Headquarters & Europe Cisbio Bioassays Phone : +33 (0)4 66 79 67 05 Fax : +33 (0)4 66 79 19 20 E-mail : [email protected] USA Cisbio US, inc. Phone : 888-963-4567 Fax : 781-687-1500 E-mail : htrfi[email protected] www.htrf.com Copyright © 2012 Cisbio Bioassays, France. HTRF®, TRACE®, and the HTRF™ logo are trademarks belonging to Cisbio Bioassays. HTRF® products are manufactured under one or more of the following patents and foreign equivalent : EP 0 180 492 / US 4,927,923 / US 5,220,012 / US 5,432,101 – EP 0 321 353 / US 5,457,185 / US 5,534,622 / US 5,346,996 / US 5,162,508 – EP 0 539 477 / US 5,512,493 – EP 0 539 435 / US 5,627,074 – EP 0 569 496 / US 5,527,684. HTRF cellular kinase assays to address ligand-gated channels: Example of the α7 nicotinic acetylcholine receptor E. Dupuis 1 , M. Partiseti 2 , M. Fink 1 , E. Trinquet 1 , G. Mathis 1 , L. Jacquemart 1 , F. Degorce 1 . 1 Cisbio Bioassays, Codolet, France - 2 Sanofi R&D, LGCR/LIT, Vitry sur Seine, France Assay principle Cellul’erk detection assay is based on a sandwich immunoassay format using an anti-phospho-ERK1/2 antibody labeled with d2 (HTRF acceptor) and an anti-ERK1/2 antibody labeled with Eu 3+ - Cryptate (HTRF donor). When the 2 HTRF® dyes are in close proximity, the excitation of the donor with a light source triggers a Fluorescence Energy Transfer (FRET) towards the acceptor, which in turn fluoresces at a specific wavelength. e specific signal modulates positively in proportion to phospho-Erk. Activation Lysis Detection e following protocols were applied to access activation and inhibition of α7 nAChR triggered by specific agonist or antagonists: - 50µl of over-expressed alpha7 acetylcholine nicotinic receptor and its associated protein Ric-3 in fresh HEK293 FlpInTREx cells (120000 cells/well) were first dispensed into a 96-well plate using growth medium with induction compound for 24h, - aſter an overnight serum starvation step, medium was removed and activation and inhibition assays were performed as follows: Activation assay - 25µl of positive allosteric modulator (PAM) PNU-120596 solution was added and incubated for 10 minutes at 37°C - 25µL of PNU-282987 (agonist) were dispensed and incubated at 37°C for 5mn. Inhibition assay - 25µl of Methyllycaconitine (MLA) and α-Bungarotoxin (antagonists) were dispensed and incubated for 20 minutes at 37°C, - 25µl of PAM (30µM) were added, then incubated at 37°C for 10mn, - 25µl of PNU-282987 (1µM) were added, then incubated at 37°C for 5mn. Detection step - medium was removed, - 50µl of supplemented lysis buffer were added, followed by 20mn at room temperature under shaking, - 16µl of the cell lysates were transferred to a 384 small volume white plate, - 2µl of Cryptate labeled conjugate and 2µl of d2-labeled conjugate were added and incubated for 2 hours at room temperature, - signal was recorded on an HTRF compatible reader. Introduction Ligand-gated ion channels (LGICs) are one type of the ionotropic receptors most representative of ion channels and implicated in several neuropsychiatric and neurodegenerative diseases. e α7 nicotinic acetylcholine receptor (α7 nAChR) , assembled as homopentameric complexes with five α7 subunits forming the pore, is the most studied ion channel of the ligand-gated channel family. According to the literature, several kinase phosphorylation pathways are α7 nAChR- mediated, for example Jak-2, Akt, STAT-3 and Erk1/2. STAT-3 α7 nAchR Ca 2+ STAT-3 Bci-2 Jak-2 NF-KB-IKB PI3 kinase Akt Ras p38 MEK CREB ERK 1/2 Here we investigate the applicability of measuring phospho-Erk to address ligand-gated ion channels (LGICs), and more precisely α7 nAChR. Phosphorylated Erk1/2 measurement using the Cellul’erk kit was compared to calcium signaling (FlexStation) using several specific pharmacological compounds including PNU- 120596 (positive allosteric modulator: PAM), PNU-282987 (agonist), and Methyllycaconitine and α-Bungarotoxin (antagonists). Cellul’erk is a ready-to-use cell-based assay from Cisbio Bioassays based on HTRF technology. Using a simple protocol, this