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A Chemical Biology Approach Using Primary Human Cell Systems and Co-cultures
for Understanding Target Biology
Ellen L. Berg, PhDScientific Director, BioSeek, a division of DiscoveRx
Physiologically Relevant Target StrategiesSLAS 2015, Washington DC
11 February 2015
• Problem:
- Pharmaceutical productivity is too low
- We are swimming in oceans of data
• A need for new approaches
- Better physiological relevance
- More predictive of clinical effects
Challenges in Drug Discovery
We need better data, not more data
2
Target-Based Drug Discovery - Challenges
• Target validation
- Biology has a modular architecture
- Function depends on “context”
• Target selectivity (poly-pharmacy)
- Most drugs interact with more than one target
- Targets interact with one another3
Solution: Primary Human Cell Systems
• BioMAP® Profiling Platform:
- In Vitro testing in primary human cell-
based tissue and disease models
• Chemical biology approach
- Data-driven research methodology
- Large scale chemical biology datasets
• Applications in drug discovery
- Compound /target validation
- Translational biology
- Drug mechanisms of action – in context of disease
4
BioMAP® Technology Platform
BioMAP®
Assay Systems
Reference
Profile Database
Predictive
Informatics Tools
Human primary cells
Disease-models
> 50 systems
Biomarker responses to drugs
are stored in the database
> 3000 drugs and agents
Custom informatics tools are
used to predict clinical outcomes
High Throughput Human Biology
5
BioMAP® Systems – Key Features
Primary human cell types
Physiologically relevant “context”
Complex activation settings
Co-cultures
Translational biomarker endpoints
6
Feature Mouse Man
Lifespan 2 Years 70 Years
Size 60 g 60 kg
EnvironmentAnimal facility,
cage-matesOutside world, people,
animals, etc.
Why Human?
Key differences:DNA repair mechanisms
Control of blood flow, hemostasis
Immune system status
7
Closer to the disease process
Downstream of multiple pathways and integrate information
“Decision-making”
Used by clinicians to guide therapy - Provide clinical “line of sight”
Why Translational Biomarkers?
mRNA,epigenome
Phospho-sites, intracellular proteins,
metabolome
Cell surface,secreted molecules
8
Primary Human Cell Systems Panels3C 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
Profile compounds
across a panel of assays
9
Panel of Primary Human Cell SystemsBioMAP® Predictive Tox Panel
3C 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
! ! ! ! !
10
• Challenges:
- Cells and assays are expensive
- Primary cells are variable
• Solutions:
- Standardized methods, automated and run at scale – strict QA
- Methods to manage variation • Donor qualification, donor pools
• Plate based normalization
- Singlicate endpoint measurements, but• Multiple concentrations (4+) per compound
• Multiple endpoints per assay system
• Multiple assay systems per compound
Experimental Design
11
BioMAP Profile of Positive Control
• 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
12
Reproducibility of Profiles
• 16 Experiments over many months
• Pairwise correlation of profiles (Pearson’s) were > 0.8
BioMAP Systems
Readout Parameters (Biomarkers)
Houck, K.A., J. Biomolecular Screening, 2009, 14:1054-66.13
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
Diversity of BioMAP Profile PatternsProfile Shapes & Concentration-Response Patterns
InactiveActive – Sharp dose-response
Active – Dose resistantActive – Selectively
14
Rapamycin (mTOR) Genistein (multi-target)
Dose ResistanceA Compound “Characteristic”
• “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 > 60,000x
15
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−0.4
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0.2
0.3
0.4
0.5
0.6
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−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
3C 4H LPS SAg BE3C CASM3C HDF3CGF KF3CT
BioMAP Profiling: Example ProfileTrametinib (MekinistTM) MEK Kinase Inhibitor
Lo
g e
xp
res
sio
n r
ati
o
(Dru
g/V
eh
icle
co
ntr
ol)
Control (no drug)
95%
significance
envelope
BioMAP Systems
Biomarker Endpoints
Concentration
Response
Cytotoxicity Readouts
16
• Trametinib is approved for the treatment of metastatic melanoma
• Common side effects are rash, diarrhea, peripheral edema, fatigue, and dermatitis• Skin toxicity requiring hospitalization occurred in 6% of patients treated with trametinib, most
commonly for secondary infections of the skin or severe skin toxicity (METRIC Phase III study)
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9
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TIM
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−1.5
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−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
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Am
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10
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P−
9
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B
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
3C 4H LPS SAg BE3C CASM3C HDF3CGF KF3CT
BioMAP Profiling: Example ProfileTrametinib (MekinistTM) MEK Kinase Inhibitor
Lo
g e
xp
res
sio
n r
ati
o
(Dru
g/V
eh
icle
co
ntr
ol)
BioMAP Systems
17
• Activities relevant to the role of MEK kinase in the cell cycle- MEK kinase (MAP2K) is a member of the MAPK signaling cascade
- Key activities include: inhibition of endothelial cell, T cell, smooth muscle cell and fibroblast proliferation
EC
Prolif. T cell
Prolif.
