PhosTRAKPhosTRAK™™: A Homogeneous: A Homogeneous3-Minute Phosphatase Assay3-Minute Phosphatase Assay

Michael J. Corey, Ph.D.

Current Affiliation:

CellExSys, Inc.

How Shall We Measure How Shall We Measure Phosphatase Activity?Phosphatase Activity?

• Detect dephosphorylated product– There’s no good, fast way to do

this

– Usually complex operationally

– Tends to be expensive

– Unnatural substrates--easy to run, difficult to interpret

• Detect free phosphate

– Malachite Green assay is slow, insensitive

– Other methods are operationally complex

– Background is a problem

PhosTRAK PhilosophyPhosTRAK Philosophy

• Time, space, and labor are valuable

• Instrument time is valuable

• Statistical significance is predictive

• Natural substrates are good

• Two steps are sometimes one too many

Side-by-Side Comparison of Side-by-Side Comparison of PhosTRAK and Malachite GreenPhosTRAK and Malachite Green

PhosTRAK Malachite Green

Limit of Detection 3-10 pmol 40-125 pmol

Assay time 3 minutes 20-90 minutes

Operationalcharacteristics

Mix, read Mix, wait, add reagent, wait, read

Phosphate Detection by PhosTRAKPhosphate Detection by PhosTRAK

1 10 100 1000 100000.1

1

10

100

1000

Phosphate (pmol)

RLU/Second

Homogeneous PhosTRAK Assay ofHomogeneous PhosTRAK Assay of-Phosphatase: Substrate Dependence-Phosphatase: Substrate Dependence

0 20 40 60 800

25

50

75+ phosphatase- phosphatase

[Casein] (μg/mL)

RLU/Second

Detection of Calcineurin Activity by Detection of Calcineurin Activity by PhosTRAK (Timepoint Assay)PhosTRAK (Timepoint Assay)

0 1 2 3 40

1

2

3

4

[Calcineurin] (Units)

RLU/second

60’ reaction, 30oC

Inhibition of Calcineurin by Autoinhibitory Inhibition of Calcineurin by Autoinhibitory Peptide (Measured with PhosTRAK)Peptide (Measured with PhosTRAK)

Inhibition of Calcineurin byAutoinhibitory Peptide

(Measured with PhosTRAK)

0 10 20 300.0

0.5

1.0

1.5

[Autoinhibitory Peptide] (μM)

RLU/Second

15’ preincubation, 17’ reaction

Autoactivation of Native Calcineurin Autoactivation of Native Calcineurin Observed by Homogeneous PhosTRAKObserved by Homogeneous PhosTRAK

No Enzyme +Native Bovine Calcineurin

CD45 Substrate DependenceCD45 Substrate DependenceMeasured by Homogeneous PhosTRAKMeasured by Homogeneous PhosTRAK

0 25 50 75 100 125 1500

2500

5000

7500

Literature Km = 14 μMMeasured Km = 22 μM

[Fisher Phosphopeptide] (μM)

RLU/Second

LAR Phosphatase Substrate Dependence LAR Phosphatase Substrate Dependence (Homogeneous Assay)(Homogeneous Assay)

LARP

0 25 50 75 100 125

0

2

4 no LARP

1.4 U LARP

[Phosphopeptide] (μM)

RLU/Second

PhosTRAK Operational PhosTRAK Operational CharacteristicsCharacteristics

• Start to read immediately after initiation (3 minutes plenty)

– Today’s luminometers can read a plate in 2 seconds

• Cocktail

– On ice, full cocktail slowly converts dynamic background to static (Pi to ATP)--otherwise fine for two hours

– Stable if enzyme cocktail is withheld and added before assay

• Data reduction

– Linear fit if you don’t have automatic injector

– Single read fine if you do

• Waste--no problem

Challenges and DrawbacksChallenges and Drawbacks

• Phosphate contamination of G3P is responsible for most of the background--should be an easy separation

• Must avoid other sources of phosphate

• Molecules that inhibit the coupled enzymes can yield false hits

• Inhibitor and validation data are lacking

PossibilitiesPossibilities

• Use in HTS for phosphatase inhibitors--targeted libraries appear especially suitable

• (Hypothetical) Measurement of T-cell activation, using either CD45 or calcineurin

• Phosphate monitoring in various systems

SummarySummary

• PhosTRAK is a 3-minute homogeneous phosphatase assay suitable for use in HTS with many protein phosphatases

• Based on phosphate detection; uses natural substrates

• No waste issue

• Requires luminometer

AcknowledgmentsAcknowledgments

• Eva Corey– POWERPOINT EXPERT– (Also Assistant Professor in Urology at UW)

• Bob Kinders (Alidex, Inc.) and Bob Vessella (UW) for facilities and advice

Contact InformationContact Information

Michael J. Corey

CellExSys, Inc.1100 Olive Way, Suite 100Seattle, WA 98101

206-521-4846

Email: coreym@targen.com