Berg ellen cal epa 29 oct2014

BioMAP®

Systems for

Investigative Toxicology & Safety

Assessment

Ellen L. Berg, PhD

Scientific Director, BioSeek a division of DiscoveRx

California EPA

Sacramento CA

29 October 2014

• Part 1: Background on BioSeek Methodology

- Challenges in Toxicology and Safety Assessment

- The BioMAP® Platform

• Part 2: Applications in pesticide prioritization, hazard identification and risk assessments

- EPA ToxCastTM

Agenda

• Toxicity mechanisms are diverse

- Few toxicity targets have been identified

• Animals poor predictors of human toxicity

- Species differences

Challenges in Safety Assessment

Feature Mice Man

Lifespan 2 Years 70 Years

Size 60 g 60 kg

EnvironmentAnimal facility, cage-mates

Outside world, people, animals, etc.

Limitations of Animal Models

Key differences:

DNA repair mechanisms

Control of blood flow, hemostasis

Immune system status

• Toxicity mechanisms are diverse

- Few toxicity targets have been identified

• Animals poor predictors of human toxicity

- Species differences

• New tools

- Opportunity for in vitro testing to transform predictive toxicology and risk assessment

Challenges in Safety Assessment

DATA

BIGDATA

Why now ?

• Advances in high throughput technologies

• Availability of “omics” data

BIG

DATA

What is ?

• VOLUME

• VELOCITY

• VARIETY

BIG

DATA

What is ?

Large datasets

Terabytes+Integration of

diverse data

Data Driven Research

OLD or


OLD

NEW

or

010101010101010101001010101010101

100101001110010100110100101001110

001000100011101001010001000100011

111010010101010101000110100101010

101010101010001110101010000110100

010000110100010001001111010010101

001000100011101001011101010101010

101010101010001110100101010101010

111010010101010101000110100101010

101010101010001110101010000110100

010000110100010001001111010010101

001000100011101001011101010101010

001000100011101001010001000100011

010101010101010101001010101010101

100101001110010100110100101001110

001000100011101001010001000100011

111010010101010101000110100101010

101010101010001110101010000110100

010000110100010001001111010010101

001000100011101001011101010101010

101010101010001110100101010101010

111010010101010101000110100101010

101010101010001110101010000110100

010000110100010001001111010010101

001000100011101001011101010101010

001000100011101001010001000100011

010101010101010101001010101010101

100101001110010100110100101001110

001000100011101001010001000100011

111010010101010101000110100101010

101010101010001110101010000110100

010000110100010001001111010010101

001000100011101001011101010101010

101010101010001110100101010101010

111010010101010101000110100101010

101010101010001110101010000110100

010000110100010001001111010010101

001000100011101001011101010101010

001000100011101001010001000100011

Hypothesis 1

Hypothesis 2

Hypothesis 3

Hypothesis 4 . . .


IssuesMany hypotheses are generated

Each hypothesis requires validation

Validation requires both computational and

“domain” expertise


Solutions

Incorporate “domain” expertise upfront

Integrate external data to validate hypotheses

BioMAP® Technology Platform

BioMAP®

Assay Systems

Reference

Profile Database

Predictive

Informatics Tools

> 40 Human Primary Cell Models

>1000s of Reference Chemicals, Agents

Analysis and Data Mining Tools

High-throughput Human Biology

14

BioMAP® Systems – Key Features

15

Primary human cell types

Physiologically relevant “context”

Complex activation settings

Co-cultures

Translational biomarker endpoints

Closer to the disease process

Downstream of multiple pathways and integrate information

“Decision-making”

Used by clinicians to guide therapy

Predictive

Benefits of Translational Biomarkers

mRNA,epigenome

Phospho-sites, intracellular proteins,

metabolome

Cell surface,secreted molecules

16

BioMAP® Systems for ToxCast3C 4H LPS SAg BE3C CASM3C HDF3CGF KF3CT

Endothelial Cells

Endothelial Cells

PBMC + Endothelial

Cells

PBMC + Endothelial

Cells

Bronchial epithelial cells

Coronary artery SMC

FibroblastsKeratinocytes + Fibroblasts

Th1 Th2 TLR4 TCR Th1 Th1 Th1 + GF Th1 + TGF

Acute Inflammation E-selectin, IL-8

E-selectin, IL-1a, IL-8, TNF-

a, PGE2 IL-8 IL-1a

IL-8, IL-6, SAA

IL-8 IL-1α

Chronic Inflammation

VCAM-1, ICAM-1, MCP-1, MIG

VCAM-1, Eotaxin-3,

MCP-1

VCAM-1, MCP-1

MCP-1, E-selectin, MIG

IP-10, MIG, HLA-DR

MCP-1, VCAM-1,MIG, HLA-

DR

VCAM-1, IP-10, MIG

MCP-1, ICAM-1, IP-10

Immune Response HLA-DR CD40, M-CSFCD38, CD40, CD69, T cell

Prolif., Cytotox.HLA-DR M-CSF M-CSF

Tissue Remodeling uPAR, MMP-1, PAI-1, TGFb1, SRB, tPA, uPA

uPAR,

Collagen III, EGFR, MMP-1, PAI-1, Fibroblast

Prolif., SRB, TIMP-1

MMP-9, SRB, TIMP-2, uPA,

TGFβ1

Vascular Biology

TM, TF, uPAR, EC

Proliferation, SRB, Vis

VEGFRII, uPAR, P-

selectin, SRB

Tissue Factor, SRB

SRB

TM, TF, LDLR, SMC

Proliferation, SRB

Vascular Biology,

Cardiovascular

Disease, Chronic

Inflammation

Asthma, Allergy,

Oncology,

Vascular Biology

Cardiovascular

Disease, Chronic

Inflammation,

Infectious Disease

Autoimmune

Disease, Chronic

Inflammation,

Immune Biology

COPD,

Respiratory,

Epithelial Biology

Vascular Biology,

Cardiovascular

Inflammation,

Restenosis

Tissue Remodeling,

Fibrosis, Wound

Healing

Skin

Biology,Psoriasis,

Dermatitis

En

dp

oin

t Ty

pe

s

Disease / Tissue Relevance

BioMAP System

Primary Human Cell Types

Stimuli

! ! ! ! !

Endothelial Cells

Bronchial Epithelial Cells

Keratinocytes

Smooth Muscle Cells

Dermal Fibroblasts

Peripheral Blood Mononuclear Cells

BioMAP® Systems for ToxCast3C 4H LPS SAg BE3C CASM3C HDF3CGF KF3CT

Endothelial Cells

Endothelial Cells

PBMC + Endothelial

Cells

PBMC + Endothelial

Cells

Bronchial epithelial cells

Coronary artery SMC

FibroblastsKeratinocytes + Fibroblasts

Th1 Th2 TLR4 TCR Th1 Th1 Th1 + GF Th1 + TGF

Acute Inflammation E-selectin, IL-8

E-selectin, IL-1a, IL-8, TNF-

a, PGE2 IL-8 IL-1a

IL-8, IL-6, SAA

IL-8 IL-1α

Chronic Inflammation

VCAM-1, ICAM-1, MCP-1, MIG

VCAM-1, Eotaxin-3,

MCP-1

VCAM-1, MCP-1

MCP-1, E-selectin, MIG

IP-10, MIG, HLA-DR

MCP-1, VCAM-1,MIG, HLA-

DR

VCAM-1, IP-10, MIG

MCP-1, ICAM-1, IP-10

Immune Response HLA-DR CD40, M-CSFCD38, CD40, CD69, T cell

Prolif., Cytotox.HLA-DR M-CSF M-CSF

Tissue Remodeling uPAR, MMP-1, PAI-1, TGFb1, SRB, tPA, uPA

uPAR,

Collagen III, EGFR, MMP-1, PAI-1, Fibroblast

Prolif., SRB, TIMP-1

MMP-9, SRB, TIMP-2, uPA,

TGFβ1

Vascular Biology

TM, TF, uPAR, EC

Proliferation, SRB, Vis

VEGFRII, uPAR, P-

selectin, SRB

Tissue Factor, SRB

SRB

TM, TF, LDLR, SMC

Proliferation, SRB

Vascular Biology,

Cardiovascular

Disease, Chronic

Inflammation

Asthma, Allergy,

Oncology,

Vascular Biology

Cardiovascular

Disease, Chronic

Inflammation,

Infectious Disease

Autoimmune

Disease, Chronic

Inflammation,

Immune Biology

COPD,

Respiratory,

Epithelial Biology

Vascular Biology,

Cardiovascular

Inflammation,

Restenosis

Tissue Remodeling,

Fibrosis, Wound

Healing

Skin

Biology,Psoriasis,

Dermatitis

En

dp

oin

t Ty

pes

Disease / Tissue Relevance

BioMAP System

Primary Human Cell Types

Stimuli

! ! ! ! !

