toxicological knowledge base (a definition) response dose “ an in computero aggregated set of the...
TRANSCRIPT
Toxicological Knowledge Base (a Definition)
Res
pon
se
Dose
“An in computero aggregated set of the most germane literature citations and biological activity datasets, together with computational models which correlate those activities with chemical structure, and, ultimately, models for risk assessment.”
Activity
Structure
Correlation
A circular process provides training sets DESIGNED to produce robust, living predictive models
EDKB Data validationand selection
Data validationand selection
Diversity analysisTraining set design
Diversity analysisTraining set design
New Data Needs &Research Hypothesis
New Data Needs &Research Hypothesis
NCTR/EPAExperiments
NCTR/EPAExperiments
Data in scientific
papers
Data in scientific
papersDevelopModels
DevelopModels
Validation:1. Cross-validation2. Testset challenge
Validation:1. Cross-validation2. Testset challenge
EDKB OBJECTIVES• Develop and validate predictive
computational models for ER and AR by:– developing appropriate data sets to train
models– examining a wide variety of models for
performance and ease of use– validating models by:
• internal validation• external validation from non-randomly selected
chemicals• external validation from randomly selected
chemicals from the 58,000 chemical EPSD
Dataset Criteria for Development of SAR models
• Develop training data set by design for– structurally diverse chemical
structures – a wide range of binding values (RBAs)
Status of ER Models
• 238 chemicals assayed (duplicate tubes; two replicates) over a six log RBA range
• Techniques developed for identification of potential binders by EDKB team
• Filters (Phase I) and 11 categorical SAR models (Phase II) finished
• Models validated against literature datasets
Status of ER Models (2)
• Models biased toward false positives to minimize false negatives
• RBA predictions made for all 58,000 chemicals “in play”
• Handout of ER model publications• ~ First two years-used literature RBAs• Subsequently used NCTR dataset
Androgen Receptor Competitive Binding AssayTwo assays evaluated:
– Ventral prostate from castrated adults•Could not obtain reproducible results
– PanVera androgen binding domain•Good saturation and competitive
binding results•Assay subsequently validated for use•Less expensive than ventral prostate•Uses no animals
Validation of PanVera Assay• PanVera protein was diluted to several
low concentrations and saturation assays were conducted with [3H]-R1881.
• Protein concentrations selected were in the range of the reported KD.
• A protein concentration in the stable KD range was used for subsequent competitive binding assays.
AR Competitive Binding Results for Steroidal Androgens
10-11
10-10
10-9
10-8
10-7
10-6
10-5
0
20
40
60
80
100
120
Androgens (set 1) R-1881 5-Androstane-3,11,17-trione 4-Androstenedione 5-Androstane-3-17-diol 5-Androstenediol 5-Androstane-3-17-diol 5-Androstan-17-ol 11-Ketotestosterone 5-Androstane 5-Dihydrotestosterone
% [3 H
]-R
-1881 B
ou
nd
Competitor Concentration (M)
AR Competitive Binding Results for Some Steroidal Estrogens
10-11
10-10
10-9
10-8
10-7
10-6
10-5
0
20
40
60
80
100
120
Steroidal Estrogens R-1881 2-Hydroxyestradiol 17-Estradiol 4-Hydroxyestradiol 17-Estradiol 3-Deoxyestradiol Estriol 3-Methylestriol Estrone Estra-1,3,5(10)-trien-3-ol
17-Estradiol,16-hydroxy-16-methyl-3-methyl ether
% [
3 H]-
R-1
881
Bo
un
d
Competitor Concentration (M)
AR Competitive Binding Results for Antiestrogens
10-11
10-10
10-9
10-8
10-7
10-6
10-5
0
20
40
60
80
100
120
Antiestrogens R-1881 Tamoxifen citrate Triphenylethylene 4-Hydroxytamoxifen ICI 164,384 Nafoxidine ICI 182,780 Clomiphene citrate
% [3 H
]-R
-1881 B
ou
nd
Competitor Concentration (M)
Status of AR AssayUp to May 9, 2001, 134 compounds were tested.To date,196 compounds have been tested
0
10
20
30
40
50
Chemical Category
Nu
mb
er o
f C
hem
ical
s
Activity Distribution for AR
0
5
10
15
20
25
30
35
40
>0 (0, -1) (-1, -2) (-2, -3) (-3, -4) SB NB
logRBA
Nu
mb
er o
f C
hem
ical
s
Comparison of NCTR Assay with EPA Assay
-3
-2
-1
0
1
2
3
-5 -4 -3 -2 -1 0 1 2 3
EPA logRBA
NC
TR
logR
BA
Progesterone
R2 = 0.83
Primary Pharmacophore Model
-3.5
-2.5
-1.5
-0.5
0.5
1.5
2.5
-3.5 -2.5 -1.5 -0.5 0.5 1.5 2.5
Actual logRBA
Cal
cula
ted
logR
BA
r2 = 0.96
CoMFA Model
De novo external validation of assays and models• EPA funded $850K contract with Batelle
Northwest for validation and comparison performance of NCTR and EPA assays and models
• Assays methods include:– uterine cytosol for ER (EPA and NCTR
assays are similar)– prostate assay for AR (EPA)– PanVera assay for AR (NCTR)
EDKB EXTERNAL FUNDING
• SOURCE AMOUNT ($)
• FDA OWH 1 185,000
• FDA OWH 2 325,000
• CMA CRADA 2 420,000
• EPA GRANT 1 425,000
• EPA IAG 3 2,000,000
• TOTAL 3,355,000
• 1 Experimental collaboration with U Missouri
• 2 Two separate awards
• 3 $500,000 for FY 2001; $1,500,000 for FY 2002-2005
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