AAdvanced cAMP Technology (ACTOne) Assay on G-Protein

Coupled Receptors (GPCRs)

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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

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-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