Slide 1 - September, 2015

Euroscreen S.A.

47 Adrienne Bolland, 6041 Gosselies, Belgium

Tel: +32 71 348 500, Fax: +32 71 348 519 Web : www.euroscreen.com

A UNIQUE ACCESS TO GPCR SCIENCES

Slide 2 - September, 2015

CONTENTS

INTRODUCTION

SERVICES

Slide 3 - September, 2015

Euroscreen S.A. at a glance

GPCR science-based research companyDual business modelDrug Development B.U. &Services B.U.40 employees (7 for CRO)Main facility (3,000 m2), in Gosselies BelgiumPrivate & Founded in 1994

Slide 4 - September, 2015

12114

4

218 satisfied customers from 23 countries

4

35

69 4

Slide 5 - September, 2015

Specialist in GPCR Services

GPCR among most druggable class of protein

30% of marketed drug hit GPCR

6 out of Top Ten drugs in US target GPCR

370 members, 250 with known activity

120 members with unknown ligands (orphan)

World market for GPCR targeting drugsto reach US$120 Billion by 2017*

* Source: Global Industry Analysts, Inc

GPCR at a glance EuroscreenFAST at glance: Recombinant proprietary cell lines for >390 receptors

(70 Orph/ 160 Orth)

>750 assays available from the shelf

World reputation for scientific and technical expertise

Tailored assay development capabilities

7 people including 2 PhDs

Top of the art readers and technologies

Providers for world pharma and biotech leaders

Slide 6 - September, 2015

Services offer

offers to its clients a complete array of services to support their Drug Discovery programs, from target validation to candidate selection. These services include :

Receptor deorphanization with our proprietary natural extracts library

High-Throughput Screening

Profiling

in vitro pharmacology for Hit-to-Lead and Lead-Optimization

Slide 7 - September, 2015

CONTENTS

INTRODUCTION

SERVICES

Slide 8 - September, 2015

Unique Target Deorphanization Platform

120 remaining Orphan receptors (70 rhodopsin like)

High potential for future drug discovery

But need of a natural ligand for TV

Few competitors in that field

Challenging activity with uncertain level of success

Deorphanization

Broadest orphan cellular tools (79)

Unique proprietary tissues extracts collection

Worldwide reputation and track record as leader in the field R&D collaboration with FTE’s based costing, upfront and milestones

Our offer

Slide 9 - September, 2015

Unique Target Deorphanization Platform: Workflow

Library collection

Assay Development

Deorphanization

Molecular Pharmacology and assay validation for further Drug Discovery

Cell line validationmRNA integrity

Cell surface expression (proprietary tag)Constitutive activity

Screening2 oGPCRs2 cell bckgrdsConfirmation

HPLCMS/MS

1,000 putative ligands750 existing tissue extracts

New extracts production based on target

Slide 10 - September, 2015

Cell line validation & assay developmentCell Background: CHO-K1, HEK-293, 1321N1, U2OS...

mRNA integrity

C1 C9 MW Ctl C3 C7 Ctl MW

Cell surface expression

Constitutive activity

0 2 20 60 200

600

20000

102030405060708090

100110

Inducer (ng/ml)

Cons

titut

ive ac

tivity

(cAM

P)

Slide 11 - September, 2015

Euroscreen tissue extracts collection

Different Extraction / Fractionation protocols Classical extracts

• Organic• Aqueous

Specific extracts• Small molecules/biogenic amines extracts• Lipid extracts• ‘Neutral pH’ extracts

New extracts production based on target

Slide 12 - September, 2015

Target Deorphanization Track Record

GPCR Target Reference: Patent application or publicationUndisclosed lipid receptor Deorphanized in 2014

GPR15(NVS-ES Collaboration Program)

WO 2015/069459A1

EBI2 (NVS-ES Collaboration Program)

WO 2010/066689A2Hannedouche et al., Oxysterols direct immune cell migration via EBI2. Nature, 2011; 475(7357): 524-7

GPR72 EP1867994B1 & US7824866B2

FPRL2 EP1607745B1Migeotte et al., Identification and characterization of an endogenous chemotactic ligand specific for FPRL2. J. Exp. Med., 2005; 201(1): 83-93.