SMC
Prolif. Fibroblast
Prolif.
CC
L2/M
CP−
1
CD
10
6/V
CA
M−
1
CD
141
/Th
rom
bo
mo
du
lin
CD
14
2/T
iss
ue
Fa
cto
r
CD
54/I
CA
M−
1
CD
62
E/E−
Sele
cti
n
CD
87
/uP
AR
CX
CL
8/IL−
8
CX
CL
9/M
IG
HL
A−
DR
Pro
life
rati
on
SR
B
CC
L2/M
CP−
1
CC
L26
/Eo
tax
in−
3
CD
10
6/V
CA
M−
1
CD
62P
/P−
sele
cti
n
CD
87
/uP
AR
SR
B
VE
GF
R2
CC
L2/M
CP−
1
CD
10
6/V
CA
M−
1
CD
14
2/T
iss
ue
Fa
cto
r
CD
40
CD
62
E/E−
Sele
cti
n
CX
CL
8/IL−
8
IL−
1alp
ha
M−
CS
F
sP
GE
2
SR
B
sT
NF−
alp
ha
CC
L2/M
CP−
1
CD
38
CD
40
CD
62
E/E−
Sele
cti
n
CD
69
CX
CL
8/IL−
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−
1alp
ha
MM
P−
1
PA
I−I
SR
B
tPA
uP
A
CC
L2/M
CP−
1
CD
10
6/V
CA
M−
1
CD
141
/Th
rom
bo
mo
du
lin
CD
14
2/T
iss
ue
Fa
cto
r
CD
87
/uP
AR
CX
CL
8/IL−
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/IL−
8
CX
CL
9/M
IG
EG
FR
M−
CS
F
MM
P−
1
PA
I−I
Pro
life
rati
on
_72
hr
SR
B
TIM
P−
1
CC
L2/M
CP−
1
CD
54/I
CA
M−
1
CX
CL
10
/IP−
10
IL−
1alp
ha
MM
P−
9
SR
B
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
3C 4H LPS SAg BE3C CASM3C HDF3CGF KF3CT
CC
L2/M
CP−
1
CD
10
6/V
CA
M−
1
CD
141
/Th
rom
bo
mo
du
lin
CD
14
2/T
iss
ue
Fa
cto
r
CD
54/I
CA
M−
1
CD
62
E/E−
Sele
cti
n
CD
87
/uP
AR
CX
CL
8/IL−
8
CX
CL
9/M
IG
HL
A−
DR
Pro
life
rati
on
SR
B
CC
L2/M
CP−
1
CC
L26
/Eo
tax
in−
3
CD
10
6/V
CA
M−
1
CD
62P
/P−
sele
cti
n
CD
87
/uP
AR
SR
B
VE
GF
R2
CC
L2/M
CP−
1
CD
10
6/V
CA
M−
1
CD
14
2/T
iss
ue
Fa
cto
r
CD
40
CD
62
E/E−
Sele
cti
n
CX
CL
8/IL−
8
IL−
1alp
ha
M−
CS
F
sP
GE
2
SR
B
sT
NF−
alp
ha
CC
L2/M
CP−
1
CD
38
CD
40
CD
62
E/E−
Sele
cti
n
CD
69
CX
CL
8/IL−
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−
1alp
ha
MM
P−
1
PA
I−I
SR
B
tPA
uP
A
CC
L2/M
CP−
1
CD
10
6/V
CA
M−
1
CD
141
/Th
rom
bo
mo
du
lin
CD
14
2/T
iss
ue
Fa
cto
r
CD
87
/uP
AR
CX
CL
8/IL−
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/IL−
8
CX
CL
9/M
IG
EG
FR
M−
CS
F
MM
P−
1
PA
I−I
Pro
life
rati
on
_72
hr
SR
B
TIM
P−
1
CC
L2/M
CP−
1
CD
54/I
CA
M−
1
CX
CL
10
/IP−
10
IL−
1alp
ha
MM
P−
9
SR
B
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
3C 4H LPS SAg BE3C CASM3C HDF3CGF KF3CT
BioMAP Profiling: Example ProfileTrametinib (MekinistTM) MEK Kinase Inhibitor
Lo
g e
xp
res
sio
n r
ati
o
(Dru
g/V
eh
icle
