Two GPCRCase Studies with the IP-One Euand Tb Assay Kits · Two GPCRCase Studies with the IP-One...

Two GPCR Case Studies with the IP-One Eu and Tb Assay KitsA Positive Modulator and an Inverse Agonist Project

Martin Graf, F. Hoffmann-La Roche

2SBS 2008, CisBio Tutorial

Nutley

Palo AltoBasel

Penzberg

The Pharma Research Sites of Roche


Dyslipidemia VascularDiseases

Obesity

Diabetes

Discovery Research at Roche BaselVascular & Metabolic Diseases


Discovery Research at Roche BaselNeurosciences

Bipolar DisorderAlzheimer‘sDisease

Depression


IP-One Assay Principle

cell lysis

anti-IP1

agonist

Gq

IP3

membraneGPCRGPCR

IP1

d2

PLCPIP2

IP2

IP1

myo-Inositolinhibition by LiCl

Ca2+IP1

Accumulated IP1displaces IP1-d2,

reduction of FRET signal

Eu3+

337 nm

665 nm

FRET

620 nm


IP1 standard curve & agonist dose response

10000*620

665

nm

nm

RFU

RFURatio =Calculations: 100*(%)

neg

negsample

Ratio

RatioRatioDeltaF

−=

Compound [nM]

ratio

IP1 standard curve EC50 = 320 nM

Compound [nM]

IP1[nM]

agonist

agonist


GPCR1 - Positive Modulator Project

GPCR2 - Inverse Agonist Project

First Results with the Terbium Kit

Summary and Conclusion


Screening Cascade – GPCR1

Positive Modulator Project

Primary Screening

Hit Confirmation / Selectivity

Hit Validation

Intracellular Ca 2+ Measurement

(FLIPR)

IP-One Assay


Agonist Shift Assay


-20

0

20

40

60

80

100

120

140

0.01 0.10 1.00 10.00 100.00

[Agonist, uM]

PC

T_C

TR

L

Agonist alone

Agonist + 10µµµµM Compound

Agonist Shift = pEC50Agonist – pEC50Agonist+Compound


0.01 0.1 1 10 100

20

60

100

140

180

220

260

300

340

Significance of the Agonist-Shift

0

2

4

6

8

10

12

14

-0.30 -0.25 -0.20 -0.15 -0.10 -0.05 0.00 0.05 0.10 0.15 0.20 0.25 0.30 0.35

shift

#

σ=0.12

shifts larger than 2σ = 0.24 are significant

Overlay of 9 Agonist Dose Response Curves

IP1[nM]

Agonist [nM]

Histogram of the shifts

IP-One: pEC50Ago = 8.22 +/- 0.09 (n=9)

FLIPR: pEC50Ago = 8.66 +/- 0.13 (n= 320) (FLIPR 2σ = 0.37)


Agonist Shift Results of Some Hits

Agonist alone

Agonist + 10µM Compound 1Agonist + 30µM Compound 1

pEC50Agonist = 8.33

Shift at 10µµµµM = -0.15

Shift at 30µµµµM = -0.35**

Comparison:

Agonist Shifts FLIPR vs. IP-One

-0.32-0.40Cmpd 3

+0.34-0.39Cmpd 2

-0.15-0.36Cmpd 1

Shift IPone

Shift FLIPR@ 10uM

Agonist [nM]

IP1[nM]

Validation in 2nd assay is important


Screening Cascade – GPCR2

Inverse Agonist Project

Primary Screening

Hit Confirmation

Hit Validation

Fluorescence Polarization

Binding Assay

IP-One Assay to discriminate:- Agonists- Antagonists- Inverse Agonists


Optimization of Agonist and inverse Agonist Mode (1)

Compound [nM]

ratio

Compound [nM]

IP1[nM]

Agonist

Agonist

Inverse Agonist

IP1 standard curve

Inverse Agonist

Conditions: 7’500 cells/well, 2h incubation

constitutive activity


Optimization of Agonist and inverse Agonist Mode (2)

Compound [nM]IP1[nM]

Compound [nM]

IP1[nM]

