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Predictive Models for Mechanism of Action Classification from Phenotypic Assay Data – Application to Phenotypic Drug Discovery Presentation at SLAS 2014 conference in San Diego, 21 January 2014
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Predictive Models for Mechanism of Action Classification from Phenotypic Assay Data – Application to Phenotypic
Drug Discovery
Ellen L. Berg, PhD21 January 2014
SLAS 2014, San Diego, CA
• Problem:
- Drug discovery productivity is at an all time low
- We are swimming in oceans of data
• High throughput technologies
• New assay models and platforms
• Needed:
- New tools or new approaches
- Framework for integrating information
Extracting Meaning from Complex Data
The Challenge of Drug Discovery
Scale (meters)
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
3
• Human biology is complex
• Multiple modular, highly interconnected networks
Context is Key
• Target validation
- Biology has a modular architecture
- Function depends on “context”
• Target selectivity (poly-pharmacy)
- Most drugs interact with more than one target
4
BioMAP® Technology Platform
BioMAP®
Assay Systems
Reference
Profile Database
Predictive
Informatics Tools
Standardized human primary cell disease models
Database of reference profiles Analysis and data mining tools
A Primary Human Cell and Co-Culture-Based Assay Platform for PDD
5
Human primary cell-based assays
Tissue & disease models
BioMAP® Systems – Key Features
6
• Primary human cell types
• Physiologically relevant “context”
- Complex activation settings
- Co-cultures
• Translational biomarker endpoints
Feature Mice Man
Lifespan 2 Years 70 Years
Size 60 g 60 kg
EnvironmentAnimal facility, cage-mates
Outside world, people, animals, etc.
Why Human?
• Key differences between mouse and man:
- DNA repair mechanisms
- Control of blood flow, hemostasis
- Immune system status
7
• Two approaches:
- (1) Measure everything• Whole genome mRNA, proteomics, metabolomics, etc.
- (2) Measure what is “decision-making”• Translational biomarkers, known disease biomarkers, are
downstream of multiple pathways and integrate information
Why Translational Biomarkers?
mRNA,epigenome
Phospho-sites, intracellular proteins,
metabolome
Cell surface,secreted molecules
8
BioMAP Profiling: Example ProfileReference p38 MAPK Inhibitor
Lo
g e
xp
res
sio
n r
ati
o
(Dru
g/D
MS
O c
on
tro
l)
Control (no drug)
99%
significance
envelope
BioMAP Systems
Readout Parameters (Biomarkers)
Dose
Response
Cytotoxicity Readouts
9
BioMAP profiles retain shape over multiple concentrations
BioMAP Profiling: Example ProfileReference p38 MAPK Inhibitor
Lo
g e
xp
res
sio
n r
ati
o
(Dru
g/D
MS
O c
on
tro
l)
10
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
BioMAP Profiling: Example ProfileReference p38 MAPK Inhibitor
Lo
g e
xp
res
sio
n r
ati
o
(Dru
g/D
MS
O c
on
tro
l)
11
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
BioMAP Profiling: Example ProfileReference p38 MAPK Inhibitor
Lo
g e
xp
res
sio
n r
ati
o
(Dru
g/D
MS
O c
on
tro
l)
12
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
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
13
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
14
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
BioMAP Data Can Cluster CompoundsAccording to Mechanisms of Action
mTOR
PKC Activation
p38 MAPK
HMG-CoA
reductase
Calcineurin
Transcription
p38 MAPK
Calcineurin
mTOR
Mitochondrial ATPase
15
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
Consensus Profiles for Mechanism Classes
p38 MAPK inhibitor 1
p38 MAPK inhibitor 2
p38 MAPK inhibitor 3
• Profiles for target-selective compounds can be used to define a mechanism class (Berg, Yang & Polokoff, 2013)
• Consensus profile reflects target-specific biology16
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
• Dense coverage of biology
• Multiple pathway classes detected per assay
Me
chan
ism
Cla
sse
s
BioMAP Assay / Endpoints
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
−S
ele
cti
n
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
−s
ele
cti
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
−S
ele
cti
n
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
−S
ele
cti
n
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
Cyclopamine 13.333 u...