co
ntr
ol)
BioMAP Systems
18
• Activities relevant to the role of MEK kinase in cancer- Angiogenesis (PMID: 10209122)
• Decreased EC proliferation; increased cell surface KDR (receptor for VEGF)
- Tissue remodeling and metastasis• Decreased proteolysis (uPAR, tPA, PAI-1), matrix metalloprotease (MMP1, TIMPs)
uPARuPAR
MMP1
PAI-1
tPA EGFRMMP1
PAI-1
TIMP-1
TIMP-2
EC
Prolif.
VEGFR2
CC
L2/M
CP−
1
CD
10
6/V
CA
M−
1
CD
141
/Th
rom
bo
mo
du
lin
CD
14
2/T
iss
ue
Fa
cto
r
CD
54/I
CA
M−
1
CD
62
E/E−
Sele
cti
n
CD
87
/uP
AR
CX
CL
8/IL−
8
CX
CL
9/M
IG
HL
A−
DR
Pro
life
rati
on
SR
B
CC
L2/M
CP−
1
CC
L26
/Eo
tax
in−
3
CD
10
6/V
CA
M−
1
CD
62P
/P−
sele
cti
n
CD
87
/uP
AR
SR
B
VE
GF
R2
CC
L2/M
CP−
1
CD
10
6/V
CA
M−
1
CD
14
2/T
iss
ue
Fa
cto
r
CD
40
CD
62
E/E−
Sele
cti
n
CX
CL
8/IL−
8
IL−
1alp
ha
M−
CS
F
sP
GE
2
SR
B
sT
NF−
alp
ha
CC
L2/M
CP−
1
CD
38
CD
40
CD
62
E/E−
Sele
cti
n
CD
69
CX
CL
8/IL−
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−
1alp
ha
MM
P−
1
PA
I−I
SR
B
tPA
uP
A
CC
L2/M
CP−
1
CD
10
6/V
CA
M−
1
CD
141
/Th
rom
bo
mo
du
lin
CD
14
2/T
iss
ue
Fa
cto
r
CD
87
/uP
AR
CX
CL
8/IL−
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/IL−
8
CX
CL
9/M
IG
EG
FR
M−
CS
F
MM
P−
1
PA
I−I
Pro
life
rati
on
_72
hr
SR
B
TIM
P−
1
CC
L2/M
CP−
1
CD
54/I
CA
M−
1
CX
CL
10
/IP−
10
IL−
1alp
ha
MM
P−
9
SR
B
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
3C 4H LPS SAg BE3C CASM3C HDF3CGF KF3CT
CC
L2/M
CP−
1
CD
10
6/V
CA
M−
1
CD
141
/Th
rom
bo
mo
du
lin
CD
14
2/T
iss
ue
Fa
cto
r
CD
54/I
CA
M−
1
CD
62
E/E−
Sele
cti
n
CD
87
/uP
AR
CX
CL
8/IL−
8
CX
CL
9/M
IG
HL
A−
DR
Pro
life
rati
on
SR
B
CC
L2/M
CP−
1
CC
L26
/Eo
tax
in−
3
CD
10
6/V
CA
M−
1
CD
62P
/P−
sele
cti
n
CD
87
/uP
AR
SR
B
VE
GF
R2
CC
L2/M
CP−
1
CD
10
6/V
CA
M−
1
CD
14
2/T
iss
ue
Fa
cto
r
CD
40
CD
62
E/E−
Sele
cti
n
CX
CL
8/IL−
8
IL−
1alp
ha
M−
CS
F
sP
GE
2
SR
B
sT
NF−
alp
ha
CC
L2/M
CP−
1
CD
38
CD
40
CD
62
E/E−
Sele
cti
n
CD
69
CX
CL
8/IL−
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−
1alp
ha
MM
P−
1
PA
I−I
SR
B
tPA
uP
A
CC
L2/M
CP−
1
CD
10
6/V
CA
M−
1
CD
141
/Th
rom
bo
mo
du
lin
CD
14
2/T
iss
ue
Fa
cto
r
CD
87
/uP
AR
CX
CL
8/IL−
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/IL−
8
CX
CL
9/M
IG
