,000

No. FTE/Lab 2.9 1.9

% Singlet DP Profiled 47% 20%

% Duplicate DP Profiled 15% 25%

% Full Dose Response DP Profiled 38% 55%

OUTSOURCED PROFILING 2004 2006

Estimated Market Size3 $16.3M $37.8M

Average Cost/DP $17.85 $18.88

No. DP Outsourced 23,000 56,000

% Singlet DP Profiled 2% 26%

% Duplicate DP Profiled 88% 47%

% Full Dose Response DP Profiled 10% 27%

Table 1: Pharma/biotech kinase profiling statistics1



Screening

2006 market for the primary screening of kinases(ie reagent proportion only) was estimated to bearound $80 million.

In Table 2 the principal vendors and fee-for-serv-ice providers serving the kinase screening and pro-filing market place are summarised. Companies aredivided into the following three categories basedon their kinase offering: 1) assay technology,enzymes, substrates, reagents and/or kits; 2) out-sourced assay services; and 3) turnkey instrument

platforms. The following are brief updates on thesevendor’s kinase product offerings.

Amphora Discovery Services (www.amphoracorp.com) utilises an automated and industrialisedapproach to kinase profiling and screening, basedon the Caliper LabChip® technology, proprietaryinformatics and a broad, physiologically relevant,peptide library. With all of its assays running on asingle platform and designed to be as comparable

Table 2: Vendors and fee-for-service providers serving kinase screening and profiling marketplace*

* Companies whose offerings are discussed in this article

VENDOR/PROVIDER WEBSITE ASSAY TECHNOLOGY/ENZYMES/SUBSTRATES/

REAGENTS/KITS

OUTSOURCED ASSAY SERVICES

TURNKEY INSTRUMENT PLATFORMS

Amphora Discovery Services www.amphoradiscovery.com �

BellBrook Labs www.bellbrooklabs.com �

Biofocus DPI www.glpg.com �

Caliper Life Sciences www.caliperls.com � � �

Carna Biosciences www.carnabio.com � �

Cerep www.cerep.com �

Cisbio International www.htrf.com �

DiscoveRx www.discoverx.com �

GE Healthcare www.gelifesciences.com/kinases �

Invitrogen www.invitrogen.com � �

MDS Pharma Services www.mdsps.com �

Millipore www.millipore.com � �

Molecular Devices www.moleculardevices.com �

PerkinElmer www.perkinelmer.com �

Promega www.promega.com �

ProQinase www.proqinase.com �

SpinX www.spinx-technologies.com �

TTP LabTech www.ttplabtech.com �



Screening

as possible, data quality is very high. In addition toproviding standard screening and profiling,Amphora also offers custom assay development,hit-to-lead optimisation services and access tolibraries through agreements with key chemistrysuppliers. Furthermore, Amphora’s integratedtechnology platform and high data quality lendsitself to the development of mineable databases. Bycombining focused customer or Amphora accessedcompound libraries and the capacity for rapid,orthogonal screening, Amphora is able to buildunique databases that can be mined at the primaryscreening stage for structural selectivity patterns(SAR). Based on these patterns, hit series are select-ed and followed up with potency, aqueous solubil-ity and enzymology studies to provide a completeoverview of a library’s relevance to multiple kinasetargets. The end result is a fully mineable, cus-tomer-unique database that enables chemists tomake rapid structural class selections, based onselectivity and true potency, at a very early stage inthe discovery process. This approach leads thetrend for increased data density and makes it pos-sible to merge the kinase screening and profilingprocesses (Figure 8).

BellBrook Labs’ (www.bellbrooklabs.com)Transcreener™ kinase assay uses homogeneousimmunodetection of ADP to enable robust detec-tion of kinase initial velocity over a broad range ofstarting ATP concentrations. Because it detects theinvariant product of a phosphorylation reaction, itcan be used for lipid and carbohydrate kinases aswell as protein kinases. Moreover, some of the morechallenging kinase screening methods such as meas-uring native protein phosphorylation, autophos-phorylation and ATPase activity are straightfor-ward using ADP detection. Transcreener is the onlytruly generic kinase assay method that does notdepend on coupling enzymes for signal production;instead it utilises highly selective antibodies that areable to distinguish between nucleotides on the basisof a single phosphate group. The first generationassay uses fluorescence polarisation as a detectionmode with a far red tracer to avoid interferencefrom light scattering and fluorescent compounds.More recently, the assay was formatted for timeresolved fluorescence resonance energy transfer(TR-FRET) incorporating Invitrogen’s (Carlsbad,CA) proprietary LanthaScreen™ lanthanidechelate-antennae complex technology for efficientenergy capture (Figure 9). The FRET donor – a ter-bium chelate complex – is covalently attached toBellBrook’s proprietary ADP-specific monoclonalantibody; the acceptor is a fluorescein-ADP tracer.

