1. Core Biology
Robert Lütjens, Ph.D
Addex Pharmaceuticals R&D Day
May 11, 2010
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2. Addex Tools for AM Discovery
 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
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3. Novel tools for HTS of allosteric modulators:
Assays developed for Non-GPCRs
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4. Non-GPCR AM Discovery
 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
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5. Signaling pathways of cytokine receptors
DR3 TNFR1 IL1R1 TLR
RIP1
RIP1
TRADD TRADD
TRAF5 TRAF2
TRAF5 TRAF2
Caspases
Caspases TAK1
TAK1
Measured
responses
p38
p38
IKK
IKK JNK
JNK
Death
pathway
NFB
NFB
NFB Stress
pathway pathway Cytokine
production
At Addex, we have developed unique assay systems to better
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measure allosteric modulation of receptors of interest

6. ADX Tags Series 2 for non GPCRs
Assay characteristics
Type-1 transmembrane
receptor
Ligand –induced
activation
!l
Tagged
na
si g
receptor
 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
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7. ADX Tags Series 2 for non GPCRs
Example with TNFR1 NAM Project
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
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8. ADX Tags Series 2 for TNFR1
Assay performance
Performance Selectivity Inhibition
300
350
250 TNF 250
300
A nti-T N FR 1 m A b
Relative units
TL1A
Relative units
200 250 200 E nbrel
IL1-
Relative units
200
150 150
150
100 100
100
50 50
50
0 0 0
-11 -10 -9 -8 -7 -12 -11 -10 -9 -8 -7 -6 -7 -6 -5 -4
Log [TNF], g/ml Log [com pound], g/m L Lo g [co m po und], g/m l
 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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9. ADX Tags Series 2 for TNFR1
Time-resolved output allows identification of
receptor specific signatures
250000 T
TNFR1
Tag output Intensity (I)
200000
Signature profiles
TN
150000
F
3.010 7
100000 TNFR1
Tag output Intensity (I)
DR3
50000
2.010 7
0
0 100 200 300 400 500 600 700 800
Time following TNF addition (Second) 1.010 7
250000
DR3
Tag output Intensity (I)
200000
0
100 200 300 400 500 600
150000
T T (s)
TL
100000
1A
I : Tag output (AUC)
50000 T : Time interval from agonist addition
to response peak
0
0 100 200 300 400 500 600 700 800
Time following TL1A addition (Second)
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10. ADX Tags Series 2 for TNFR1
Compounds affect receptor signature differently
ADX61410 ADX29342
3.010 7 3.010 7
TNF + DR ADX61410 TNF + DR ADX29342
DR TNF
Tag output Intensity (I)
Tag output Intensity (I)
TNF + DR anti-TNFR1 mAb DR TNF
2.010 7 2.010 7
1.010 7 1.010 7
0 0
250 300 350 400 450 500 550 250 300 350 400 450 500 550
T (s) T (s)
Both compounds inhibit TNF-induced tag2 TNFR1 activation, but ADX61410
does so without altering global TNFR1 signature profile, whereas ADX29342
strongly changes it
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11. ADX Tags Series 2 for TNFR1
Representative Compounds behave as non-
competitive antagonists (NAMs) of TNFR1
4.010 7 no compound ADX60357-2 5.010 7 no compound ADX29342-1
1.87 uM 1.87 uM
3.75 uM 3.75 uM
7.5 uM 4.010 7 7.5 uM
3.010 7 15 uM 15 uM
30 uM 30 uM
no compound no compound
RLU (AUC)
3.010 7
RLU (AUC)
2.010 7
2.010 7
EC50 EC50
no compound 1.212e-009 no compound 9.548e-010