• Challenges

- Cells and assays are expensive

- Primary cells (all cell-based assays!) are variable

- Very large number of assay components / choices• Media

• Additives

• Cell type

• Time point

• Endpoint Measurements

Experimental Design

• Solutions (compromise)

- Automation and standardized methods – microwell plates

- Cells from pools of donors, prequalified

- Single well per sample

- Multiple concentrations per compound (4+)

- 6-8 vehicle replicates, two positive controls per plate

- Normalize data within plate (Log10 ratio of compound/vehicle)

Experimental Design

Quality Control

• Quality Management System in place

- Quality Management Plan, external QA manager, controlled documents, equipment and materials tracking, SOPs, audits, training program

• Defined assay acceptance criteria

- All data provided to EPA has passed these criteria

- Vehicle control replicates meet acceptance criteria (95% of plates have CV < 20%)

• Ph I data: %CVs ranged from 0.3% to 18.5% (average = 5.3%)

- Positive controls (colchicine or no stimulation control) are similar to historical (based on Pearson similarity metric)

• Pearson test

BioMAP Profile of Colchicine

• Colchicine is an inhibitor of microtubules - It is active in every system and used as a positive control on every plate

• Colchicine profile has a distinctive pattern of activities or “shape”

BioMAP Systems

Readout Parameters (Biomarkers)

Cytotoxicity Readouts

Colchicine 1.1 μM

Lo

g e

xp

res

sio

n r

ati

o

(Dru

g/D

MS

O c

on

tro

l)

Vehicle Control

(no drug)

95%

significance

envelope

BioMAP Profile of No Stimulation Control

• “No Stimulation” condition also has a specific pattern of activities

• Each biomarker readout has a distinctive stim/non-stim ratio

Lo

g e

xp

res

sio

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(Dru

g/D

MS

O c

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tro

l)

Vehicle Control

(no drug)

95%

significance

envelope

BioMAP Systems



“No Stimulation”

Reproducibility of BioMAP ProfilesMultiple independent experiments

Profile shape and EC50’s remains the same experiment-to-experiment

Pearson correlation routinely > 0.8 – 0.9 (perfect match = 1)

12 experiments, each performed at different time, different donors, different lots of the same compound

R1 R2 R3 R4 R5 R6 R7 R8 R9 R10 R11 R12

R1 1

R2 0.95 1

R3 0.96 0.94 1

R4 0.98 0.98 0.96 1

R5 0.93 0.94 0.91 0.94 1

R6 0.96 0.96 0.93 0.97 0.98 1

R7 0.94 0.91 0.9 0.93 0.89 0.9 1

R8 0.95 0.98 0.94 0.98 0.94 0.98 0.92 1

R9 0.91 0.92 0.88 0.92 0.89 0.91 0.93 0.93 1

R10 0.88 0.9 0.81 0.89 0.93 0.93 0.85 0.91 0.83 1

R11 0.94 0.97 0.9 0.94 0.91 0.93 0.94 0.96 0.91 0.89 1

R12 0.92 0.9 0.84 0.89 0.96 0.96 0.89 0.91 0.87 0.92 0.91 1

• Cytotoxicity

- Flag compounds (concentrations) that are overtly cytotoxic

• Profiles

- Profile characteristics

- Unsupervised and supervised approaches to compare profiles

• Individual activities

- Identify statistically significant activities (non-cytotoxic concentrations)

• Correlations to external data

- MoA hypotheses and support AOPs

Analysis Flow for BioMAP Profile Data

• Cytotoxicity can be a confounding factor

- If cells are dead, changes in the levels of biomarker endpoints are non-specific

• What can we use to measure cell death?

- BioMAP cell systems are highly activated• Metabolic endpoints (e.g. alamar blue, ATP measurements) are not

selective for cell death

- Total cell protein most closely correlates with cell death: • Sulforhodamine B (SRB, a stain for total protein)

• Skehan, P., 1990, J. Natl. Cancer Inst. 82:1107

• How do we define cell death?

- SRB Log10 ratio of ≤ -0.3

- This represents a ≥ 50% loss of total protein

Overt Cell Cytotoxicity

Cytotoxicity Depends on Cell Type and Activation State

• Cytotoxicity (SRB < -0.3) is indicated by black arrows

• Potency of test agent for cytotoxicity differs depending on the system

Cytotoxicity (potency)

Cell Type

Endothelial Cells Epithelial Cells SMC Fibroblasts EC

Activation State

• Cytotoxicity is dependent on both cell type and activation state

Types of BioMAP Profiles

Inactive

Active – Sharp dose-response Active – Dose resistant

Active – Selectively

Rapamycin (mTOR) Genistein (multi-target)

Dose Resistance

• “Dose resistant” compounds have similar activity profiles over a wide range of concentrations- No sharp activity jumps; Rapamycin > Genistein

• Characteristic of approved drugs & target-selective compounds- Rapamycin is highly selective for mTOR

- Genistein has multiple targets

- The dose resistance index of Rapamycin is > 60,000x29

• EC50, Slope, Magnitude of effect

• Plateau Dose-resistance

• Cytotoxicity

Rapamycin Genistein

No effect

Max

Inhibition

Increasing Concentration Increasing Concentration

Cytotoxicity

HLA

-DR

Levels

Plateau

EC50Magnitude

Concentration Effects

• BioSeek definition of key activities (for n=1 screening data):

- Log10 ratio values outside historical control 95% significance at more than one concentration tested

- Only concentrations that are not overtly cytotoxic (SRB > - 0.3)

• Other options

- AC50 – 50% activating concentration (EPA)

- LEC – lowest effective concentration

- Add additional requirement for a 20% effect size

- Categorical• BioSeek data has 3 categories (increased, no effect and decreased)

Reporting of Activities

BioMAP Profiling: Example ProfileReference p38 MAPK Inhibitor

Lo

g e

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(Dru

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O c

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Control (no drug)

99%

significance

envelope

BioMAP Systems


Dose

Response


32

This profile shows dose-resistance – similar over a range of concentrations


Lo

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(Dru

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O c

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33

Activities relevant to the role of p38 in monocyte / Th1-type inflammation

p38 kinase is important for Th1-dependent inflammatory responses

Takanami-Ohnishi Y, et al., Essential role of p38 mitogen-activated protein kinase in contact hypersensitivity. J Biol Chem. 2002, 277:37896-903.

IL-8

HLA-DR

Monocyte

activation

IL-6IL-1aCD38HLA-DR

TNF-a


Lo

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xp

ressio

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(Dru

g/D

MS

O c

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34

Activities relevant to anti-thrombotic effects of p38 inhibitors

Tissue factor is the primary cellular initiator of coagulation

p38α deficiency impairs thrombus formation

Sakurai K, et al. Role of p38 mitogen-activated protein kinase in thrombus formation. J Recept Signal Transduct Res. 2004;24(4):283-96.