This format provides a simple two componentdetection module resulting in lower reagent costscompared with other TR-FRET detection modulesrequiring three or more binding components. Themost advantageous feature of the TR-FRET formatis that the assay signal is delayed relative to theprompt fluorescence from small organic fluors thatare typically the source of assay interference.

Biofocus DPI (www.glpg.com) the service divisionof Galapagos, has two assay development andscreening groups, located in Basel, Switzerland andCambridge, UK. These screening groups haveextensive experience in employing high-throughputscreening to find small molecules which modulatethe activity of a wide range of target classes (includ-ing, proteases, kinases, GPCRs, ion channels, phos-phatases and nuclear hormone receptors) using bio-chemical assays and cellular model-systems.

TargetsCompounds

Figure 8Amphora heat-map generatedform a customer-uniquedatabase and merged withstructures provide a powerfultool for enhanced selectivityprofiling

Figure 9: BellBrook Labs is redefining the universal HTS kinase assay using nucleotidedetection. The newest assay offering is the Transcreener kinase TR-FRET assay. Transcreeneris the only truly generic kinase assay method that does not depend on coupling enzymes forsignal production. Because it detects the invariant product of a phosphorylation reaction, itcan be used for lipid and carbohydrate kinases as well as protein kinases



Screening

Biofocus DPI has a breadth of experience in thekinase area, with >100 successful screening cam-paigns performed. The company has a proven trackrecord for identifying hit compounds using high-quality, robust assays in a range of formats, includ-ing FlashPlate, FilterPlate, SPA, FP, and FRET.Supporting the state-of-the-art screening technolo-gies is access to BioFocus DPI’s compound librariescomprising rationally designed, chemogenomic,hit-finding kinase libraries (SoftFocus®), and alarge collection of 700,000 diverse compounds. Toincrease capabilities in the kinase area further,BioFocus DPI has a commercial agreement withCarna Biosciences (Japan) which allows access to awide range of high-quality existing and bespokekinase enzymes for hit-finding and profiling.

Caliper Life Sciences (www.caliperls.com), throughits Discovery Alliances & Services group(NovaScreen Biosciences, acquired in 2005 andXenogen Biosciences, acquired in 2006), offersoutsourced kinase assays using the CaliperLabChip® platform. Its current assay portfolioconsists of 100 kinases, with a planned expansionto more than 200 assays in 2007. In addition toprotein kinases, it offers sphingosine 1 & 2 lipidkinases and is currently developing PI3 kinaseassays using the LabChip technology platform.Because the LabChip directly detects substrate andproduct, and does not require the use of antibodiesor radiometric filtration, this platform is optimalfor lipid kinases. In early 2007, the DiscoveryAlliances & Services group of Caliper will belaunching its rapid KinaseAdvisor profile. Thisdiverse panel of assays consists of 48 kinases witha “less than five day” turnaround for a completereport. The KinaseAdvisor utilises the DeskTopProfiler, Caliper’s complete solution for ‘in-the-lab’

kinase profiling, which consists of a new benchtopmicrofluidic reader (Figure 10) and ProfilerProkinase profiling kits. ProfilerPro kits consist of 48kinases in two 384 well microplates along withmatching ATP/substrate plates. You simply defrostthe plates, mix the contents, stop the reaction andread. Unlike typical outsourced kinase profilingwhich has lead times of several weeks, ProfilerProallows researchers to obtain data faster than everbefore. The Discovery Alliances & Services grouphas expanded Caliper’s kinase profiling byenabling customers to study kinase inhibitors inanticancer cell proliferation panels (with many celllines selected from the NCI 60 panel). It can alsoperform in vivo efficacy studies with subcutaneousand orthotopic xenograft oncology models usingthe Xenogen IVIS bioluminescent detection plat-form. Additionally, the Discovery Alliances &Services group has access to all of Caliper LifeSciences high-throughput automation technology,which has allowed it to help several large pharma-ceutical companies with their kinase HTS projectsin 2006. These screens ranged in size from 10,000to 500,000 compounds.

