DiscoveRx beta-arrestin and internalization assays for GPCR receptors can uncover unique pharmacology.

1. Revealing Ligand & GPCR Bias Using
A Combination of 2nd Messenger,
β-Arrestin & Internalization
EFC Assay Formats

2. DiscoveRx – Your Drug Discovery Partner
Cell-Based Assays
246 GPCR cell lines
Catalog 15 Cell Based Kinases
Products 26 NHR cell lines
6 Pathway assays
Custom
Profiling and
Assay
Screening
Development

3. DiscoveRx Core Technology
Active Enzyme Inactive Fragments Active Enzyme
Enzyme PK
PK
Complementation +
Biochemical Cell Biology
HitHunterTM Product Line PathHunterTM Product Line
• Cellular GPCR Assays
• GPCR Signaling Assays
• Cellular NHR Assays
• Generic Kinase Assays
• Cellular Kinase Assays
• Protease Technology
• Cellular Protease Assays
• Unactive Kinase Technology
• Pathway / Signaling Assays

4. Evolution of Understanding GPCR Function
1. Binding - On/ Off
Radioligand binding studies
2. Receptor Activation Through Ga
Second messenger assays
3. Receptor De-sensitization via b-Arrestin
Internalization & recruitment studies

5. Evolution of Understanding GPCR Function
4. b-Arrestin involved in desensitization Balanced Signal
but also has signaling properties
Kinases (MAPK, P3K, AKT)
Originally thought that all downstream
pathways equally activated for a given agonist
5. Novel conformations of the same Biased Signals
receptor can initiate different
signaling cascades
Now understood that agonists can
activate only a subset of signals

6. Evolution of GPCRs

7. Functional GPCR Activities & Ligand Bias
Events following ligand binding to GPCRs
Second
Messenger Internalization
Calcium Signaling
Arrestin
cAMP
Differential activation of these pathways occurs using the same ligand
(Ligand Bias)

8. GPCR Activation: Multiple Biological Processes
G-protein Activation Arrestin Recruitment Internalization
Opportunity for GRK
1 2 3
ligand bias
Calcium cAMP
•2nd Messenger Signaling •Alternate pathway •Long term desensitization
•Short term desensitization •Altered Trafficking
•Contributes to Internalization •Degradation / Recycling
Long Acting Agonists Functional Antagonists,
Avoid Unwanted Signaling
Receptor removal
Monitoring each function can aid in compound characterization

9. PathHunter™ Protein Interaction Technology
Active Enzyme Inactive Fragments Active Enzyme
PK
PK
Enzyme
Complementation
+
Weak/No activity High activity
Modified Enzyme
Complementation
for Protein
interactions +
Features:
• Generic, homogeneous approach applicable to a wide range of drug target classes
• Flexible, small peptide tag with options for high and low affinity
• Only technology successfully transitioned from biochemical to cell based format

10. GPCR Activation & b-Arrestin Binding
PathHunter™ b-Arrestin Assays
50000
ADRB2  Small protein tag (42 aa)
40000
 Dynamic protein interaction assay
30000
RLU
 Target-specific signal
20000
 Chemiluminescent or fluorescent detection
10000
 Transfers easily from benchtop to full HTS
0
10 -11 10 -10 10 -9 10 -8 10 -7 10 -6 10 -5 campaigns
Isoproterenol [M]

11. Arrestin-Based GPCR Signaling
Thaw & Plate Cells
SSTR2 (Gi) CHRM5 (Gq)
ACHR5
3500 EC50=2.0 nM EC50=3.1 μM;
3000 S/B=42 14000
S/B=5.9
2500 12000
12-48 hrs 2000 10000
RLU
RLU
1500 8000
6000
1000
4000
500
2000
0
10 -12 10 -11 10 -10 10 -9 10 -8 10 -7 10 -6 0
Somatostatin 28 10 -11 10 -10 10 -9 10 -8 10 -7 10 -6 10 -5 10 -4 10 -3
1 hr
11hr
hour Oxotremorine M
ADRB2 (Gs) Type B Glucagon (GCGR)
40000
50000
EC50=11.2 nM EC50=15.3nM;
1 hour 40000 S/B=6.6 30000 S/B=11
30000
RLU
RLU
20000
20000
10000
10000
0 0
10 -14 10 -13 10 -12 10 -11 10 -10 10 -9 10 -8 10 -7 10 -6 10 -5 10 -12 10 -11 10 -10 10 -9 10 -8 10 -7 10 -6 10 -5 10 -4
Isoproterenol [M] Glucagon [M]
Read Luminescence

