The document discusses BioMAP, a platform using primary human cell-based disease models to characterize compounds and discover their mechanisms of action and toxicity. It can assess over 2000 compounds across diverse pathways and targets. BioMAP generates biological profiles for compounds and uses these to classify compounds by similarity of mechanism. It has been used in collaborations to efficiently profile EPA ToxCast compounds and identify unexpected targets. The platform bridges molecular and cellular data to help validate targets and indications and connect to in vivo studies.

1. BioMAP Primary Human Cell-Based Systems for Drug Discovery Ellen L. Berg, PhD CSO, BioSeek, Inc. IBC Assays and Cellular Targets San Diego, CA September 26, 2008 Bio Seek

2. Goal Assay platform that provides broad assessment of compound activity Efficacy Safety Approaches: mRNA profiling, proteomics Biochemical assays Cell-based and high content assays Problems: What biological system do you measure? What do you do with the results? Can you make a decision? Alternative: Focus on what you really want: disease and tissue biology

3. Presentation Overview BioMAP Human Cell Systems Platform Primary human cell-based disease models Compound characterization Correlation with in vivo biology Clinical indication selection, biomarker discovery Mechanism of action discovery Toxicity assessment EPA - ToxCast TM program

4. BioMAP ® Technology Bridging the Gap Scale (meters) (Time) 10 -9 M 10 -8 M 10 -7 M 10 -6 M 10 -5 M 10 -4 M 10 -3 M 10 -2 M 10 -1 M 1 M Human exposure Molecular targets 10 -6 sec 10 2 sec 10 4 sec 10 5 sec 10 8 sec BioMAP Technology Biological Complexity: A challenge to drug discovery molecules pathways cells tissues humans

5. BioMAP ® Technology Platform BioMAP is highly complementary to biochemical target and phenotypic screening Assays Human primary cells Disease-like culture conditions LPS BF4T SM3C Profile Database Informatics Biological responses to drugs and stored in the database Specialized informatics tools are used to mine and analyze biological data

6. BioMAP Systems are cell-based assays engineered to model complex human disease biology Human primary cells Co-cultures, multiple stimulation factors, activated cells Quantitative protein readouts - biomarkers Pharmacological relevance of systems and biomarkers validated with known drugs BioMAP ® Technology Platform Assays Human primary cells Disease-like culture conditions LPS BF4T SM3C

7. Assay endpoints include human clinical biomarkers and risk factors (proteins) Cytokines, chemokines Adhesion and growth receptors Biological mediators (prostaglandins, etc.) Proteases, enzymes (MMPs, plasminogen activators) Others (hemostatic factors, matrix components) Involved in cell-cell communication -- outside the cell BioMAP ® Technology Platform Assays Human primary cells Disease-like culture conditions LPS BF4T SM3C

8. Assays Human primary cells Disease-like culture conditions Profile Database Biological responses to drugs and stored in the database BioMAP ® Technology Platform > 2000 agents Approved drugs Experimental compounds Bioactive agents Toxic compounds LPS BF4T SM3C

9. Panel of Current BioMAP Systems Inflammation / Autoimmune Disease / Respiratory / Cardiovascular Primary Human Cell Types Disease Relevance BioMAP ® System Endothelial cells (EC) Th1/ Th2 inflammation, allergy, asthma, dermatitis, angiogenesis, wound healing, restenosis, atherosclerosis (coronary art.) EC + Peripheral Blood Mononuclear Cells Th1 inflammation, psoriasis, COPD fibrosis, monocyte and T cell responses EC + Macrophages Macrophage responses, arthritis, COPD, fibrosis EC + Mast cells Asthma, allergy, dermatitis, fibrosis EC + Smooth Muscle Cells Vascular biology, restenosis, atherosclerosis EC + Th2 blasts Allergy, asthma Fibroblasts Arthritis, asthma, dermatitis, fibrosis, psoriasis, wound healing Myofibroblasts Fibrosis, COPD, wound healing Keratinocytes Psoriasis, dermatitis, wound healing Keratinocytes + Fibroblasts Psoriasis, dermatitis, wound healing Bronchial Epithelial Cells Th1 and Th2 inflammation Allergy, asthma, fibrosis, COPD Bronchial Epithelial Cells + Fibroblasts Asthma, allergy, fibrosis, COPD Smooth Muscle Cells Vascular inflammation, asthma, COPD, fibrosis (coronary artery SMC) LPS SAg HPNo 3C 4H Mphg MCIgE HTh2 HSM3C HDF3CGF HDF3C HDFNo HDF3CT HDFT BF4T SM3C BE3C BE4T K3CT MyoF KFNo KF3CT CA3C CASM3C

