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Novartis Institutes for Novartis Institutes for BioMedicalBioMedical ResearchResearchRochdi BouhelalSBS Sept. 2005

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A new HTRF inositol A new HTRF inositol phosphate assay to phosphate assay to monitor Gmonitor Gqq coupled coupled GPCRs responsesGPCRs responses

— Comparative study calcium mobilisation / IP1

Ina Hammerl, Stéphane Martinez, Monique Amoravain and Rochdi Bouhelal

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GPCRs: A universal communication systemGPCRs: A universal communication system

α

β

γEffectorsystem

PeptidesMonoamineslipids Photons

Protons (e.g.OGR1)Ions (Ca2+)

Signal Transduction mechanisms — cGMP PDE, — Adenylate cyclase (cAMP) & Protein kinase A — Phospholipase C , IP3 , calcium & Protein kinase C,— MAP kinase pathways, — Ion channel channels conductance

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— Several assay formats exist to monitor all events in the GPCR activation cascade with a high throughput.

— While such assays to monitor cAMP and calcium levels were developed the past 10 years and are now widely used in the pharmaceutical industry, current IP technologies are limited by their low throughput and safety issues.

— Need of assays amenable to HTS or MTS in the lead discovery process.— Recently, a homogeneous HTRF assay was developed by Cisbiowhich measures IP1 the last component of the PIP2 degradation pathways. — A study was initiated at Novartis with the objective to validate this novel assay format and to evaluate its usefulness in our discovery processes.

Need for a HTS assay format for IP formationNeed for a HTS assay format for IP formationNeed for a HTS assay format for IP formation

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Study descriptionStudy descriptionStudy description

— The assay was assessed in various cell systems HEK293 and CHOK1 cell expressing native GPCRs CCL39 cell expressing the recombinant human parathyroid calcium sensing receptor

(HupCaR).

— Pharmacological characterization of GPCRs with agonists and antagonists. — In addition, Novartis libraries are tested in the HupCaR calcium mobilization assay using the FLIPR technology and the HTRF IP1 accumulation assay using an imaging reader (Viewlux)—Aim: Comparison of hit rates obtained in the two assays and the suitability of the IP1 assay as a primary or secondary assay for GPCR screening.

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HTRF readout

γ

PLCß

αq β

PIP3

DAG +

γαi

β γαs β

α*q

ACαi* αs*

ATP

- +

cAMP

IP3

OHOHHO

HOHO

OPO3H

OHOHH2PO3O

H2PO3OHO

OPO3H2

IP1Ino

sitol

phos

phate

brea

kdow

n casca

de

IP2O

PO3H2HO

HOH2O3P O

OHOH

Inhibitionby LiCl

Gq signaling pathways

OHOHHO

HOHO

OH

IP3

Ca++ FLIPR readout (Fluo-4)

HTRF readout

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GPCR

OHOHHO

HOHO

OPO3H

IP1

FRET

Cell Lysis + HTRF reagents

NO FRET

IP1

IP1

•A fluorescent analog of IP1•A monoclonal antibody against IP1

IP1

IP1

basedbased on unique on unique andandproprietaryproprietary reagentsreagents

IPIP--oneone assayassay principleprinciple

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Methods: HTRF & Calcium assays

Cell seeding— 10 000 cells/well/50 ul media in a white tissue culture treated 384 MTP — 24 h incubation at 37 °C and 5 % CO2

Cell stimulation— Cell media removal — Cells stimulation with agonists / compounds— diluted in HBS Buffer with (LiCL 50 mM)

for 30 min— addition of HTRF reagents and

incubation for 1 h

HTRF Reading:— read plate in Viewlux at 665 and 620 nm

Cell seeding• 10 000 cells/well/50 ul media in a black tissue• culture treated 384 MTP with clear bottom.• 24 h incubation at 37 °C and 5 % CO2

Cell loading:• After removing cell media, load cells with Fluo4 • incubate 1 h at 37°C and 5 % CO2

Cell washing:• Remove remaining Fluo4 by washing plates

Cell stimulation & Calcium Reading• Read plate in FLIPR at 525 nm duringcompound injection

HTRF / IP1 FLIPR Fluo4 calcium

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Endogenous P2YR and M3R in HEK293 cellsEndogenous P2YR and M3R in HEK293 cellsEndogenous P2YR and M3R in HEK293 cells

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Endogenous Muscarinic M3R and P2YR in HEK293 cells

1E-3 0.01 0.1 1 10 100 10000.0

0.5

1.0

1.5

2.0

2.5

3.0 Carbachol EC50=2.08 µΜ ATP EC50=0.41 µΜ

Cal

cium

(nor

mal

ised

fluo

resc

ence

)

