LANCE Ultra cAMP: A New, Two-Component TR-FRET cAMP Assay for HTS of Gs- and Gi-Coupled Receptors

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
cAMP Detection
+ 5 µL Compound(s)
+ 5 µL Cells
- -12-11-10 -9 -8 -7 -6 -5 -4 -3 -2
Read on the EnVision®
500
0
5
LANCE Ultra
Norepinephrine
Company C
Company D
S/B
EC50
(nM)
S/B
EC50
(nM)
S/B
EC50
(nM)
Isoproterenol
18.8
0.29
7.1
0.41
2.2
1.9
Norepinephrine
20.7
3.0
6.4
4.6
2.1
15.4
10,000
Formoterol
19.4
9.7
6.3
11.9
2.0
53.3
5,000
BRL 37344
5.3
54.9
2.3
101
1.3
464
Salmeterol
9.2
335
3.6
375
1.5
752
25,000
Formoterol
20,000
BRL 37344
15,000
- -12-11-10 -9 -8 -7 -6 -5 -4 -3 -2
Salmeterol
- -12-11-10 -9 -8 -7 -6 -5 -4 -3 -2
Log [Agonist] (M)
3,000
0
- -11
-10
-9
-8
-7
-6
-5
2,000
1,500
1,000
500
Log [Agonist] (M)
6
-10
-9
-8
-7
CXCL11
CXCL10
CXCL9
80,000
60,000
2,000
1,500
1,000
500
0
- -11 -10
-9
-8
-7
-6
-5
-4
Incubate
1h
20,000
-6
- -11
-5
-10
-9
-8
-7
-6
-9
-8
-7
-6
-5
7
1,500
AM 251
SR141716
LY 320135
1,250
1,000
750
LANCE Ultra
Company C
500
Log [Antagonist] (M)
S/B
EC50
(nM)
S/B
EC50
(nM)
S/B
EC50
(nM)
CXCL11
6.3
1.7
3.7
3.6
2.9
3.4
CXCL10
4.7
27.3
3.5
47.7
2.3
11.1
CXCL9
5.3
130
4.3
924
1.8
51.8
S/B
IC50 (nM)
S/B
IC50 (nM)
AM 251
5.0
11.9
3.5
11.2
SR141716
4.4
36.6
3.7
35.9
LY 320135
2.4
149
2.3
191
Agonist Responses in CHO
Cells Expressing
Gs-hMC4 Receptors
-8
-7
-6
-5
-4
-3
Company C (dynamic 2 kit)
5,000 cells, 2.5 µM Forskolin
+ 1 µM WIN55,212-2
AM 251
SR141716
LY 320135
2,500
2,000
1,500
1,000
500
0
- -10 -9
-8
-7
-6
30
-5
-4
Log [Antagonist] (M)
-3
40
50
60
Cells
Agonist
Agonist + Antagonist
Detection
time
Mean
SD
%CV
Mean
SD
%CV
Mean
SD
%CV
1h
15518
952
6.1
1505
83
5.5
10080
945
9.4
O/N
12412
746
6.0
1363
111
8.1
8506
709
8.3
Cells/Agonist
S/B
10.3
Z'
0.78
9.1
0.77
Agonist + Antagonist/
Agonist
S/B
Z'
6.7
0.64
6.2
0.66
LANCE Ultra
Company C
IC50 (nM)
IC50 (nM)
AM 251
120
55.9
SR141716
163
161
LY 320135
277
382
9
Standard Curve Stability Over Time
LANCE Ultra cAMP kit
Company C (dynamic 2 kit)
25,000
20,000
15,000
10,000
5,000
0
- -11
-10
-9
-8
-7
-6
-5
4,500
4,000
3,500
3,000
2,500
2,000
1,500
1,000
500
0
1h
O/N
- -11
Log [cAMP] (M)
-10
-9
-8
-7
-6
-5
Log [cAMP] (M)
LANCE Ultra
Company C
Detection
time
S/B
IC50 (nM)
S/B
IC50 (nM)
1h
68.1
1.4
19.5
4.3
O/N
68.1
1.5
12.7
4.1
15,000
Melanotan II
NDP--MSH
-MSH
12,500
10,000
7,500
10
5,000
Conclusions
2,500
• The LANCE Ultra cAMP kit showed the highest assay
sensitivity and signal window: IC50 value of 28 fmoles and
S/B ratio of ~70 in a cAMP standard curve. These two assay
parameters were stable following overnight incubation.
