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AAdvanced cAMP Technology (ACTOne) Assay on G-Protein
Coupled Receptors (GPCRs)
1
2
Biogenic Amines
Amino Acids and Ions Lipids
Peptides and Proteins
Others
q s i
N E1 E2 E3
I1 I2 GDP
I3 C
Signal Transduction Pathway with GPCR
GTP GTP GTP
[Ca++] [cAMP] [cAMP]
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GPCR
cAMP
Reporter assay BDTMACTOne
cAMP Assays Available on Market
Alpha Screen ELISA
G s
[ ]
G i
LANCE
LANCE: A homogenous TR-FRET assay
Alpha Screen: A bead based non-radioactive Amplified Luminescent Proximity Homogeneous Assay
Antibody based assays
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BBDTMACTOne
Living cell Real time High Sensitivity Big assay window World wide patent issued Well accepted by pharmaceutical industry
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Overview of ACTOne Biosensor
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ACTOne Assay Flow Chart
Grow a confluent monolayer of cells in microplates
O/N at 37 C with CO2
Add ACTOne fluorescent dye
2 hours at room temperature
Read baseline with a Pathway Imager, or fluorescence reader
End Point Assay
Add compound
Read with a Pathway Imager, or fluorescence reader
~30 min at room temperature
Grow a confluent monolayer of cells in microplates
O/N at 37 C with CO2
Add ACTOne fluorescent dye
2 hours at room temperature
Read on a Pathway, FLIPR, or FLEXStation
Kinetic Assay
The BD ActOne assay is a homogenous assay without washing steps. The assay can be performed as an end point assay or a kinetic assay.
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NECA
0
100
200
300
400
500
0 10 20 30 40 50 60
Time (min)%
Incr
ease
30 uM10 uM3 uM1 uM0.3 uM0.1 uM0.03 uMBuffer
Kinetic Assay End Point Assay
ACTOne: Fast Response and Stable Signal
0 50 100 150 2000
50
100
150
200
250
% In
crea
se
Time (sec)
300 nM SST 100 nM SST 30 nM SST 10 nM SST 3 nM SST 1 nM SST Buffer 1 uM ISO
SST = somatostatin, agonist of SSTR NECA: Agonist of A2b
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ACTOne Has Higher Sensitivity Over ELISA
0.001 0.01 0.1 1 10 100 1000-10
10
30
50
70
90
110ELISAACTOne
NECA (uM)%
of M
axim
a R
espo
nse
0.001 0.01 0.1 1 10 100 1000-10
10
30
50
70
90
110
ELISAACTOne
FSK (uM)
%of
Max
ima
Res
pons
e
ActOne technology shows improved sensitivity compared to ELISA by measuring localized cAMP generation at the cytoplasmic membrane.
FSK: Foskolin, Activator of adenylate cyclase (AC)
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ACTOne Is More Sensitive Than Reporter Assay
0.01 0.1 1 10 100
0.0
0.2
0.4
0.6
0.8
1.0 N
orm
aliz
ed R
eado
ut
[Forskolin (uM)]
ACTOne CRE-Luc
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0 30 60 90 120 150 180
0
40
80
120
160
200
240
F/F0
%
Time (sec)0 30 60 90 120 150 180
0
40
80
120
160
200
240
Time (sec)0 30 60 90 120 150 180
020406080
100120140
Time (sec)
NECA Isoproterenol PGE1
Adenosine A2B -adrenoceptor Prostanoid EP2
300 100 30 10 3
1 0.3 0
nM 3000 1000 300 100
30 10 3 0
nM 30 10 3 1
0.3 0.1 0.03 0
M
0.1 1 10 100 10000
40
80
120
160
200
Isoproterenol (nM)
EC50 = 2.0 nM EC50 = 57 nM
1 10 100 1000 100000
40
80
120
160
200
F/F0 %
PGE1 (nM)10 100 1000 10000 100000
0
20
40
60
80
100
120
NECA (nM)
EC50 = 540 nM
ACTOne is Excellent to Detect Endogenous GPCRs
Receptor:
Ligand:
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ACTOne Works Well with Recombinant Gs-GPCR
0.01 0.1 1 10 100 1000
1
2
3
4
5
6
7
8 30 min
Ft/F
0
Dopamine (nM)
Parental Cell Line
DRD1 Stable Clone
12
-1000
2300
5600
8900
12200
15500
18800
22100
25400
28700
32000
0 0 1 2 3 4 5 6 7 8
Samples in Duplicate
Flu
ores
cenc
e U
nit
Agonist
Antagonist
Known agonist Testing Compounds
Non-reacting compound
ACTOne Can Screen Both Agonist and Antagonist in One Assay
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-Adrenoceptor Agonist and Antagonist Detection
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0
50
100
150
200
250
Buffer 1 uM Iso
Flu
ore
scen
ce in
cre
ase %
1, Z' = 0.792, Z' = 0.883, Z' = 0.624, Z' = 0.845, Z' = 0.586, Z' = 0.79
0
50
100
150
200
250
300
350
400
Buffer 1 uM IsoFl
uore
scen
ce in
crea
se %
1, Z' = 0.822, Z' = 0.563, Z' = 0.884, Z' = 0.515, Z' = 0.696, Z' = 0.657, Z' = 0.948, Z' = 0.89
Well-to-well and Experiment-to-experiment Variability
Kinetic Assay Endpoint Assay
ACTOne: Excellent S/N Ratio and Reproducibility
Z’ = 1- 3*(SD1+SD2)/(Fmax-Fmin) Z’ = 1.0: Ideal. This is approached when you have a huge dynamic range with tiny standard deviations. Z-factors can never actually equal 1.0 and can certainly never be greater than 1.0. Z’ between 0.5-1.0: An excellent assay. Z’ between 0-0.5: A marginal assay. Z’ less than 0: The signal from the positive and negative controls overlap, making the assay essentially useless for screening purposes.