Tissue

Factor


Lo

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(Dru

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MS

O c

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35

Activities relevant to side effects – clinical finding: skin rash

Upregulation of VCAM and ITAC are characteristic of skin hyperreactivity

Melikoglu M, et al., Characterization of the divergent wound-healing responses occurring in the pathergy reaction and normal healthy volunteers. J Immunol. 2006, 177:6415-21.

ITAC

VCAM

MMP1

VCAM

36

BioMAP® Analyses

Predictive

Informatics Tools

Custom informatics tools are

used to predict clinical outcomes

Similarity SearchUnsupervised analysis

Mechanism ClassificationSupervised analysis

Clinical AssociationsMechanism of action

37

BioMAP® Reference Database

BioMAP®

Reference Database

Biomarker responses to drugs

are stored in the database

>3000 drugs

• More than 3000 agents

- Drugs – Clinical stage, approved, and failed

- Experimental Chemicals - Research tool

compounds, environmental chemicals,

nanomaterials

- Biologics – Antibodies, cytokines, factors,

peptides, soluble receptors

• Availability of reference data

- Key reference data are published and have

been made available (Berg, 2010; Berg,

2013)

Similarity Analysis of Profiles

Highly correlated Similar

Pearson’s correlation of r > 0.7

Low correlation Not similar

Pearson’s correlation of r < 0.7

38

Microtubule

Stabilizers

Mitochondrial

ET chain

Retinoids

Hsp90

CDK

NFkB

MEK

DNA

synthesis

JNK

Protein

synthesis

Microtubule

Destabilizers

Estrogen R

PI-3K

Ca++

Mobilization

BioMAP Data Can Cluster CompoundsAccording to Mechanisms of Action

mTOR

PKC Activation

p38 MAPK

HMG-CoA

reductase

Calcineurin

Transcription

39

Each circle represents a compound tested at a single dose

Lines are drawn between compounds whose profiles are similar (r > 0.7)

Figure adopted from Berg, JPTox Meth. 2010

Microtubule

Stabilizers

Mitochondrial

ET chain

Retinoids

Hsp90

CDK

NFkB

MEK

DNA

synthesis

JNK

Protein

synthesis

Microtubule

Destabilizers

Estrogen R

PI-3K

Ca++

Mobilization


mTOR

PKC Activation

p38 MAPK

HMG-CoA

reductase

Calcineurin

Transcription

p38 MAPK

Calcineurin

mTOR

Mitochondrial ATPase

40

Each circle represents a compound tested at a single dose

Lines are drawn between compounds whose profiles are similar (r > 0.7)

Figure adopted from Berg, JPTox Meth. 2010

Microtubule

Stabilizers

Mitochondrial

ET chain

Retinoids

Hsp90

CDK

NFkB

MEK

DNA

synthesis

JNK

Protein

synthesis

Microtubule

Destabilizers

Estrogen R

PI-3K

Ca++

Mobilization


mTOR

PKC Activation

p38 MAPK

HMG-CoA

reductase

Calcineurin

Transcription

Mechanism of Action

(On-Target)

Pathway

Relationships

41

Consensus Profiles for Mechanism Classes

p38 MAPK inhibitor 1



Consensus profile reflects target-specific biology

Can define mechanism class

42

1 1 1 1 1 1 1 1 1

Mechanism Class Consensus Profiles

AhRAgonist

CalcineurinInhibitor

EGFRInhibitor

EPAgonist

ERAgonist

GRAgonist(Full)

H1Antagonist

HDACInhibitor

HMG-CoAReductaseInhibitor

Hsp90Inhibitor

IKK2Inhibitor

IL-17AAgonist

JAKInhibitor

MEKInhibitor

MicrotubuleDisruptor

MicrotubuleStabilizer

MitochondrialInhibitor

mTORInhibitor

p38MAPKInhibitor

PDEIVInhibitor

PI3KInhibitor

PKC(c+n)Inhibitor

ProteasomeInhibitor

RAR/RXRAgonist

SRCa++ATPaseInhibitor

SrcFamilyInhibitor

TNF-alphaAntagonist

VitaminDReceptorAgonist

Patterns reflect “mechanism class” or target biology

Reproducible patterns permit building of classifiers for automated mechanism assignment

Me

chan

ism

Cla

sse

s

BioMAP Assay / Endpoints

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−1.5

−1.4

−1.3

−1.2

−1.1

−1.0

−0.9

−0.8

−0.7

−0.6

−0.5

−0.4

−0.3

−0.2

−0.1

0.0

0.1

0.2

0.3

0.4

0.5

0.6

0.7

0.8

0.9

1.0

Lo

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Profiles

Cyclopamine 40 uM

Cyclopamine 13.333 u...