10. Broad Coverage of Pathways / Mechanisms Cancer HDAC Hsp90 Proteasome EGFR NF  B PI-3K  /AKT Mek CDK RAR/RXR Ras/MAPK TGF  Microtubule Jak/Stat Tie2 R Mitochondrial function Inflammation / Autoimmune Calcineurin, TCR Glucocorticoid R Prostaglandin, leukotriene TNF-  IL-10, IL-4, IL-17 NF  B IL-1, IFN  , IFN  p38 kinase Jak/Stat (Jak1, 2, 3, Tyk2) Lck kinase, PI-3K  , PCK  mTOR JNK Asthma/Allergy H1-Receptor  2 Adrenergic cAMP/PDE PAF IL-4, IL-13 Cardiovascular ACE  2 Adrenergic Ca++ Channel Cholesterol Antioxidant Metabolism PPAR  PPAR  GR LXR FXR Estrogen receptor Androgen receptor HMG-CoA reductase AMPK GSK3  … .and many others (>2000 drugs/compounds in reference database)

11. BioMAP Profiling: Example Profile Reference p38 Inhibitor Log expression ratio (Drug/DMSO control) BioMAP activity profiles are robust and reproducible Profiles retain shape over multiple concentrations Control (no drug) 99% significance envelope BioMAP Systems Readout Parameters (Biomarkers) Dose Response Cytotoxicity Readouts

12. Key Features Consistent with Known Biology Reference p38 Inhibitor Log expression ratio (Drug/DMSO control) Tissue factor IL-8 HLA-DR Monocyte activation ITAC VCAM TIMP-2 Thrombomodulin MMP1 IL-1  CD38 T cell activation p38 MAP kinase regulates many biological processes 99% significance envelope BioMAP Systems

13. Key Features Consistent with Known Biology Role of p38 MAP kinase in Th1-type inflammation Log expression ratio (Drug/DMSO control) HLA-DR Activities relevant to the role of p38 in Th1-type inflammation Takanami-Ohnishi Y, Amano S, Kimura S, Asada S, Utani A, Maruyama M, Osada H, Tsunoda H, Irukayama-Tomobe Y, Goto K, Karin M, Sudo T, Kasuya Y. Essential role of p38 mitogen-activated protein kinase in contact hypersensitivity. J Biol Chem. 2002 Oct 4;277(40):37896-903. CD38 T cell activation Monocyte activation IL-8 IL-1  99% significance envelope BioMAP Systems

14. Key Features Consistent with Known Biology Role of p38 MAP kinase in thrombus formation Log expression ratio (Drug/DMSO control) Tissue factor Thrombomodulin Activities relevant to the role of p38 in thrombus formation Sakurai K, Matsuo Y, Sudo T, Takuwa Y, Kimura S, Kasuya Y. Role of p38 mitogen-activated protein kinase in thrombus formation. J Recept Signal Transduct Res. 2004;24(4):283-96. 99% significance envelope BioMAP Systems

15. Key Features Consistent with Known Biology Activities relevant to side effects Log expression ratio (Drug/DMSO control) Activities relevant to side effects Melikoglu M, Uysal S, Krueger JG, Kaplan G, Gogus F, Yazici H, Oliver S. Characterization of the divergent wound-healing responses occurring in the pathergy reaction and normal healthy volunteers. J Immunol. 2006 Nov 1;177(9):6415-21. Potential pro-inflammatory effects in some tissue settings Activation of stress pathway ITAC VCAM 99% significance envelope BioMAP Systems

16. Classification by Similarity of Biological Mechanisms Multidimensional Scaling - Function Similarity Map I  B  IKK-2 CDK  2 Adrenergic PI3-K MEK 1/2 Lck/calcineurin steroid ACE Histamine H1 statins mTOR p38 MAPK Hsp90

17. Classification by Similarity of Biological Mechanisms Multidimensional Scaling - Function Similarity Map I  B  IKK-2 CDK  2 Adrenergic PI3-K MEK 1/2 Lck/calcineurin steroid ACE Histamine H1 statins mTOR p38 MAPK Hsp90 Mechanism of Action (On-Target) Off-Target Secondary Activities Pathway Relationships

18. NFkappaB Activation Inhibitor Strong profile in BioMAP systems - Unknown Mechanism of Action 6-Amino-4-(4-phenoxyphenylethylamino)quinazoline “ Potent inhibitor of NFkappaB activation” M. Tobe et al. Bioorg. Med. Chem., 2003, 11:383

