Identification of thyroid hormone receptor active compounds using a quantitative high-throughput screening platform.
Identification of thyroid hormone receptor active compounds using a quantitative high-throughput screening platform.
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DOI：
10.2174/2213988501408010036
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发表时间：
2014
期刊：
影响因子：
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通讯作者：
Murk AJ
中科院分区：
文献类型：
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作者：
Freitas J;Miller N;Mengeling BJ;Xia M;Huang R;Houck K;Rietjens IM;Furlow JD;Murk AJ
为了使 GH3.TRE-Luc 报告基因细胞系适用于定量高通量筛选 (qHTS) 平台，我们将报告基因测定小型化为 1536 孔板格式。对药理活性化合物库 (LOPAC) 和国家毒理学计划 (NTP) 1408 种化合物库中的 1280 种化学物质进行了分析，以确定潜在的甲状腺激素受体 (TR) 激动剂和拮抗剂。在测试的 2688 种化合物中，当相对于最大三碘甲状腺原氨酸 (T3) 诱导，阳性命中截止值被定义为 ≥10% 功效时，8 种被评为潜在 TR 激动剂，并且其中只有一种化合物达到 ≥20% 功效。激动剂检测中呈阳性的一类常见化合物是类视黄醇，例如全反式视黄酸，它们可能通过类视黄醇-X 受体（TR 的异二聚体伴侣）发挥作用。确定了五种潜在的 TR 拮抗剂，包括抗过敏药物曲尼司特和抗焦虑药物 SB 205384，以及一些细胞毒性化合物，如 5-氟尿嘧啶。没有一种非活性化合物在结构上与 T3 相关，也没有其他地方报道为甲状腺激素干扰物，因此没有检测到假阴性。低效 (>100μM) TR 激动剂均不类似于 T3 或 T4，因此它们可能不会直接结合在受体的配体结合袋中。对于 TR 激动剂，在 qHTS 中，100 µM 效力≥20% 的命中截止值可能会避免识别出生理相关性较低或没有的阳性结果。小型化 GH3.TRE-Luc 检测为内分泌干扰的体外测试电池提供了一个有希望的补充，并且考虑到测试呈阳性的化合物百分比较低，其高通量性质是未来毒理学筛选的重要优势。
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.