Identification of thyroid hormone receptor active compounds using a quantitative high-throughput screening platform.
To adapt the use of GH3.TRE-Luc reporter gene cell line for a quantitative high-throughput screening (qHTS) platform, we miniaturized the reporter gene assay to a 1536-well plate format. 1280 chemicals from the Library of Pharmacologically Active Compounds (LOPAC) and the National Toxicology Program (NTP) 1408 compound collection were analyzed to identify potential thyroid hormone receptor (TR) agonists and antagonists. Of the 2688 compounds tested, eight scored as potential TR agonists when the positive hit cut-off was defined at ≥10% efficacy, relative to maximal triiodothyronine (T3) induction, and with only one of those compounds reaching ≥20% efficacy. One common class of compounds positive in the agonist assays were retinoids such as all-trans retinoic acid, which are likely acting via the retinoid-X receptor, the heterodimer partner with the TR. Five potential TR antagonists were identified, including the antiallergy drug tranilast and the anxiolytic drug SB 205384 but also some cytotoxic compounds like 5-fluorouracil. None of the inactive compounds were structurally related to T3, nor had been reported elsewhere to be thyroid hormone disruptors, so false negatives were not detected. None of the low potency (>100µM) TR agonists resembled T3 or T4, thus these may not bind directly in the ligand-binding pocket of the receptor. For TR agonists, in the qHTS, a hit cut-off of ≥20% efficacy at 100 µM may avoid identification of positives with low or no physiological relevance. The miniaturized GH3.TRE-Luc assay offers a promising addition to the in vitro test battery for endocrine disruption, and given the low percentage of compounds testing positive, its high-throughput nature is an important advantage for future toxicological screening.
Division 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.
Jaime
J
Freitas
National Center for Advancing Translational Sciences, National Institutes of Health, Bethesda, MD, 20892, USA.
Nicole
N
Miller
Department of Neurobiology, Physiology, and Behavior, University of California, Davis 95616, USA.
Brenda J
BJ
Mengeling
National Center for Advancing Translational Sciences, National Institutes of Health, Bethesda, MD, 20892, USA.
Menghang
M
Xia
National Center for Advancing Translational Sciences, National Institutes of Health, Bethesda, MD, 20892, USA.
Ruili
R
Huang
National Center for Computational Toxicology, Office of Research and Development, U.S. Environmental Protection Agency, Research Triangle Park, North Carolina 27711, USA.
Keith
K
Houck
Division of Toxicology, Wageningen University, Tuinlaan 5, 6703 HE Wageningen, The Netherlands.
Ivonne M C M
IM
Rietjens
Department of Neurobiology, Physiology, and Behavior, University of California, Davis 95616, USA.
J David
JD
Furlow
Division of Toxicology, Wageningen University, Tuinlaan 5, 6703 HE Wageningen, The Netherlands ; Subdepartment of Environmental Technology, Wageningen University, and Wageningen-IMARES, Axis Z (Building number 118), Room TT.1.100, Bornse Weilanden 96708, WG Wageningen, The Netherlands.
Albertinka J
AJ
Murk
Endocrine disruption; pituitary cells; quantitative high-throughput screening; reporter gene assay; retinoid-X receptor.; thyroid hormone receptor