Cyclopamine 4.444 uM


3C 4H LPS SAg BE3C CASM3C HDF3CGF KF3CT

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CD

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CC

L2

/MC

P−

1

CD

10

6/V

CA

M−

1

CD

14

1/T

hro

mb

om

od

uli

n

CD

14

2/T

iss

ue

Fa

cto

r

CD

87

/uP

AR

CX

CL

8/I

L−

8

CX

CL

9/M

IG

HL

A−

DR

IL−

6

LD

LR

M−

CS

F

Pro

life

rati

on

Se

rum

Am

ylo

id A

SR

B

CD

10

6/V

CA

M−

1

Co

llag

en

III

CX

CL

10

/IP−

10

CX

CL

8/I

L−

8

CX

CL

9/M

IG

EG

FR

M−

CS

F

MM

P−

1

PA

I−I

Pro

life

rati

on

_7

2h

r

SR

B

TIM

P−

1

CC

L2

/MC

P−

1

CD

54

/IC

AM−

1

CX

CL

10

/IP−

10

IL−

1a

lph

a

MM

P−

9

SR

B

TG

F−

be

taI

TIM

P−

2

uP

A

−1.5

−1.4

−1.3

−1.2

−1.1

−1.0

−0.9

−0.8

−0.7

−0.6

−0.5

−0.4

−0.3

−0.2

−0.1

0.0

0.1

0.2

0.3

0.4

0.5

0.6

0.7

0.8

0.9

1.0

Lo

g R

atio

Profiles

Cyclopamine 40 uM





CC

L2

/MC

P−

1

CD

10

6/V

CA

M−

1

CD

14

1/T

hro

mb

om

od

uli

n

CD

14

2/T

iss

ue

Fa

cto

r

CD

54

/IC

AM−

1

CD

62

E/E−

Se

lec

tin

CD

87

/uP

AR

CX

CL

8/I

L−

8

CX

CL

9/M

IG

HL

A−

DR

Pro

life

rati

on

SR

B

Vis

ua

l

CC

L2

/MC

P−

1

CC

L2

6/E

ota

xin−

3

CD

10

6/V

CA

M−

1

CD

62

P/P−

se

lecti

n

CD

87

/uP

AR

SR

B

VE

GF

R2

CC

L2

/MC

P−

1

CD

10

6/V

CA

M−

1

CD

14

2/T

iss

ue

Fa

cto

r

CD

40

CD

62

E/E−

Se

lec

tin

CX

CL

8/I

L−

8

IL−

1a

lph

a

M−

CS

F

sP

GE

2

SR

B

sT

NF−

alp

ha

CC

L2

/MC

P−

1

CD

38

CD

40

CD

62

E/E−

Se

lec

tin

CD

69

CX

CL

8/I

L−

8

CX

CL

9/M

IG

PB

MC

Cy

toto

xic

ity

Pro

life

rati

on

SR

B

CD

87

/uP

AR

CX

CL

10

/IP−

10

CX

CL

9/M

IG

HL

A−

DR

IL−

1a

lph

a

MM

P−

1

PA

I−I

SR

B

TG

F−

be

taI

tPA

uP

A

CC

L2

/MC

P−

1

CD

10

6/V

CA

M−

1

CD

14

1/T

hro

mb

om

od

uli

n

CD

14

2/T

iss

ue

Fa

cto

r

CD

87

/uP

AR

CX

CL

8/I

L−

8

CX

CL

9/M

IG

HL

A−

DR

IL−

6

LD

LR

M−

CS

F

Pro

life

rati

on

Se

rum

Am

ylo

id A

SR

B

CD

10

6/V

CA

M−

1

Co

llag

en

III

CX

CL

10

/IP−

10

CX

CL

8/I

L−

8

CX

CL

9/M

IG

EG

FR

M−

CS

F

MM

P−

1

PA

I−I

Pro

life

rati

on

_7

2h

r

SR

B

TIM

P−

1

CC

L2

/MC

P−

1

CD

54

/IC

AM−

1

CX

CL

10

/IP−

10

IL−

1a

lph

a

MM

P−

9

SR

B

TG

F−

be

taI

TIM

P−

2

uP

A

−1.5

−1.4

−1.3

−1.2

−1.1

−1.0

−0.9

−0.8

−0.7

−0.6

−0.5

−0.4

−0.3

−0.2

−0.1

0.0

0.1

0.2

0.3

0.4

0.5

0.6

0.7

0.8

0.9

1.0

Lo

g R

atio

Profiles

Cyclopamine 40 uM





CC

L2

/MC

P−

1

CD

10

6/V

CA

M−

1

CD

14

1/T

hro

mb

om

od

uli

n

CD

14

2/T

iss

ue

Fa

cto

r

CD

54

/IC

AM−

1

CD

62

E/E−

Se

lec

tin

CD

87

/uP

AR

CX

CL

8/I

L−

8

CX

CL

9/M

IG

HL

A−

DR

Pro

life

rati

on

SR

B

Vis

ua

l

CC

L2

/MC

P−

1

CC

L2

6/E

ota

xin−

3

CD

10

6/V

CA

M−

1

CD

62

P/P−

se

lecti

n

CD

87

/uP

AR

SR

B

VE

GF

R2

CC

L2

/MC

P−

1

CD

10

6/V

CA

M−

1

CD

14

2/T

iss

ue

Fa

cto

r

CD

40

CD

62

E/E−

Se

lec

tin

CX

CL

8/I

L−

8

IL−

1a

lph

a

M−

CS

F

sP

GE

2

SR

B

sT

NF−

alp

ha

CC

L2

/MC

P−

1

CD

38

CD

40

CD

62

E/E−

Se

lec

tin

CD

69

CX

CL

8/I

L−

8

CX

CL

9/M

IG

PB

MC

Cy

toto

xic

ity

Pro

life

rati

on

SR

B

CD

87

/uP

AR

CX

CL

10

/IP−

10

CX

CL

9/M

IG

HL

A−

DR

IL−

1a

lph

a

MM

P−

1

PA

I−I

SR

B

TG

F−

be

taI

tPA

uP

A

CC

L2

/MC

P−

1

CD

10

6/V

CA

M−

1

CD

14

1/T

hro

mb

om

od

uli

n

CD

14

2/T

iss

ue

Fa

cto

r

CD

87

/uP

AR

CX

CL

8/I

L−

8

CX

CL

9/M

IG

HL

A−

DR

IL−

6

LD

LR

M−

CS

F

Pro

life

rati

on

Se

rum

Am

ylo

id A

SR

B

CD

10

6/V

CA

M−

1

Co

llag

en

III

CX

CL

10

/IP−

10

CX

CL

8/I

L−

8

CX

CL

9/M

IG

EG

FR

M−

CS

F

MM

P−

1

PA

I−I

Pro

life

rati

on

_7

2h

r

SR

B

TIM

P−

1

CC

L2

/MC

P−

1

CD

54

/IC

AM−

1

CX

CL

10

/IP−

10

IL−

1a

lph

a

MM

P−

9

SR

B

TG

F−

be

taI

TIM

P−

2

uP

A

−1.5

−1.4

−1.3

−1.2

−1.1

−1.0

−0.9

−0.8

−0.7

−0.6

−0.5

−0.4

−0.3

−0.2

−0.1

0.0

0.1

0.2

0.3

0.4

0.5

0.6

0.7

0.8

0.9

1.0

Lo

g R

atio

Profiles

Cyclopamine 40 uM





CC

L2

/MC

P−

1

CD

10

6/V

CA

M−

1

CD

14

1/T

hro

mb

om

od

uli

n

CD

14

2/T

iss

ue

Fa

cto

r

CD

54

/IC

AM−

1

CD

62

E/E−

Se

lec

tin

CD

87

/uP

AR

CX

CL

8/I

L−

8

CX

CL

9/M

IG

HL

A−

DR

Pro

life

rati

on

SR

B

Vis

ua

l

CC

L2

/MC

P−

1

CC

L2

6/E

ota

xin−

3

CD

10

6/V

CA

M−

1

CD

62

P/P−

se

lecti

n

CD

87

/uP

AR

SR

B

VE

GF

R2

CC

L2

/MC

P−

1

CD

10

6/V

CA

M−

1

CD

14

2/T

iss

ue

Fa

cto

r

CD

40

CD

62

E/E−

Se

lec

tin

CX

CL

8/I

L−

8

IL−

1a

lph

a

M−

CS

F

sP

GE

2

SR

B

sT

NF−

alp

ha

CC

L2

/MC

P−

1

CD

38

CD

40

CD

62

E/E−

Se

lec

tin

CD

69

CX

CL

8/I

L−

8

CX

CL

9/M

IG

PB

MC

Cy

toto

xic

ity

Pro

life

rati

on

SR

B

CD

87

/uP

AR

CX

CL

10

/IP−

10

CX

CL

9/M

IG

HL

A−

DR

IL−

1a

lph

a

MM

P−

1

PA

I−I

SR

B

TG

F−

be

taI

tPA

uP

A

CC

L2

/MC

P−

1

CD

10

6/V

CA

M−

1

CD

14

1/T

hro

mb

om

od

uli

n

CD

14

2/T

iss

ue

Fa

cto

r

CD

87

/uP

AR

CX

CL

8/I

L−

8

CX

CL

9/M

IG

HL

A−

DR

IL−

6

LD

LR

M−

CS

F

Pro

life

rati

on

Se

rum

Am

ylo

id A

SR

B

CD

10

6/V

CA

M−

1

Co

llag

en

III

CX

CL

10

/IP−

10

CX

CL

8/I

L−

8

CX

CL

9/M

IG

EG

FR

M−

CS

F

MM

P−

1

PA

I−I

Pro

life

rati

on

_7

2h

r

SR

B

TIM

P−

1

CC

L2

/MC

P−

1

CD

54

/IC

AM−

1

CX

CL

10

/IP−

10

IL−

1a

lph

a

MM

P−

9

SR

B

TG

F−

be

taI

TIM

P−

2

uP

A

−1.5

−1.4

−1.3

−1.2

−1.1

−1.0

−0.9

−0.8

−0.7

−0.6

−0.5

−0.4

−0.3

−0.2

−0.1

0.0

0.1

0.2

0.3

0.4

0.5

0.6

0.7

0.8

0.9

1.0

Lo

g R

atio

Profiles

Cyclopamine 40 uM





CC

L2

/MC

P−

1

CD

10

6/V

CA

M−

1

CD