12. Highlights of PathHunter™ Arrestin Format
CHO A2 ADRB2 Clone Ligand Rank Order
Salbutamol
1750 Isoproterenol
 Stochiometric Signal Generation 1500
1250
(-) Epinephrine
Deoxyepinephrine
Clenbuterol
Compound Efficacy 1000
RLU
Salmeterol
750 Dobutamine
500 Formoterol
Antagonist Mode 250
0
Isoetharine
Metaproterenol
10 -11 10 -10 10 -9 10 -8 10 -7 10 -6 10 -5 10 -4 10 -3 Ritodrine
Agonist [M] Terbutaline
CHO A2 GLP2R
 Predictable Pharmacology 10000
7500
RLU
5000
 Specificity
2500
0
10 -12 10 -11 10 -10 10 -9 10 -8 10 -7 10 -6 10 -5
 Serum samples GLP 2 (1-33) human [M]
• Chemokines 8000
7000
• Bioactive lipids 6000
20%
40%
FBS
FBS
5000 60% FBS
• Antibody Supernatants
RLU
4000 80% FBS
3000
2000
1000
0
10 -8 10 -7 10 -6 10 -5 10 -4 10 -3
Anti-CCL20 mAb [g/mL] + 10nM CCL20

13. PathHunter Performance
ADRB2
>50% are 9X or better
S:B Ratio
Clenbuterol
Formoterol
Isoproterenol
PathHunter clones
(Receptors tested with reference agonist)
 Real-time analysis
 Signal to noise

14. Arrestin is G-protein Independent
Basis for Arrestin Ligand Bias cAMP Assay Arrestin Assay
CHO Gi FPRL1 CHO Arrestin FPRL1
1250 60000
(+) Pertussis tox
 G-protein inhibitor 1000 50000
40000
(-) Pertussis tox
750
independent (+) Pertussis tox
RLU
RLU
(-) Pertussis tox 30000
500
20000
250
10000
0 0
10 -14 10 -13 10 -12 10 -11 10 -10 10 -9 10 -8 10 -7 10 -6 10 -12 10 -11 10 -10 10 -9 10 -8 10 -7 10 -6 10 -5
WKYMVm-NH2 [M] + 20 M Forskolin WKYMVm-NH2 [M]
CXCR7 30000 C5L2
40000
 Arrestin in the 30000
20000
RLUs
absence of G-protein
RLU
20000
10000
activation 10000
0 0
10 -13 10 -12 10 -11 10 -10 10 -9 10 -8 10 -7 10 -6 10 -11 10 -10 10 -9 10 -8 10 -7 10 -6 10 -5 10 -4
SDF1a [M] Complement C5a [M]
Lambertus et al. J. Biomol Screening (2009)
Arrestin binding and internalization can occur in the absence of G-protein activity

15. Second Messenger
 Native Gi, Gs, and Gq coupled cell lines
 HitHunter® biochemical reagents for cAMP detection
Complex pharmacology- Agonists, antagonists, partial agonists & allosterism
Differentiating compounds - Large assay windows, ideal for difficult Gi targets
Miniaturizable
 Fluorescent and Aequorin-based calcium detection

16. Arrestin Biased Agonism
AGTR1 Ca++
Reference agonist Reference agonist
1500
4000 1250
1000
3000
RLU
RLU
750
2000
500
1000 250
0
0 10 -15 10 -14 10 -13 10 -12 10 -11 10 -10 10 -9 10 -8
10 -13 10 -12
10 -11
10 -10
10 -9
10 -8
10 -7
Angiotensin II [M]
Angiotensin II [M]
4000
Biased agonist SII Biased agonist SII
1500
3000 1250
1000
RLU
RLU
2000
750
500
1000
250
0 0
10 -11 10 -10 10 -9 10 -8 10 -7 10 -6 10 -5 10 -4 10 -11 10 -10 10 -9 10 -8 10 -7 10 -6 10 -5 10 -4
[Sar1, Ile4,8]-Angiotensin II [M] [Sar1, Ile4,8]-Angiotensin II [M]