assay is a robust, reliable and rapid alternative to more conventional technologies such as electrophysiology or Calcium flux measurement. Determination of pharmacokinetic parameters: Cellul’erk kit compared to FlexStation Activation of α7 nAChR: EC50 determination Inhibition of α7 nAChR: IC50 determination Alpha-bungarotoxin MLA Non transfected cells Nicotinic alpha7 -12 -11 -10 -9 -8 -7 -6 -5 0 50 00 10 000 15 000 20 000 25 000 30 000 log [compound]M HTRF ratio Cellul’erk kit -12 -11 -10 -9 -8 -7 -6 -5 -4 0 10 20 30 40 50 60 70 80 90 Max-Min log [compound]M FlexStation HTRF ratio -10.0 -9.5 -9.0 -8 .5 -8.0 -7.5 - 7.0 -6 .5 -6.0 0 5000 10000 15000 20000 25000 30000 35000 Cellul’erk kit log [PNU282987] M Max-Min -12 -11 -10 -9 -8 -7 -6 -5 -4 0 10 20 30 40 50 60 70 80 FlexStation log [PNU282987] M Log EC50 -7.558 S/N 20 EC50 2.766e-008 Log EC50 -7.523 EC50 3.001e-008 MLA alpha-Bgt Log IC50 -7.189 -8.672 IC50 6.466e-008 2.131e-009 MLA alpha-Bgt Log IC50 -7.449 -9.177 S/N 21 16 IC50 3.55e-008 6.658e-010 Compound EC50 IC50 cellul’erk kit (HTRF) FlexStation (calcium flux) cellul’erk kit (HTRF) FlexStation (calcium flux) PNU-282987 (agonist) 28nM 30nM - - MLA (antagonist) - - 36nM 64nM a-Bgt (antagonist) - - 0.7nM 2nM Concentration-effect curves of α7 nAChR ligand-induced were obtained - for Cellul’erk, by plotting HTRF ratio values (ratio of fluorescence of the acceptor over that of the donor) - for FlexStation, by plotting maximal–minimal fluorescence signals obtained with the Fluo-4 probe (a fluorescent permeant sensor sensitive to Ca 2+ variation flux ) Values are expressed as means ± SEM in triplicate and calculated with GraphPad. e above results show that the functional response is the same when measuring Ca ++ entrance through the channel or detecting phosphorylated erk; therefore ligand-gated ion channels can be studied at different levels of the agonist- mediated activation cascade. Read Stimulation step Detection step Transfer In this study, we demonstrate that Cellul’erk is a powerful, sensitive and robust tool for measuring Erk phosphorylation pathway mediated by ligand-gated ion channels. Pharmacological results are consistent with the literature (3) (4) and perfectly matched those obtained with FlexStation, moreover very robust S/N windows are measured. is homogeneous, ready-to-use HTRF assay is very simple to handle, no washing steps are required, making it even more user-friendly for measuring phosphorylated Erk1/2. Highly miniaturizable this assay is tailored for both research and high-throughput screening. Using this common simple protocol, Cisbio Bioassays has developed a panel of cell-based assays (Akt, Stat3, Jak2, IKKB, p38) dedicated to study the behavior of a compound toward a signaling pathway. ese homogeneous assays represent a reliable and rapid alternative to classical technologies like Western Blot and ELISA. e robustness of HTF signal makes them particularly adapted to a variety of applications run on therapeutic areas and HTS labs. References 1] Arredondo J, Chernyavsky AI, Jolkovsky DL, Pinkerton KE, Grando SA. FASEB J. 20(12):2093-101;2006 [2] Bencherif M.Acta Pharmacol Sin. 30 (6):702-714;2009 [3] El Kouhen R, Hu M, Anderson DJ, Li J, Gopalakrishnan. Br J Pharmacol. 156(4):638-48;2009. [4] Gubbins EJ, Gopalakrishnan M, Li J. Brain Res. 1328:1-11; 2010 Conclusion
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Headquarters & Europe
Cisbio BioassaysPhone : +33 (0)4 66 79 67 05Fax : +33 (0)4 66 79 19 20E-mail : [email protected]
USA
Cisbio US, inc.Phone : 888-963-4567Fax : 781-687-1500E-mail : [email protected]
www.htrf.comCopyright © 2012 Cisbio Bioassays, France. HTRF®, TRACE®, and the HTRF™ logo are trademarks belonging to Cisbio Bioassays. HTRF® products are manufactured under one or more of the following patents and foreign equivalent : EP 0 180 492 / US 4,927,923 / US 5,220,012 / US 5,432,101 – EP 0 321 353 / US 5,457,185 / US 5,534,622 / US 5,346,996 / US 5,162,508 – EP 0 539 477 / US 5,512,493 – EP 0 539 435 / US 5,627,074 – EP 0 569 496 / US 5,527,684.