GPR43 WO 2003/057730A1Le Poul et al., Functional characterization of human receptors for short chain fatty acids and their role in polymorphonuclear cell activation. J. Biol. Chem., 2003; 278(28): 25481-9

GPR7 and GPR8 WO 1995/12670A1 Brézillon et al., Identification of natural ligands for orphan G protein-coupled receptors GPR7 and GPR8. J. Biol. Chem., 2003; 278(2): 776 - 83.

CHEMERIN Receptor WO 2003/006996A2Wittamer et al., Specific recruitment of antigen-presenting cells by chemerin, a novel processed ligand from human inflammatory fluids. J. Exp. Med., 2003; 198(7): 977-85

P2Y13/GPR86 WO 2003/014731A2Communi et al., Identification of a novel human ADP receptor coupled to Gi. J. Biol. Chem., 2001; 276(44): 41479-41485

NPFF2 Kotani et al., Functional characterization of a human receptor for neuropeptide FF and related peptides. Br. J. Pharmacol., 2001; 133(1): 138-44.

GPR54 Kotani et al., The metastasis suppressor gene KiSS-1 encodes Kisspeptins, the natural ligands of the orphan G protein-coupled receptor GPR54. J. Biol. Chem., 2001; 276(37): 34631-6.

HCC-1 Detheux et al., Natural proteolytic processing of hemofiltrate CC chemokine 1 generates a potent CC chemokine receptor (CCR)1 and CCR5 agonist with anti-HIV properties. J. Exp. Med., 2000; 192(10): 1501-8

P2Y11 WO 1999/02675A1Communi et al., Cloning of a human purinergic P2Y receptor coupled to phospholipase C and adenylyl cyclase. J. Biol. Chem., 1997; 272(51): 31969-73

CCR5 WO 1997/32019A2Samson et al., Molecular cloning and functional expression of a new human CC-chemokine receptor gene. Biochemistry, 1996; 35(11):3362-7

ORL1 (Nociceptin Receptor) WO 1997/07208A1 Meunier et al., Isolation and structure of the endogenous agonist of opioid receptor-like ORL1 receptor. Nature, 1995; 377(6549):532-5

P2Y4 WO 1997/19170A1Communi et al., Cloning, functional expression and tissue distribution of the human P2Y6 receptor. Biochem Biophys Res Commun., 1996; 222(2):303-8

17 successful GPCR deorphanizations over the last 20 years

Slide 13 - September, 2015

in vitro Pharmacology Platform

Cell lines & assays development

Cloning & building of the cell lines from scratch

Assay development fitting client robustness and sensitivity criteria

Fast Turn Around Time (TAT): 30 days for a cell line 15 days for cellular assay

Exclusive or non-exclusive assay development

Non GPCR assay development capabilities

High Troughput Screening (HTS)

Functional (orthosteric or allosteric) or R*

Integrated offer with assay validation, HTS, confirmation and dose-response

Compounds handling capabilities

Up to 300,000 compounds in 15 working days, 384-well plate

Client or Euroscreen full or subset library

H2L support & profiling

Hit to Lead and Lead optimization support with

fast TAT (5 working days)