17. PathHunter is Ideal for Investigating Ligand Bias
GCGR cAMP
Glucagon
2000 Glucagon
1250
DHG+
300nM Glucagon DHG
1000 1500
RLU
750 1000
RLU
500
500
250
0
0 10 -13 10 -12 10 -11 10 -10 10 -9 10 -8 10 -7 10 -6 10 -5 10 -4
10 -12 10 -11 10 -10 10 -9 10 -8 10 -7 10 -6 10 -5 10 -4
Ligand [M]
Ligand [M]
DHG is a ligand for the GCGR receptor--
antagonist in Arrestin but a partial agonist in cAMP

18. Lessons Learned
7TMR Signaling
Both G-protein dependent & independent pathways
Linked but not dependent pathways
Can be modulated independently by compounds
PathHunter™ Arrestin Technology
Broadly applicable measure of arrestin signaling
Ideal for difficult targets and orphans
HTS friendly
Opportunity for multiplexed measurements

19. GPCR Internalization
Applicable to the majority of
GPCRs
Reduces the number of cell
surface receptors acutely
Recycled
Targeted for degradation
Functional antagonism
Compartmentalized signaling
has been observed

20. PathHunter™ GPCR Internalization Assays:
2 Convenient Platforms. Same Answer. Does the receptor internalize?
Activated Internalization
EA EA
Arrestin Recruitment
EA
EA
EA
EA
Endo
EA
Complemented Enzyme
Complemented Enzyme
- Gain-of-signal, large S:B
- Arrestin dependent - Gain-of-signal, large S:B
-Short-term desensitization at - Arrestin dependent
membrane - Long-term desensitization at
endosome

21. PathHunter™ Activated GPCR Internalization Assays:
Broadly Applicable
Neurotensin Chemokine Muscarinic
1000
750
750
500
RLU
RLU
7500
500
250
250
5000
RLU
0 0
10 -13 10 -12 10 -11 10 -10 10 -9 10 -8 10 -7 10 -6 10 -10 10 -9 10 -8 10 -7 10 -6 10 -5 10 -4 10 -3
2500
CCL3 [M] Oxotremorine-M [M]
NTSR1 0
10 -12 10 -11 10 -10 10 -9 10 -8 10 -7 10 -6 10 -5
CCR5 M5
Neurotensin [M]
 Tested across 35 different families
BOTTOM
NTSR1
177.4
TOP 6785
 Over 65 targets available (over 80 by 3/1)
LOGEC50
HILLSLOPE
-8.850
1.651
EC50 1.4128e-009
 Building out specific panels
S/B = 47.0
 Compound selectivity
 Timing, magnitude, cell type specificity

22. GPCR “Trio Project” For Ligand Bias
24 targets tested in all 3 formats, average of 5 agonists per target
Pharmacology differences observed in 11 of 24
Expected overlap between cAMP/Calcium and Arrestin
Only 1% of ligands are completely biased, 5-10% show some bias
More differences with internalization
Currently expanding studies and following up on “biased” ligands
22

23. DiscoveRx Trio Initiative
CXCR2 CCR1 BDK2
No Bias detected Efficacy Bias Potency Bias
Calcium
2nd Messenger
cAMP cAMP
125
125
150
CXCL1 CCL3
100 Lys-Bradykinin
% cAMP inhibition
125 CCL23
% cAMP Inhibition
% Activation
100 CXCL2 Bradykinin
CXCL3 100 75
75
[Phe8"Psi"BDK
CXCL5 75 50
50 CXCL6
Interleukin-8 50
25
25 25
0 0 0
1
10
0
1
1
10 0
01
01
10 00
0. 1
00
01
1
01
1
1
10
0
00
01
1
01
1
1
10
0
00
0
10
00
0
00
0.
00
10
0.
00
10
00
0.
10
00
0
00
0.
00
10
0.
00
10
0.
00
0.
0.
10
0.
0.
0.
0.
Agonist + 15 M Forskolin Agonist + 15 M Forskolin Agonist
Arrestin Arrestin Arrestin
150 150 150
CXCL1 CCL3
125 125
Arrestin
125 CXCL2 CCL23 Lys-Bradykinin
% Activation
% Activation
% Activation
100 CXCL3 100 100 Bradykinin
CXCL5 [Phe8"Psi"-BDK
75 75 75
CXCL6
50 Interleukin-8 50 50
25 25 25
0 0 0
0
0
0
0
00
00
00
00
.0
.0
.0
.0
1
1
10
0
1
01
00
01
0
01
1
1
0. 1
1
10
0
01
00
10 00
00
0.
1.
2.
3.
10
00
00
0.
-4
-3
-2
-1
10
00
00
0.
10
00
0.
00
10
0
00
0.
10
0.
00
10
0.
0.
Agonist
0.
Agonist
Agonist
Internalization
Internalization Internalization
Internalization
150 125
CXCL1 600
125 CXCL2 CCL3
500 100 Lys-Bradykinin
% Activation
100 CXCL3 CCL23
% Activation
Bradykinin
% Activation
CXCL5 400 75
75 [Phe8"Psi"-BDK
CXCL6
50 300 50
Interleukin-8
25 200
25
0 100
0
0
1
1
1
10
0
01
01
00
10
00
0.
10
00
0.
0.
0.
1
10
1
0
1
01
00
10 00
01
00
0. 1
1
1
10
0
1
01
00
01
0
10
00
00
0.
10
00
00
0.
10
10
00
00
0
0.
00
0.
10
0.
10
00
0.
Agonist
0.
0.
Agonist
Agonist