0
- -13 -12 -11 -10
-9
-8
-7
-6
Log [Agonist] (M)
0
- -10 -9
20
LANCE Ultra cAMP kit
1,000 cells
250
Log [Antagonist] (M)
-4
Company D
Log [Agonist] (M)
3,000
- -11 -10
Company C
-5
Company C (dynamic 2 kit)
5,000 cells, 2.5 µM Forskolin
+ 1 µM WIN55,212-2
LANCE Ultra cAMP kit
2,500 cells, 2.5 µM Forskolin
+ 1 µM WIN55,212-2
900
800
700
600
500
400
300
200
100
0
LANCE Ultra
40,000
Antagonist Responses in CHO Cells Expressing
Gi-hCB1 Receptors
2,500
10
100,000
Log [Agonist] (M)
LANCE Ultra cAMP kit
2,500 cells, 2.5 µM Forskolin
+ 1 µM WIN55,212-2
0
1h
O/N
0
0
- -11
NDP--MSH (0.5 nM)
+ SHU9119 (30 µM)
Detection
time
TR-FRET Signal (665 nm)
6,000
NPD--MSH (0.5 nM)
# Wells
Tr-FRET Signal (665 nm)
9,000
2,500
Luminescence (cps)
12,000
TR-FRET Signal (665 nm)
15,000
Cells
15,000
12,500
10,000
7,500
5,000
2,500
0
Company D (XS+ kit)
5,000 cells, 20 µM Forskolin
Company C (dynamic 2 kit)
5,000 cells, 1 µM Forskolin
LANCE Ultra cAMP kit
2,000 cells, 300 nM Forskolin
20,000
17,500
Log [Agonist] (M)
Agonist Responses in CHO Cells Expressing Gi-hCXCR3 Receptors
Log [Antagonist] (M)
Each kit was used with the cell density giving the highest S/B
ratio. All reagents were prepared and dispensed according to
manufacturer’s recommendations. Experiments with all kits were
performed side-by-side with the same batch of frozen cells and
using the same serially diluted solutions of the cAMP standard,
forskolin, agonists and antagonists. For antagonist assays, agonist
(Gs-GPCR) or forskolin/agonist (Gi-GPCR) were used at their EC90
(fluorescence values). All assays were conducted manually in 384well format. Signal was detected with the EnVision reader in TRFRET or luminescence mode.
Isoproterenol
30,000
0
Log [Agonist] (M)
+ 5 µL Eu-cAMP
+ 5 µL ULight-anti-cAMP
Incubate
30 min
1,000
35,000
TR-FRET Signal (665 nm)
0
Luminescence (cps)
3,000
TR-FRET Signal (665 nm)
TR-FRET Signal (665 nm)
6,000
In the presence of free cAMP
Protocol (384-well Format)
Cell Stimulation
9,000
1,500
LANCE Ultra cAMP kit
1,000 cells
TR-FRET Signal (665 nm)
3
12,000
2,000
cAMP (fmoles)
In the absence of free cAMP
15,000
Company D (HS+ kit)
1,000 cells
2,500
TR-FRET Signal (665 nm)
LANCE Ultra cAMP Assay Principle
Company C (dynamic 2 kit)
7,500 cells
18,000
TR-FRET Signal (665 nm)
2
LANCE Ultra cAMP kit
2,000 cells
TR-FRET Signal (665 nm)
Guanosine triphosphate binding protein-coupled receptors
(GPCRs) represent one of the largest and most important classes
of pharmaceutical drug targets. We have developed a secondgeneration LANCE® time-resolved fluorescence resonance energy
transfer (TR-FRET) immunoassay designed to measure cAMP
produced upon modulation of adenylyl cyclase activity by
activated GPCRs. The homogeneous two-component LANCE Ultra
cAMP assay kit is based on the competition between europium
chelate-labeled cAMP and cellular cAMP for binding to high-affinity
anti-cAMP monoclonal antibodies labeled with the ULight™ dye.
Here we present data comparing the performance in 384-well
format of the new LANCE Ultra cAMP kit with that of alternative
cAMP assay technologies, namely a TR-FRET assay (dynamic 2 kit
from Company C) and two Enzyme Fragment Complementation
assays (XS+ and HS+ kits from Company D). These cAMP assay
technologies were evaluated for their ability to detect agonist- or
antagonist-induced cAMP responses in suspension cells expressing
endogenous (β-adrenergic) or recombinant receptors (Gs-MC4;
Gi-CXCR3; Gi-CB1). The LANCE Ultra kit yields a cAMP assay with
the highest sensitivity and signal window than any tested: IC50
value of 28 fmoles and signal-to-background (S/B) ratio of 70 in a
cAMP standard curve. These features allow using fewer cells
compared to other cAMP detection platforms. Results of cell-based
assays indicated that all four cAMP platforms provide comparable
assay pharmacology with the expected rank order of agonist or
antagonist potency. However, in every application, the LANCE
Ultra outperformed the other cAMP platforms in terms of S/B
ratio. This key advantage, combined to a stable pharmacology,
simple assay protocol and unique kit format for every application,
makes the LANCE Ultra cAMP assay a superior HTS technology
suitable for both Gs- and challenging Gi-coupled receptors.
Z’ Study in CHO Cells Expressing
Gs-hMC4 Receptors
8
Agonist Responses in SK-N-MC Cells Expressing Endogenous Gs-coupled
β-adrenergic Receptors
TR-FRET Signal (665 nm)
4
Abstract
cAMP (fmoles)
1
Company C (dynamic 2 kit)
5,000 cells
2,000
Melanotan II
NDP--MSH
-MSH
1,500
1,000
• The LANCE Ultra technology enabled the development of
robust cAMP assays suitable for HTS, as indicated by the
high and stable Z’ values obtained.
500
0
- -13 -12 -11 -10
-9
-8
-7
-6
Log [Agonist] (M)
LANCE Ultra
Melanotan II
NDP-α-MSH
α-MSH
S/B
10.9
10.8
9.0
EC50 (nM)
0.02
0.11
0.16
Company C
S/B
4.4
4.0
3.8
• Expected pharmacological profiles and ranking of compound
potency were obtained with all four cAMP kits tested.
However, the LANCE Ultra technology consistently provided
higher S/B ratios while using fewer cells.
EC50 (nM)
0.04
0.15
0.20
• These key advantages, combined with a simple assay
protocol and unique kit format, make the LANCE Ultra cAMP
assay a superior HTS technology suitable for both Gs- and
Gi-coupled GPCRs.
PerkinElmer, Inc., 940 Winter Street, Waltham, MA USA (800) 762-4000 or (+1) 203 925-4602 www.perkinelmer.com