Toxicological Knowledge Base (a Definition) Response 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 validation and selection Data validation and selection Diversity analysis Training set design Diversity analysis Training set design New Data Needs & Research Hypothesis New Data Needs & Research Hypothesis NCTR/EPA Experiments NCTR/EPA Experiments Data in scientific papers Data in scientific papers Develop Models Develop Models Validation: 1. Cross-validation 2. Testset challenge Validation: 1. Cross-validation 2. 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 Assay Two 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 [ 3 H]-R1881. Protein concentrations selected were in the range of the reported K D. A protein concentration in the stable K D range was used for subsequent competitive binding assays.

AR Competitive Binding Results for Steroidal Androgens

AR Competitive Binding Results for Some Steroidal Estrogens

AR Competitive Binding Results for Antiestrogens

Status of AR Assay Up to May 9, 2001, 134 compounds were tested. To date,196 compounds have been tested Number of Chemicals

Activity Distribution for AR Number of Chemicals

Comparison of NCTR Assay with EPA Assay

Primary Pharmacophore Model

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 TOTAL3,355,000 1 Experimental collaboration with U Missouri 2 Two separate awards 3 $500,000 for FY 2001; $1,500,000 for FY

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