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ACTOne on High Throughput Screening
The Initial Screen of MC4R Cells with 3,500 Compounds
0
100
200
300
400
0 1000 2000 3000
Well # (arbitrary)
%
Incr
ease
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GPCR
G s G i
GPCR
cAMP cAMP
cAMP binds to CNG
Detection: Florescence Change
cAMP
ACTOne for Gi-GPCR
(CNG)
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0 200 400 600 800 0
50
100
150
200
250
% In
crea
se
Time (sec)
Buffer
100 nM Dopamine
1 Dopamine
Dopamine Inhibits Forskolin Induced cAMP Activation in DRD2 Stable Cells (Kinetic Assay)
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Endpoint Assay for Gi-Coupled DRD2
0.01 0.1 1 10 100 10000
20406080
100120140160180200220240260280
% In
crea
se
Dopamine (nM)
DRD2
Load Fluoresce Dye
2 hours
Add FSK and Dopamine Mixture
30 min
Read Plate
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10-13 10-12 10-11 10-10 10-9 10-8 10-7 10-6 10-5 10-40.5
1.5
2.5
3.5
4.5
5.5
6.5
7.5
DopaQPRNPASNPADHX
Compound (M)
Ft/F
0
ACTOne Detects Partial Agonist of DRD2
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Forskolin, 3uM Gs, Isoproterenol 1uM
Gq, Carbachol 10uM
Gi, Dopamine 300nM
Buffer
ACTOne Differentiates Gq, Gs and Gi Pathway
0 100 200 300 0.35
0.65
0.95
1.25
Buffer
Fura
-2 ra
tio
Gs
Gq
Gi
1 2 sec.
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List of ACTOne Stable Clones
Transduced Gi-coupled receptors (22) Cannabinoid receptor 1 (CB1) Dopamine Receptor 2 (DRD2) Somatostatin Receptor 5 (SSTR5) Adenosine A1 Receptor (ADORA1) Chemokine (C-C motif) receptor 5 (CCR5) Melanin-concentrating Hormone Receptor 1 (MCHR1) Cannabinoid receptor 2 (CB2) Glutamate receptor, metabotropic 8 (GRM8) Opioid receptor, kappa 1 (OPRK1) Adenosine A3 receptor (ADORA3) Glutamate receptor, metabotropic 8 (GRM8) Neuropeptide Y Receptor Y1 (NPY1R) Neuropeptide Y Receptor Y2 (NPY2R) Sphingosine-1-phosphate receptor 1 (EDG1) Glutamate receptor, metabotropic 7 (GRM7) Glutamate receptor, metabotropic 2 (GRM2) Chemokine (C-X-C motif) receptor 4 (CXCR4) Opiate receptor-like 1 (OPRL1) Angiotensin II receptor-like 1 (AGTRL1) Opioid receptor, mu 1 (OPRM1) Glutamate receptor, metabotropic 4 (GRM4) Neuropeptide Y Receptor Y4 (NPY4R)
Transduced Gs coupled receptors (34) Vasoactive Intestinal Peptide Receptor 2 (VIPR2) Melanocortin 4 Receptor (MC4R) Melanocortin 5 Receptor (MC5R) Parathyroid Hormone Receptor 1 (PTHR1) Glucagon Receptor (GCGR) Dopamine Receptor 1 (DRD1) Prostaglandin E Receptor 4 (EP4) Vasoactive Intestinal Peptide Receptor 1 (VIPR1) Gastric Inhibitor Peptide Receptor (GIPR) Dopamine Receptor 5 (DRD5) Parathyroid Hormone Receptor 2 (PTHR2) 5-hydroxytryptamine (serotonin) receptor 6 (HTR4) Corticotropin Releasing Hormone Receptor 2 (CRHR2) Adenylate Cyclase Activating Polypeptide 1 Receptor type I (ADCYAP1R1) Secretin Receptor (SCTR) Follicle Stimulating Hormone Receptor (FSHR) 5-hydroxytryptamine (serotonin) receptor 6 (HTR6) 5-hydroxytryptamine (serotonin) receptor 7 (HTR7B) Glucagon-like peptide 2 receptor (GLP2R) Thyroid stimulating hormone receptor (TSHR) Melanocortin 3 Receptor (MC3R) Arginine Vasopressin Receptor 2 (AVPR2) Prostaglandin I2 Receptor (PTGIR) Glucagon-like-1 Receptor (GLPR1) Corticotropin Releasing Hormone Receptor 1 (CRHR2) Calcitonin receptor-like (CALCRL) Adenosine A2b receptor (ADORA2B) Beta2 Andrenergic Receptor (ADRB2) Prostaglandin D2 receptor (PTGDR) Prostaglandin E receptor 2 (PTGER2) Melanocortin 1 Receptor (MC1R) Calcitonin receptor (CALCR) Adenosine A2a receptor (ADORA2a) Amylin 3 receptor (Amy3R)
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Measures physiological levels of cAMP in living cells Excellent signal to noise ratio and reproducibility Fast Response and stable signal allowing kinetic or
endpoint assay formats Screen agonists and antagonists in one assay. Advantage to detect endogenous GPCRs Powerful deorphanizing tool to help study novel receptors
Summary of ACTOne on GPCRS