Cyclopamine 4.444 uM
Cyclopamine 1.482 uM
3C 4H LPS SAg BE3C CASM3C HDF3CGF KF3CT
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
−S
ele
cti
n
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
−s
ele
cti
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
−S
ele
cti
n
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
−S
ele
cti
n
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
Cyclopamine 13.333 u...
Cyclopamine 4.444 uM
Cyclopamine 1.482 uM
3C 4H LPS SAg BE3C CASM3C HDF3CGF KF3CT
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
−S
ele
cti
n
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
−s
ele
cti
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
−S
ele
cti
n
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
−S
ele
cti
n
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
Cyclopamine 13.333 u...
Cyclopamine 4.444 uM
Cyclopamine 1.482 uM
3C 4H LPS SAg BE3C CASM3C HDF3CGF KF3CT
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
−S
ele
cti
n
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
−s
ele
cti
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
−S
ele
cti
n
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
−S
ele
cti
n
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
Cyclopamine 13.333 u...
Cyclopamine 4.444 uM
Cyclopamine 1.482 uM
3C 4H LPS SAg BE3C CASM3C HDF3CGF KF3CT
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
−S
ele
cti
n
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
−s
ele
cti
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
−S
ele
cti
n
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
−S
ele
cti
n
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
Cyclopamine 13.333 u...
Cyclopamine 4.444 uM
Cyclopamine 1.482 uM
3C 4H LPS SAg BE3C CASM3C HDF3CGF KF3CT
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
−S
ele
cti
n
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
−s
ele
cti
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
−S
ele
cti
n
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
−S
ele
cti
n
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
Cyclopamine 13.333 u...
Cyclopamine 4.444 uM
Cyclopamine 1.482 uM
3C 4H LPS SAg BE3C CASM3C HDF3CGF KF3CT
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
−S
ele
cti
n
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
−s
ele
cti
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
−S
ele
cti
n
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
−S
ele
cti
n
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
Cyclopamine 13.333 u...
Cyclopamine 4.444 uM
Cyclopamine 1.482 uM
3C 4H LPS SAg BE3C CASM3C HDF3CGF KF3CT
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
−S
ele
cti
n
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
−s
ele
cti
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
−S
ele
cti
n
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
−S
ele
cti
n
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
Cyclopamine 13.333 u...
Cyclopamine 4.444 uM
Cyclopamine 1.482 uM
3C 4H LPS SAg BE3C CASM3C HDF3CGF KF3CT
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
−S
ele
cti
n
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
−s
ele
cti
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
−S
ele
cti
n
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
−S
ele
cti
n
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
Cyclopamine 13.333 u...
Cyclopamine 4.444 uM
Cyclopamine 1.482 uM
3C 4H LPS SAg BE3C CASM3C HDF3CGF KF3CT
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
−S
ele
cti
n
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
−s
ele
cti
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
−S
ele
cti
n
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
−S
ele
cti
n
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
Cyclopamine 13.333 u...
Cyclopamine 4.444 uM
Cyclopamine 1.482 uM
3C 4H LPS SAg BE3C CASM3C HDF3CGF KF3CT
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
−S
ele
cti
n
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
−s
ele
cti
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
−S
ele
cti
n
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
−S
ele
cti
n
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
Cyclopamine 13.333 u...
Cyclopamine 4.444 uM
Cyclopamine 1.482 uM
3C 4H LPS SAg BE3C CASM3C HDF3CGF KF3CT
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
−S
ele
cti
n
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
−s
ele
cti
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
−S
ele
cti
n
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
−S
ele
cti
n
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
Cyclopamine 13.333 u...
Cyclopamine 4.444 uM
Cyclopamine 1.482 uM
3C 4H LPS SAg BE3C CASM3C HDF3CGF KF3CT
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
−S
ele
cti
n
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
−s
ele
cti
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
−S
ele
cti
n
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
−S
ele
cti
n
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
Cyclopamine 13.333 u...