Functional selectivity profiling

Diversity Family Therapeutic Tailored

Bridging assays Cytokine release Chemotaxis Insulin release NEFA release

Slide 14 - September, 2015

Cell Lines and Assay DevelopmentRhesus monkey Chemokine CCR2 receptor assay development and comparison with

human receptor

Rhesus monkey CCR2 Human CCR2

Agonist functional

assay

Antagonist functional

assay

log[ligand], M1x10-12 1x10-10 1x10-8

%A

ctiv

atio

n

0

20

40

60

80

100

120

MCP-1 EC50 : 2.1 nM

log[ligand], M1x10-9 1x10-7 1x10-5

%In

hibi

tion

-20

0

20

40

60

80

100

RS102895IC50 : 248 nM

log[ligand], M1x10-12 1x10-9

%A

ctiv

atio

n

0

20

40

60

80

100 MCP-1 EC50 : 1.4 nM

log[ligand], M1x10-11 1x10-8 1x10-5

%In

hibi

tion

-20

0

20

40

60

80

100RS102895IC50 : 185 nM

Cloning, transfection, validation

TAT: 6 weeks for stable pool

Slide 15 - September, 2015

Log [Ligand], M1x10-15 1x10-13 1x10-11 1x10-9

%A

ctiv

atio

n

-100

102030405060708090

100

Cell Lines and Assay DevelopmentSNAP-GLP-1R cell line development for cAMP, binding and internalization readouts

cAMP assay

GLP-1 (7-36) EC50 : 0.04 nM

Log [Ligand], M1x10-12 1x10-10 1x10-8 1x10-6

%In

tern

lizat

ion

0

20

40

60

80

100

Internalization assay

GLP-1 (7-36) EC50 : 2.5 nM

Log[Ligand], M1x10-12 1x10-10 1x10-8 1x10-6

%B

indi

ng

0

20

40

60

80

100

Binding assay

GLP-1 (7-36) IC50 : 4.8 nM

Cloning, transfection, validation

TAT: 6 weeks for stable pool

Slide 16 - September, 2015

Cell Lines and Assay DevelopmentGTPg35S SP assay development for Gas-coupled Adenosine A2A receptor

Log[Ligand], M1x10-11 1x10-9 1x10-7 1x10-5

%A

ctiv

atio

n

-20

0

20

40

60

80

100

120

Log[Ligand], M1x10-12 1x10-10 1x10-8 1x10-6

%In

hibi

tion

-20

0

20

40

60

80

100

120

Agonist functional assay Antagonist functional assay

NECA EC50 : 27.1 nM

ZM241382IC50 : 6.75 nM

Slide 17 - September, 2015

Cell Lines and Assay DevelopmentNon-GPCR Assay Development : TRPA1

Agonist functional assay Antagonist functional assay

log[ligand], M1x10-8 1x10-6 0.0001

%A

ctiv

atio

n

-10

10

30

50

70

90

110

log[ligand], M1x10-11 1x10-8 1x10-5

%In

hibi

tion

-20

0

20

40

60

80

100AITC EC50 : 14 µM

A 967079IC50 : 170 nM

Cloning, transfection, validation

TAT: 6 weeks for stable pool

Slide 18 - September, 2015

Cell Lines and Assay Development

Cloning, transfection, validation

TAT: 6 weeks for stable pool

Non-GPCR Assay Development : SRD5a

Inhibition of Steroid 5-a-Reductase (SRD5a)-catalyzed testosterone degradation

log[ligand], M1x10-8 1x10-7 1x10-6 1x10-5

%In

hibi

tion

0

20

40

60

80

100

120 Linolenic AcidIC50 : 529 nM

Slide 19 - September, 2015

Cell Lines and Assay Development

Molecular Pharmacology

Residence time experiments

Bridging assay

Insulin Secretion from rat pancreatic islets

0 10 20 30 40 50 60 70 80 90 100 110 120 130 140 150 1600

100

200

300

400

500

600

1,1 nM risperidone3,6 nM risperidone10,8 nM risperidone

kon risperidone = 4,4 107 M-1 min -1

koff risperidone = 0,036 min-1

Time (min)

Spec

ific b

indi

ng (c

pm)

Human Dopamine D2L

0 2 4 6 8 10 12 14 16 18 200

50100150200250300350400450

ControlGLP1 10-6MGLP1 10-7M

****

/

++

** p<0.01 vs control/ p<0.05 vs control+ p<0.05 vs control

[Glucose] mM

Insu

lin s

ecet

ion

(ng/

mL)

