Transcript Document

Stress Response Pathway Ensemble:
A New Paradigm in High Throughput Toxicity Screening
Steve Simmons
US EPA Office of Research and Development
RTP, NC
The McKim Conferences
September 2008
Office of Research and Development
NHEERL, Neurotoxicology Division, Cellular and Molecular Toxicology Branch
Outline
• The Challenge Before Us
• Stress Response Pathways as Toxicity Pathways
• Stress Response Assays
• Implementation to HTS
• What does this have to with QSAR?
Office of Research and Development
NHEERL, Neurotoxicology Division, Cellular and Molecular Toxicology Branch
Problem/Challenge
• Large number of environmental compounds that currently
need characterization and prioritization for further
screening
• Limited testing resources
• Current approaches are slow, laborious, and expensive
• Ethical need to reduce animal use in toxicity testing
• In vitro –omics approaches too expensive for screening
applications and data interpretation problematic
• Multiple alternative approaches needed, assay cost to be
minimized
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Solution
• Rapid, inexpensive, reproducible and predictive
assays that allow for effective screening of large
numbers of compounds to enable prioritization for
further characterization
• In vitro assays amenable to high-throughput
screening, preferably using human cells and
tissues that are significantly more economical and
practical than –omic methods and traditional in vivo
testing methods
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Toxicity Pathways
Chemical Characterization
Toxicity Testing
•Poor definition
•Number unknown
•Extended research effort
Toxicity Pathways
• Evaluation of perturbations in toxicity pathways
rather than apical endpoints
• Emphasis on high-throughput approaches
using cell lines, preferably of human origin
• Use of medium-throughput assays of more
integrated cellular responses
Toxicity
Pathways
Targeted Testing
Dose-Response and
Extrapolation Modeling
Targeted Testing
• Testing conducted to evaluate metabolites, assess
target tissues, and develop understanding of
affected cellular processes at genomics level
• Limited types and duration of in vivo studies,
focusing on up to 14-day exposures
• More extensive testing for representative
compounds in novel chemical classes
Adapted from Toxicity testing in the twenty-first century: a vision and strategy: NRC, July 2007
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NHEERL, Neurotoxicology Division, Cellular and Molecular Toxicology Branch
Toxicity Pathways
• Are all cellular pathways potential toxicity pathways?
• If so, are all pathways created equal, or are some more
important than others?
• How do you determine importance/priority?
 Cover the most chemical space?
 Easiest to model?
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5
Stress Responses Sense Perturbations
Exposure
Tissue Dose
Adapted from: Toxicity Testing in the Twenty-first
Century: A Vision and a Strategy,
National Research Council. 2007.
Biologic Interaction
Perturbation
Normal
Biologic
Inputs
Biologic
Function
Reversible
Adaptive Stress
Response
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Early
Cellular
Changes
Cell
Injury
Irreversible
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Morbidity and
Mortalilty
Adaptive Stress-Response Pathways
• Protective signaling pathways activated in
response to environmental insults such as
chemical toxicity
• Present in all cells and highly conserved
• Broad indicators of cellular toxicity
• Triggered at low doses before more apical effects
such as cell death or apoptosis
• A few (<10) key cellular stress pathways identified
• Pathways well-characterized and classified
mechanistically mode of action info??
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Adaptive Stress Response Pathways
Oxidative stress
DNA damage
Heat shock
ER stress
Hypoxia/Anoxia
Inflammation
Heavy metal stress
Osmotic stress*
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NHEERL, Neurotoxicology Division, Cellular and Molecular Toxicology Branch
Well-defined Mechanisms :
MOA information?
Oxidative Stress
Genotoxic Stress
Heat Shock
ER Stress
Hypoxia
Inflammation
Metal Response
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Stress Pathway Architecture
Stress
Transducers
Sensor
TF
Target Genes
Nucleus
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NHEERL, Neurotoxicology Division, Cellular and Molecular Toxicology Branch
Example:
Nrf2-mediated Oxidative Stress Response
Oxidative
Stress
PKC
ERK2
JNK1
p38
Keap1
Nrf2
Hmox1
NQO1
GSTA2
Nucleus
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Biological Centrality of Stress Responses:
Pathway
inducer
Sensor
Tr. Fact.
Sens. K/O
TF K/O
Dbl. K/O
Ox stress
Oxygen
radicals
Keap-1
Nrf-2
Emb. leth
Viable*
viable
Genotoxic
DNA
damage
Mdm2
p53
Emb. leth
Viable*
viable
Hypoxia
Oxygen
deprivation
VHL
HIF-1
Emb. leth
Emb. leth
???
Heat
shock
Protein
denatn.