EG
FR
M−
CS
F
MM
P−
1
PA
I−I
Pro
life
rati
on
_72
hr
SR
B
TIM
P−
1
CC
L2/M
CP−
1
CD
54/I
CA
M−
1
CX
CL
10
/IP−
10
IL−
1alp
ha
MM
P−
9
SR
B
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
3C 4H LPS SAg BE3C CASM3C HDF3CGF KF3CT
BioMAP Profiling: Example ProfileTrametinib (MekinistTM) MEK Kinase Inhibitor
Lo
g e
xp
res
sio
n r
ati
o
(Dru
g/V
eh
icle
co
ntr
ol)
BioMAP Systems
19
• Activities relevant to side effects – risk of infections- Immunosuppression
• Inhibition of T cell proliferation and T cell activation
T Cell
Proliferation
T cell
activation
CC
L2/M
CP−
1
CD
10
6/V
CA
M−
1
CD
141
/Th
rom
bo
mo
du
lin
CD
14
2/T
iss
ue
Fa
cto
r
CD
54/I
CA
M−
1
CD
62
E/E−
Sele
cti
n
CD
87
/uP
AR
CX
CL
8/IL−
8
CX
CL
9/M
IG
HL
A−
DR
Pro
life
rati
on
SR
B
CC
L2/M
CP−
1
CC
L26
/Eo
tax
in−
3
CD
10
6/V
CA
M−
1
CD
62P
/P−
sele
cti
n
CD
87
/uP
AR
SR
B
VE
GF
R2
CC
L2/M
CP−
1
CD
10
6/V
CA
M−
1
CD
14
2/T
iss
ue
Fa
cto
r
CD
40
CD
62
E/E−
Sele
cti
n
CX
CL
8/IL−
8
IL−
1alp
ha
M−
CS
F
sP
GE
2
SR
B
sT
NF−
alp
ha
CC
L2/M
CP−
1
CD
38
CD
40
CD
62
E/E−
Sele
cti
n
CD
69
CX
CL
8/IL−
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−
1alp
ha
MM
P−
1
PA
I−I
SR
B
tPA
uP
A
CC
L2/M
CP−
1
CD
10
6/V
CA
M−
1
CD
141
/Th
rom
bo
mo
du
lin
CD
14
2/T
iss
ue
Fa
cto
r
CD
87
/uP
AR
CX
CL
8/IL−
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/IL−
8
CX
CL
9/M
IG
EG
FR
M−
CS
F
MM
P−
1
PA
I−I
Pro
life
rati
on
_72
hr
SR
B
TIM
P−
1
CC
L2/M
CP−
1
CD
54/I
CA
M−
1
CX
CL
10
/IP−
10
IL−
1alp
ha
MM
P−
9
SR
B
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
3C 4H LPS SAg BE3C CASM3C HDF3CGF KF3CT
CC
L2/M
CP−
1
CD
10
6/V
CA
M−
1
CD
141
/Th
rom
bo
mo
du
lin
CD
14
2/T
iss
ue
Fa
cto
r
CD
54/I
CA
M−
1
CD
62
E/E−
Sele
cti
n
CD
87
/uP
AR
CX
CL
8/IL−
8
CX
CL
9/M
IG
HL
A−
DR
Pro
life
rati
on
SR
B
CC
L2/M
CP−
1
CC
L26
/Eo
tax
in−
3
CD
10
6/V
CA
M−
1
CD
62P
/P−
sele
cti
n
CD
87
/uP
AR
SR
B
VE
GF
R2
CC
L2/M
CP−
1
CD
10
6/V
CA
M−
1
CD
14
2/T
iss
ue
Fa
cto
r
CD
40
CD
62
E/E−
Sele
cti
n
CX
CL
8/IL−
8
IL−
1alp
ha
M−
CS
F
sP
GE
2
SR
B
sT
NF−
alp
ha
CC
L2/M
CP−
1
CD
38
CD
40
CD
62
E/E−
Sele
cti
n
CD
69
CX
CL
8/IL−
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−
1alp
ha
MM
P−
1
PA
I−I
SR
B
tPA
uP
A
CC
L2/M