Slide 20 - September, 2015

High Throughput Screening : Capabilities

Functional (orthosteric or allosteric):• Gq-coupling or Ca2+ channel: Aequorin, IPOne HTRF• Gi-coupling: cAMP HTRF, GTPg35S binding• Gs-coupling: cAMP HTRF• 7TM: ERK1/2 HTRF, Internalization

Radioligand binding (3H or 125I) Filtration or SPA

Up to 300,000 compounds in 15 business days, 384-well plate

Client or Euroscreen full or subset library

Slide 21 - September, 2015

High Throughput Screening : Equipment Liquid Handling

3 Minitrak stations (96/384, 96-384, 384-384) Tecan Genesis + gripper/carousel

+ 2 Multidrop stations

1 Janus station

Plate readers

2 FDSS6000 (96 & 96/384)luminescence

Tecan F200Pro (96/384)FI, FP, TR-FRET, lumi, BRET

Envision (96/384)FI, laser TR-FRET, lumi

TopCount (96/384)Scintillation counting

Slide 22 - September, 2015

High Throughput Screening : tailored offer

Determination of Z’ and assay stability to fit with Client criteria

DMSO tolerance

Frozen cells batch validation if possible

Miniscreen if necessary

HTS (up to 300k cpds/15 days), Cherry picking, Hit confirmation, DRC integrated offer

Validation of secondary assays & counterscreening

> 20 receptors screened

Slide 23 - September, 2015

High Throughput Screening : track record

Aequorin:• Orexin 1 & 2 antagonist• FFA receptors agonist & antagonist• mGluR5 NAM• CCR2b, CCR5, CXCR2, CXCR3, CX3CR1 agonist & antagonist• S1P1 agonist & PAM• S1P5 agonist & antagonist

cAMP:• GABAb PAM• 5-HT6 agonist & antagonist • GLP-1, GIP & GLP-2 agonist

ERK1/2 & GTPg35S:• FFA receptors agonist & antagonist

Slide 24 - September, 2015

Hit-to-Lead support

Repetitive assay with data delivery in 5 business days for rapid SAR Significant discounts offered based on frequency/volume/duration MSR determination for long-term studies Flexibility in data reporting to fit with your in-house requirements

Log[Ligand], M1x10 -12 1x10 -9 1x10 -6

%A

ctiv

atio

n

0

20

40

60

80

100 DAMGO EC50: 2nM

-1 0 . 0

-9 . 5

-9 . 0

-8 . 5

-8 . 0

-7 . 5

-7 . 01 2 3 4 5 6 7 8 9 1 0 1 1 1 2 1 3

OP3 receptor GTPg35S agonist assay

Slide 25 - September, 2015

Profiling

Preset profile (functional)

Tailored profile

57 targets diversity, natural coupling, A+/A-

Family targets (21 ChR, mGluR, 5-HTR, Orphan) Therapeutic areas (pain, inflammation, metabolism, CNS)

Slide 26 - September, 2015

Profiling 57 targets diversity, natural coupling, A+/A-FAST Diversity Profile Natural coupling Assay FAST Diversity Profile Natural coupling Assay

Serotonin 5-HT1A Gi/o cAMP Dopamine D1 Golf, Gs cAMP

Serotonin 5-HT1B Gi/o GTPgS Dopamine D2 Gi/o GTPgS

Serotonin 5-HT2A Gq/11 AEQ Dopamine D3 Gi/o GTPgS

Serotonin 5-HT2B Gq/11 AEQ Dopamine D4.4 Gi/o GTPgS

Serotonin 5-HT2Cne Gq/11 AEQ Endothelin ETA Gq/11, Gs AEQ

Serotonin 5-HT3 Ion channel AEQ Endothelin ETB Gq/11, Gi/o AEQ

Serotonin 5-HT4e Gs cAMP Ghrelin GHS-R Gq/11 AEQSerotonin 5-HT5 Gi/o AEQ Histamine H1 Gq/11 AEQ