1.87 uM 1.346e-009 1.87 uM 8.337e-010
1.010 7
3.75 uM 9.482e-010 3.75 uM 7.267e-010
7.5 uM 9.928e-010
1.010 7
7.5 uM 7.395e-010
15 uM 7.246e-010 15 uM 7.406e-010
30 uM 3.753e-010 30 uM 5.977e-010
no compound 8.853e-010 no compound 8.521e-010
0 0
-11 -10 -9 -8 -7 -11 -10 -9 -8 -7
Log [TNF], g/ml Log [TNF], g/ml
5.010 7 no compound ADX52945-1 5.010 7 no compound ADX85142-1
1.87 uM 1.87 uM
3.75 uM 3.75 uM
4.010 7 7.5 uM 4.010 7 7.5 uM
15 uM 15 uM
30 uM 30 uM
no compound no compound
RLU (AUC)
3.010 7
RLU (AUC)
3.010 7
2.010 7 2.010 7
EC50 EC50
no compound 8.259e-010
no compound 6.951e-010
1.87 uM 8.103e-010
1.87 uM 5.447e-010
3.75 uM 7.300e-010
1.010 7 3.75 uM 5.214e-010
7.5 uM 8.333e-010 1.010 7
7.5 uM 4.240e-010
15 uM 8.878e-010
15 uM 3.683e-010
30 uM 7.596e-010
no compound 8.883e-010
30 uM 3.682e-010
0 no compound 6.328e-010
-11 -10 -9 -8 -7 0
Log [TNF], g/ml -11 -10 -9 -8 -7
Log [TNF], g/ml

12. ADX Tags Series 2 for TNFR1
Identification of compounds with PAM or NAM
effects
R1 R2
A Linker 1 B A Linker 2 B
ADX29342 ADX10612
120 80
% enhancement over buffer
100
% max response (AUC)
60
Tag Output
80
Tag Output
60 40
40
20
20 ADXtag2-IL1R at IL1- EC80 ADXtag2-IL1R at IL1- EC20
ADXtag2-TNFR1 at TNF EC80 ADXtag2-TNFR1 at TNF EC20
0 0
-7 -6 -5 -4 -7 -6 -5 -4
Log [ADX29342], (M) Log [ADX10612], (M)
Existence of a functional switch from negative to
positive TNFR1 modulation in identified series
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13. ADX Tags Series 2
Applicable to variety of receptor targets
Assays developed for various receptor families :
ADX Tags Series 2 for DR3 ADX Tags Series 2 for BMP-2R ADX Tags Series 2 for TrkB
125000000
6000000
400000
100000000
Relative Units
Relative units
Relative Units
300000 75000000
4000000
50000000
200000
2000000 25000000
100000
0
-10 -9 -8 -7 -6 -5
0 0 Log [compound], g/ml or M
-10 -9 -8 -7 -6 -5 -4 -11 -10 -9 -8 -7 -6 -5
Log [compound], g/mL Log [BMP-2], g/ml BDNF, EC50 = 25ng/ml
TL1A, EC50 = 25 ng/ml K252a on EC80 of BDNF, IC50 = 70 nM
BMP-2, EC50 = 86 ng/ml
DCR3, IC50 = 4.22 g/ml EC80 100ng/ml
Window signal/noise >50x Window signal/noise >15x Window signal/noise >10x
Z’ (EC20) > 0.4
Preparation for HTS ongoing
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14. ADX Tags Series 2 for non GPCRs
Summary
 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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15. Conclusion
 Addex has developed a set of unique biological tools, addressing
GPCR 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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16. Addex Biological Patent Portfolio
Assay Details of filing
APRA (Accessory Protein GB 0900860
Relocalization Assay) Application filed 2009
EP09178233
Phoenyx
Application filed 2009
EP10160519
ProxyLite
Application filed 2010
ADX Tags Series 1 ongoing
EP10162068
ADX Tags Series 2
Application filed 2010
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17. Addex R&D Day 2010
11:00-11:15 Welcome & Overview – Vincent Mutel, CEO
11:15-11:45 Allosteric Platform – Robert Lutjens, Head of Core Biology
11:45-12:30 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 Lunch
13:30-13:50 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 Mutel
15:00-15:45 Tour of Addex & Closing Reception
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18. allosteric modulators for human health
www.addexpharma.com
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