BioMAP® Primary Human Cell-Based Systems for Drug Discovery

BioMAP Primary Human Cell-Based Systems for Drug

Discovery

Ellen L. Berg, PhD CSO, BioSeek, Inc.

IBC Assays and Cellular TargetsSan Diego, CA

September 26, 2008 BioSeek

BioSeek

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

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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 - ToxCastTM program

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BioSeek

BioMAP® Technology Bridging the Gap

Scale (meters) (Time)

molecules pathways cells tissues humans

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 exposureMolecular targets

10-6 sec 102 sec 104 sec 105 sec 108 sec

BioMAP Technology

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Biological Complexity: A challenge to drug discovery

BioSeek

BioMAP® Technology Platform

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

BioMAP is highly complementary to biochemical target

and phenotypic screening

BioMAP is highly complementary to biochemical target

and phenotypic screening

BioSeek

• BioMAP Systems are cell-based assays BioMAP Systems are cell-based assays engineered to model complex human engineered to model complex human disease biologydisease 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


Assays

LPS

BF4T

SM3C


BioSeek

• Assay endpoints include human clinical Assay endpoints include human clinical biomarkers and risk factors (proteins)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


Assays

LPS

BF4T

SM3C


BioSeek

AssaysAssays


LPS

BF4T

SM3C

Profile DatabaseProfile Database

Biological responses to drugs and stored in the database


• > 2000 agents> 2000 agents

• Approved drugsApproved drugs

• Experimental Experimental

compoundscompounds

• Bioactive agentsBioactive agents

• Toxic compoundsToxic compounds

BioSeek

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 + MacrophagesMacrophage responses, arthritis, COPD, fibrosis

EC + Mast cells Asthma, allergy, dermatitis, fibrosis

EC + Smooth Muscle CellsVascular biology, restenosis, atherosclerosis

EC + Th2 blasts Allergy, asthma

FibroblastsArthritis, asthma, dermatitis, fibrosis, psoriasis, wound healing

Myofibroblasts Fibrosis, COPD, wound healing

Keratinocytes Psoriasis, dermatitis, wound healing

Keratinocytes + Fibroblasts Psoriasis, dermatitis, wound healing

Bronchial Epithelial CellsTh1 and Th2 inflammation Allergy, asthma, fibrosis, COPD

Bronchial Epithelial Cells + Fibroblasts Asthma, allergy, fibrosis, COPD

Smooth Muscle CellsVascular inflammation, asthma, COPD, fibrosis (coronary artery SMC)

Panel of Current BioMAP Systems Inflammation / Autoimmune Disease / Respiratory / Cardiovascular

LPS SAg HPNo

3C 4H

Mphg MCIgE

HTh2HSM3C

HDF3CGFHDF3C HDFNo

HDF3CTHDFT

BF4T SM3C

BE3C BE4T

K3CT MyoF

KFNoKF3CT

CA3C

CASM3C

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Broad Coverage of Pathways / Mechanisms

Cancer • HDAC• Hsp90• Proteasome• EGFR• NFB• 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• NFB• 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•PPARPPAR•GR•LXR•FXR•Estrogen receptor•Androgen receptor•HMG-CoA reductase•AMPK•GSK3

…….and many others (>2000 drugs/compounds in reference database).and many others (>2000 drugs/compounds in reference database)

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BioMAP Profiling: Example ProfileReference p38 Inhibitor

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Control (no drug)

99% significance envelope

BioMAP Systems

Readout Parameters (Biomarkers)

DoseResponse

• BioMAP activity profiles are robust and reproducible

• Profiles retain shape over multiple concentrations

Cytotoxicity Readouts

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Key Features Consistent with Known BiologyReference p38 Inhibitor

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BioMAP Systems

Tissuefactor

IL-8

HLA-DRMonocyteactivation

ITACVCAM

TIMP-2

Thrombomodulin

MMP1IL-1CD38

T cellactivation

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• p38 MAP kinase regulates many biological processes

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Key Features Consistent with Known BiologyRole of p38 MAP kinase in Th1-type inflammation

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BioMAP Systems

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 cellactivation

Monocyteactivation

IL-8 IL-1

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Key Features Consistent with Known BiologyRole of p38 MAP kinase in thrombus formation

Lo

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(Dru

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BioMAP Systems

Tissuefactor

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.

