Core Biology Activities - Addex 2009 R&D Day

Core Biology Robert Lütjens Addex Pharmaceuticals R&D Day July 16, 2009

Novel Approaches for GPCR AM Discovery

Development of New Screening Assays ,[object Object],Ideal setup allows a dynamic and proximal measurement of receptor activation, without changing the homeostasis of the system Promiscuous or chimeric G prot Yes No Yes Ca 2+ flux PDE inhibitors Yes No No, stop assays cAMP levels Requires False positives Proximal to Receptor Dynamic Type of Signal

Addex cAMP Biosensor ,[object Object],[object Object],[object Object],[object Object],BIOSENSOR X PHOENYX 0 0% STOP ASSAY % of max response to 100 nM isoproterenol 30 Time (min) 5 10 15 25% 50% 75% 100% cAMP Levels

ADX Tags Series 1 ,[object Object],[object Object],[object Object]

ADX Tags Series 1.1 Plasma membrane C Activation A) Tag GPCR N BP B) Tag binding partners of activated receptor signal ! N BP BP

ADX Tags Series 1.2 Plasma membrane C Activation A) Tag GPCR B) Tag binding partners of inactive receptor BP BP BP NO signal! N N signal ! signal ! N

ADX Tags Series 1 for GPCRs ,[object Object],[object Object],[object Object]

Novel Approaches for non-GPCR AM Discovery

APRA Accessory Protein Relocalization Assay ,[object Object],[object Object],[object Object]

Visualizing TNF Receptors activation APRA for TNFR1 TNFR1 RIP1 TNF Clustering Quantifiable by Optical Microscopy control Patent Application filed in Jan’09 Endogenous Receptor Prevents ligand-independent receptor triggering TNF, 15min, 63X Control, 15min, 63X Tagged Accessory protein

Pharmacology of TNFR1 APRA Assay Spots/Cell Clone #49-E2 Log TNF concentration (g/ml) Performance Log Compound concentration (g/ml) Selectivity Inhibition Log Compound concentration (g/ml) Window signal/noise > 15x Z’ (EC 80 ) > 0.6 -14 -13 -12 -11 -10 -9 -8 -7 -6 0 1 2 3 4 5 6 7 8 9 TNF TL1A IL1  CD40L TRAIL anti-FAS Poly I:C LPS Enbrel 16.6 ng/ml IC 50 Anti-TNFR1 70.3 ng/ml IC 50 -10 -9 -8 -7 -6 -5 -4 -3

ADX Tags Series 1 for Non-GPCRs ,[object Object],[object Object],ACTIVATION Type 1 trans- membrane receptor NO signal! A) Tag receptor signal ! signal ! B) Tag binding partner OR

ADX Tags Series 2 ,[object Object],[object Object],ACTIVATION Type 1 trans- membrane receptor Tag receptor signal !

ADX Tags Series 2 Applied to TNFR1 ,[object Object],[object Object],[object Object],[object Object],[object Object],TNF response on cells expressing TNFR1-TAG2 media 172 nM 2.6 nM 0 100 200 300 400 500 7000 6000 5000 4000 3000 Relative Units Time (seconds)

Novel Approaches to drive the Medicinal Chemistry of Allosteric Modulators

[object Object],Allosteric Modulators: complex pharmacological behavior 100 50 Log [compound] 0 50 Log [compound] 0 % of max PAM response 100 % of max NAM response drc partial PAM drc full PAM drc partial NAM drc full NAM

Negative Allosteric Modulation endogenous ligand cell membrane outside cell inside cell active site negative allosteric modulator (NAM) AM binding site COOH

Positive Allosteric Modulation endogenous ligand positive allosteric modulator (PAM) active site cell membrane outside cell inside cell COOH

Silent Allosteric Modulation endogenous ligand cell membrane outside cell inside cell active site silent allosteric modulator (SAM) COOH

FBBA Fluorescence-Based Binding Assay ,[object Object],[object Object],[object Object],[object Object],[object Object],[object Object],[object Object]

FBBA setup: Plasma membrane Fluorescently-Tagged Receptor Time Receptor Fluorescence N SNAP FLUORESCENCE 1 st addition: « Cold » ADX cpd A) 2 nd addition: Tagged ADX cpd B) B) A)

FBBA: Compound MoA Determination ,[object Object],[object Object],[object Object],EC20 GLP-1 Functional Measure FBBA Measure

Addex novel assays ,[object Object],[object Object],[object Object],[object Object],[object Object],[object Object],[object Object],[object Object],[object Object]

Core Biology Activities - Addex 2009 R&D Day

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