[Agonist] µM0.01 0.1 1 10 100 1000

8000

9000

10000

11000

12000

13000

14000

15000

16000

Carbachol EC50 = 17.4 µMATP EC50 = 6.8 µM

Rat

io 6

65 n

M /

620

nM

[Agonist] µM

— Carbachol and ATP are more potent in the calcium assay— Curve shift factors 8 and 16 for carbachol and ATP— Differences between efficacies in the two assay systems are also reflected.— Calcium mobilisation assay is more sensitive (amplification step)in particular with low receptor expression (endogenous receptors)

Agonist effectsHTRF / IP1 FLIPR Fluo4 calcium60,000 cells, 24 h 10,000 cells, 24h

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Endogenous Muscarinic M3R in HEK293 cells

1E-3 0.01 0.1 1 10 100 1000 10000

0.0

0.5

1.0

1.5

Cal

cium

(nor

mal

ised

fluo

resc

ence

)

IC50 = 0.13 nΜ

[Atropine] nM0.01 0.1 1 10 100 1000 10000

8000

9000

10000

11000

12000

13000

14000

15000

16000

Rat

ion

665

nm /

620

nm

[Atropine] nM

IC50 = 9.7 nΜ

— Atropine potency higher in calcium assay (75 fold)— Reflects differences of agonist concentrations and receptor number used

Carbachol 100 µM in IP1 / 15 µM in Calcium 60,000 cells in IP1 / 10,000 in calcium

Conclusion: No major difference in antagonist potency expected

100 mM Carbachol 15 mM Carbachol

HTRF / IP1 FLIPR Fluo4 calcium

Antagonist effects

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Endogenous P2Y receptors in CHOK1 cells: Agonist effects

1E-4 1E-3 0.01 0.1 1 10 1000.0

0.5

1.0

1.5

2.0

2.5

3.0

Cal

cium

(nor

mal

ised

fluo

resc

ence

)

ATP EC50=0.42 µΜ UTP EC50=0.36 µΜ

[Agonist] µM

1E-3 0.01 0.1 1 10 100 1000

12000

13000

14000

15000

16000

ATP EC50 = 1.6 µM UTP EC50 = 2.1 µMR

atio

665

nm

/ 62

0 nm

[Agonist] µM

HTRF / IP1, 60,000 cells

FLIPR Fluo4 calcium, 10,000 cells

25.05.0525.05.05

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Recombinant human calcium sensing receptor(HupCaR) in CCL39 cells

Recombinant human calcium sensing receptorRecombinant human calcium sensing receptor((HupCaRHupCaR) in CCL39 cells) in CCL39 cells

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The parathyroid calcium sensing receptor The parathyroid calcium sensing receptor The parathyroid calcium sensing receptor

— A Gq coupled receptor highly expressedIn the parathyroid gland— Uses circulating calcium as an “agonist”— Can be blocked by allosteric

negative modulators— Controls PTH release— Role in bone formation

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0.1 1 10

0.0

0.5

1.0

1.5

2.0

2.5

3.0

Calcium EC50=1.41 mM

Cal

cium

(nor

mal

ised

fluo

resc

ence

)

[Calcium] mM0.1 1 10 100

4000

5000

6000

7000

8000

Rat

io 6

65 n

M /

620

nM

[Calcium] mM

EC50 = 2.9 mM

10000 cells/well, 08.07.0510000 cells/well, 08.07.05

HTRF / IP1 FLIPR Fluo4 calcium

Effect of calcium on IP1 & Calcium mobilisation

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HupCaR: Evaluation of HTRF IP1 assay parameters: cell density

0.1 1 10 100

6000

8000

10000

12000

14000

16000

Rat

io 6

65 n

m /

620

nm

[Calcium] mM

60 000 cells/well EC50 = 5.5 mM 40 000 cells/well EC50 = 5.8 mM 20 000 cells/well EC50 = 5.7 mM 10 000 cells/well EC50 = 4.8 mM

0.1 1 10 1006000

8000

10000

12000

14000

Rat

io 6

65 n

m /

620

nm

[Calcium] mM

40 000 cells/well EC50 = 7.7 mM 30 000 cells/well EC50 = 7.0 mM 20 000 cells/well EC50 = 6.2 mM 10 000 cells/well EC50 = 5.2 mM

Cell growth in plate over 24 h Cell growth in plate over 48 h

— Potencies unchanged— Cell density can be reduced to 5000-10000 cells /well over 24 or 48 h