Hsp-90
HSF-1
HSF-3
Emb. leth
Viable*
???
Inflammat.
TNF, LPS
IkB
NFkB
Emb. leth
Viable*
???
Metals
Heavy
metals
(none)
Zn activat.
MTF-1
NA
Emb. leth
???
♀ infertile
* Disease susceptibility and normal functions compromised
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Stress-Response Pathways
1. Stress pathways share a common pattern of
organization
2. Stimulation of a stress pathway results in activated
transcription factor
3. The transcription factor serve as a nodal point for
multiple “toxicity pathways”
4. Activated transcription factor up-regulates unique
target genes
Regulatory elements of target genes can be used to
measure pathway activation!
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Genomic vs. Synthetic Promoters
sensitivity vs. specificity
AP-1 Sp1
-12kb
CEBPa Nrf2 CREB
NF-kB Maf
AP-1
Heme Oxygenase-1
Maf
MTF-1 Nrf2
Sp1 NF-kB
AP-2
AP-1 MTF-1 +1
CREB
Distal Enhancer 2
Distal Enhancer 1
Nrf2
Nrf2
Nrf2
ARE
ARE
ARE
Sp1
Proximal Enhancer
Proximal Promoter
Basal Promoter
Nrf2 Synthetic
Multimerized Response Elements
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Constructing Stress-Responsive
Reporter Genes
Stress Promoters
Reporter ORF
Stress Pathway
Genotoxic stress
Oxidative stress
Heat shock (protein unfolding)
Endoplasmic reticulum stress
Inflammatory response
Hypoxia
Heavy metals exposure
GeneReporters
Promoters
p21, GADD45A,
Firefly
Luciferasep53-RE
Hmox-1,
Nrf2-RE, NQO1
Renilla
Luciferase
hsp70, HSF1-RE
Secreted
Metridia Luciferase
Grp78, XBP1-RE
Fluorescent
Proteins
IL-8, NFkB-RE
Secreted
Alkaline Phosphatase
Grp94, HIF1-RE, Hi95
Beta-Glucoronidase
MT-1, MT-2A, MTF1-RE
Beta-Lactamase
Beta-Galactosidase
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Why Reporter Genes?
• Why not measure stress protein levels?
 Stability- rapid turnover
 Throughput
• Why not measure transcripts by microarray or qPCR?
 Cost
 Throughput
 Remember… we need to assay 1000s of chemicals and
establish dose-response information
 Reporter genes meet all of the HTS requisites at the right
price
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What Makes a Good HTS Assay?
• Miniaturization: 96-, 384-, 1536-well formats
 minimizes compound requirements and waste
 lowers screening cost for consumables
 dose-response and time course data
• Assay Performance
 Signal-to-background
 Coefficient of variance
 ↑ Signal/Background, ↓ CV = ↑ Z’ score
• Reagent availability, cost, compatibility w/ library
• Relevance
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Typical Assay Conditions
• Stable cells seeded overnight in multi-well assay plates
• Cell treated with compounds for pre-determined time
• Luciferase activity normalized to GFP viability per well
HEK293T-GFP cells
8
log fold change over controls
6
4
5' LTR
SV40
ARE-Nrf2
AP-1
hsp70
MT-2A
2
0
-2
-4
-6
-8
0.1
0.3
1
3
10
30
100
300
1000
[CdCl2] uM
Inactivation
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Activation
Stress Signatures
Compound #1
30
30
25
25
Nrf2
hsp70
p53
VEGF
Grp78
NFkB
MT2
15
10
5
20
Activity
20
Activity
Compound #2
Nrf2
hsp70
p53
VEGF
Grp78
NFkB
MT2
15
10
5
0
0
-8
-6
-4
Compound log M
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-8
-3
Compound log M
19
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20
Throwing Off Pharmaco-philosophy
• Disparity between the mandates of pharma industry and
regulatory toxicology
 Acceptance of false positive/negatives
 Known targets vs. unknown mechanisms
 $$$
• Much of the efforts to-date to implement HTS tox testing
has adopted pharma tools and pharma thinking
 Blunt tests for cytotoxicity vs. sensitive assays for
“drug-able” targets
 Single dose testing (usually determined by solubility)
 Overly-conservative criteria for calling “hits”
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NHEERL, Neurotoxicology Division, Cellular and Molecular Toxicology Branch
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Throwing Off Pharmaco-philosophy
• What is needed:
 Reduced reliance on loss-of-signal assays
 Better understanding of mechanisms/pathways
 Sensitive assays to measure mechanistic endpoints
 Chemical libraries at higher concentrations
 Dose-response information
 Establish criteria for determining “actives”
 $$$
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NHEERL, Neurotoxicology Division, Cellular and Molecular Toxicology Branch
22
Chemical Library Construction
• Chemicals are assembled in groups of several hundreds
• Diverse chemical properties including solubility
• Pharma libraries use constrained property ranges
 Molecular weight
 log P
• DMSO is currently the solvent of choice
• Compound with lowest solubility determines solubility limit
for the entire library
• This increases chances for false negatives
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23
5
1nM-100uM
activity
4
3
signal
viability
2
1
True Negative or
NOEL?