Cyclopamine 4.444 uM
Cyclopamine 1.482 uM
3C 4H LPS SAg BE3C CASM3C HDF3CGF KF3CT
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
−S
ele
cti
n
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
−s
ele
cti
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
−S
ele
cti
n
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
−S
ele
cti
n
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
Cyclopamine 13.333 u...
Cyclopamine 4.444 uM
Cyclopamine 1.482 uM
3C 4H LPS SAg BE3C CASM3C HDF3CGF KF3CT
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
−S
ele
cti
n
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
−s
ele
cti
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
−S
ele
cti
n
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
−S
ele
cti
n
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
Cyclopamine 13.333 u...
Cyclopamine 4.444 uM
Cyclopamine 1.482 uM
3C 4H LPS SAg BE3C CASM3C HDF3CGF KF3CT
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
−S
ele
cti
n
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
−s
ele
cti
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
−S
ele
cti
n
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
−S
ele
cti
n
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
Cyclopamine 13.333 u...
Cyclopamine 4.444 uM
Cyclopamine 1.482 uM
3C 4H LPS SAg BE3C CASM3C HDF3CGF KF3CT
17
• Can we use these signatures to build classifiers?
- Predictive models for specific mechanism classes would enable automated mechanism assignments
• Certain mechanisms are known to be associated with certain human outcomes (safety and/or toxicity)
- Automated mechanism assignment could be used as a tool to help in compound assessment and prioritization
- Triage of phenotypic drug discovery hits
Consensus Profiles – Phenotypic Signatures
18
• Generate reference dataset
- Compounds from 28 mechanism classes• Well characterized, target selective
- Test in 8 BioMAP systems• Multiple concentrations
• Build a series of Two-class models using support vector machines
- Use profiles in the “known” class versus a “null” set
- Model output is “decision value”
Automated Mechanism Class Assignment -Predictive Models
19
• Test reference data set in each model
- PPV – positive predictive value, for a given decision value cut off, what fraction (percentage) of profiles are correctly classified? (=TP/(TP+FP))
- Sensitivity – for a given decision value, what percentage of profiles were assigned to the class? (=TP/(TP+FN))
Assessing Model Performance
MitochondrialInhibitor
p38 MAPKInhibitor
Berg, Yang and Polokoff, 201320
Prediction Results – p38 MAPK
• Among Phase II chemicals (800) tested as part of the EPA’s ToxCast program:
- The 2 named p38 MAPK inhibitors were both classified as p38 MAPK inhibitors (highest decision values were 1.01 and 0.91)
- Manuscript submitted (Nicole Kleinstreuer, Keith Houck et al)
• Final results will depend on disclosure of target mechanisms for compounds donated by ToxCastPharma partners
• Screening and library characterization
• Triage of hits/actives from discovery programs
- Phenotypic drug discovery programs
• Elucidation of Adverse (Efficacy) Outcome Pathways
- Connecting initiating events (targets) with clinical outcomes
Applications
22
2323
Source Library Type Conc. % Active % Cytotoxic
A Secreted proteins 10 mg/ml 18% 0%
B Peptides 1 mM 7% 0.2%
C Kinase 3 mM 25% 6%
D Diversity 3.3 mM 31% 6%
E Natural Product 5 mM 50% 11%
F Kinase 1.6 mM 66% 1%
Screening and Library Characterization
• Phenotypic assays higher hit rates
• Small molecule libraries and collections can be prioritized
Mechanism Classification for Triage of Phenotypic Actives
Environmental BioactivesKinase Focused Collection
Mitochondria
Microtubule
cAMPElevator
mTOR
Proteasome
AhR
EGFR
Other
Unclassified
Mitochondria
Microtubule
cAMPElevator
mTOR
Proteasome
AhR
EGFR
Other
Unclassified
• ~50% of phenotypic actives from two collections could be classified
• Mitochondrial and microtubule inhibitors are common mechanisms in both sets of compounds
24
Adverse Outcome Pathway Framework
MIEKey
EventAdverse
OutcomeKey
EventKey
Event
Molecular
Initiating EventClinical Effect
• Framework for integrating mode of action hypotheses to outcomes for chemical risk assessment (OECD)
• Focused on the clinical outcome
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
• Dense coverage of biology
• Multiple pathway classes detected per assay
Me
chan
ism
Cla
sse
s
BioMAP Assay / Endpoints
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
−S
ele
cti
n
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
−s
ele
cti
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
−S
ele
cti
n
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
−S
ele
cti
n
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
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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
Cyclopamine 13.333 u...
