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1 Core Biology Robert Lütjens, Ph.D Addex Pharmaceuticals R&D Day May 11, 2010
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Core Biology
Robert Lütjens, Ph.D
Addex Pharmaceuticals R&D DayMay 11, 2010
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Addex has developed new proprietary tools, measuring molecular changes at the receptor level following its activation by the binding of its ligand
Those tools were conceived to assess the real kinetic changes associated with receptor allosteric modulation and, by doing so, remove off-target contribution in assay readout
Addex Tools for AM Discovery
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Novel tools for HTS of allosteric modulators:Assays developed for Non-GPCRs
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Current available biological tool arsenal is even poorer than for GPCRs, and has not been developed with allosteric modulators in mind
The most receptor-proximal measurements rely upon events happening downstream in cascades, sometimes hours after receptor activation. Readouts are carried out at integration nodes in canonical pathways, common to numerous signalling cascades triggered by various ligands
Non-GPCR AM Discovery
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Signaling pathways of cytokine receptors
CaspasesCaspases
NFBNFB
Death pathway
DR3
TRADD
TNFR1
TRADD
IL1R1 TLR
RIP1RIP1
TRAF5TRAF5 TRAF2TRAF2
TAK1TAK1
p38p38IKKIKK
NFBpathway
JNKJNK
Stresspathway Cytokine
production
Measuredresponses
At Addex, we have developed unique assay systems to better measure allosteric modulation of receptors of interest
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Addex proprietary dynamic & receptor proximal assay performed with live cells Monitors conformational and/or multimerization alterations Tag positioning may or may not require truncation of receptors HTS & Medchem 384-w plates compatible
Type-1 transmembrane receptor
Taggedreceptor sig
nal !
Ligand –inducedactivation
ADX Tags Series 2 for non GPCRsAssay characteristics
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Project objective:
Identify orally available, safe, selective, centrally and peripherally active small molecule antagonist (Negative Allosteric Modulators) targeting TNFR1 for the treatment of peripheral and central inflammatory conditions, as well as neurodegenerative diseases
ADX Tags Series 2 for non GPCRsExample with TNFR1 NAM Project
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Performance Selectivity Inhibition
-7 -6 -5 -40
50
100
150
200
250
300
A nti-T NFR1 mA bE nbrel
Lo g [co m po und], g/m l
Rel
ativ
e un
its
-11 -10 -9 -8 -70
50
100
150
200
250
Log [TNF], g/ml
Rel
ativ
e un
its
-12 -11 -10 -9 -8 -7 -60
50
100
150
200
250
300
350
Log [compound], g/mL
Rel
ativ
e un
its
TNFTNFTL1ATL1AIL1IL1--
ADX Tags Series 2 for TNFR1Assay performance
Window signal/noise >25x, Z’ (EC80) > 0.6 Assay is TNFR1 selective HTS successfully performed Hit rate was 0.6% at > 50% inhibition
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100 200 300 400 500 6000
1.0107
2.0107
3.0107
TNFR1DR3
T (s)
Tag
outp
ut In
tens
ity (I
)
0 100 200 300 400 500 600 700 8000
50000
100000
150000
200000
250000
Time following TNF addition (Second)
Tag
outp
ut In
tens
ity (I
) TNFR1
I : Tag output (AUC) T : Time interval from agonist addition to response peak
T
0 100 200 300 400 500 600 700 8000
50000
100000
150000
200000
250000DR3
Time following TL1A addition (Second)
Tag
outp
ut In
tens
ity (I
)
T
Signature profilesTNFTL1A
ADX Tags Series 2 for TNFR1Time-resolved output allows identification of receptor specific signatures
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250 300 350 400 450 500 5500
1.0107
2.0107
3.0107
TNF + DR ADX29342DR TNF
T (s)
Tag
outp
ut In
tens
ity (I
)Both compounds inhibit TNF-induced tag2 TNFR1 activation, but ADX61410 does so without altering global TNFR1 signature profile, whereas ADX29342 strongly changes it
250 300 350 400 450 500 5500
1.0107
2.0107
3.0107
TNF + DR ADX61410DR TNFTNF + DR anti-TNFR1 mAb
T (s)
Tag
outp
ut In
tens
ity (I
)ADX Tags Series 2 for TNFR1Compounds affect receptor signature differently
ADX29342ADX61410
ADX Tags Series 2 for TNFR1Representative Compounds behave as non-competitive antagonists (NAMs) of TNFR1
-11 -10 -9 -8 -70
1.0107
2.0107
3.0107
4.0107
5.0107 no compound1.87 uM3.75 uM7.5 uM15 uM30 uMno compound
EC50no compound 9.548e-010
EC50
1.87 uM 8.337e-010
EC50
3.75 uM 7.267e-010
EC50
7.5 uM 7.395e-010
EC50
15 uM 7.406e-010
EC50
