An overview of the issues and approaches in selecting the best targets for drug discovery and validating them. Given at the Drug Discovery Forum held at the Royal Society, London and organised by the Academy of Medical Sciences

1. Target Validation Academy of Medical Sciences London 1 st Dec 2006 Mike Romanos VP Discovery Technology GlaxoSmithKline R&D Stevenage

4. Drug Discovery Process and Target Validation Continuum Gene to target Target to Lead Lead to candidate Candidate to FTIH FTIH to PoC PoC to Commit to Phase III Phase III File & Launch Lifecycle mgt Target selection Lead validation Compound on/off target effects Refined validation vs product profile TARGET VALIDATION Translational medicine

5. Essential Tools: Biological Systems Animal models of disease Human tissues Disease-relevant cell models Humans

6. Essential Tools: Target Modulation Transgenics RNAi Tool compounds Biopharmaceutical tools TT TT

7. Essential Tools: Analytical Methods Genetics Transcriptomics Quantitative PCR Histology Proteomics Bioinformatics

10. Compound-based Functional Characterisation of FXR Vehicle + GW4064 Mouse liver Primary human hepatocytes Vehicle + GW4064 or CDCA FXR transcriptome

11. FXR: Master Regulator of Bile Acid Homeostasis cholesterol Liver Intestine blood bile acid bile bile acid RXR FXR bile acid RXR FXR bile acid

12. Efficacy of GW4064 in Cholestasis Model # p<0.05 cw Normal * p<0.05 cw ANIT Bile Acids Bilirubin 0 200 400 600 800 Bile acid (µmol/L) 0 1 2 3 4 5 6 Total bilirubin (mg/dL) * # # Enzyme activity (U/L) 0 500 1000 1500 2000 ALT AST # # * * Liu et al., JCI , 112 , 1678 (2003) Normal ANIT ANIT+GW4064 ANIT+TUDCA

16. Mouse VR1 Knockout No response to capsaicin – no overt pathology No hyperalgaesia induced by inflammatory insult (but no effect on mechanical hypersensitivity) -8.0 -7.0 -6.0 -5.0 -4.0 -3.0 -2.0 -1.0 0.0 1.0 2.0 Capsaicin (KO) Vehicle (KO) Capsaicin (Het) Vehicle (Het) Capsaicin (WT) Vehicle (WT) Mean Maximum Temp Change (Centigrade )

18. VR1 expression is up-regulated in chronic inflamed rectum A proliferation of VR1 positive nerve terminals is seen in chronic inflamed gut (oesophagus) C22, 1:5000 Human Disease Tissue: VR1 in Visceral Pain rectal mucosa Normal Hyper 0.0 0.1 0.2 0.3 0.4 0.5 0.6 VR1 immunoreactivity % area p = 0.0286 Mann Whitney U

21. siRNA Target Identification in Inflammatory Diseases Assays selected to reflect key disease mechanisms Mast Cells: IgER mediated histamine release (Rhinitis) Dendritic Cells: CD40 mediated IL-12 release (Asthma) Macrophages: IC mediated TNF  release (RA) Epithelial Cells: Oxidative stress mediated cytokine release/apoptosis (COPD) T Cells: CD3/CD28 mediated IL-5 and IL-13 release (Asthma) siRNA

22. siRNA for High Throughput Screen BioFocus Collaboration: Biology-driven Target Discovery

23. Validation of Target A and B in Primary Human CD4+ Cells Using siRNA Target A (TCR response) Target B (antigen response) Profound inhibition of key cytokines in Rheumatoid Arthritis (TNF  ) and Asthma (IL13) * Selective inhibition of Asthma ‘pathological’ cytokine production 0 1000 2000 3000 4000 5000 6000 7000 TNFa IL13 pg/ml Control T Target A IL13 Control Target B 0 1000 2000 3000 4000 5000 pg/ml

24. Target Validation in Human Disease Tissue Targeting Allergen Specific T Cell Responses in Asthma Effect of drug on secretion of Cytokines and other Mediators Supernatant allergic asthmatic biopsy material Allergen ± Drug p=0.02 Unchallenged allergen challenged allergen challenged +drug Effect of tool compound to Target A on IL-5 release

28. Clinical improvement Clinical decline Model-adjusted mean Change from baseline in ADAS-cog by ApoE4 status Pharmacogenetic Stratification Enables Observation of Clinical Efficacy: Roziglitazone in AD