Carna Biosciences (www.carnabio.com) hasreleased its renewed kinase profiling service inwhich it offers a new assay platform called ‘mobil-ity shift assay’. This new profiling service offerscustomers more robust and reliable test data thanever. The service is low-cost, has a high throughputcapability and uses optimised substrates and high-ly active kinases. So far 150 kinases have been test-ed and confirmed as available on this platform. Inaddition, this year Carna is planning to add at least50 more kinases to the list. Also, 70 other types ofkinases are continuously available on its presentplatforms, such as ELISA and IMAP. Therefore,more than 220 kinases will be available on thisnew profiling service. All of the kinases used onCarna’s profiling service are produced in-houseand are advantaged in their high-accuracy andhigh-quality (without any contaminated kinasederived from host cell).

Cerep’s (www.cerep.com) kinase platform has beendeveloped by turning basic research into concreteindustrial applications. A comprehensive andcoherent hit-to-lead platform was developed inorder to meet the exacting requirements needed forthe identification and characterisation of the activ-ity and selectivity of kinase inhibitors early in thedrug discovery process. Cerep’s strategy has beento expand its kinase assays to cover the kinome interms of diversity. This ever-expanding range of

Figure 10Caliper Life Sciences’ DeskTop

Profiler



Screening

kinase assays is backed by industrial concepts: con-stant quality high throughput profiling, homoge-neous and robust data delivery, turnaround timeand cost reducing. It has allowed Cerep to offer anExpress Kinase panel with 45 kinases profiled inone day. Another new offering last year and con-tinuing in 2007 is Cerep’s ‘stand alone’ assay devel-opment service. Using this customer-oriented-development service, clients can implement theirproprietary assays or have a specific test developedexclusively. With this service, clients can consoli-date assay development and screening without cut-ting quality. Cerep’s kinase assays will continue togrow this year and it plans to evolve its profilingoffers and implement various critical signallingpathways. A panel of cellular kinase assays willalso be developed in 2007 using various cellularmodels for hit confirmation and pathway profiling.

The growing interest in screening and profilingkinases has prompted the development of many newassay technologies. Cisbio’s (www.htrf.com) univer-sal HTRF® KinEASE™ platform developed in part-nership with Millipore, uses universal biotinylatedsubstrates with just one monoclonal antibody,labelled with europium cryptate (EuK) for a sensi-tive, specific detection of the phospho-peptide. Now,the initial HTRF® KinEASE™ assay for screeningserine/threonine kinases (STKs) has been extendedto include tyrosine kinases (TKs). The KinEASE TKuses a choice of two substrates containing a singlephosphorylation site recognised by a Eu(K) labelledanti phospho-tyrosine antibody. After the kinasereaction, the addition of the antibody and strepta-vidin XL665 to capture the biotinylated substrateallows FRET to occur. This signal increases propor-tionally with kinase activity and the amount of thekinase added. KinEASE TK has already been vali-dated on more than 50 tyrosine kinases and the opti-mal substrate determined for each. This single assaysystem allows even more tyrosine kinases to bescreened. A TK reaction buffer, especially optimisedfor optimal signal to background and suitable forany tyrosine kinases, will be associated with otherreagents in a kit. Figure 11 shows excellent signal tobackground ratios obtained with a panel of tyrosinekinases (cytosolic and receptor) using the optimalsubstrate. In the next development step, a HTRF®KinEASE™ platform currently allowing more than150 serine/threonine or tyrosine kinases to bescreened, will be extended to a new kinase family,the MAP kinases.

DiscoveRx (www.discoverx.com) offers kinasebinding assays for kinases with little or no activity,

or where the substrate is unavailable. With thisnew assay technology, direct or indirect binding ofthe inhibitor to the kinase rather than functionalactivity is measured. Currently there is a growingneed to screen for inhibitors that bind to inactiveand low activity kinases due to the potential foraugmented kinase inhibitor selectivity at patholog-ically important mutant forms of the enzyme.DiscoveRx’s kinase binding assay platform utilisesEFC technology to homogeneously measure bind-ing of inhibitors to the ATP binding site. In theassay, the ED-inhibitor conjugate and compoundscompete for binding to the ATP binding site. If theconjugate is not bound to the kinase, it is free torecombine with EA (inactive EFC detectionenzyme), resulting in an active ß-gal enzyme, which