Serotonin 5-HT6 Gs cAMP Histamine H2 Gs cAMP

Serotonin 5-HT7 Gs cAMP Histamine H3 Gi/o GTPgS

Adenosine A1 Gi/o cAMP Motilin MTL Gq/11 AEQ

Adenosine A2A Gs cAMP Muscarinic M1 Gq/11 AEQ

Adenosine A3 Gi/o cAMP Muscarinic M2 Gi/o GTPgS

Adrenergic beta1 Gs cAMP Muscarinic M3 Gq/11 AEQ

Adrenergic beta2 Gs cAMP Muscarinic M4 Gi/o GTPgS

Adrenergic  alpha1A Gq/11 AEQ Muscarinic M5 Gq/11 AEQ

Adrenergic  alpha1B Gq/11 AEQ Nociceptin OFQ (NOP) Gi/o GTPgS

Adrenergic  alpha2A Gi/o GTPgS Neurokinin NK1 Gq/11 AEQ

Adrenergic  alpha2B Gi/o GTPgS Neurokinin NK2 Gq/11 AEQ

Adrenergic  alpha2C Gi/o GTPgS Neuropeptide NPY1 Gi/o cAMP

Angiotensin AT1 Gq/11 AEQ Opioid OP1 Gi/o GTPgS

Bradykinin B1 Gq/11 AEQ Opioid OP2 Gi/o GTPgS

Cannabinoid CB1 Gi/o cAMP Opioid OP3 Gi/o GTPgS

Cannabinoid CB2 Gi/o cAMP Somatostatin sst4 Gi GTPgS

Cholecystokinin CCK1 Gq/11, Gs AEQ Urotensin-II Gq/11 AEQ

Cholecystokinin CCK2 Gq AEQ Vasopressin V1a Gq/11 AEQ

Calcitonin CGRP Gq/11, Gs AEQ Vasopressin V1b Gq/11 AEQ

Chemokine CXCR1 Gi/o GTPgS VPAC1 Gs AEQLeukotrien CysLT1 Gq/11 AEQ

Slide 27 - September, 2015

Profiling Family targets (21 ChR, also available for murine receptors)

J113863 (CCR1)

020406080

100120

RS102895 (CCR2)

0

20

40

60

80

100

BMSCCR2 22 (CCR2)

0

20

40

60

80

100

RS504393 (CCR2)

020406080

100

CCR1CCR2

CCR3CCR4

CCR5CCR6

CCR7CCR8

CCR9

CCR10

CXCR1

CXCR2

CXCR3

CXCR4

CXCR5

CXCR6

CX3CR1

XCR1

AMD3100 (CXCR4)

020406080

100

CCR1CCR2

CCR3CCR4

CCR5CCR6

CCR7CCR8

CCR9

CCR10

CXCR1

CXCR2

CXCR3

CXCR4

CXCR5

CXCR6

CX3CR1

XCR1

SB225002 (CXCR2)

020406080

100120

SB265610 (CXCR2)

0

20

40

60

80

100

T487 (CXCR3)

0

20

40

60

80

100

Slide 28 - September, 2015

Profiling Family targets (8 mGluR, natural coupling for all but mGluR3)

-11 -10 -9 -8 -7 -6 -5 -4 -3 -20

100200300400500600700800900

1000

L-AP4 EC50 = 247 µMMMPIP IC50 = 109 nMAMN082 EC50 = 24 nM

Log[ligand], M

Del

ta F

-12 -11 -10 -9 -8 -7 -6 -5 -4 -3 -20

5000

10000

15000

20000

25000 LY369268 EC50 = 6,7 nMGlutamate EC50 = 242 nMDCG IV EC50 = 27 nM

LY341495 IC50 = 1 nMMSOP IC50 = 6,7 nM

Log[ligand], M

Lum

ines

cenc

e (R

LU)