24. Uncover Biased Ligands Using the Complete
DiscoveRx GPCR Portfolio
2nd Messenger Arrestin Internalization
Prolonged Prolonged
G-protein Desensitization
Biased Signaling
activation Functional
Antagonism
Delta Opioid Receptor Case Study
• Opioid system controls responses to pain
- OPRD1 (hDOR) activation alleviates persistent pain
- Desensitization leads to persistent pain
• Receptor responds to endogenous & synthetic ligands
• hDOR undergoes rapid internalization
• Compound-specific differences in re-sensitization have been identified

25. SNC-80 Results in OPRD1 Desensitization
• SNC-80 showed 95% inhibition slower
sustained for 30 minutes
• Faster & stronger desensitization
vs endogenous enkephalins
• Fresh ligand could not restore
signal

26. SNC-80 Results in Prolonged OPRD1
Internalization
•SNC-80 identified as a
strongly internalizing
compound
•Agonist recycling is
compound-specific

27. In vivo Effects of a Non-Internalizing Ligand
First 2nd
ARMS390 and SNC80 are full agonists Challenge Challenge
by GTPgS
SNC80 –EC50: 122nM
ARMS390-EC50: 170nM
Summary
•SNC80 is a strongly
internalizing ligand
•SNC80 behaves as a
functional antagonist
in vivo

28. Differential GPCR Biology: OPRD1
2nd Messenger Arrestin Internalization
200000
2000
25000
20000 1500
150000
15000
RLU
RLU
RLU
100000 1000
10000
50000 500
5000
0 0 0
10 -14 10 -13 10 -12 10 -11 10 -10 10 -9 10 -8 10 -7 10 -6 10 -5 10 -12 10 -11 10 -10 10 -9 10 -8 10 -7 10 -6 10 -5 10 -4 10 -3 10 -12 10 -11 10 -10 10 -9 10 -8 10 -7 10 -6 10 -5 10 -4 10 -3
Agonist [M] + 20 M Forskolin Agonist [M] Agonist [M]
 DAMGO is a weak agonist  DAMGO weakly recruits Arrestin  SNC-80 strong Arrestin-
compared to SNC-80  SNC-80 strong Arrestin response mediated internalization vs
DAMGO

29. More Information - CCKAR
•Full agonist in calcium
•Similar in vivo half lives
•Why is a compound that
is less potent, more active
in vivo?

30. Relevant, Predictive, Informative
High Content Compound Analysis
Arrestin
Endo
400000
sCCK8 7000
A-71623 6000 sCCK8
300000 A-71623
5000
RLU
200000 4000
RLU
3000
100000
2000
1000
0
10 -12 10 -11 10 -10 10 -9 10 -8 10 -7 10 -6 10 -5 10 -4 0
10 -12 10 -11 10 -10 10 -9 10 -8 10 -7 10 -6 10 -5
[M]
[M]
Working hypothesis: A-71623 has prolonged duration of action due to poor internalization.
Moving the 2nd Messenger Arrestin Internalization
industry……. Potency Efficacy Potency Efficacy Potency Efficacy
Compound X 10nM Full 5nM Full 5nM Partial
Compound Y 50nM Full 35nM Partial 35nM Full
Compound Z 20nM Partial 100nM Full 100nM Full