Figure 11: Signal to background obtained with Cisbio’s HTRF® KinEASE™ Tyr using a panelof 10 tyrosine kinase/substrate pairs. The kinase concentration (50ng/well), the substrate(1µM), the streptavidin XL665 (62.5nM), the ready to use TK antibody-Eu(K) and incubationtime (30min) were all kept constant. Each assay was performed in 50µL for the kinasereaction and 50µL added for the detection giving a final volume of 100µL. Straightforwardminiaturisation reduces the final volume to <4µL by simply keeping each reagent’s finalconcentration constant

Figure 12DiscoveRx assay principle



Screening

hydrolyses the luminescent substrate. The signalproduced by active EFC enzyme is proportional tothe amount of compound bound to your kinase.The HitHunter Kinase Binding Assay platform cur-rently includes four ready-to-use kits (p38alpha,PKC/CAMKII, c-Kit/ABL/SRC and LCK) as wellas a profiling service that will demonstrate bindingcharacteristics of your kinase and identify the bestED-inhibitor conjugate for screening. If a customerprovides DiscoveRx with a sample of their kinase

of interest, they will run it against a panel of ED-ligand conjugates and send customers the kinasebinding data, enabling them to select the optimalligand to perform a screen using DiscoveRx’s novelkinase binding assay technology (Figure 12).

GE Healthcare (www.gelifesciences.com/kinases)offers the choice of radioactive or non-radioactiveassay formats for researchers involved in kinasescreening and profiling. Products include SPA beads– recognised as the gold standard for radiometrickinase screening, and the HitHunter™ and ADPaccumulation range of kinase assays. HitHunterand ADP accumulation kinase assays offer homo-geneous chemiluminescence and fluorescence basedsolutions for all phases of kinase-targeted therapeu-tic campaigns from target validation and assaydevelopment to hit identification and lead optimi-sation. A choice of assay technologies means specif-ic, generic and inactive kinase targets can be evalu-ated for activity, potency and selectivity. HitHunterEnzyme Fragment Complementation Kinase Assaysare antibody based assays with high specificity forthe kinase reaction product. Standard kinase reac-tion conditions facilitate the assessment of func-tional activity in a broad range of kinases while theenzymatically amplified signal has minimal back-ground and does not produce artifacts caused bynon-specific binding. When little or no informationis known about the kinase substrate and its associ-ated antibody, HitHunter Kinase Binding Assayscan be used to measure and characterise the inter-action of inhibitors with an inactive kinase in acompetitive binding assay format without the needfor a peptide substrate, antibody or ATP. Thechemiluminescent signal produced is unaffected bynaturally fluorescing compounds and can be readon imagers or PMT-based plate readers. ADPQuest™, ADP Quest HS and ADP Hunter™ Plusassays measure the accumulation of ADP, a productof kinase enzyme activity. Unlike alternative gener-ic approaches that monitor the depletion of ATPfrom a kinase reaction, these assays follow theproduct of the reaction in a convenient fluorescencebased gain-of-signal format (Figure 13).Invitrogen’s (www.invitrogen.com) SelectScreen™Kinase Profiling Service was launched in 2004 aspart of the company’s major expansion of itskinase biology platform. The screening service hasgrown dramatically since launch and Invitrogennow offers partners the ability to screen lead com-pounds, SAR series and focused library collectionsagainst a panel of >220 kinases. The foundation ofthe service is Invitrogen’s industry leading libraryof purified protein kinase targets and its robust

Figure 13: GE Healthcare’s range of assay technologies address all phase of kinase-targetedtherapeutic campaigns

Correlation of Invitrogen SelectScreen™ biochemical and cell-based pathway profiling data. Kinase targets can be interrogated withboth biochemical and cellular assays with the Invitrogen SelectScreen™ service. A. Schematic representation of the classical mitogenicsignalling pathway activated by EGF. B. PD153035 IC50 determination using the AP-1-bla ME-180 CellSensor® cell line. C. Comparisonof the biochemical IC50 value for inhibition of EGFR (ErbB1) with the cell-based IC50 value in SelectScreen™ service. This data showsthe expected reduction in cellular potency of an ATP competitive inhibitor

