1. LANCE Ultra cAMP: A New, Two-Component TR-FRET cAMP Assay for HTS of Gs- and Gi-
Coupled Receptors
Nancy Gauthier, Julie Blouin, Mireille Caron, Philippe Roby, Lucille Beaudet & Jaime Padrós
1 Abstract 4 Agonist Responses in SK-N-MC Cells Expressing Endogenous Gs-coupled 8 Z’ Study in CHO Cells Expressing
Guanosine triphosphate binding protein-coupled receptors β-adrenergic Receptors Gs-hMC4 Receptors
(GPCRs) represent one of the largest and most important classes
of pharmaceutical drug targets. We have developed a second- LANCE Ultra cAMP kit Company C (dynamic 2 kit) Company D (HS+ kit) LANCE Ultra cAMP kit
generation LANCE® time-resolved fluorescence resonance energy 2,000 cells 7,500 cells 1,000 cells 1,000 cells
transfer (TR-FRET) immunoassay designed to measure cAMP
TR-FRET Signal (665 nm)
TR-FRET Signal (665 nm)
TR-FRET Signal (665 nm)
18,000 2,500 35,000 20,000
produced upon modulation of adenylyl cyclase activity by
Luminescence (cps)
Isoproterenol LANCE Ultra Company C Company D 17,500 Cells
15,000 30,000
activated GPCRs. The homogeneous two-component LANCE Ultra 2,000
25,000
Norepinephrine
S/B
EC50
S/B
EC50
S/B
EC50 15,000 NPD--MSH (0.5 nM)
12,000 (nM) (nM) (nM)
cAMP assay kit is based on the competition between europium 1,500 20,000
Formoterol
Isoproterenol 18.8 0.29 7.1 0.41 2.2 1.9
12,500 NDP--MSH (0.5 nM)
+ SHU9119 (30 µM)
chelate-labeled cAMP and cellular cAMP for binding to high-affinity 9,000
1,000 15,000
BRL 37344
Norepinephrine 20.7 3.0 6.4 4.6 2.1 15.4
10,000
Salmeterol 7,500
anti-cAMP monoclonal antibodies labeled with the ULight™ dye. 6,000
10,000 Formoterol 19.4 9.7 6.3 11.9 2.0 53.3 5,000
Here we present data comparing the performance in 384-well 3,000 500
5,000 BRL 37344 5.3 54.9 2.3 101 1.3 464 2,500
format of the new LANCE Ultra cAMP kit with that of alternative 0 0 0 Salmeterol 9.2 335 3.6 375 1.5 752 0
cAMP assay technologies, namely a TR-FRET assay (dynamic 2 kit - -12-11-10 -9 -8 -7 -6 -5 -4 -3 -2 - -12-11-10 -9 -8 -7 -6 -5 -4 -3 -2 - -12-11-10 -9 -8 -7 -6 -5 -4 -3 -2 0 10 20 30 40 50 60
# Wells
from Company C) and two Enzyme Fragment Complementation Log [Agonist] (M) Log [Agonist] (M) Log [Agonist] (M)
Detection Cells Agonist Agonist + Antagonist
assays (XS+ and HS+ kits from Company D). These cAMP assay time Mean SD %CV Mean SD %CV Mean SD %CV
technologies were evaluated for their ability to detect agonist- or 1h 15518 952 6.1 1505 83 5.5 10080 945 9.4
antagonist-induced cAMP responses in suspension cells expressing
endogenous (β-adrenergic) or recombinant receptors (Gs-MC4;
5 Agonist Responses in CHO Cells Expressing Gi-hCXCR3 Receptors O/N 12412 746 6.0 1363 111 8.1 8506 709 8.3
Gi-CXCR3; Gi-CB1). The LANCE Ultra kit yields a cAMP assay with Detection Cells/Agonist
Agonist + Antagonist
/Agonist
the highest sensitivity and signal window than any tested: IC50 time
S/B Z' S/B Z'
value of 28 fmoles and signal-to-background (S/B) ratio of 70 in a LANCE Ultra cAMP kit Company C (dynamic 2 kit) Company D (XS+ kit) 1h 10.3 0.78 6.7 0.64
2,000 cells, 300 nM Forskolin 5,000 cells, 1 µM Forskolin 5,000 cells, 20 µM Forskolin O/N 9.1 0.77 6.2 0.66
cAMP standard curve. These features allow using fewer cells
TR-FRET Signal (665 nm)
TR-FRET Signal (665 nm)
compared to other cAMP detection platforms. Results of cell-based 15,000 2,500 100,000
Luminescence (cps)
CXCL11
assays indicated that all four cAMP platforms provide comparable 12,000 80,000 LANCE Ultra Company C Company D
9
2,000 CXCL10
assay pharmacology with the expected rank order of agonist or
9,000 1,500 60,000
CXCL9 S/B
EC50
(nM)
S/B
EC50
(nM)
S/B
EC50
(nM) Standard Curve Stability Over Time
antagonist potency. However, in every application, the LANCE CXCL11 6.3 1.7 3.7 3.6 2.9 3.4
Ultra outperformed the other cAMP platforms in terms of S/B 6,000 1,000 40,000
CXCL10 4.7 27.3 3.5 47.7 2.3 11.1 LANCE Ultra cAMP kit Company C (dynamic 2 kit)
ratio. This key advantage, combined to a stable pharmacology,
TR-FRET Signal (665 nm)
TR-FRET Signal (665 nm)
3,000 500 20,000 CXCL9 5.3 130 4.3 924 1.8 51.8 25,000 4,500
simple assay protocol and unique kit format for every application, 4,000 1h
0 0 20,000 O/N
makes the LANCE Ultra cAMP assay a superior HTS technology 0 3,500
- -11 -10 -9 -8 -7 -6 -5 - -11 -10 -9 -8 -7 -6 -5 - -11 -10 -9 -8 -7 -6 -5 3,000
suitable for both Gs- and challenging Gi-coupled receptors.
