Tox21

Tox21 screens a chemical library to identify modulators of thyroid hormone receptors (TRs) and assess potential thyroid hormone disruption relevant to neurodevelopmental and reproductive toxicities.

Key Features:

• High-Throughput Screening: Screening of the Tox21 chemical collection comprising 8,305 unique structures to identify compounds that modulate TR activity as agonists or antagonists.
• Quantitative Reporter Gene Assay: Use of a quantitative high-throughput cell-based reporter gene assay to detect TR agonist and antagonist activities.
• Orthogonal Assays for Characterization: Active compounds are further characterized using mammalian one-hybrid assays, coactivator recruitment assays, and a high-throughput fluorescent imaging nuclear receptor translocation assay.
• Identification of Agonists and Indirect Activators: Recovery of known agonist reference chemicals and identification of a novel direct agonist, betamipron, with indirect TR activation detected via RXR agonist assays implicating the retinoid-X-receptor (RXR) heterodimer partner.
• Challenges in Antagonist Identification: Antagonist identification was confounded by cytotoxicity and non-TR-specific mechanisms in transactivation assays, although mefenamic acid, diclazuril, and risarestat were confirmed as antagonists.

Scientific Applications:

• Assessment of Structural Diversity: Evaluation of the structural diversity of environmentally relevant chemicals that can directly bind or modulate TRs.
• Prioritization of Thyroid Axis Targets: Evidence of limited structural diversity for direct TR ligands supports prioritizing other potential target sites within the thyroid hormone axis for bioactivity screening to identify thyroid axis disruptors.

Methodology:

An integrated approach combining quantitative high-throughput screening of the Tox21 chemical collection with multiple orthogonal assays (mammalian one-hybrid, coactivator recruitment, and nuclear receptor translocation assays) was used for characterization and validation of TR modulators.