Figure 14Invitrogen SelectScreen kinase

profiling service



Screening

Z’-LYTE™ kinase assay platform. The currentcollection of kinase targets has been assembledbased on therapeutic relevance, phylogeneticdiversity and representation of key signallingpathways. All the reagents utilised in the serviceare manufactured according to strict qualityguidelines within Invitrogen’s Discovery SciencesUnit in Madison, Wisconsin, USA. In addition toensuring the utmost integrity in assay reagents,Invitrogen has invested significantly in people,process and automation to execute screening cam-paigns. To keep up with the rapid demand forscreening services, Invitrogen opened a secondscreening facility in Scotland in July 2006. Nowclients have access to identical services at bothsites and engage in real-time communications withInvitrogen’s global screening leaders. Invitrogenhas also recently launched a cellular pathwayscreening service, SelectScreen™ Cell-basedPathway Profiling Service, to enable clients to

interrogate kinase targets in their native cellularenvironment as well as assessing the effect of com-pounds across signalling pathway space. In 2007,Invitrogen plans to expand the breadth of molec-ular and cellular assays on to its SelectScreen™platform, as well as building out screening servic-es for other major target classes (Figure 14).

Last year, Molecular Devices (www.molecularde-vices.com) introduced a new detection mode, TR-FRET, to the IMAP platform for kinases, phospho-diesterases and phosphatases. IMAP TR-FRETutilises all of the current components of the IMAPFP system plus a terbium (Tb) labelled TR-FRETdonor (‘Tb Donor’) tightly associated with theIMAP binding entity (Figure 15). When a fluores-cent-labelled substrate gets phosphorylated, it canbind in proximity to the Tb donor, enabling energytransfer. Both fluorescein (FAM) and TAMRA aresuitable fluorescent labels. IMAP TR-FRET com-bines the advantages of IMAP FP (no antibodies,homogeneous format, easy assay development,scalability, high throughput) with those of TR-FRET mode (broad range of substrate size and con-centration, low background due to time resolvedmode). IMAP TR-FRET extends the range ofmolecular weights for kinase substrates from smallpeptides up to large proteins. For example, MEK1can be assayed using inactive ERK2 labelled withfluorescein as substrate. Fluorescent-labelled strep-tavidin can now be used to detect phosphorylationof biotinylated peptide substrates (Figure 16). Thelatter makes IMAP TR-FRET compatible withexisting biotinylated substrate libraries for sub-strate identification. IMAP TR-FRET further sup-ports the expansion of the IMAP technology fornon-protein kinases like glucose kinase and sphin-gosine kinases, emerging drug targets for whichIMAP is one of very few HTS-friendly technologies.

Phosphodiesterases (PDEs) are another hard-to-screen target class enabled through IMAP. Therecent availability of recombinant PDEs (EMDBiosciences) combined with IMAP’s high assayfidelity and throughput should accelerate drug dis-covery in this area.

MDS Pharma Services (www.mdsps.com) providesa comprehensive line of kinase assay services toaddress the in vitro and in vivo aspects of drug dis-covery screening and profiling. MDS offers a wideselection of kinases (greater than 170) in radiomet-ric, fluorescent and cell-based formats. Clients mayselect from its radiometric ‘gold standard’ assays(currently 155 kinase assays) with a turnaroundtime of two weeks or its FastKinase™ fluorescence

Figure 16Molecular Devices IMAP TR-

FRET using streptavidin fordetection. Enzyme assays for

Pim1 used 300nM PKCpseudosubstrate-derived

peptide (biotin-ERMRPRKRQGSVRRRV-NH2)and 100µM ATP, reaction time

1h at RT. FAM-Streptavidin(Anaspec) 30nM

0.001 0.01 0.1 10

100

200

300

400

500

0.3µM Biotin-substrate

Pim1 [U/ml]

corr

. T

R-F

RE

T/T

b *

10,

000

PO4

kinase

ATPfluorescent peptide substrate(Tyr, Ser or Thr)

phospho-peptideproduct

phosphatase

IMAP bindingreagent

PO4

PO4

MIII

lowTR-FRET

highTR-FRET

Tb-donor

sensitiser

MIII PO4

Tb

linker

Figure 15: Molecular Devices IMAP TR-FRET assay for kinases. When fluorescent substratesbecome phosphorylated products, they can bind to the trivalent metals on the IMAP bindingreagent that also bears a terbium donor. Energy transfer occurs between donor andfluorophore on the product