HTRF cellular kinase assays to address ligand-gated channels: Example of the α7 nicotinic acetylcholine receptorE. Dupuis1, M. Partiseti2, M. Fink1, E. Trinquet1, G. Mathis1, L. Jacquemart1, F. Degorce1.
1Cisbio Bioassays, Codolet, France - 2Sanofi R&D, LGCR/LIT, Vitry sur Seine, France
Assay principleCellul’erk detection assay is based on a sandwich immunoassay format using an anti-phospho-ERK1/2 antibody labeled with d2 (HTRF acceptor) and an anti-ERK1/2 antibody labeled with Eu3+- Cryptate (HTRF donor). When the 2 HTRF® dyes are in close proximity, the excitation of the donor with a light source triggers a Fluorescence Energy Transfer (FRET) towards the acceptor, which in turn fluoresces at a specific wavelength. The specific signal modulates positively in proportion to phospho-Erk.
Activation Lysis Detection
The following protocols were applied to access activation and inhibition of α7 nAChR triggered by specific agonist or antagonists:
- 50µl of over-expressed alpha7 acetylcholine nicotinic receptor and its associated protein Ric-3 in fresh HEK293 FlpInTREx cells (120000 cells/well) were first dispensed into a 96-well plate using growth medium with induction compound for 24h, - after an overnight serum starvation step, medium was removed and activation and inhibition assays were performed as follows:
Activation assay
- 25µl of positive allosteric modulator (PAM) PNU-120596 solution was added and incubated for 10 minutes at 37°C
- 25µL of PNU-282987 (agonist) were dispensed and incubated at 37°C for 5mn.
Inhibition assay
- 25µl of Methyllycaconitine (MLA) and α-Bungarotoxin (antagonists) were dispensed and incubated for 20 minutes at 37°C, - 25µl of PAM (30µM) were added, then incubated at 37°C for 10mn, - 25µl of PNU-282987 (1µM) were added, then incubated at 37°C for 5mn.
Detection step
- medium was removed, - 50µl of supplemented lysis buffer were added, followed by 20mn at room temperature under shaking, - 16µl of the cell lysates were transferred to a 384 small volume white plate, - 2µl of Cryptate labeled conjugate and 2µl of d2-labeled conjugate were added and incubated for 2 hours at room temperature, - signal was recorded on an HTRF compatible reader.
IntroductionLigand-gated ion channels (LGICs) are one type of the ionotropic receptors most representative of ion channels and implicated in several neuropsychiatric and neurodegenerative diseases. The α7 nicotinic acetylcholine receptor (α7 nAChR) , assembled as homopentameric complexes with five α7 subunits forming the pore, is the most studied ion channel of the ligand-gated channel family.
According to the literature, several kinase phosphorylation pathways are α7 nAChR-mediated, for example Jak-2, Akt, STAT-3 and Erk1/2.
STAT-3
α7 nAchR
Ca2+
STAT-3
Bci-2
Jak-2
NF-KB-IKB
PI3kinase
Akt
Ras
p38
MEK
CREB
ERK 1/2
Here we investigate the applicability of measuring phospho-Erk to address ligand-gated ion channels (LGICs), and more precisely α7 nAChR.