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HTRF signal stability over timeHTRF signal stability over timeHTRF signal stability over time

0.1 1 10 1004000

5000

6000

7000

8000

9000

10000

[Calcium] mM

Rat

io 6

65 n

M /

620

nMIP1 detection over 24 h

Immediate measurement EC50 = 3.6 mM after 24 h EC50 = 3.4 mM

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Antagonist effects in the HTRF & Calcium assays

1E-4 1E-3 0.01 0.1 1 10 100

0.0

0.5

1.0

1.5

2.0

2.5

Compound 1 IC50=1.11 µM Compound 2 IC50=0.31 µM Compound 3 IC50=2.12 µM Compound 4 IC50=1.36 µM Compound 5 IC50=1.08 µM Compound 6 IC50=0.66 µM Compound 7 IC50=2.66µM

Fluo

resc

ence

dF/

F

[Antagonist] µM

3.06 ± 1.141.88 ± 1.137

1.32 ± 1.432.51 ± 1.876

1.32 ± 1.04 0.61 ± 0.215

1.98 ± 0.542.91 ± 0.814

1.75 ± 0.841.23 ± 0.383

0.66 ± 0.511.00 ± 0.672

0.74 ± 0.320.58 ± 0.021

IC50 (µM) n =3

Calcium (FLIPR) IP1 (HTRF) Compound ID

1E-4 1E-3 0.01 0.1 1 10 1005000

5500

6000

6500

7000

7500

8000

Rat

io 6

65 n

m /

620

nm

[Antagonist] µM

Compound 1 IC50=0.60µM Compound 2 IC50=0.24µM Compound 3 IC50=1.46µM Compound 4 IC50=2.56µM Compound 5 IC50=0.83µM Compound 6 IC50=0.45µM Compound 7 IC50=3.18µM

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1 10 100

-0.20.00.20.40.60.81.01.21.41.61.82.0

RKL001 10 µM EC50 = 3.0 mM 3 µM EC50 = 2.3 mM 1 µM EC50 = 1.9 mM 0.31 µM EC50 = 1.8 mM 0.10 µM EC50 = 1.4 mM 0.031 µM EC50 = 1.3 mM 0.001 µM EC50 = 1.1 mM Control EC50 = 1.3 mM

Nor

mal

ized

Flu

ores

cenc

e dF

/F

[Calcium] mM

1 10 100

6500

7000

7500

8000

8500

9000

9500

10000

10500RKL001

10 µM EC50 = 3.0 mM 3 µM EC50 = 6.0 mM 1 µM EC50 = 5.3 mM 0.31 µM EC50 = 4.7 mM 0.10 µM EC50 = 4.7 mM 0.031 µM EC50 = 4.7 mM 0.001 µM EC50 = 4.2 mM Control EC50 = 4.5 mM

Rat

io 6

65 n

m /

620

nM

[Calcium] mM

Antagonist effects: RKL001

— Varying blocker concentration depresses maximal activity without majochanges in calcium EC50

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IP1 in HTSIP1 in HTSIP1 in HTS

Study aim

— Use the IP one assay in a productive screening campaign— Comparison to calcium mobilization screen— 7744 natural product tested

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TimeBaseline measurement

Agonist Addition

Antagonist FLIPR assay set-up

Offline addition of antagonist

FLIPR measurement

Compound Addition orbuffer control

FLIPR assay types: Data AnalysisFLIPR assay types: Data Analysis

— Fbasal or Fb = Fluorescence before agonist injection — Fmax = Fluorescence maximum or peak— dF/F = Normalised Fluorescence = (Fmax - Fb) / Fb

— Fb sample/average Fb controls = high ratio indicates autofluorescence/ toxicity of compound

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FLIPR data handling for antagonist assaysFLIPR data handling for antagonist assaysFLIPR data handling for antagonist assays

Fb

Fmax

Agonist = calcium

Two values of fluorescence calcium responses are exported

Fb corresponding to the value prior to agonist injection, Fmax, the fluorescence at the signal peak.

From these the values two parameters were then calculated and exported Calculate dF/F = Fm – Fb / Fb for High controls H, Low controls (L, Buffer) and Samples (S).Activity (A) expressed as a percent of the maximal stimulation induced by agonist .