-12 -11 -10 -9 -8 -7 -6 -5 -4 -3 -20

5000

10000

15000

20000

Glutamate EC50 = 381 nMQuisqualate EC50 = 26 nM(1S,3R)-ACDP EC50 = 6,1 µMJNJ16259685 IC50 = 8 nMCPCCOet IC50 = 4,1 µMYM298198 IC50 = 202 nMCPPHA coad. EC50 = 1,75 µM

Log[ligand], M

Lum

ines

cenc

e (R

LU)

-12 -11 -10 -9 -8 -7 -6 -5 -4 -3 -20

100200300400500600700800900

100011001200 LY379268 EC50 = 4,3 nM

LY341495 IC50 = 3,4 nMMSOP IC50 = 7,6 µM

(1S,3R)-ACPD EC50 = 15 µMGlutamate EC50 = 4,2 µMDCG IV EC50 = 260 nM

LY487379 EC50 = 84 nM

Log [Ligand, M]

Del

ta F

-11 -10 -9 -8 -7 -6 -5 -4 -3 -20

2500

5000

7500

10000

L-AP4 EC50 = 440 nMSIB1893 EC50 = 7.15 µMVUO15041 EC50 = 890 nMLY341495 IC50 = 7.9 µM

Log[ligand] , M

(35S)

GTP

g S b

ound

(cpm

)

mGlu1 mGlu2

mGlu8

mGlu4

mGlu7

mGlu3

-12 -11 -10 -9 -8 -7 -6 -5 -40

10000

20000

30000

40000Glutamate EC50= 310 nMFenobam IC50= 144 nM

MPEP IC50= 15.3 nMBromo-MPEPy IC50= 3.9 nMMMPEP IC50= 3.5 nM

Log[ligand], M

Lum

ines

cenc

e R

LU)

mGlu6mGlu5

-13 -12 -11 -10 -9 -8 -7 -6 -5 -4 -3 -20

500

1000

1500

2000

L-AP4 EC50 = 786 nMGlutamate EC50 = 4,78 µMDCG-IV EC50 = 14,4 µMLY341495 IC50 = 153 nM

Log[ligand] , M

(35S)

GTP

g S b

ound

(cpm

)

-11 -10 -9 -8 -7 -6 -5 -4 -3 -2 -10

250

500

750

1000

1250

L-AP4 EC50 = 1 µMGlutamate EC50 = 17 µMLY341495 IC50 = 119 nM

Log[ligand], M

delta

F

Slide 29 - September, 2015

Profiling Tailored profile - Osanatant

-120

-100

-80

-60

-40

-20

0

20

40

60

5HT1A

5HT1B

5-HT6 A1

ALPHA2C AT1

BETA1C3A CB1

CB2CCK2

CCR2CGRP

CRF1

CRTH2

CXCR1

CXCR2

CXCR4 D2 D3 DPETA

FPRL1

GHS-R

GPR40

GPR41 H2 H3HM74

LTB4 M1 M2 M3MCH2

MT2NK2

NK3OP1

OP2OP3

OX1

OXER1

P2Y11

PAR2SST1

SST2SST3

SST4SST5

AgonistAntagonistCerep Binding

Osanatant, an NK3 antagonist was tested at 5 µM in customized functional profiling, using 24 aequorin, 5 cAMP and 20 GTPgS assays. Data were compared to an industry standard binding profile performed at 10 µM, when available. In addition to customized GPCR selection, the functional profiling gives information on agonist or antagonist activity and is also available for positive allosteric modulator testing

Std

Slide 30 - September, 2015

Euroscreen FAST Key Drivers

Expertise>50 years cumulated experience

FlexibilityTo fit with customer’s needs

ReactivityAverage TAT of 10.5 business days

ProximityStraight communication with lab

Beyond compound testing, look for an extension of your lab!