INVESTIGATING KINASE ACTIVITY IN A CELLULAR

CONTEXTE.TrinquetVP Product DevelopmentSLAS meeting, Cisbio Tutorial January 2013, Orlando

Protein phosphorylations are key signalling events

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Phospho-proteins

Signalling cascades

Cellular responses(proliferation, cell death…)

Dysregulationmutation

Diseases(cancers, inflammation…)

Common techniques to monitor protein phosphorylation

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Western Blot

ELISA ELISA-like techniques

Homogeneousbead-based techniques

Cell lysates

A straightforward HTRF® assay platform to monitor proteinphosphorylation using adherent or suspended cells

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HTRF assays are fast & convenient compare to othertechniques

Mix & Read

Time & Labor Saving

BetterRobustness

HTRF® & WB are well correlated, but HTRF® oftendisplays a superior sensitivity

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39 19 9.6 4.8 2.4 1.2 0.6 0.3 0.150

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X 103 cells

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MEK1 (45kDa)

Western blot (ECL reagents)

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Phospho-MEK1

HTRF Phospho-MEK1 signal (Delta F%)

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EC

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Phospho-MEK1 (Hela cells)Sensitivity comparison

(cells / well)

Cellular kinase HTRF Western-blot

P-ERK 200 1960

P-AKT 195 3125

P-Stat3 750 750

P-p38 MAPK 1250 10000

P-IKKb 580 46500

P-mTOR 1250 1250

P-CREB 6250 6250

P-MEK1 600 2400

P-MEK1/2 1200 1200

P-JNK 3120 3120

P-c Jun 2500 20000

HTRF® & ELISA show similar results & sensitivity

Experimental conditions Assay Window Compound EC50 (M)

Cellular kinase assay Cell line Compound HTRF ELISA HTRF E LISA

P-Stat3 (Y705) Hela IFNa 7.7 5.3 10.7E-10 5.9E-10

P-p38 MAPK (T180-Y182) Hela anisomycin 10.8 3.5 7.6E-8 8.7E-8

P-IKKb (Ser177/181) Hela IL1b 3.5 3.6 2.48E-11 3.58E-11

P-mTOR (S2448) HEK293 rapamycin 2.4 2.2 5.5E-6 4.7E-6

P-CREB (S133) HEK293 Forskolin 4.1 4.6 1.4E-6 1.1E-6

P-MEK1 (S217/S222) Hela EGF 3.4 2.8 7E-10 1E-9

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Phospho-IKKb (Hela cells)

HTRF® is the most sensitive homogeneous technique to monitor protein phosphorylation

Experimental conditions Assay Window Compound EC50 (M)

Cellular kinase assay Cell line Compound HTRF Surefire® H TRF Surefire®

P-Stat3 (Y705) Hela IFNa 7.7 3 10.7E-10 1.3E-9

P-p38 MAPK (T180-Y182) Hela anisomycin 10.8 13.5 7.6E-8 6.0E-8

P-IKKb (Ser177/181) Hela IL1b 3.5 1.4 2.48E-11 1.79E-11

P-mTOR (S2448) HEK293 rapamycin 2.4 1 5.5E-6 -

P-CREB (S133) HEK293 Forskolin 4.1 2.5 1.4E-6 3.6E-9

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Surefire signal

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Surefire signal

Phospho-IKKb (Hela cells)Phospho-p38 (Hela cells)

High reagents quality enables long-term programs

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Batch to batch reproducibility(Phospho-P38 – HeLa cells)

Reagent stability

Freeze/Thaw tolerance(Phospho-IKKb – HeLa cells)

Long term stability(Phospho-mTor – HEK293)

No Lys

ate 1/8

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+3 Months

QC after production

+ 6 Months

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Cell background compatibility

• HTRF® assays are usually compatible with human, mouse & rats cell lines

• HTRF® assays can work either with adherent or suspended cells

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log [Erlotinib] M

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log [FSK] M

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P-CREB Human cells (A431)

P-CREB Mouse cells (NIH3T3)

P-CREB Rat cells (PC12)

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log [Anisomycin] (M)

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log [IL1-beta] (M)

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P-JNK Adherent cells (HEK293)

P-JNK Suspended cells (Jurkat)

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Flexibility in assays format

Photo courtesy of Greiner Bio-One

384 sv well20µl

96 well50µl

Phosphorylation StepVolume of cell lysates

Detection StepVolume of detection

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Flexibility in assays format

Photo courtesy of Greiner Bio-One

96 Half well40µl

384 sv well20µl

1536 well5-10µl

96 well50µl

6 well500µl

24 well250µl

T1753-5ml

T751.5ml

T250.5ml

Ø10cm1.5ml

Ø3.5cm500µl

384 well20µl

Phosphorylation StepVolume of cell lysates

Detection StepVolume of detection

Assay robustness

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Assay equilibrium

Assay equilibrium + 20H

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Assay equilibrium

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P-STAT3 assay on HeLa Cells

HTRF Surefire®

• HTRF® signal shows no degradation over time

• HTRF® allows multiple readouts

HTRF® assays are amenable to HTS

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Phospho-MEK1Jurkat Cells – 20kcells/well

• Flexible use of HTRF kits due to high reagent quality

• Cell background compatibility

• Flexibility in assays format

• Assay robustness

• HTS amenable

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HTRF® Phospho assay main feature allow their use under a large variety of formats for drug research

Total protein detection is now possible with the sameplatform

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• Accurate determination of compound MOA- Assessment of protein expression- Assessment of protein phosphorylation

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Total P

roteinH

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Phospho & Total ERKA431 cells – 5’ EGF

Phospho & Total protein detection can be done with the same cell lysate

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HTRF® is as sensitive as WB to monitor total protein level

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StimulatedUnstimulated

Blk 586 1172 2344 4688 9375 18750 37500 75000 1500000

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StimulatedUnstimulated

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Phospho-ERK 1/2 - A431

Cells/well

Total ERK 1/2 - A431

Sensitivity comparison(Number of cells)

Target HTRF WB

Stat3 1172 1172

MEK1 9375 4688

ERK 1/2 1172 4688

Total proteins can be quantitatively detected in severalspecies

• Validated on human, mouse, rat and hamster cell lines

• Quantitative protein detection

Negative A431 Hela NIH3T3 CHO-K1 HEK293 Jurkat0

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Total ERK

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S/B

HTRF® assay are available for a large set of targets

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Phospho-protein Total protein

Phospho-Erk1/2 (Cellul’erk) Total ERK1/2

Phospho-AKT (Ser473)

Phospho-AKT (Thr308)

Phospho-JNK (Thr183/Tyr185)

Phospho-c Jun (Ser63)

Phospho-STAT3 (Tyr705) Total-STAT3

Phospho-p38 MAPK (Thr180/Tyr182)

Phospho-IKKb (Ser177/181)

Phospho-mTOR (Ser2448)

Phospho-CREB (Ser133)

Phospho-EGFR

Phospho-MEK1 (Ser218/222) Total-MEK1

Phospho-MEK1/2 (Ser218/222)

• Mix & Read solutions

• Faster & equally or more sensitive than othertechniques

• Cost efficient

• Amenable to all steps of drug research

Cisbio’s experts can design a custom assay for yourtarget of interest

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• Fast assay development

• More than 25 custom assays developed in 2012

• Success rate in 2012: 90%

Assay optimization

Assay transferand/or

implementationwith dedicated

expert

Confidentialityagreement

Paperstudy

Go / No Go

Assayfeasibilitystudy

Best pair analysis

Assay report

Reagent sourcing& labeling

Go / No Go

Data review

Custom kit supplies

Thank you