CP−
1
CD
10
6/V
CA
M−
1
CD
141
/Th
rom
bo
mo
du
lin
CD
14
2/T
iss
ue
Fa
cto
r
CD
87
/uP
AR
CX
CL
8/IL−
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
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−0.4
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0.4
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0.6
0.7
0.8
0.9
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BioMAP Profiling: Example ProfileTrametinib (MekinistTM) MEK Kinase Inhibitor
Lo
g e
xp
res
sio
n r
ati
o
(Dru
g/V
eh
icle
co
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BioMAP Systems
20
• Activities relevant to side effects – risk of infections- Immunosuppression
• Inhibition of T cell proliferation and T cell activation
- Inhibition of acute inflammatory markers IL-1α, IL-8 and TNFα
T Cell
Proliferation
T cell
activation
IL-8 IL-1a
TNF-aIL-8
IL-1aIL-8
IL-8IL-1a
CC
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alp
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E/E−
Sele
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−0.7
−0.6
−0.5
−0.4
−0.3
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−0.1
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0.1
0.2
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0.5
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6
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llag
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−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
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3C 4H LPS SAg BE3C CASM3C HDF3CGF KF3CT
BioMAP Profiling: Example ProfileTrametinib (MekinistTM) MEK Kinase Inhibitor
Lo
g e
xp
res
sio
n r
ati
o
(Dru
g/V
eh
icle
co
ntr
ol)
BioMAP Systems
21
• Activities relevant to side effects – skin rash- Upregulation of VCAM and IP-10 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.
IP-10VCAMVCAM
• Confirm expected target activities
• Identify unexpected activities
- Target or off-target
• Evaluate suitability for in vivo preclinical studies
- Safety window
- Dose-resistance
• Guide in vivo preclinical studies
- Dose selection
- Identify new biomarkers or pathways to track
How Can These Data Help in TDD?
22
Alert!!
