Abstract
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 (>100 M) 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.
| Original language | English (US) |
|---|---|
| Pages (from-to) | 36-46 |
| Number of pages | 11 |
| Journal | Current Chemical Genomics and Translational Medicine |
| Volume | 8 |
| Issue number | 1 |
| DOIs | |
| State | Published - Jan 1 2014 |
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Keywords
- Endocrine disruption
- Pituitary cells
- Quantitative high-throughput screening
- Reporter gene assay
- Retinoid-X receptor
- Thyroid hormone receptor
ASJC Scopus subject areas
- Biochemistry
- Molecular Medicine
- Molecular Biology
- Genetics
Cite this
Identification of thyroid hormone receptor active compounds using a quantitative high-throughput screening platform. / Freitas, Jaime; Miller, Nicole; Mengeling, Brenda J.; Xia, Menghang; Huang, Ruili; Houck, Keith; Rietjens, Ivonne M.C.M.; Furlow, John; Murk, Albertinka J.
In: Current Chemical Genomics and Translational Medicine, Vol. 8, No. 1, 01.01.2014, p. 36-46.Research output: Contribution to journal › Article
}
TY - JOUR
T1 - Identification of thyroid hormone receptor active compounds using a quantitative high-throughput screening platform
AU - Freitas, Jaime
AU - Miller, Nicole
AU - Mengeling, Brenda J.
AU - Xia, Menghang
AU - Huang, Ruili
AU - Houck, Keith
AU - Rietjens, Ivonne M.C.M.
AU - Furlow, John
AU - Murk, Albertinka J.
PY - 2014/1/1
Y1 - 2014/1/1
N2 - 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 (>100 M) 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.
AB - 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 (>100 M) 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.
KW - Endocrine disruption
KW - Pituitary cells
KW - Quantitative high-throughput screening
KW - Reporter gene assay
KW - Retinoid-X receptor
KW - Thyroid hormone receptor
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UR - http://www.scopus.com/inward/citedby.url?scp=84989270930&partnerID=8YFLogxK
U2 - 10.2174/2213988501408010036
DO - 10.2174/2213988501408010036
M3 - Article
C2 - 24772387
AN - SCOPUS:84989270930
VL - 8
SP - 36
EP - 46
JO - Current Chemical Genomics and Translational Medicine
JF - Current Chemical Genomics and Translational Medicine
SN - 2213-9885
IS - 1
ER -