31. Uncover Biased Ligands Using the DiscoveRx
Complete GPCR Offering: CHRM2
CHRM2
cAMP CHRM2 Arrestin CHRM2 Internalization CHRM2
12500
12500
150000
10000
10000
7500 100000
RLU
7500
RLU
5000 RLU
5000
50000
2500
2500
0
0 0
10 -10 10 -9 10 -8 10 -7 10 -6 10 -5 10 -4 10 -10 10 -9 10 -8 10 -7 10 -6 10 -5 10 -4 10 -3 10 -2
10 -10.0 10 -7.5 10 -5.0 10 -2.5 10 0.0
[M] [M] [M]
 Oxo-M gives a functional response  Oxo-M shows weak  Oxo-M shows strong Arrestin-
 Behaves as an agonist b-Arrestin recruitment mediated internalization

32. Uncover Biased Ligands Using the DiscoveRx
Complete GPCR Offering: NTSR1
NTSR1
Calcium NTSR1 Arrestin NTSR1
Internalization NTSR1
350 1250000
70000
300
1000000 60000
250
50000
200 750000
RLU
RLU
40000
RLU
150 500000 30000
100 20000
250000
50 10000
0 0 0
10 -12 10 -11 10 -10 10 -9 10 -8 10 -7 10 -6 10 -5 10 -4 10 -12 10 -11 10 -10 10 -9 10 -8 10 -7 10 -6 10 -5 10 -4 10 -12 10 -11 10 -10 10 -9 10 -8 10 -7 10 -6 10 -5 10 -4
[M]
[M] [M]
 Kinetensin behaves as a weak  Kinetensin is a weak  Kinetensin results in weak
agonist agonist, similar EC50s internalization
*Currently building out data sets and algorithms (in collaboration)
for Ligand Bias quantification*

33. Why Are PathHunter Internalization Assays Such
An Important Drug Discovery Tool?
• Receptor Internalization Influences Agonist Efficacy
-Fewer receptors at the cell surface, reduces potency
-Selective and non-selective agonists can be compared
• New classes of drugs can be developed
- Functional Antagonists that remove receptors from cell surface
can reduce unwanted side effects
- Non-recycling receptors may be better targets for chronic diseases
(multiple administrations)
• Molecular Pharmacology
- Determine a direct link between internalization/localization &
function for a target

34. DiscoveRx GPCR Portfolio Summary
2nd Messenger Arrestin Internalization
Prolonged Prolonged
G-protein Desensitization
Biased Signaling
activation Functional
Antagonism
 Multi-Mode GPCR characterization provides:
 Extensive ligand analysis – identification of biased ligands
 Potential to select ligands with specific properties and durations
 Correlate biological function with biochemical characterization
 Similar Studies
• OPRM1 - μ- Opioid receptors: correlation of agonist efficacy for signalling with
ability to activate internalization: McPherson et al. Mol Pharm July 2010
• EP4 - Functional selectivity of natural and synthetic prostaglandin EP4 receptor
ligands: Leduc et al. JPET July 2009

35. Biased Ligand Discovery
with DiscoveRx
Monitor GPCR activity through multiple 7TMR
signaling pathways and uncover novel, biased ligands
• Largest GPCR menu covering all 7TMR signaling
pathways -- 2nd messenger, arrestin & internalization)
• Preferred supplier offering all technology platforms in
the same robust, easy-to-use, HTS-friendly format
• Provide “Next Generation” GPCR platforms that can be
used explore novel receptor biology including
internalization and dimerization

36. DiscoveRx Comprehensive GPCR Portfolio
Thank
You!
GPCR Pathway DRX Products Advantages
Gi/Gs Native Gi/Gs cell lines, Native, non-force coupled cell lines with chemiluminescent
HitHunter™ cAMP XS+ detection
Gq Native Gq cell lines, Native, non-force coupled cell lines with fluorescent detection
HitHunter™ Calcium No Wash PLUS
Gq HitHunter™ IP3 Secondary confirmation of Calcium response
G-protein PathHunter™ Arrestin De-orphanization, uncover novel pharmacology biased ligand
Independent discovery
Signaling
PathHunter™ Activated GPCR Monitor activated GPCR internalization without imaging, biased
Internalization Assays ligand discovery
PathHunter™ GPCR Dimerization Assays Specific and quantitative measure of GPCR heterodimers