Cyclopamine 4.444 uM
Cyclopamine 1.482 uM
3C 4H LPS SAg BE3C CASM3C HDF3CGF KF3CT
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
−S
ele
cti
n
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
−s
ele
cti
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
−S
ele
cti
n
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
−S
ele
cti
n
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
Cyclopamine 13.333 u...
Cyclopamine 4.444 uM
Cyclopamine 1.482 uM
3C 4H LPS SAg BE3C CASM3C HDF3CGF KF3CT
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
−S
ele
cti
n
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
−s
ele
cti
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
−S
ele
cti
n
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
−S
ele
cti
n
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
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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
Cyclopamine 13.333 u...
Cyclopamine 4.444 uM
Cyclopamine 1.482 uM
3C 4H LPS SAg BE3C CASM3C HDF3CGF KF3CT
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
−S
ele
cti
n
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
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xin−
3
CD
10
6/V
CA
M−
1
CD
62
P/P
−s
ele
cti
n
CD
87
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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
−S
ele
cti
n
CX
CL
8/I
L−
8
IL−
1a
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a
M−
CS
F
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GE
2
SR
B
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NF−
alp
ha
CC
L2
/MC
P−
1
CD
38
CD
40
CD
62
E/E
−S
ele
cti
n
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
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a
MM
P−
1
PA
I−I
SR
B
TG
F−
be
taI
tPA
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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
Cyclopamine 13.333 u...
Cyclopamine 4.444 uM
Cyclopamine 1.482 uM
3C 4H LPS SAg BE3C CASM3C HDF3CGF KF3CT
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
−S
ele
cti
n
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
−s
ele
cti
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
−S
ele
cti
n
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
−S
ele
cti
n
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
Cyclopamine 13.333 u...
Cyclopamine 4.444 uM
Cyclopamine 1.482 uM
3C 4H LPS SAg BE3C CASM3C HDF3CGF KF3CT
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
−S
ele
cti
n
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
−s
ele
cti
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
−S
ele
cti
n
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
−S
ele
cti
n
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
Cyclopamine 13.333 u...
Cyclopamine 4.444 uM
Cyclopamine 1.482 uM
3C 4H LPS SAg BE3C CASM3C HDF3CGF KF3CT
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
−S
ele
cti
n
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
−s
ele
cti
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
−S
ele
cti
n
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
−S
ele
cti
n
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
Cyclopamine 13.333 u...
Cyclopamine 4.444 uM
Cyclopamine 1.482 uM
3C 4H LPS SAg BE3C CASM3C HDF3CGF KF3CT
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
−S
ele
cti
n
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
−s
ele
cti
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
−S
ele
cti
n
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
−S
ele
cti
n
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
Cyclopamine 13.333 u...
Cyclopamine 4.444 uM
Cyclopamine 1.482 uM
3C 4H LPS SAg BE3C CASM3C HDF3CGF KF3CT
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
−S
ele
cti
n
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
−s
ele
cti
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
−S
ele
cti
n
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
−S
ele
cti
n
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
Cyclopamine 13.333 u...
Cyclopamine 4.444 uM
Cyclopamine 1.482 uM
3C 4H LPS SAg BE3C CASM3C HDF3CGF KF3CT
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
−S
ele
cti
n
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
−s
ele
cti
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
−S
ele
cti
n
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
−S
ele
cti
n
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
Cyclopamine 13.333 u...
