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Identification of thyroid hormone receptor active compounds using a quantitative high-throughput screening platform.

Jaime Freitas1, Nicole Miller2, Brenda J Mengeling3

  • 1Division of Toxicology, Wageningen University, Tuinlaan 5, 6703 HE Wageningen, The Netherlands ; Group of Cell Activation and Gene Expression, Institute for Molecular and Cellular Biology, University of Porto, Porto, Portugal.

Current Chemical Genomics and Translational Medicine
|April 29, 2014
PubMed
Summary

A miniaturized reporter gene assay was developed for high-throughput screening of thyroid hormone receptor (TR) agonists and antagonists. This assay identified potential TR modulators among tested chemical libraries, aiding endocrine disruption research.

Keywords:
Endocrine disruptionpituitary cellsquantitative high-throughput screeningreporter gene assayretinoid-X receptor.thyroid hormone receptor

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Area of Science:

  • Endocrinology
  • Toxicology
  • Molecular Biology

Background:

  • Thyroid hormone receptors (TRs) are crucial for development and metabolism.
  • Endocrine-disrupting chemicals pose risks to human health.
  • High-throughput screening (HTS) methods are needed to identify TR modulators.

Purpose of the Study:

  • To adapt the GH3.TRE-Luc reporter gene assay for quantitative high-throughput screening (qHTS).
  • To identify potential TR agonists and antagonists using a miniaturized 1536-well plate format.
  • To evaluate the utility of this assay for toxicological screening.

Main Methods:

  • Miniaturization of the GH3.TRE-Luc assay to a 1536-well plate format.
  • Screening of 2688 compounds from LOPAC and NTP collections.
  • Analysis of compounds for TR agonist and antagonist activity relative to triiodothyronine (T3).

Main Results:

  • Eight potential TR agonists were identified, with one showing ≥20% efficacy.
  • Retinoids, such as all-trans retinoic acid, were among the identified TR agonists.
  • Five potential TR antagonists were identified, including tranilast and SB 205384.
  • Cytotoxic compounds like 5-fluorouracil were also flagged as potential antagonists.
  • No structural similarity to T3/T4 was observed in low-potency agonists, suggesting indirect mechanisms.

Conclusions:

  • The miniaturized GH3.TRE-Luc assay is suitable for qHTS of TR modulators.
  • The assay effectively identified potential endocrine disruptors.
  • A hit cut-off of ≥20% efficacy at 100 µM is recommended for TR agonists to ensure physiological relevance.
  • This assay is a valuable addition to in vitro testing batteries for endocrine disruption screening.