0
5
4
3
2
1
0
1nM-100mM
signal
viability
activity
activity
-9 -8 -7 -6 -5 -4 -3 -2 -1
Compound log M
-9 -8 -7 -6 -5 -4 -3 -2 -1
Compound log M
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NHEERL, Neurotoxicology Division, Cellular and Molecular Toxicology Branch
5
4
3
2
1
0
1nM-100mM
signal
viability
-9 -8 -7 -6 -5 -4 -3 -2 -1
Compound log M
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Abandoning Single-dose Screening
• Quantitative HTS (qHTS) employs chemical libraries in
1536-well plate format
• Each library constrained to a single 1536-well plate; library
is titrated across multiple plates
1
A
B
C
D
E
F
G
H
I
J
K
L
M
N
O
P
Q
R
S
T
U
V
W
X
Y
Z
AA
AB
AC
AD
AE
AF
2
3
4
5
6
7
8
9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48
92uM
Titrated positive control
Fixed positive control
Vehicle control
Additional positive control
Compound Area
1.2nM
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25
Using HTS Assays in Mechanistic Research
EC50
Caspase 3/7 assay
(Gain of signal)
IC50
Cell TiterGlo (ATP)
(Loss of signal)
Huang et al. Chem. Res. Toxicol. 2008, 21, 659–667
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NTP 1408 Chemicals
Hierarchal Clustering
23 Clusters
Estrogenics
Antineoplastics
Hormone Antagonists
Cytostatics
Huang et al. Chem. Res. Toxicol. 2008, 21, 659–667
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“While the goal of the clustering is to generate a
hypothesis about a compound’s specific mechanism
of action, the broad nature of these cytotoxicity assays
likely prevents any detailed understanding of the
molecular basis of the toxic effect; the inclusion of or
confirmation of activity in other, more mechanistic
assays would obviously improve this aspect of the
current study.”
Stress pathway assays move us a step in this direction
Huang et al. Chem. Res. Toxicol. 2008, 21, 659–667
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NHEERL, Neurotoxicology Division, Cellular and Molecular Toxicology Branch
28
Progress to Date
• Engineered 20+ assays covering most of the key stress
pathways; filling in gaps with new assays
• Miniaturizing assay to 1536-well format
• Screened two assays (Nrf2 and hsp70) with two
libraries: NTP and EPA; preliminary re-clustering
• Screening additional assays in phased manner
• New chemical libraries
• NTP-B 1408: Summer 2009
• EPA-B 1408: Summer 2009; EPA-C 1408: Under consideration
• Adding primary cells models: human hepatocytes,
rodent renal proximal tubule cells, etc.
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Beyond In Vitro
control
0.2uM
5uM
Blechinger SR, Warren JT Jr, Kuwada JY, Krone PH.
Developmental toxicology of cadmium in living embryos of a
stable transgenic zebrafish line.
Environ Health Perspect. 2002 Oct;110(10):1041-6.
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125uM
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In Vitro
Alt. Species
QSAR
Screening and Prioritization
Enhanced Predictivity
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NHEERL, Neurotoxicology Division, Cellular and Molecular Toxicology Branch
31
Summary and Conclusions
• Stress pathway assay ensemble to generate “stress
signatures”
• Clustering by biological response in in vitro assays:
structural similarities???
• Can this type of information improve QSAR models?
• Current HTS assays measure typically blunt responses:
mechanisms will need further delineation
• As we move forward with HTS testing, we need to move
from the pharma approach to maximize information
gains that will useful for toxicology
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NHEERL, Neurotoxicology Division, Cellular and Molecular Toxicology Branch
Acknowledgements
US EPA Neurotoxicology Division
Ram Ramabhadran
Chun-Yang Fan
Jeanene Olin
Theresa Freudenrich
Helen Carlsen
NIH Chemical Genomic Center
Chris Austin
Jim Inglese
Menghang Xia
Ruili Huang
Sunita Shukla
The Hamner Institutes
Rusty Thomas
Open Biosystems (Thermo-Fisher)
John Wakefield
Attila Seyhan
US EPA, National Center for
Computational Toxicology
Keith Houck
David Dix
Office of Research and Development
NHEERL, Neurotoxicology Division, Cellular and Molecular Toxicology Branch