Prioritize
Predict
Confirm
• Testing Drug Combinations
- Preclinical testing of drug combinations to preview in vivo
effects
• Elucidating Mechanisms of Toxicity
- Data mining chemical biology datasets
- Connect target biology in knowledge frameworks built around
clinical outcomes
Case Studies
23
• Challenges for studying drug combinations:
- System must include both targets
- Physiologically relevant setting (ideally all human)
- Suitably robust to capture combination effects
• Case Example
- BioMAP Oncology systems that model tumor-host
microenvironments
- Trametinib (MEK kinase inhibitor) + Dabrafenib (Braf inhibitor)
• Combination approved for treatment of melanoma
Drug Combinations
25
Trametinib + Dabrafenib Combination
Dabrafenib
Trametinib
• Combination approved by the
US FDA June 2014 for the
treatment of BRAF V600E
mutation-positive unresectable
or metastatic melanoma
• Improved patient responses
• Reduced incidence of cutaneous
squamous cell carcinoma
Human In Vitro Models of Tumor MicroenvironmentBioMAP CRC Oncology Systems
System Primary Human Cell TypesDisease / Tissue
RelevanceBiomarker Readouts
StroHT29
HT-29 colon adenocarcinoma cell
line + Primary Human Fibroblasts
+ PBMC
Oncology: Host Tumor-
Stromal Microenvironment
sVEGF, MMP9, TIMP2, tPA, uPA, uPAR, collagen I,
collagen III, PAI-1, SRB, sIL-2, pCyt, sIL-6, sIL-10,
sIFNγ, sTNFα, sIL-17A, sGranzyme B, Keratin 20,
CEACAM5, IP-10, VCAM-1
VascHT29
HT-29 colon adenocarcinoma cell
line + Primary Human Endothelial
cells + PBMC
Oncology: Host Tumor-
Vascular Microenvironment
CD40, CD69, uPAR, collagen IV, MCP-1, VCAM-1,
pCyt, SRB, sIL-2, sIL-6, sIL-10, sIFNγ, sTNFα, sIL-
17A, sGranzyme B, CEACAM5, Keratin 20, IP-10,
MIG
• Biomarker Endpoints:
• Immunomodulation: IL-2, IL-6, IL-10, IL-4, IFNγ, CD40, CD69, IL-17, Granzyme B
• Inflammation: TNFα, MCP-1, VCAM, CXCL9/MIG,
• Metastasis / Remodeling: MMP9, TIMP2, Collagens I, III, IV, uPA, uPAR, PAI-1
• Angiogenesis / Fibrinolysis: uPA, uPAR, PAI-1, VEGF
• Tumor specific markers: CEACAM5, CK2028
Dabrafenib (B-raf) Trametinib (MEK) Dabrafenib +Trametinib
29
Combination Study Example: Dabrafenib (B-Raf) + Trametinib (MEK Inhibitor)
Dabrafenib (B-raf) Trametinib (MEK) Dabrafenib +Trametinib
• Combination effects of Dabrafenib (B-raf) and Trametinib (MEK)- Tumor cell marker (CEACAM5) is reduced only in the combination (green
arrow)
- Consistent with the combination being more efficacious against tumors in vivo
30
Combination Study Example: Dabrafenib (B-Raf) + Trametinib (MEK Inhibitor)
Dabrafenib (B-raf) Trametinib (MEK) Dabrafenib +Trametinib
• Combination effects of Dabrafenib (B-raf) and Trametinib (MEK)- Tumor cell marker (CEACAM5) is reduced only in the combination
- Consistent with the combination being more efficacious against tumors in vivo
- Reduced levels of Inflammatory endpoints; collagen III (grey arrows)
- Consistent with reduced Trametinib-related skin side effects (Flaherty, 2012, NEJM 367:1694) and reduced skin proliferative disorders
31
Combination Study Example: Dabrafenib (B-Raf) + Trametinib (MEK Inhibitor)
• GOAL: To develop a cost-effective approach for efficiently prioritizing the toxicity testing of thousands of chemicals
• Profiling in BioMAP Systems since 2007: • > 1100 Chemicals Profiled
• > 300,000 Datapoint Chemical Biology Dataset for ToxCast
EPA ToxCastTM Program
• Patterns in the data Insights
Cluster of Chemicals Identified by SOMKey Feature: Increased Tissue Factor
• Cluster of chemicals defined by their BioMAP signature
- Key feature: Increased Tissue Factor (TF) in BioMAP 3C system
Nicole Kleinstreuer, et al., NBT, 2014
Tissue Factor
• Phenotypic signature of compounds in SOM cluster #57
- Box and whisker plot for cluster 57 representing a signature for AhR activation
• Compounds: AhR Agonists
- 85% of members of clusters 57, 67 (adjacent in the 10X10 SOM) were active in an AhR reporter gene assay (examples shown here).