14

1/T

hro

mb

om

od

uli

n

CD

14

2/T

iss

ue

Fa

cto

r

CD

54

/IC

AM−

1

CD

62

E/E−

Se

lec

tin

CD

87

/uP

AR

CX

CL

8/I

L−

8

CX

CL

9/M

IG

HL

A−

DR

Pro

life

rati

on

SR

B

Vis

ua

l

CC

L2

/MC

P−

1

CC

L2

6/E

ota

xin−

3

CD

10

6/V

CA

M−

1

CD

62

P/P−

se

lecti

n

CD

87

/uP

AR

SR

B

VE

GF

R2

CC

L2

/MC

P−

1

CD

10

6/V

CA

M−

1

CD

14

2/T

iss

ue

Fa

cto

r

CD

40

CD

62

E/E−

Se

lec

tin

CX

CL

8/I

L−

8

IL−

1a

lph

a

M−

CS

F

sP

GE

2

SR

B

sT

NF−

alp

ha

CC

L2

/MC

P−

1

CD

38

CD

40

CD

62

E/E−

Se

lec

tin

CD

69

CX

CL

8/I

L−

8

CX

CL

9/M

IG

PB

MC

Cy

toto

xic

ity

Pro

life

rati

on

SR

B

CD

87

/uP

AR

CX

CL

10

/IP−

10

CX

CL

9/M

IG

HL

A−

DR

IL−

1a

lph

a

MM

P−

1

PA

I−I

SR

B

TG

F−

be

taI

tPA

uP

A

CC

L2

/MC

P−

1

CD

10

6/V

CA

M−

1

CD

14

1/T

hro

mb

om

od

uli

n

CD

14

2/T

iss

ue

Fa

cto

r

CD

87

/uP

AR

CX

CL

8/I

L−

8

CX

CL

9/M

IG

HL

A−

DR

IL−

6

LD

LR

M−

CS

F

Pro

life

rati

on

Se

rum

Am

ylo

id A

SR

B

CD

10

6/V

CA

M−

1

Co

llag

en

III

CX

CL

10

/IP−

10

CX

CL

8/I

L−

8

CX

CL

9/M

IG

EG

FR

M−

CS

F

MM

P−

1

PA

I−I

Pro

life

rati

on

_7

2h

r

SR

B

TIM

P−

1

CC

L2

/MC

P−

1

CD

54

/IC

AM−

1

CX

CL

10

/IP−

10

IL−

1a

lph

a

MM

P−

9

SR

B

TG

F−

be

taI

TIM

P−

2

uP

A

−1.5

−1.4

−1.3

−1.2

−1.1

−1.0

−0.9

−0.8

−0.7

−0.6

−0.5

−0.4

−0.3

−0.2

−0.1

0.0

0.1

0.2

0.3

0.4

0.5

0.6

0.7

0.8

0.9

1.0

Lo

g R

atio

Profiles

Cyclopamine 40 uM





CC

L2

/MC

P−

1

CD

10

6/V

CA

M−

1

CD

14

1/T

hro

mb

om

od

uli

n

CD

14

2/T

iss

ue

Fa

cto

r

CD

54

/IC

AM−

1

CD

62

E/E−

Se

lec

tin

CD

87

/uP

AR

CX

CL

8/I

L−

8

CX

CL

9/M

IG

HL

A−

DR

Pro

life

rati

on

SR

B

Vis

ua

l

CC

L2

/MC

P−

1

CC

L2

6/E

ota

xin−

3

CD

10

6/V

CA

M−

1

CD

62

P/P−

se

lecti

n

CD

87

/uP

AR

SR

B

VE

GF

R2

CC

L2

/MC

P−

1

CD

10

6/V

CA

M−

1

CD

14

2/T

iss

ue

Fa

cto

r

CD

40

CD

62

E/E−

Se

lec

tin

CX

CL

8/I

L−

8

IL−

1a

lph

a

M−

CS

F

sP

GE

2

SR

B

sT

NF−

alp

ha

CC

L2

/MC

P−

1

CD

38

CD

40

CD

62

E/E−

Se

lec

tin

CD

69

CX

CL

8/I

L−

8

CX

CL

9/M

IG

PB

MC

Cy

toto

xic

ity

Pro

life

rati

on

SR

B

CD

87

/uP

AR

CX

CL

10

/IP−

10

CX

CL

9/M

IG

HL

A−

DR

IL−

1a

lph

a

MM

P−

1

PA

I−I

SR

B

TG

F−

be

taI

tPA

uP

A

CC

L2

/MC

P−

1

CD

10

6/V

CA

M−

1

CD

14

1/T

hro

mb

om

od

uli

n

CD

14

2/T

iss

ue

Fa

cto

r

CD

87

/uP

AR

CX

CL

8/I

L−

8

CX

CL

9/M

IG

HL

A−

DR

IL−

6

LD

LR

M−

CS

F

Pro

life

rati

on

Se

rum

Am

ylo

id A

SR

B

CD

10

6/V

CA

M−

1

Co

llag

en

III

CX

CL

10

/IP−

10

CX

CL

8/I

L−

8

CX

CL

9/M

IG

EG

FR

M−

CS

F

MM

P−

1

PA

I−I

Pro

life

rati

on

_7

2h

r

SR

B

TIM

P−

1

CC

L2

/MC

P−

1

CD

54

/IC

AM−

1

CX

CL

10

/IP−

10

IL−

1a

lph

a

MM

P−

9

SR

B

TG

F−

be

taI

TIM

P−

2

uP

A

−1.5

−1.4

−1.3

−1.2

−1.1

−1.0

−0.9

−0.8

−0.7

−0.6

−0.5

−0.4

−0.3

−0.2

−0.1

0.0

0.1

0.2

0.3

0.4

0.5

0.6

0.7

0.8

0.9

1.0

Lo

g R

atio

Profiles

Cyclopamine 40 uM





CC

L2

/MC

P−

1

CD

10

6/V

CA

M−

1

CD

14

1/T

hro

mb

om

od

uli

n

CD

14

2/T

iss

ue

Fa

cto

r

CD

54

/IC

AM−

1

CD

62

E/E−

Se

lec

tin

CD

87

/uP

AR

CX

CL

8/I

L−

8

CX

CL

9/M

IG

HL

A−

DR

Pro

life

rati

on

SR

B

Vis

ua

l

CC

L2

/MC

P−

1

CC

L2

6/E

ota

xin−

3

CD

10

6/V

CA

M−

1

CD

62

P/P−

se

lecti

n

CD

87

/uP

AR

SR

B

VE

GF

R2

CC

L2

/MC

P−

1

CD

10

6/V

CA

M−

1

CD

14

2/T

iss

ue

Fa

cto

r

CD

40

CD

62

E/E−

Se

lec

tin

CX

CL

8/I

L−

8

IL−

1a

lph

a

M−

CS

F

sP

GE

2

SR

B

sT

NF−

alp

ha

CC

L2

/MC

P−

1

CD

38

CD

40

CD

62

E/E−

Se

lec

tin

CD

69

CX

CL

8/I

L−

8

CX

CL

9/M

IG

PB

MC

Cy

toto

xic

ity

Pro

life

rati

on

SR

B

CD

87

/uP

AR

CX

CL

10

/IP−

10

CX

CL

9/M

IG

HL

A−

DR

IL−

1a

lph

a

MM

P−

1

PA

I−I

SR

B

TG

F−

be

taI

tPA

uP

A

CC

L2

/MC

P−

1

CD

10

6/V

CA

M−

1

CD

14

1/T

hro

mb

om

od

uli

n

CD

14

2/T

iss

ue

Fa

cto

r

CD

87

/uP

AR

CX

CL

8/I

L−

8

CX

CL

9/M

IG

HL

A−

DR

IL−

6

LD

LR

M−

CS

F

Pro

life

rati

on

Se

rum

Am

ylo

id A

SR

B

CD

10

6/V

CA

M−

1

Co

llag

en

III

CX

CL

10

/IP−

10

CX

CL

8/I

L−

8

CX

CL

9/M

IG

EG

FR

M−

CS

F

MM

P−

1

PA

I−I

Pro

life

rati

on

_7

2h

r

SR

B

TIM

P−

1

CC

L2

/MC

P−

1

CD

54

/IC

AM−

1

CX

CL

10

/IP−

10

IL−

1a

lph

a

MM

P−

9

SR

B

TG

F−

be

taI

TIM

P−

2

uP

A

−1.5

−1.4

−1.3

−1.2

−1.1

−1.0

−0.9

−0.8

−0.7

−0.6

−0.5

−0.4

−0.3

−0.2

−0.1

0.0

0.1

0.2

0.3

0.4

0.5

0.6

0.7

0.8

0.9

1.0

Lo

g R

atio

Profiles

Cyclopamine 40 uM





CC

L2

/MC

P−

1

CD

10

6/V

CA

M−

1

CD

14

1/T

hro

mb

om

od

uli

n

CD

14

2/T

iss

ue

Fa

cto

r

CD

54

/IC

AM−

1

CD

62

E/E−

Se

lec

tin

CD

87

/uP

AR

CX

CL

8/I

L−

8

CX

CL

9/M

IG

HL

A−

DR

Pro

life

rati

on

SR

B

Vis

ua

l

CC

L2

/MC

P−

1

CC

L2

6/E

ota

xin−

3

CD

10

6/V

CA

M−

1

CD

62

P/P−

se

lecti

n

CD

87

/uP

AR

SR

B

VE

GF

R2

CC

L2

/MC

P−

1

CD

10

6/V

CA

M−

1

CD

14

2/T

iss

ue

Fa

cto

r

CD

40

CD

62

E/E−

Se

lec

tin

CX

CL

8/I

L−

8

IL−

1a

lph

a

M−

CS

F

sP

GE

2

SR

B

sT

NF−

alp

ha

CC

L2

/MC

P−

1

CD

38

CD

40

CD

62

E/E−

Se

lec

tin

CD

69

CX

CL

8/I

L−

8

CX

CL

9/M

IG

PB

MC

Cy

toto

xic

ity

Pro

life

rati

on

SR

B

CD

87

/uP

AR

CX

CL

10

/IP−

10

CX

CL

9/M

IG

HL

A−

DR

IL−

1a

lph

a

MM

P−

1

PA

I−I

SR

B

TG

F−

be

taI

tPA

uP

A

CC

L2

/MC

P−

1

CD

10

6/V

CA

M−

1

CD

14

1/T

hro

mb

om

od

uli

n

CD

14

2/T

iss

ue

Fa

cto

r

CD

87

/uP

AR

CX

CL