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Key Features Consistent with Known BiologyActivities relevant to side effects

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BioMAP Systems

• 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

ITACVCAM

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Classification by Similarity of Biological MechanismsMultidimensional Scaling - Function Similarity Map

IB

IKK-2

CDK

2 AdrenergicPI3-K

MEK 1/2

Lck/calcineurin

steroid

ACE

Histamine H1

statins

mTOR

p38 MAPK

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Hsp90

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Classification by Similarity of Biological MechanismsMultidimensional Scaling - Function Similarity Map

IB

IKK-2

CDK

2 AdrenergicPI3-K

MEK 1/2

Lck/calcineurin

steroid

ACE

Histamine H1

statins

mTOR

p38 MAPK

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Mechanism of Action(On-Target)

Hsp90

Off-TargetSecondary Activities

PathwayRelationships

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NFkappaB Activation InhibitorStrong profile in BioMAP systems - Unknown Mechanism of Action

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• 6-Amino-4-(4-phenoxyphenylethylamino)quinazoline “Potent inhibitor of NFkappaB activation” M. Tobe et al. Bioorg. Med. Chem., 2003, 11:383

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NFkappaB Activation InhibitorDoes not cluster with core NFB pathway inhibitors

IB

IKK-2

p38 MAP kinase

Glucocorticoids

TNF Antagonist

IL1 Antagonist

Core NFBPathway

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NFkappaB Activation InhibitorDatabase Search Reveals Unexpected Mechanism

IB

IKK-2

p38 MAP kinase

Glucocorticoids

TNF Antagonist

IL1 Antagonist

Core NFBPathway

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MitochondrialInhibitors

F(0)F(1)ATPase

Complex I

BioSeek

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

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MicrotubuleStabilizers

Mito ATPase

Retinoids

Hsp90

CDK

HDAC

NFB

MEK

DNASynth.

XIAP

Proteinsynthesis

mTOR

MicrotubuleDestabilizers

Estrogen R

PI-3KCa++

Mobilization

Classification of Toxic Agents By Mechanism BioMAP Profiling Clusters Toxic Agents by Mechanism

BioSeek

BioSeek - EPA ToxCast Project

BioSeek

BioSeek - EPA ToxCastTM Project (Phase I)

320 ToxCastTM Compounds

219 (68%)Active

8 BioMAP Systems

87 Biomarkers

4 Concentrations

BioSeek

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)

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Reference CompoundsBioMAP Profiling Distinguishes Reference Compounds

Colchicine

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Dose-Response Relationships

• Rapamycin mTOR inhibitor

Target-specific

Profile is relatively

dose-independent

• Genistein Isoflavone

Multiple targets

Profile is relatively

dose-dependent

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Diversity of Mechanisms

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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



Tryfloxystrobin, 40 M

Tryfloxystrobin, 13.33 M

Tryfloxystrobin, 4.44 M

BioSeek

Trifluoxystrobin

Reference Database Search Identifies MOA

Tryfloxystrobin

Search results:

• Optimized search algorithms use combination of metrics

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Trifluoxystrobin

Reference Database Search Identifies MOA

Search results:

• Optimized search algorithms use combination of metrics

• Confirms mechanism of action

• Of >3000 reference profiles searched, only 7

compounds are significantly similar

• All are known inhibitors of mitochondrial function

Tryfloxystrobin

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• 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

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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

BioSeek


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cAMP Elevation

DNA Alkylation

NFB

Tubulin Inhibition

Mitochondrial Dysfunction

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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

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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

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Documents

BioMAP® Primary Human Cell-Based Systems for Drug Discovery