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BioMAP<sup>®</sup> Primary Human Cell-Based Systems for Drug Discovery

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Presentation at IBC conference on Assays &amp; Cellular Targets, San Diego, CA, Sept 26, 2008

Presentation at IBC conference on Assays &amp; Cellular Targets, San Diego, CA, Sept 26, 2008


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  • 2 1
  • BioMAP systems are complex primary human cell based disease models that can be used directly for phenotypic screening. The most attractive feature of this platform, however, is it’s ability of the platform to provide target and pathway mechanisms of action. This enables reverse pharmacology of bioactive agents and drugs as well as in depth characterization of leads for identifying on versus off-target biology, which in turn impact safety and also clinical indication selection.
  • BioMAP systems are complex primary human cell based disease models that can be used directly for phenotypic screening. The most attractive feature of this platform, however, is it’s ability of the platform to provide target and pathway mechanisms of action. This enables reverse pharmacology of bioactive agents and drugs as well as in depth characterization of leads for identifying on versus off-target biology, which in turn impact safety and also clinical indication selection.
  • The complexity of biological systems makes it difficult to predict outcomes from both target-based as well as phenotypic drug discovery efforts. The BioMAP platform of cell-based assay platform designed to include more of the biological complexity of human disease, but yet in a practical format with sufficient throughput to be used in early discovery. So what do we mean by biological complexity?
  • So, we have developed systems that cover a broad range of cell and tissue biology relevant to inflammation, autoimmune, lung and CV disease.
  • So what does a BioMAP profile look like?
  • I’ve annotated some of the key features of this p38 inhibitor so that you can read them.
  • Many of these activities are consistent with the known role of p38 MAP kinase in Th1-type inflammation.
  • Other activities are relevant to the known role of p38 in thrombus formation
  • And still other activities may be relevant to certain side effects reported for this class of compounds. So you can see how we can interpret the BioMAP profile of an individual compound, connecting activities with in vivo biology.
  • Manuscript submitted to Nature Medicine. Second manuscript will probably be submitted to Nature Biotechnology.
  • Transcript

    • 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
    • 38.