Phosphorylated Erk1/2 measurement using the Cellul’erk kit was compared to calcium signaling (FlexStation) using several specific pharmacological compounds including PNU-120596 (positive allosteric modulator: PAM), PNU-282987 (agonist), and Methyllycaconitine and α-Bungarotoxin (antagonists).
Cellul’erk is a ready-to-use cell-based assay from Cisbio Bioassays based on HTRF technology. Using a simple protocol, this assay is a robust, reliable and rapid alternative to more conventional technologies such as electrophysiology or Calcium flux measurement.
Determination of pharmacokinetic parameters: Cellul’erk kit compared to FlexStationActivation of α7 nAChR: EC50 determination Inhibition of α7 nAChR: IC50 determination
Alpha-bungarotoxinMLANon transfected cells Nicotinic alpha7
-12 -11 -10 -9 -8 -7 -6 -50
5000
10000
15000
20000
25000
30000
log [ compound] M
HT
RF
ratio
Cellul’erk kit
-12 -11 -10 -9 -8 -7 -6 -5 -40
102030405060708090
Max
-Min
log [ compound] M
FlexStation
HT
RF
ratio
-10.0 -9.
5-9.
0-8
.5-8.
0-7.
5- 7
.0 -6.5
-6.0
0
5000
10000
15000
20000
25000
30000
35000Cellul’erk kit
log [PNU282987] M
Max
-Min
-12 -11 -10 -9 -8 -7 -6 -5 -40
10
20
30
4050
60
7080
FlexStation
log [PNU282987] M
Log EC50 -7.558S/N 20
EC50 2.766e-008Log EC50 -7.523
EC50 3.001e-008
MLA alpha-BgtLog IC50 -7.189 -8.672
IC50 6.466e-008 2.131e-009
MLA alpha-BgtLog IC50 -7.449 -9.177
S/N 21 16IC50 3.55e-008 6.658e-010
CompoundEC50 IC50
cellul’erk kit (HTRF)
FlexStation (calcium flux)
cellul’erk kit (HTRF)
FlexStation (calcium flux)
PNU-282987 (agonist) 28nM 30nM - -
MLA (antagonist) - - 36nM 64nM
a-Bgt (antagonist) - - 0.7nM 2nM
Concentration-effect curves of α7 nAChR ligand-induced were obtained
- for Cellul’erk, by plotting HTRF ratio values (ratio of fluorescence of the acceptor over that of the donor) - for FlexStation, by plotting maximal–minimal fluorescence signals obtained with the Fluo-4 probe (a fluorescent permeant sensor sensitive to Ca2+ variation flux )
Values are expressed as means ± SEM in triplicate and calculated with GraphPad.
The above results show that the functional response is the same when measuring Ca++ entrance through the channel or detecting phosphorylated erk; therefore ligand-gated ion channels can be studied at different levels of the agonist-mediated activation cascade.
Read
Stimulation step Detection step
Transfer
In this study, we demonstrate that Cellul’erk is a powerful, sensitive and robust tool for measuring Erk phosphorylation pathway mediated by ligand-gated ion channels.
Pharmacological results are consistent with the literature (3) (4) and perfectly matched those obtained with FlexStation, moreover very robust S/N windows are measured. This homogeneous, ready-to-use HTRF assay is very simple to handle, no washing steps are required, making it even more user-friendly for measuring phosphorylated Erk1/2.
Highly miniaturizable this assay is tailored for both research and high-throughput screening. Using this common simple protocol, Cisbio Bioassays has developed a panel of cell-based assays (Akt, Stat3, Jak2, IKKB, p38) dedicated to study the behavior of a compound toward a signaling pathway. These homogeneous assays represent a reliable and rapid alternative to classical technologies like Western Blot and ELISA. The robustness of HTF signal makes them particularly adapted to a variety of applications run on therapeutic areas and HTS labs.
References1] Arredondo J, Chernyavsky AI, Jolkovsky DL, Pinkerton KE, Grando SA. FASEB J. 20(12):2093-101;2006 [2] Bencherif M.Acta Pharmacol Sin. 30 (6):702-714;2009 [3] El Kouhen R, Hu M, Anderson DJ, Li J, Gopalakrishnan. Br J Pharmacol. 156(4):638-48;2009. [4] Gubbins EJ, Gopalakrishnan M, Li J. Brain Res. 1328:1-11; 2010
Conclusion
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