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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
13
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
14
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
19
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
20
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
21
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
22
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
23
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
24
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
25
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
26
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
27
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 ?
28
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 ?
29
AcknowledgementsAcknowledgementsAcknowledgements
NovartisK. SeuwenD. Gabriel
Cisbio France
E. TrinquetM. FinkP. SeguinJ-L Tardieu
30
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