Screening

polarisation based platform of 94 kinases.FastKinase™ enables the rapid profiling of largernumbers of compounds and guarantees a five-dayturnaround time with the flexibility of single con-centration testing (in duplicate) or upfront IC50determination. In addition, PathTrak™ cellularsolutions assays enable clients to predict kinaseefficacy and selectivity in the native cellular envi-ronment. PathTrak™ detects phosphorylationstates of endogenuos intracellular proteins withoutthe need for over-expression. The assays measurechanges in phospho-protein levels due to com-pound mediated inhibition of kinase activity in sig-nalling pathways. MDS Pharma Services is work-ing on expanding its kinase testing services toinclude two new FastKinase™ assay packages(tyrosine kinase specific and kinome diversity pan-els), additional PathTrak™ assays, multiplexedkinase testing and high-content analysis.

Kinase activity measured using radiolabelled ATP, asused in Millipore’s (www.millipore.com)KinaseProfiler™ continues to be the gold standardto which non-radiometric formats are compared.However, rapid and cost-effective medium or large-scale profiling with this method is not always prac-tical. HTRF™ is the most widely used present-daykinase screening technology in biotech and pharma-ceutical companies and avoids difficulties associatedwith waste management of a radioactive assay for-mat. To address the need to profile large compoundlibraries and chemical arrays in a commonly usedformat, Millipore is introducing a new service:KinaseProfiler HTRF. The service is based on assaysdeveloped in partnership with Cisbio using onemonoclonal antibody for serine/threonine kinases

(STKs) and one for tyrosine kinases (TKs). To dateMillipore’s KinaseProfiler HTRF development teamhas validated assays for more than 80 STKs and 30TKs. Cisbio has demonstrated activity for an addi-tional 24 STKs and 30 TKs that are currently indevelopment to support screening services availableto Millipore’s clients. Figure 17 shows the inhibitiondata generated by 60 kinases in both the radiomet-ric and HTRF formats at 10mM ATP and 1mMstaurosporine. Figure 18 shows the inhibition dataof both formats in the presence of 30 kinaseinhibitors. Both graphs demonstrate the consistencybetween the two assay formats. Combined with fol-low-up IC50 determinations performed in the radio-metric assay, customers will have access to a com-prehensive solution for their large-scale kinase pro-filing needs. The new HTRF-based KinaseProfilerservice will be available for general access early this year.

PerkinElmer (www.perkinelmer.com) has recentlyintroduced LANCE® Ultra, enhanced LANCETR-FRET assays which utilise ULight™, a newlight-insensitive, small molecular weight red-shift-ed emission acceptor dye. ULight provides theability to directly label small molecules such aspeptides and cAMP. New products include bothspecific and generic kinase substrates and antibod-ies directly labelled with the ULight acceptor dye,and an array of Europium chelate donor dyelabelled anti-phospho-antibodies (Figure 19).PerkinElmer has also introduced a new collectionof SureFire™ cell-based kinase assays in collabo-ration with TGR BioSciences of Thebarton,Australia. These assays are based upon HTS-proven AlphaScreen® technology, enabling them

Figure 17: Kinase activity was measured at 10µM ATPand 1µM staurosporine. Each data point represents 1 of60 kinases tested in the Millipore’s KinaseProfilerradiometric (KP) and HTRF formats. Significancebetween the 2 data sets was p < 0.0001

Figure 18: BRSK2 activity was measured at 10µM ATPeach data points represents the % inhibition obtainedagainst 1 of 30 inhibitors tested in the Millipore’sKinaseProfiler radiometric (KP) and HTRF formats.Significance between the 2 data sets was p < 0.0001

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to work with very large molecules such as fulllength endogenous proteins expressed at physio-logically natural levels. AlphaScreen SureFireassays are suited for screening a wide range of tar-get kinases and detecting activated, phosphorylat-ed protein kinases in whole cells. UsingAlphaScreen SureFire technology, researchers caninterrogate a diverse range of targets such asGPCRs, RTKs, growth factor and cytokine recep-tors and receptors involved in the inflammatoryand stress response. Fully validated kits are avail-able for ERK 1/2 and MEK 1 in the ras/raf path-way, AKT and p70S6K in the PI3K pathway, andJNK, p38MAPK and STAT-3. AdditionalPerkinElmer product introductions include a newglutathione-coated donor bead. Utilised in con-junction with AlphaScreen acceptor bead offer-ings, researchers can screen full-length substratesexpressed as GST fusions without further need forchemical modification such as biotinylation. Thenew donor bead type greatly reduces the possibil-ity of biotin mimetic compound interference.

