Robust array-based coregulator binding assay predicting ERα-agonist potency and generating binding profiles...

Area of Science:

• Toxicology
• Biochemistry
• Chemical Safety Testing

Background:

• Endocrine-disrupting chemicals pose risks, necessitating reliable safety testing.
• In vitro alternatives to animal testing are crucial for evaluating estrogen receptor (ER) signaling interference.
• An ERα-coregulator peptide binding assay shows promise for replacing traditional uterotrophic assays.

Purpose of the Study:

• To determine the reproducibility and robustness of an in vitro ERα-coregulator binding assay.
• To assess the assay's ability to predict estrogenicity and classify ERα endocrine disruptors.
• To evaluate the correlation between binding affinity, potency, and structural characteristics of tested compounds.

Main Methods:

• Validated an in vitro assay profiling ligand-induced binding of ERα to coregulator peptides.
• Tested 14 model compounds recommended for laboratory proficiency testing.
• Assessed reproducibility using median coefficient of variation and correlation coefficients (R²).
• Compared ERα-coregulator binding potency with ER binding affinity and transactivation assay results.
• Utilized hierarchical clustering to analyze structural similarities among compounds.

Main Results:

• The ERα-coregulator binding assay demonstrated high reproducibility (median CV = 5.0%) and excellent correlation (R² = 0.993) in duplicate measurements.
• The assay correctly predicted estrogenicity for 13 out of 14 tested compounds.
• Excellent correlations were observed between ERα-coregulator binding potency, ER binding affinity (R² = 0.96), and transactivation potency (R² = 0.94).
• Hierarchical clustering successfully grouped structurally related compounds and distinguished between different steroid A-ring structures.

Conclusions:

• The ERα-coregulator binding assay is a reproducible and robust method for identifying ERα agonists and antagonists.
• The assay accurately predicts estrogenicity and reflects structural similarities of ERα agonists.
• This in vitro assay holds significant potential for high-fidelity identification and classification of ERα endocrine disruptors, serving as a valuable alternative to animal testing.