15,000
2,500
Log [Agonist] (M) Log [Agonist] (M) Log [Agonist] (M) 2,000
10,000
1,500
5,000 1,000
2 LANCE Ultra cAMP Assay Principle 500
7
0 0
6 Antagonist Responses in CHO Cells Expressing Gi- Agonist Responses in CHO - -11 -10 -9 -8
Log [cAMP] (M)
-7 -6 -5 - -11 -10 -9 -8
Log [cAMP] (M)
-7 -6 -5
In the absence of free cAMP In the presence of free cAMP
hCB1 Receptors Cells Expressing Gs-hMC4 Detection
time
LANCE Ultra Company C
S/B IC50 (nM) S/B IC50 (nM)
Receptors 1h 68.1 1.4 19.5 4.3
O/N 68.1 1.5 12.7 4.1
LANCE Ultra cAMP kit
LANCE Ultra cAMP kit Company C (dynamic 2 kit) 1,000 cells
Tr-FRET Signal (665 nm)
2,500 cells, 2.5 µM Forskolin 5,000 cells, 2.5 µM Forskolin 15,000
Melanotan II
+ 1 µM WIN55,212-2 + 1 µM WIN55,212-2 12,500
TR-FRET Signal (665 nm)
TR-FRET Signal (665 nm)
1,500 NDP--MSH
2,500
AM 251 10,000 -MSH
1,250
2,000 SR141716 7,500
1,500
1,000 LY 320135
5,000 10 Conclusions
3 Protocol (384-well Format) 1,000
750
LANCE Ultra Company C 2,500
500
S/B IC50 (nM) S/B IC50 (nM) 0 • The LANCE Ultra cAMP kit showed the highest assay
sensitivity and signal window: IC50 value of 28 fmoles and
500 250 - -13 -12 -11 -10 -9 -8 -7 -6
AM 251 5.0 11.9 3.5 11.2
Cell Stimulation cAMP Detection Read on the EnVision® Log [Agonist] (M)
0 0 S/B ratio of ~70 in a cAMP standard curve. These two assay
- -11 -10 -9 -8 -7 -6 -5 -4 - -10 -9 SR141716
parameters were stable following overnight incubation.
-8 -7 -6 -5 -4 -3 4.4 36.6 3.7 35.9
+ 5 µL Compound(s) + 5 µL Eu-cAMP Company C (dynamic 2 kit)
+ 5 µL Cells + 5 µL ULight-anti-cAMP Log [Antagonist] (M) Log [Antagonist] (M) LY 320135 2.4 149 2.3 191 5,000 cells
TR-FRET Signal (665 nm)
Incubate Incubate 2,000 • Expected pharmacological profiles and ranking of compound
LANCE Ultra cAMP kit Company C (dynamic 2 kit) Melanotan II
30 min 1h
2,500 cells, 2.5 µM Forskolin 5,000 cells, 2.5 µM Forskolin NDP-a-MSH potency were obtained with all four cAMP kits tested.
1,500
+ 1 µM WIN55,212-2 + 1 µM WIN55,212-2 a-MSH However, the LANCE Ultra technology consistently provided
900 3,000 1,000 higher S/B ratios while using fewer cells.
800 AM 251
Each kit was used with the cell density giving the highest S/B 2,500
cAMP (fmoles)
cAMP (fmoles)
SR141716
700
• The LANCE Ultra technology enabled the development of
ratio. All reagents were prepared and dispensed according to 600 2,000 LY 320135 500
robust cAMP assays suitable for HTS, as indicated by the
manufacturer’s recommendations. Experiments with all kits were 500
400
1,500 LANCE Ultra Company C 0 high and stable Z’ values obtained.
performed side-by-side with the same batch of frozen cells and 300 1,000
- -13 -12 -11 -10 -9 -8 -7 -6
using the same serially diluted solutions of the cAMP standard, 200
500
IC50 (nM) IC50 (nM)
Log [Agonist] (M)
• These key advantages, combined with a simple assay
forskolin, agonists and antagonists. For antagonist assays, agonist 100 AM 251 120 55.9 LANCE Ultra Company C
protocol and unique kit format, make the LANCE Ultra cAMP
0 0
(Gs-GPCR) or forskolin/agonist (Gi-GPCR) were used at their EC90 - -11 -10 -9 -8 -7 -6 -5 -4 - -10 -9 -8 -7 -6 -5 -4 -3 SR141716 163 161
Melanotan II
S/B
10.9
EC50 (nM)
0.02
S/B
4.4
EC50 (nM)
0.04 assay a superior HTS technology suitable for both Gs- and
(fluorescence values). All assays were conducted manually in 384- Log [Antagonist] (M) Log [Antagonist] (M) LY 320135 277 382 NDP-α-MSH 10.8 0.11 4.0 0.15 Gi-coupled GPCRs.
well format. Signal was detected with the EnVision reader in TR- α-MSH 9.0 0.16 3.8 0.20
FRET or luminescence mode.
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