Tissue Factor
Kleinstreuer, 2014, Nature Biotechnology, 32:583-91.35
Cluster of Chemicals Identified by SOMAryl Hydrocarbon Receptor Agonists
Tissue Factor (TF)Primary Cellular Initiator of Blood Coagulation
RW Colman 2006 J. Exp. Med
Blood
Coagulation
36
Thrombosis
• Pathologic setting – aberrant coagulation thrombosis
- The formation of a blood clot (coagulation) within a vein
- Clinical manifestations• Deep vein thrombosis (DVT), stroke, and pulmonary embolism thrombi
break off and get lodged in the lung
• Ebola – consumptive coagulopathy
Thrombosis Can Also Be Pathologic
Smooth muscle cells
Endothelial cells
Vessel Lumenplatelets in fibrin clot
38
• Aryl Hydrocarbon receptor agonists- PAHs, Benz(a)anthracene
- Smoking (Cigarette smoke extract)
• mTOR inhibitors- Everolimus (Baas, 2013, Thromb Res 132:307)
• Anti-Estrogens / SERMS, oral contraceptives- Tamoxifen, Clomiphene, Cyproterone
• Second generation anti-psychotics- Clozapine
• Others- Crizotinib (oncology drug)
Mechanisms / Drugs Associated with Thrombosis-Related Side Effects
All show increased Tissue Factor levels in the BioMAP 3C System
39
• Leverage our large chemical biology database of >3800 compounds
• Search the database for all compounds / test agents that increase TF in the 3C system
- How common is this activity?
- What are the mechanisms represented?
- Is there a connection that helps us better understand the regulation of thrombosis?
Are These Mechanisms Connected?
40
Analysis of Reference CompoundsTest Agents Mechanism
Confidence in
Mechanism 2-Mercaptobenzothiazole AhR agonist High
3-Hydroxyfluorene AhR agonist High
Benzo(b)fluoranthene AhR agonist High
C.I Solvent yellow 14 AhR agonist High
FICZ AhR agonist High
Abiraterone CYP17A Inhibitor High
Ketoconazole CYP17A Inhibitor High
Clomiphene citrate Estrogen R Antagonist High
Histamine H1R agonist High
Histamine Phosphate H1R agonist High
Cobalt(II) Chloride Hexahydrate HIF-1α Inducer High
Tin(II) Chloride HIF-1α Inducer High
Chloroquine Phosphate Lysosome Inhibitor High
Primaquine Diphosphate Lysosome Inhibitor High
Temsirolimus mTOR Inhibitor High
Torin-1 mTOR Inhibitor High
Torin-2 mTOR Inhibitor High
Bryolog PKC activator High
Bryostatin PKC activator High
Bryostatin 1 PKC activator High
Phorbol 12-myristate 13-acetate PKC activator High
Phorbol 12,13-didecanoate PKC activator High
Picolog PKC activator High
3,5,3-Triiodothyronine Thyroid H R agonist Good
Concanamycin A Vacuolar ATPase Inhibitor Good
Mifamurtide NOD2 agonist Good
Oncostatin M OSM R agonist Good
Ethanol Organic Solvent Good
PAz-PC Oxidized phospholipid Good
Z-FA-FMK Cysteine protease Inhibitor Good
8-Hydroxyquinoline Chelating agent Unknown
A 205804 ICAM, E-selectin inhibitor Unknown
AZD-4547 FGFR Inhibitor Unknown
Crizotinib ALK, c-met Inhibitor Unknown
Desloratadine H1R antagonist Unknown
Dodecylbenzene Industrial chemical Unknown
Fenaminosulf Fungicide Unknown
GDC-0879 B-Raf Inhibitor Unknown
GW9662 PPARγ agonist Unknown
Imatinib PDGFR, c-Kit, Bcr-Abl Inhibitor Unknown
KN93 CaMKII Inhibitor Unknown
Linoleic Acid Ethyl Ester Fatty Acid Unknown
Mancozeb Fungicide Unknown
MK-2206 AKT Inhibitor Unknown
Mometasone furoate GR agonist Unknown
N-Ethylmaleimide Alkylating agent Unknown