Drug screening against kinase targets is a difficulttask that typically involves several different assaysdepending on the kinases being screened. Havinga single detection platform to screen all kinasessimplifies the screening process significantly.Promega’s (www.promega.com) Kinase-Glo® PlusLuminescent Kinase Assay can be used to screenvirtually any kinase/kinase substrate combination.This homogeneous assay is performed in a singlewell of a 96-, 384- or 1536-well plate by adding avolume of reagent equal to the volume of a com-pleted kinase reaction and measuring lumines-cence. The luminescent format is preferred over

B. Wortmannin Titration in PI3-K with 10µM ATP Using KGP

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Figure 19: PerkinElmer LANCE Ultra assay principle

Figure 20: Panel A shows IC50 data for PI3-kinase (PI3-K), a lipid kinase, generated using Promega’s Kinase-Glo Plus with a variety of lipid substrates. Usingphosphoinositide as a substrate (Panel B), the PI3-K inhibitor Wortmannin was used to generate an IC50 curve


fluorescence because compound autofluorescenceis no longer an issue. Kinase-Glo Plus sensitivelymonitors the depletion of ATP in a kinase reac-tion. The luminescence-based assay format offersexcellent signal-to-background ratios, and rou-tinely delivers Z’ factor values >0.7. The assay canbe performed at ATP concentrations up to100µM. Custom formulations that are linear up to500µM ATP are also available. The Kinase-GloPlus Assay can easily be used to generate dosecurves without the need to specifically label pep-tides or use radioisotopes (Figure 20).

In vitro profiling of drug candidates is a substantialpart of all drug development projects. Compoundsat late stages of pre-clinical development especiallyrequire extensive profiling against growing panelsof targets and counter targets since the selectivityprofile of a lead compound is an important param-eter for decision processes regarding further actionsleading to clinical trials. Moreover, broad profilingof lead compounds allows the identification ofunknown targets and therefore sometimes mayopen insight on the molecular mechanism of a drugeffect. ProQinase’s (www.proqinase.com) novel invitro kinase profiling service FreeSelect is optimallydesigned for IC50 characterisation of a single testcompound. This service allows the customer toobtain broad IC50 data on a growing set of cur-rently 170 different recombinant protein kinases(Figure 21). Using a novel modular set-up, a robot-ic system performs highly reproducible IC50 meas-urements over a concentration range of 12 half-log-arithmic dilutions of a single compound against 32kinases in parallel. The technology is based on aradiometric kinase assay, which is still the goldstandard in kinase assay technology. This method isbased on the use of 33P-ATP in combination withscintillant-coated microtiter plates. In contrast toother in vitro profiling technologies that require dif-ferent assay conditions for different kinases,FreeSelect allows IC50 measurement under identicalassay conditions for all kinases.

SpinX Technologies (www.spinx-technologies.com)has developed a bench-top instrument (SpinX Lab)integrating liquid handling and fluorescent detec-tion to perform assays in nanolitre volumes (Figure22). The system uses disposable microfluidic cardswhich are combined into a 384-well microplate for-mat so that standard liquid handlers can be used toload the different assay components. Each assaycomponent is loaded on to a single well in concen-trated stock solution, preserving compound andreagent integrity. These wells act as input reservoirsleading into a network of microfluidic structureswhere, according to the user-defined protocol, thedifferent assay components are brought together toperform dilutions, set up the biochemical reactions,and read the result. The system is presently beingvalidated for Early Access sites using a variety ofhomogeneous enzymatic assays with fluorescentreadout. For kinase profiling, it offers the uniqueadvantage that any compound need only be loaded

Figure 22SpinX Technologies’ bench topinstrument the SpinX Lab

Figure 21: IC50 profile of akinase inhibitor with 141different recombinant proteinkinases. The data weregenerated with ProQinase’sFreeSelect service, which isoptimally designed for singlecompound IC50-profiling

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into a single well from which serial dilution in100% DMSO is performed in the microfluidicstructures, with the resulting solutions distributedto different assays in parallel, improving ease of useand precision. Current developments on the SpinXsystem address two emerging trends in the kinaseprofiling market: the growing importance of time-resolved fluorescence as a preferred assay formatand an increasing use of Ki determinations. Toaddress the former, SpinX is developing a time-resolved fluorescence module to complement itsprompt fluorescence readout. For the latter, thetechnology is uniquely suited to integrating allassay preparation steps required to enable automat-ed Ki as well as mode-of-action studies.