8/I

L−

8

CX

CL

9/M

IG

HL

A−

DR

IL−

6

LD

LR

M−

CS

F

Pro

life

rati

on

Se

rum

Am

ylo

id A

SR

B

CD

10

6/V

CA

M−

1

Co

llag

en

III

CX

CL

10

/IP−

10

CX

CL

8/I

L−

8

CX

CL

9/M

IG

EG

FR

M−

CS

F

MM

P−

1

PA

I−I

Pro

life

rati

on

_7

2h

r

SR

B

TIM

P−

1

CC

L2

/MC

P−

1

CD

54

/IC

AM−

1

CX

CL

10

/IP−

10

IL−

1a

lph

a

MM

P−

9

SR

B

TG

F−

be

taI

TIM

P−

2

uP

A

−1.5

−1.4

−1.3

−1.2

−1.1

−1.0

−0.9

−0.8

−0.7

−0.6

−0.5

−0.4

−0.3

−0.2

−0.1

0.0

0.1

0.2

0.3

0.4

0.5

0.6

0.7

0.8

0.9

1.0

Lo

g R

atio

Profiles

Cyclopamine 40 uM





CC

L2

/MC

P−

1

CD

10

6/V

CA

M−

1

CD

14

1/T

hro

mb

om

od

uli

n

CD

14

2/T

iss

ue

Fa

cto

r

CD

54

/IC

AM−

1

CD

62

E/E−

Se

lec

tin

CD

87

/uP

AR

CX

CL

8/I

L−

8

CX

CL

9/M

IG

HL

A−

DR

Pro

life

rati

on

SR

B

Vis

ua

l

CC

L2

/MC

P−

1

CC

L2

6/E

ota

xin−

3

CD

10

6/V

CA

M−

1

CD

62

P/P−

se

lecti

n

CD

87

/uP

AR

SR

B

VE

GF

R2

CC

L2

/MC

P−

1

CD

10

6/V

CA

M−

1

CD

14

2/T

iss

ue

Fa

cto

r

CD

40

CD

62

E/E−

Se

lec

tin

CX

CL

8/I

L−

8

IL−

1a

lph

a

M−

CS

F

sP

GE

2

SR

B

sT

NF−

alp

ha

CC

L2

/MC

P−

1

CD

38

CD

40

CD

62

E/E−

Se

lec

tin

CD

69

CX

CL

8/I

L−

8

CX

CL

9/M

IG

PB

MC

Cy

toto

xic

ity

Pro

life

rati

on

SR

B

CD

87

/uP

AR

CX

CL

10

/IP−

10

CX

CL

9/M

IG

HL

A−

DR

IL−

1a

lph

a

MM

P−

1

PA

I−I

SR

B

TG

F−

be

taI

tPA

uP

A

CC

L2

/MC

P−

1

CD

10

6/V

CA

M−

1

CD

14

1/T

hro

mb

om

od

uli

n

CD

14

2/T

iss

ue

Fa

cto

r

CD

87

/uP

AR

CX

CL

8/I

L−

8

CX

CL

9/M

IG

HL

A−

DR

IL−

6

LD

LR

M−

CS

F

Pro

life

rati

on

Se

rum

Am

ylo

id A

SR

B

CD

10

6/V

CA

M−

1

Co

llag

en

III

CX

CL

10

/IP−

10

CX

CL

8/I

L−

8

CX

CL

9/M

IG

EG

FR

M−

CS

F

MM

P−

1

PA

I−I

Pro

life

rati

on

_7

2h

r

SR

B

TIM

P−

1

CC

L2

/MC

P−

1

CD

54

/IC

AM−

1

CX

CL

10

/IP−

10

IL−

1a

lph

a

MM

P−

9

SR

B

TG

F−

be

taI

TIM

P−

2

uP

A

−1.5

−1.4

−1.3

−1.2

−1.1

−1.0

−0.9

−0.8

−0.7

−0.6

−0.5

−0.4

−0.3

−0.2

−0.1

0.0

0.1

0.2

0.3

0.4

0.5

0.6

0.7

0.8

0.9

1.0

Lo

g R

atio

Profiles

Cyclopamine 40 uM





CC

L2

/MC

P−

1

CD

10

6/V

CA

M−

1

CD

14

1/T

hro

mb

om

od

uli

n

CD

14

2/T

iss

ue

Fa

cto

r

CD

54

/IC

AM−

1

CD

62

E/E−

Se

lec

tin

CD

87

/uP

AR

CX

CL

8/I

L−

8

CX

CL

9/M

IG

HL

A−

DR

Pro

life

rati

on

SR

B

Vis

ua

l

CC

L2

/MC

P−

1

CC

L2

6/E

ota

xin−

3

CD

10

6/V

CA

M−

1

CD

62

P/P−

se

lecti

n

CD

87

/uP

AR

SR

B

VE

GF

R2

CC

L2

/MC

P−

1

CD

10

6/V

CA

M−

1

CD

14

2/T

iss

ue

Fa

cto

r

CD

40

CD

62

E/E−

Se

lec

tin

CX

CL

8/I

L−

8

IL−

1a

lph

a

M−

CS

F

sP

GE

2

SR

B

sT

NF−

alp

ha

CC

L2

/MC

P−

1

CD

38

CD

40

CD

62

E/E−

Se

lec

tin

CD

69

CX

CL

8/I

L−

8

CX

CL

9/M

IG

PB

MC

Cy

toto

xic

ity

Pro

life

rati

on

SR

B

CD

87

/uP

AR

CX

CL

10

/IP−

10

CX

CL

9/M

IG

HL

A−

DR

IL−

1a

lph

a

MM

P−

1

PA

I−I

SR

B

TG

F−

be

taI

tPA

uP

A

CC

L2

/MC

P−

1

CD

10

6/V

CA

M−

1

CD

14

1/T

hro

mb

om

od

uli

n

CD

14

2/T

iss

ue

Fa

cto

r

CD

87

/uP

AR

CX

CL

8/I

L−

8

CX

CL

9/M

IG

HL

A−

DR

IL−

6

LD

LR

M−

CS

F

Pro

life

rati

on

Se

rum

Am

ylo

id A

SR

B

CD

10

6/V

CA

M−

1

Co

llag

en

III

CX

CL

10

/IP−

10

CX

CL

8/I

L−

8

CX

CL

9/M

IG

EG

FR

M−

CS

F

MM

P−

1

PA

I−I

Pro

life

rati

on

_7

2h

r

SR

B

TIM

P−

1

CC

L2

/MC

P−

1

CD

54

/IC

AM−

1

CX

CL

10

/IP−

10

IL−

1a

lph

a

MM

P−

9

SR

B

TG

F−

be

taI

TIM

P−

2

uP

A

−1.5

−1.4

−1.3

−1.2

−1.1

−1.0

−0.9

−0.8

−0.7

−0.6

−0.5

−0.4

−0.3

−0.2

−0.1

0.0

0.1

0.2

0.3

0.4

0.5

0.6

0.7

0.8

0.9

1.0

Lo

g R

atio

Profiles

Cyclopamine 40 uM





CC

L2

/MC

P−

1

CD

10

6/V

CA

M−

1

CD

14

1/T

hro

mb

om

od

uli

n

CD

14

2/T

iss

ue

Fa

cto

r

CD

54

/IC

AM−

1

CD

62

E/E−

Se

lec

tin

CD

87

/uP

AR

CX

CL

8/I

L−

8

CX

CL

9/M

IG

HL

A−

DR

Pro

life

rati

on

SR

B

Vis

ua

l

CC

L2

/MC

P−

1

CC

L2

6/E

ota

xin−

3

CD

10

6/V

CA

M−

1

CD

62

P/P−

se

lecti

n

CD

87

/uP

AR

SR

B

VE

GF

R2

CC

L2

/MC

P−

1

CD

10

6/V

CA

M−

1

CD

14

2/T

iss

ue

Fa

cto

r

CD

40

CD

62

E/E−

Se

lec

tin

CX

CL

8/I

L−

8

IL−

1a

lph

a

M−

CS

F

sP

GE

2

SR

B

sT

NF−

alp

ha

CC

L2

/MC

P−

1

CD

38

CD

40

CD

62

E/E−

Se

lec

tin

CD

69

CX

CL

8/I

L−

8

CX

CL

9/M

IG

PB

MC

Cy

toto

xic

ity

Pro

life

rati

on

SR

B

CD

87

/uP

AR

CX

CL

10

/IP−

10

CX

CL

9/M

IG

HL

A−

DR

IL−

1a

lph

a

MM

P−

1

PA

I−I

SR

B

TG

F−

be

taI

tPA

uP

A

CC

L2

/MC

P−

1

CD

10

6/V

CA

M−

1

CD

14

1/T

hro

mb

om

od

uli

n

CD

14

2/T

iss

ue

Fa

cto

r

CD

87

/uP

AR

CX

CL

8/I

L−

8

CX

CL

9/M

IG

HL

A−

DR

IL−

6

LD

LR

M−

CS

F

Pro

life

rati

on

Se

rum

Am

ylo

id A

SR

B

CD

10

6/V

CA

M−

1

Co

llag

en

III

CX

CL

10

/IP−

10

CX

CL

8/I

L−

8

CX

CL

9/M

IG

EG

FR

M−

CS

F

MM

P−

1

PA

I−I

Pro

life

rati

on

_7

2h

r

SR

B

TIM

P−

1

CC

L2

/MC

P−

1

CD

54

/IC

AM−

1

CX

CL

10

/IP−

10

IL−

1a

lph

a

MM

P−

9

SR

B

TG

F−

be

taI

TIM

P−

2

uP

A

−1.5

−1.4

−1.3

−1.2

−1.1

−1.0

−0.9

−0.8

−0.7

−0.6

−0.5

−0.4

−0.3

−0.2

−0.1

0.0

0.1

0.2

0.3

0.4

0.5

0.6

0.7

0.8

0.9

1.0

Lo

g R

atio

Profiles

Cyclopamine 40 uM





CC

L2

/MC

P−

1

CD

10

6/V

CA

M−

1

CD

14

1/T

hro

mb

om

od

uli

n

CD

14

2/T

iss

ue

Fa

cto

r

CD

54

/IC

AM−

1

CD

62

E/E−

Se

lec

tin

CD

87

/uP

AR

CX

CL

8/I

L−

8

CX

CL

9/M

IG

HL

A−

DR

Pro

life

rati

on

SR

B

Vis

ua

l

CC

L2

/MC

P−

1

CC

L2

6/E

ota

xin−

3

CD

10

6/V

CA

M−

1

CD

62

P/P−

se

lecti