Cyclopamine 4.444 uM
Cyclopamine 1.482 uM
3C 4H LPS SAg BE3C CASM3C HDF3CGF KF3CT
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
−S
ele
cti
n
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
−s
ele
cti
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
−S
ele
cti
n
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
−S
ele
cti
n
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
Cyclopamine 13.333 u...
Cyclopamine 4.444 uM
Cyclopamine 1.482 uM
3C 4H LPS SAg BE3C CASM3C HDF3CGF KF3CT
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
−S
ele
cti
n
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
−s
ele
cti
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
−S
ele
cti
n
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
−S
ele
cti
n
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
Cyclopamine 13.333 u...
Cyclopamine 4.444 uM
Cyclopamine 1.482 uM
3C 4H LPS SAg BE3C CASM3C HDF3CGF KF3CT
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
−S
ele
cti
n
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
−s
ele
cti
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
−S
ele
cti
n
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
−S
ele
cti
n
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
Cyclopamine 13.333 u...
Cyclopamine 4.444 uM
Cyclopamine 1.482 uM
3C 4H LPS SAg BE3C CASM3C HDF3CGF KF3CT
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
−S
ele
cti
n
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
−s
ele
cti
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
−S
ele
cti
n
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
−S
ele
cti
n
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
Cyclopamine 13.333 u...
Cyclopamine 4.444 uM
Cyclopamine 1.482 uM
3C 4H LPS SAg BE3C CASM3C HDF3CGF KF3CT
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
−S
ele
cti
n
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
−s
ele
cti
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
−S
ele
cti
n
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
−S
ele
cti
n
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
Cyclopamine 13.333 u...
Cyclopamine 4.444 uM
Cyclopamine 1.482 uM
3C 4H LPS SAg BE3C CASM3C HDF3CGF KF3CT
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
−S
ele
cti
n
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
−s
ele
cti
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
−S
ele
cti
n
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
−S
ele
cti
n
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
Cyclopamine 13.333 u...
Cyclopamine 4.444 uM
Cyclopamine 1.482 uM
3C 4H LPS SAg BE3C CASM3C HDF3CGF KF3CT
26
VCAM
An Adverse Outcome Pathway for Skin Rash
MIEKey
EventAdverse
Outcome
Inhibition of
p38 MAPKUpregulation
of VCAMSkin Rash
MIE
Inhibition of
MEK
Inflammatory
Cell
Recruitment?
Key Event
Key Event
JNK Pathway
Activation?
Molecular
Initiating EventClinical Effect
HDF3CGF
In vitro
disease model
• Phenotypic data sets from primary human cell and co-culture models can be used to classify mechanisms of action
- Assays are sufficiently reproducible
- Mechanisms are distinguishable
• Applications
- Screening & Library characterization
- Triage of discovery program hits
- Outcome pathway knowledge
- Phenotypic drug discovery
Summary
28
• Using biological systems to discover new drugs
- Target agnostic approach
• Neoclassic Drug Discovery
- The combination of using biologically complex model systems & high throughput approaches (JAL & EB, 2013)
- Screening assays that are extraordinarily well characterized• Tool compounds
• Omics and genetic technologies
- Integration of target-based and phenotypic drug discovery
• Please attend:
- Phenotypic Drug Discovery SIG, Wednesday 8:00 AM
Phenotypic Drug Discovery
29
DiscoveRx Solutions: an Integrated Portfolio
HDAC/HMT
De
pth
of
Co
vera
ge
100%
0%
28
4
38
9 21 2
3 40
GPCRs Kinases NHRs Pathways Bromodomains
Primary Human Cell Profiling
1,118 assays covering 741 druggable targets 30
• BioSeek
- Mark A. Polokoff
- Alison O’Mahony
- Jian Yang
- Antal Berenyi
Acknowledgements
• EPA
- Keith Houck
- Nicole Kleinstreuer
31
BioSeek, A Division of DiscoveRx
310 Utah, Suite 100
South San Francisco, CA 94030
650-416-7600
Ellen L. Berg, PhD
www.biomapsystems.com
CONTACTS