PP3 SRC Kinase Inhibitor Unknown
Primidone GABA R agonist Unknown
Sulindac Sulfide NSAID Unknown
Terconazole Anti-fungal Unknown
Tris(1,3-dichloro-2-propyl) phosphate Flame retardant Unknown
TX006146 Unknown Unknown
TX006237 Unknown Unknown
TX011661 Unknown Unknown
U-73343 Unknown Unknown
UO126 MEK Inhibitor Unknown
ZK-108 PI-3K Inhibitor (βγ-selective) Unknown
Mechanisms that Increase TF
AhR Agonist
CYP17A Inhibitor
Estrogen R Antagonist
H1R Agonist
HIF-1α Inducer
Lysosomal Inhibitor
mTOR Inhibitor
PKC Activator
Thyroid H R Agonist
Vacuolar ATPase Inhibitor
NOD2 Agonist
OSM R Agonist
41
• Increased TF is an uncommon activity
• 55/3187 compounds (1.7%)
Implicate Autophagy
Berg, et al., IJMS, 2015
Autophagy
• Intracellular self-degradation system
• Cellular response to nutrient deprivation
• Also contributes to recycling of dysfunctional organelles, handling of protein aggregates, bacteria and viruses42
• Summary
- Mechanistic Hypothesis: thrombosis-related side effects are associated with alterations in the process of autophagy that increase TF cell surface levels
- In moderation, during nutrient deprivation, an increase in TF leading to the recruitment of nutrient-rich platelets to a tissue sites would be a beneficial response
• Next Step:
- Incorporation of these data in a knowledge framework
Tissue Factor, Autophagy & Thrombosis
49
Adverse Outcome Pathway (AOP)Knowledge Framework
MIEKey
EventAdverse
OutcomeKey
EventKey
Event
Molecular
Initiating EventClinical Effect
• Framework for integrating mode of action hypotheses to outcomes for chemical risk assessment (OECD)- http://www.oecd.org/chemical safety/testing/adverse-outcome-pathways-
molecular-screening-and-toxicogenomics.htm
• Focused on the clinical outcome- Anchored at both ends
50
An AOP for DVT
MIEKey
EventAdverse
Outcome
Activation of
AhR
Upregulation
of Tissue
Factor
Deep Vein
Thrombosis
Initiation of
Coagulation
Key Event
Key Event
Molecular
Initiating EventClinical Effect
Increase in
Autophagic
Vacuolization
HDF3CGF
In vitro
disease model
3C
3C 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
! ! ! ! !
51
MIE
Inhibition of
Estrogen R
• Profiling across primary human cell systems can be applied in target-based drug discovery for:
- Defining characteristics of good drugs• Confirming expected target activities
• Identifying unexpected activities (target or off-target)
• Concentration-response characteristics (dose resistance)
- Guide in vivo preclinical and clinical studies• Dose selection, safety window
• Identify new biomarkers or pathways to track
• Preview drug combination effects
• Applications at the organizational level:
- Connect target biology across programs and therapeutic areas
- Improve safety prediction early in discovery
- Facilitate opportunities for new indication discovery
In Conclusion
52
• BioSeek
- Mark A. Polokoff
- Dat Nguyen
- Xitong Li
- Antal Berenyi
- Alison O’Mahony
• NIEHS (ILS)
- Nicole Kleinstreuer
Acknowledgements
• EPA
- Keith Houck
- Richard Judson
- David Dix
- Bob Kavlock
- David Reif
- Matt Martin
- Ann Richard
- Tom Knudsen
53
Contact:
Ellen L. Berg, PhD,
Scientific Director
BioSeek, a division of DiscoveRx
310 Utah Avenue, Suite 100
South San Francisco, CA 94080
+1-650-416-7621
54