n

CD

87

/uP

AR

SR

B

VE

GF

R2

CC

L2

/MC

P−

1

CD

10

6/V

CA

M−

1

CD

14

2/T

iss

ue

Fa

cto

r

CD

40

CD

62

E/E−

Se

lec

tin

CX

CL

8/I

L−

8

IL−

1a

lph

a

M−

CS

F

sP

GE

2

SR

B

sT

NF−

alp

ha

CC

L2

/MC

P−

1

CD

38

CD

40

CD

62

E/E−

Se

lec

tin

CD

69

CX

CL

8/I

L−

8

CX

CL

9/M

IG

PB

MC

Cy

toto

xic

ity

Pro

life

rati

on

SR

B

CD

87

/uP

AR

CX

CL

10

/IP−

10

CX

CL

9/M

IG

HL

A−

DR

IL−

1a

lph

a

MM

P−

1

PA

I−I

SR

B

TG

F−

be

taI

tPA

uP

A

CC

L2

/MC

P−

1

CD

10

6/V

CA

M−

1

CD

14

1/T

hro

mb

om

od

uli

n

CD

14

2/T

iss

ue

Fa

cto

r

CD

87

/uP

AR

CX

CL

8/I

L−

8

CX

CL

9/M

IG

HL

A−

DR

IL−

6

LD

LR

M−

CS

F

Pro

life

rati

on

Se

rum

Am

ylo

id A

SR

B

CD

10

6/V

CA

M−

1

Co

llag

en

III

CX

CL

10

/IP−

10

CX

CL

8/I

L−

8

CX

CL

9/M

IG

EG

FR

M−

CS

F

MM

P−

1

PA

I−I

Pro

life

rati

on

_7

2h

r

SR

B

TIM

P−

1

CC

L2

/MC

P−

1

CD

54

/IC

AM−

1

CX

CL

10

/IP−

10

IL−

1a

lph

a

MM

P−

9

SR

B

TG

F−

be

taI

TIM

P−

2

uP

A

−1.5

−1.4

−1.3

−1.2

−1.1

−1.0

−0.9

−0.8

−0.7

−0.6

−0.5

−0.4

−0.3

−0.2

−0.1

0.0

0.1

0.2

0.3

0.4

0.5

0.6

0.7

0.8

0.9

1.0

Lo

g R

atio

Profiles

Cyclopamine 40 uM





CC

L2

/MC

P−

1

CD

10

6/V

CA

M−

1

CD

14

1/T

hro

mb

om

od

uli

n

CD

14

2/T

iss

ue

Fa

cto

r

CD

54

/IC

AM−

1

CD

62

E/E−

Se

lec

tin

CD

87

/uP

AR

CX

CL

8/I

L−

8

CX

CL

9/M

IG

HL

A−

DR

Pro

life

rati

on

SR

B

Vis

ua

l

CC

L2

/MC

P−

1

CC

L2

6/E

ota

xin−

3

CD

10

6/V

CA

M−

1

CD

62

P/P−

se

lecti

n

CD

87

/uP

AR

SR

B

VE

GF

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CC

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10

6/V

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14

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CD

62

E/E−

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tin

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8

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NF−

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38

CD

40

CD

62

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69

CX

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8

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87

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6

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F

Pro

life

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on

Se

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Am

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SR

B

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10

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M−

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llag

en

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CL

10

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10

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CL

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8

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CL

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EG

FR

M−

CS

F

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P−

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PA

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Pro

life

rati

on

_7

2h

r

SR

B

TIM

P−

1

CC

L2

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P−

1

CD

54

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AM−

1

CX

CL

10

/IP−

10

IL−

1a

lph

a

MM

P−

9

SR

B

TG

F−

be

taI

TIM

P−

2

uP

A

−1.5

−1.4

−1.3

−1.2

−1.1

−1.0

−0.9

−0.8

−0.7

−0.6

−0.5

−0.4

−0.3

−0.2

−0.1

0.0

0.1

0.2

0.3

0.4

0.5

0.6

0.7

0.8

0.9

1.0

Lo

g R

atio

Profiles

Cyclopamine 40 uM





CC

L2

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P−

1

CD

10

6/V

CA

M−

1

CD

14

1/T

hro

mb

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od

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14

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54

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CD

62

E/E−

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tin

CD

87

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AR

CX

CL

8/I

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8

CX

CL

9/M

IG

HL

A−

DR

Pro

life

rati

on

SR

B

Vis

ua

l

CC

L2

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P−

1

CC

L2

6/E

ota

xin−

3

CD

10

6/V

CA

M−

1

CD

62

P/P−

se

lecti

n

CD

87

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AR

SR

B

VE

GF

R2

CC

L2

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P−

1

CD

10

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M−

1

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14

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40

CD

62

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tin

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CL

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8

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2

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38

CD

40

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62

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69

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8

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1

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6

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F

Pro

life

rati

on

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Am

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SR

B

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10

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llag

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10

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10

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CL

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8