Cell-based high-content assays are most widelyapplied for secondary profiling of kinase activityafter the majority of compounds have been dis-missed. Their application in HTS poses significantchallenges mainly due to the low throughput ofexisting technologies. For high-content kinaseassays there is also the added complication thatfixation protocols required for immunodetectionare multi-step and time consuming. Laser-scan-ning fluorescence microplate cytometers, such asTTP LabTech’s (www.ttplabtech.com) theAcumen® eX3 combine the object-recognitioncapabilities of microscope-based CCD imagerswith the fast read speeds of bulk fluorescencereaders. This instrument has been applied to theprimary screening of kinases and their role in theregulation of the cell cycle. Where anti-phosphok-inase antibodies are employed, protein kinase acti-vation can be detected using single colour proto-cols by the emergence of fluorescence staining incells2. In contrast, methods using anti-kinase anti-

bodies or GFP-tagged kinases report activation byquantifying the translocation of enzyme withineach cell, most commonly from cytoplasm tonucleus (Figure 23). The whole well scanningcapability of microplate cytometers allows highcontent analysis of every cell in every well at highthroughput. This has many benefits for kinaseprofiling including overcoming the problems ofvariable stimulation and random cell distributionoften observed following immunostaining.Additionally, whole well scanning enables normal-isation of biological responses to total cell number,offering a simple toxicity or proliferation readoutwith every test. Additionally, the introduction ofmultilaser microplate cytometers further extendsthe range of fluorescent probes and proteins thatcan be combined in multicolour, multiplexed assayprotocols for kinase profiling.

SummaryWhereas wider coverage of the kinome in terms ofdiversity was the principal focus of kinase profilersa few years ago1, the following trends have nowassumed amplified significance among vendor’sproduct offerings:1 Increasing use by fee-for-service providers ofautomated and industrialised approaches to kinaseprofiling and screening, offering partners compre-hensive solutions ranging from a few lead com-pounds to large-scale kinase profiling, all withenhanced turnaround times.2 Growing importance of time-resolved fluores-cence as a preferred assay format.3 Wider pursuit of universal assay approaches thatextend the breadth of single assay systems, allow-ing more kinases to be screened under identicalassay conditions.

Figure 23Virtual well view in TTPLabTech’s Acumen eX3

software showingtranslocation of a GFP-tagged

protein kinase from cytoplasmto nucleus. A: (left panel) non-translocated control; B: (right

panel) translocated

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4 Increased availability of assays that measure theaccumulation of ADP, a product of kinase enzymeactivity (as opposed to methods that focus on thedepletion of ATP).5 Greater emphasis on screening inhibitors thatbind to inactive and low activity kinases.6 Greater diversity of cellular kinase approaches,including compound mediated inhibition of kinaseactivity in signalling pathways, which enable theprediction of kinase efficacy and selectivity in thenative cellular environment.7 Emergence of several bench-top instrumentturnkey solutions based on microfluidic approach-es that greatly simplify IC50 and Ki determinations.8 Growing awareness of the potential of label-freeapproaches to address kinase assay deficiencies.

Overall the end user is spoilt for choice with therange of alternative offerings, methodologies and

approaches currently available for kinase screeningand profiling. DDW

Dr John Comley is Managing Director of HTStecLimited an independent market research consultancywhose focus is on assisting clients delivering novelenabling platform technologies (liquid handling, lab-oratory automation, detection instrumentation andassay reagent technologies) to drug discovery. Since itformation in 2003, HTStec has published 22 marketreports on drug discovery technologies and DrComley has authored 16 review articles in DrugDiscovery World. Further information on accessingthe market report ‘Kinases Screening & ProfilingTrends 2006’ can be obtained by visitingwww.htstec.com or by emailing [email protected] to receive a free copy of the Report’sExecutive Summary and Table of Contents.

References1 Comley, J (2004). Expandingthe profile of kinase panels.Drug Discovery World, 5 (4):45-56.2 Wedge, SR et al (2005).AZD2171: A Highly Potent,Orally Bioavailable, VascularEndothelial Growth FactorReceptor-2 Tyrosine KinaseInhibitor for the Treatment ofCancer. Cancer Research, 65:4389-4400.

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