Download - Esfast services presentation
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!