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CL

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FR

M−

CS

F

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Pro

life

rati

on

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2h

r

SR

B

TIM

P−

1

CC

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1

CD

54

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1

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CL

10

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10

IL−

1a

lph

a

MM

P−

9

SR

B

TG

F−

be

taI

TIM

P−

2

uP

A

−1.5

−1.4

−1.3

−1.2

−1.1

−1.0

−0.9

−0.8

−0.7

−0.6

−0.5

−0.4

−0.3

−0.2

−0.1

0.0

0.1

0.2

0.3

0.4

0.5

0.6

0.7

0.8

0.9

1.0

Lo

g R

atio

Profiles

Cyclopamine 40 uM





CC

L2

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P−

1

CD

10

6/V

CA

M−

1

CD

14

1/T

hro

mb

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14

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54

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CD

62

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tin

CD

87

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AR

CX

CL

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8

CX

CL

9/M

IG

HL

A−

DR

Pro

life

rati

on

SR

B

Vis

ua

l

CC

L2

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P−

1

CC

L2

6/E

ota

xin−

3

CD

10

6/V

CA

M−

1

CD

62

P/P−

se

lecti

n

CD

87

/uP

AR

SR

B

VE

GF

R2

CC

L2

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P−

1

CD

10

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M−

1

CD

14

2/T

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ue

Fa

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r

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40

CD

62

E/E−

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tin

CX

CL

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8

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CS

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2

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B

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NF−

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ha

CC

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P−

1

CD

38

CD

40

CD

62

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tin

CD

69

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8

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life

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10

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6

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F

Pro

life

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on

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Am

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SR

B

CD

10

6/V

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1

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10

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10

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CL

8/I

L−

8

CX

CL

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IG

EG

FR

M−

CS

F

MM

P−

1

PA

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Pro

life

rati

on

_7

2h

r

SR

B

TIM

P−

1

CC

L2

/MC

P−

1

CD

54

/IC

AM−

1

CX

CL

10

/IP−

10

IL−

1a

lph

a

MM

P−

9

SR

B

TG

F−

be

taI

TIM

P−

2

uP

A

−1.5

−1.4

−1.3

−1.2

−1.1

−1.0

−0.9

−0.8

−0.7

−0.6

−0.5

−0.4

−0.3

−0.2

−0.1

0.0

0.1

0.2

0.3

0.4

0.5

0.6

0.7

0.8

0.9

1.0

Lo

g R

atio

Profiles

Cyclopamine 40 uM





43

TF VCAM

Building Support Vector Machine Classifiers

• 88 Compounds

• 28 Target/Pathway

mechanisms

• 1-8 concentrations

• 327 Profiles

• 84 endpoints (8 BioMAP

Systems)

• Support Vector Machine

• 2-class models

• Mechanism class versus “Null”

set

• Result = Decision Value (DV)

• PPV – positive predictive value

(fraction of profiles that are correctly

classified)• PPV = TP / (TP + FP))

• Sensitivity (fraction of profiles that

are assigned to the class)• Sensitivity = TP / (TP + FN))

MitochondrialInhibitor

Microtubule Stabilizer Hsp90 Inhibitor

Classifier Performance: Examples

PDE IVInhibitor

Generate Data Set

Build Classifiers

Test Performance of Classifiers

Berg, Yang & Polokoff, 2013, J. Biomol Screen. 18:1260.

• AhR agonist (Aryl Hydrocarbon)

• Calcineurin

• EGFR (Epidermal Growth Factor R)

• SERCA (SR Ca++ ATPase)

• EP agonist

• Estrogen R agonist

• Glucocorticoid R agonist

• H1R Antagonist (Histamine)

• HDAC

• HMG-CoA-Reductase

• Hsp90 Inhibitor

• IKK2

• IL-17 R agonist

• JAK

Confidential45

List of Classifiers (SVM Mechanism Models)

• MEK

• Microtubule Disruptor

• Microtubule Stabilizer

• Mitochondrial Inhibitor

• mTOR

• p38 MAPK

• PDE IV (Phosphodiesterase

• PI3K

• PKC (c+n)

• Proteasome

• RAR-RXR agonist

• Src family

• TNF (Tumor Necrosis Factor)

• VDR agonist (Vitamin D R)

Berg, Yang & Polokoff, 2013, J. Biomol Screen. 18:1260.

• Chemical profiling in human cell systems generates activity profiles that can be used to:

- Group chemicals into bioactivity classes

- Generate MoA hypotheses

- Identify activities that may correlate with in vivo outcomes

• High throughput in vitro data is most informative when combined with external information

- Known targets

- In vivo effects

Summary

• Application for predictive toxicology and risk assessment must also include:

- Exposure - level and route

- Distribution

- Metabolism – inactivation or transformation

• Test agent issues

- Chemical stability and purity

- Solubility

- Mixtures

- Polypharmacy

Challenges and Considerations

BioSeek, A Division of DiscoveRx

310 Utah, Suite 100

South San Francisco, CA 94080

650-416-7600

Ellen L. Berg, PhD

[email protected]

www.biomapsystems.com

CONTACTS

Date post:	19-Jul-2015
Category:	Health & Medicine
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Berg ellen cal epa 29 oct2014

Health & Medicine