— A (%) = (S-H) /(H-L)* 100 — A quality parameter (Rb) which indicates whether the compounds acted through a physiologically relevant mechanism)

Rb = Fb,s / Fb,H— This parameter is used to reject compounds showing a sustained activity (toxicity / autofluoresence)

Control

Fluorescent compounds

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Calcium receptor HupCaR miniscreen - HTRF & Calcium assayCalcium Calcium receptorreceptor HupCaRHupCaR miniscreenminiscreen -- HTRF & Calcium HTRF & Calcium assayassay

-40 -20 0 20 400

500

1000

1500

2000

2500

3000

3500

4000

4500

IP1 Ca2+

Freq

uenc

y

Bin

Analysis of 7744 compounds from Napr collectionin IP1 and Calcium measurement

28375075165Ca++

122417IP1Hit number

-70-60-50-40-30Threshold

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MiniscreenMiniscreen: Data quality: Data quality

1.30.48 IC50, Anta [µM]

1.97 ± 0.53 n=74.94 ± 3.39 n=9EC50,Ca [mM]

Ca2+IP1

0.56 ± 0.100.85 ± 0.05Z’

45.014.23*sd

0.66 ± 0.060.86 ± 0.05Z

15.024.74sd

-0.57-0.45mean % change

Ca2+IP1

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IP1 and Calcium: Compound interference IP1 and Calcium: Compound interference IP1 and Calcium: Compound interference

— Frequent hitters are compounds scoring positive in all FLIPR screening campaigns due to their toxicity, their fluorescence at 488 nM or their interaction with common pathways.

— These compounds are not detected in the HTRF IP one assay

Compound IP1 % change % change Ratio Rb

1 6 -59 1.342 -4 -64 2.033 -3 -85 2.134 -3 -102 3.655 0 -102 4.076 -3 -100 4.097 -3 -102 3.968 0 -102 4.579 -2 -102 4.0410 1 -93 3.811 5 -102 4.3812 3 -100 3.0213 -1 -93 2.3314 -4 -72 1.4515 -1 -72 1.5816 -3 -97 1.7717 -12 -102 2.5918 -3 -91 2.42

Calcium

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IP1 and Calcium: Common hits IP1 and Calcium: Common hits IP1 and Calcium: Common hits

Compound IP1 % change % change Ratio Rb

1 -37 -78 0.752 -31 -93 0.823 -22 -50 0.704 -35 -78 0.735 -34 -68 0.736 -30 -74 0.747 -38 -65 0.77

Calcium

— Hits interfering with the receptor of common mechanism

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IP1 and Calcium: PS hits in calciumIP1 and Calcium: PS hits in calciumIP1 and Calcium: PS hits in calcium

Compound IP1 % change % change Ratio Rb

1 -9 -54 0.912 1 -88 1.003 13 -67 0.864 3 -55 0.715 7 -63 0.936 2 -58 1.247 1 -55 1.028 -1 -64 0.719 -2 -72 0.7010 3 -62 0.6311 4 -86 0.6612 11 -67 0.7013 3 -54 0.7814 -2 -55 0.8515 -1 -88 0.8116 -3 -56 0.8417 -2 -70 0.6218 -1 -61 1.0519 2 -71 0.9820 -5 -72 0.5421 13 -53 1.0522 12 -52 1.2123 6 -73 0.71

Calcium

— A number of compounds are found only in the calcium assaywithout modification of the calcium baseline

Common FLIPR hits, i.e found in several FLIPR assay (

False positives in calcium assay; Known for FLIPR assays

Other mechanisms ?

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Conclusion / outlookConclusion / outlookConclusion / outlook

— IP1-one is a useful HTS assay using IP1 as a reporter for IP3Homogenous assay. Some parameters were optimised (Cell density & culture

conditionSimilar data obtained compared to calcium FLIPR with a slightly higher sensitivity for

calcium (amplification mechanisms)Hit rates lower

Can be used for secondary screening to exclude false positives and FLIPR specific hits

More robust. Better assay quality in productive screening set-up

— Further studiesMore studies with controlled conditions with endogenous GPCRs.

More data to be produced with other recombinant system (SMW agonist GPCR assay with competitive antagonists. Potential calcium interference with IP1 assay ?

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AcknowledgementsAcknowledgementsAcknowledgements

NovartisK. SeuwenD. Gabriel

Cisbio France

E. TrinquetM. FinkP. SeguinJ-L Tardieu

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0.1 1 10 1004000

5000

6000

7000

8000

9000

10000

11000

12000

Rat

io 6

65 n

M /

620

nM

[Calcium] mM0.1 1 10 100

0.0

0.5

1.0

1.5

2.0

2.5

3.0

3.5

4.0

Fluo

resc

ence

dF/

F[Calcium] mM

The calcium sensing receptor HupCaR

HTRF / IP1 FLIPR Fluo4 calcium

EC50 = 4.79 mM EC50 = 3.0 mM