19. NFkappaB Activation Inhibitor Does not cluster with core NF  B pathway inhibitors I  B  IKK-2 p38 MAP kinase Glucocorticoids TNF  Antagonist IL1  Antagonist Core NF  B Pathway

20. NFkappaB Activation Inhibitor Database Search Reveals Unexpected Mechanism I  B  IKK-2 p38 MAP kinase Glucocorticoids TNF  Antagonist IL1  Antagonist Mitochondrial Inhibitors F(0)F(1) ATPase Complex I Core NF  B Pathway

21. NFkappaB Activation Inhibitor BioMAP profiling provides mechanism of action Mitochondrial energy production inhibition MOA is not consistent with therapeutic hypothesis Inhibition of NFkappaB suggests inflammatory conditions MOA suggests clinical application in metabolic disease or cancer Targeting mitochondrial energy production is also a safety concern

22. Microtubule Stabilizers Mito ATPase Retinoids Hsp90 CDK HDAC NF  B MEK DNA Synth. XIAP Protein synthesis mTOR Microtubule Destabilizers Estrogen R PI-3K Ca ++ Mobilization Classification of Toxic Agents By Mechanism BioMAP Profiling Clusters Toxic Agents by Mechanism

23. BioSeek - EPA ToxCast Project

24. BioSeek - EPA ToxCast TM Project (Phase I) 320 ToxCast TM Compounds 219 (68%) Active 8 BioMAP Systems 87 Biomarkers 4 Concentrations

25. BioMAP Profiles of Positive Control (Colchicine) Replicates Overlay of BioMAP profiles of positive controls (colchicine) Each replicate represents a separate plate (template) 99% Significance envelope is shown (grey shading)

26. Reference Compounds BioMAP Profiling Distinguishes Reference Compounds Colchicine

27. Dose-Response Relationships Rapamycin mTOR inhibitor Target-specific Profile is relatively dose-independent Genistein Isoflavone Multiple targets Profile is relatively dose-dependent

28. Diversity of Mechanisms

29. Diversity of Mechanisms

30. BioMAP Profiles of 2 Compounds in Example Cluster Pyraclostrobin and Tryfloxystrobin are strobilium herbicides BioMAP profiles are highly similar to one another Tryfloxystrobin Pyraclostrobin Pyraclostrobin, 13.33  M Pyraclostrobin, 4.44  M Pyraclostrobin, 1.48  M Tryfloxystrobin, 40  M Tryfloxystrobin, 13.33  M Tryfloxystrobin, 4.44  M

31. Reference Database Search Identifies MOA Tryfloxystrobin Search results: Optimized search algorithms use combination of metrics Trifluoxystrobin

32. Reference Database Search Identifies MOA Of >3000 reference profiles searched, only 7 compounds are significantly similar All are known inhibitors of mitochondrial function Search results: Optimized search algorithms use combination of metrics Confirms mechanism of action Tryfloxystrobin Trifluoxystrobin

33. Similarity of profiles indicates similarity of mechanism Benomyl is a known inhibitor of microtubule function Paclitaxel is an anti-mitotoic that interferes with tubulin function Benomyl and Fludioxonil: Similarity to Paclitaxel

34. Mancozeb and Maneb: Similarity to Chlorambucil Mancozeb and Maneb are dithiocarbamate fungicides Chlorambucil is a DNA alkylating agent Nitrogen mustard chemotherapeutic agent (used in CLL) Side effects include bone marrow suppression, GI, CNS effects, hepatotoxicity

35. Diversity of Mechanisms cAMP Elevation DNA Alkylation NF  B Tubulin Inhibition Mitochondrial Dysfunction

36. Summary BioMAP profiling provides efficient characterization of compounds across a broad range of human biology Inflammation biology, cardiovascular, lung, skin, cancer, etc. Many pathways and targets covered Direct bridge to in vivo studies Compound / target validation for therapeutic indication, biomarker discovery Connects molecular, biochemical, & cell-based data to in vivo study results Rapid identification of target mechanisms Useful for identification of unexpected or secondary targets, safety assessment

37. Acknowledgements BioSeek Eric Kunkel Jennifer Melrose Dat Nguyen Elen Rosler Stephanie Tong Jian Yang Antal Berenyi David Patterson Jonathan Bingham EPA Keith Houck David Dix Stanford Eugene Butcher Rob Tibshirani Trevor Hastie