Best Condition for Agonist Mode: 7’500 cells/well, 1h incubation

Best Condition for inverse Agonist Mode: 7’500 cells/well, 4h incubation

Agonist:EC50 = 10 nM

Agonist:

EC50 = 8.5 nM

Inverse Agonist:EC50 = 105nM


Cell Number and Incubation time Optimization

in order to cover dynamic range of the standard curve


Agonist (nM)0.001 0.1 10 1000

IP-O

ne

(nM

)

0

200

400

600

800

1000

1200

1400

1600

1800

2000

2200

Comparison of an Antagonist and an Inverse Agonist

Agonist Dose Response+ Antagonist+ inverse Agonist

Inverse Agonist (nM)0.001 0.1 10 1000

IP-O

ne

(nM

)

0

200

400

600

800

1000

1200

1400

1600

1800

2000

2200

Antagonist (nM)0.001 0.1 10 1000

IP-O

ne

(nM

)

0

200

400

600

800

1000

1200

1400

1600

1800

2000

2200

EC80 agonist + Antagonist

Antagonist only

EC80 agonist + inverse Agonist

inverse Agonist only


0

5000

10000

15000

20000

25000

30000

35000

40000

45000

50000

290 300 310 320 330 340 350 360 370 380 390

Wavelength (nm)

Mo

l. E

xtin

c. C

oef

. (M

-1.c

m-1

)

Europium Cryptate Lumi4-Tb Cryptate LanthaScreen Terbium Chelate

Terbium Cryptate

Europium Cryptate

Excitation Spectrum Europium and Terbium

YAG-Laser (355nm)


plate::explorer system and plate::vision reader

• Co-development Carl Zeiss Jena and F. Hoffmann-La Roche • Support and Maintenance now by Perkin Elmer


Tb and Eu - different platesStandard curves measured on plate::vision reader

IP1 [nM]

Delta F %

Terbium:EC50IP1 = 83 nM

Europium:EC50IP1 = 160 nM

Tb vs Eu on plate::vision Tb in different plates on plate::vision

IP1 [nM]Delta F %

Corning black clear bottom (3712): EC50IP1 = 83 nM

Corning white clear bottom (3707): EC50IP1 = 97 nM

Greiner white clear bottom (780098): EC50IP1 = 98 nM


Different Readers

plate::vision (PerkinElmer) EC50IP1 = 83 nM

NanoScan (IOM) EC50IP1 = 130nM

EnVision (PerkinElmer) EC50IP1 = 96 nM

Tb standard curves in different readers

IP1 [nM]

Delta F %


Cellular Assay with Terbium Assay Kit GPCR2 – inverse agonist project – comparison with Eu

Compound [nM]IP1[nM]

7’500 cells/well, 4h incubation,Plate: 384 white Greiner 781080,

Discovery reader

Agonist:

EC50 = 8.5 nM


Terbium Kit Europium Kit

7’500 cells/well, 4h incubation,Plate: 384 black Corning 3712,

Plate::vision reader

Agonist:

EC50 = 6.9 nM


Optimized inverse agonist conditions

Compound [nM]

IP1[nM]


Summary - Conclusion

Cisbio IP-One Kit

� simple to use and robust

� cell concentration / incubation time needs to be optimized

� GPCR 1: Highly reproducible assay is crucial for optimization of positive modulator

� GPCR 2: Profiling and mode of action: agonist, antagonist, inverse agonist

Terbium Reagents

� Spectral properties fit very well with plate::vision reader

� 2-4 times more sensitive than Eu-assay

� enables us to use IP-One in primary screening


Acknowledgments

Hoffmann-La Roche, Basel

• Michel Dietz

• Thilo Enderle

• Doris Roth

• Ramona Schäfer

• Veronique Schirmer

Cisbio

• Stéphane Martinez

• Jean-Luc Tardieu

• Eric Trinquet


We Innovate Healthcare

Two GPCRCase Studies with the IP-One Euand Tb Assay Kits · Two GPCRCase Studies with the IP-One...

Documents

Transcript of Two GPCRCase Studies with the IP-One Euand Tb Assay Kits · Two GPCRCase Studies with the IP-One...