30 uM 5.977e-010
EC50
no compound 8.521e-010
Log [TNF], g/ml
RLU
(AUC
)
ADX29342-1
-11 -10 -9 -8 -70
1.0107
2.0107
3.0107
4.0107 no compound1.87 uM3.75 uM7.5 uM15 uM30 uMno compound
EC50no compound 1.212e-009
EC50
1.87 uM 1.346e-009
EC50
3.75 uM 9.482e-010
EC50
7.5 uM 9.928e-010
EC50
15 uM 7.246e-010
EC50
30 uM 3.753e-010
EC50
no compound 8.853e-010
Log [TNF], g/ml
RLU
(AUC
)
ADX60357-2
-11 -10 -9 -8 -70
1.0107
2.0107
3.0107
4.0107
5.0107 no compound1.87 uM3.75 uM7.5 uM15 uM30 uMno compound
EC50no compound 6.951e-010
EC50
1.87 uM 5.447e-010
EC50
3.75 uM 5.214e-010
EC50
7.5 uM 4.240e-010
EC50
15 uM 3.683e-010
EC50
30 uM 3.682e-010
EC50
no compound 6.328e-010
Log [TNF], g/ml
RLU
(AUC
)
ADX85142-1
-11 -10 -9 -8 -70
1.0107
2.0107
3.0107
4.0107
5.0107 no compound1.87 uM3.75 uM7.5 uM15 uM30 uMno compound
EC50no compound 8.259e-010
EC50
1.87 uM 8.103e-010
EC50
3.75 uM 7.300e-010
EC50
7.5 uM 8.333e-010
EC50
15 uM 8.878e-010
EC50
30 uM 7.596e-010
EC50
no compound 8.883e-010
Log [TNF], g/ml
RLU
(AUC
)
ADX52945-1
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Existence of a functional switch from negative to positive TNFR1 modulation in identified series
-7 -6 -5 -40
20
40
60
80
100
120
Log [ADX29342], (M)
Tag O
utput
% m
ax re
spon
se (A
UC)
ADX29342
AR1
BLinker 1
-7 -6 -5 -40
20
40
60
80
Log [ADX10612], (M)Ta
g O
utpu
t%
enh
ance
men
t ove
r buf
fer
ADXtag2-IL1R at IL1- EC80ADXtag2-TNFR1 at TNF EC80
ADXtag2-IL1R at IL1- EC20ADXtag2-TNFR1 at TNF EC20
ADX Tags Series 2 for TNFR1Identification of compounds with PAM or NAM effects
ADX10612
A BLinker 2
R2
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Assays developed for various receptor families :
ADX Tags Series 2Applicable to variety of receptor targets
ADX Tags Series 2 for BMP-2RADX Tags Series 2 for DR3 ADX Tags Series 2 for TrkB
Window signal/noise >50x Window signal/noise >10xZ’ (EC20) > 0.4Preparation for HTS ongoing
-10 -9 -8 -7 -6 -50
25000000
50000000
75000000
100000000
125000000
BDNF, EC50 = 25ng/ml
K252a on EC80 of BDNF, IC50 = 70 nM
EC80 100ng/ml
Log [compound], g/ml or M
Rel
ativ
e U
nits
Window signal/noise >15x
-11 -10 -9 -8 -7 -6 -50
100000
200000
300000
400000
BMP-2, EC50 = 86 ng/ml
Log [BMP-2], g/ml
Rel
ativ
e U
nits
-10 -9 -8 -7 -6 -5 -40
2000000
4000000
6000000
TL1A, EC50 = 25 ng/mlDCR3, IC50 = 4.22 g/ml
Log [compound], g/mL
Rel
ativ
e un
its
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ADX Tags Series 2 for non GPCRsSummary
Successfully applied to members of the TNF receptor superfamily(TNFR1, DR3), as well as of the TGF receptor superfamily(BMP-2R), and of the FGF receptor superfamily (TrkB)
Approach is applicable to all type 1 single-pass transmembrane receptors, numerous of these representing attractive drug targets
Assays successfully amenable to HTS (TNFR1, TrkB and DR3 ongoing) and used to test compounds synthesized within project (TNFR1 NAM project)
Patent filed
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Conclusion
Addex has developed a set of unique biological tools, addressingGPCR as well as non-GPCR drug targets
These tools have been developed to measure proximally the changes linked to receptor activation
Signal output allows a time-dependent analysis of the receptor dynamics, which is crucial for allosteric modulator discovery
With those tools in hand, we are strongly moving ahead towards our objectives which are to identify allosteric modulators for all types of receptors
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Addex Biological Patent Portfolio
EP10162068Application filed 2010
ADX Tags Series 2
ongoingADX Tags Series 1
EP10160519Application filed 2010
ProxyLite
EP09178233Application filed 2009
Phoenyx
GB 0900860Application filed 2009
APRA (Accessory Protein Relocalization Assay)
Details of filingAssay
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Addex R&D Day 201011:00-11:15 Welcome & Overview – Vincent Mutel, CEO11:15-11:45 Allosteric Platform – Robert Lutjens, Head of Core Biology11:45-12:30 Selected Discovery Programs
• mGluR4 PAM – Emmanuel Le Poul, Head of CNS• GLP1R PAM – Vincent Mutel, CEO • IL1R1 NAM – Laurent Galibert, Head of Inflammation
12:30 – 13:30 Lunch13:30-13:50 ADX71943 – Sonia Poli, Head of Non-Clinical Development 13:50-14:50 ADX48621
• PD-LID background & context – Tom Babic, i3 Research• Status & Plan – Charlotte Keywood, Chief Medical Officer
14:50-15:00 Closing Remarks – Vincent Mutel15:00-15:45 Tour of Addex & Closing Reception
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www.addexpharma.com
allosteric modulators for human health
