Oak Ridge Institute for Science and Education, Oak Ridge, Tennessee 37830, United States.
Center for Computational Toxicology and Exposure, Great Lakes Toxicology and Ecology Division, U.S. Environmental Protection Agency, Office of Research and Development, Duluth, Minnesota 55804, United States.
Chem Res Toxicol. 2024 Oct 21;37(10):1670-1681. doi: 10.1021/acs.chemrestox.4c00215. Epub 2024 Sep 11.
Transthyretin (TTR) is one of the serum binding proteins responsible for transport of thyroid hormones (TH) to target tissue and for maintaining the balance of available TH. Chemical binding to TTR and subsequent displacement of TH has been identified as an end point in screening chemicals for potential disruption of the thyroid system. To address the lack of data regarding chemicals binding to TTR, we optimized an assay utilizing the fluorescent probe 8-anilino-1-napthalenesulfonic acid (ANSA) and the human protein TTR to screen over 1500 chemicals from the U.S. EPA's ToxCast ph1_v2, ph2, and e1k libraries utilizing a tiered approach. Testing of a single high concentration (target 100 μM) resulted in 888 chemicals with 20% or greater activity based on displacement of ANSA from TTR. Of these, 282 chemicals had activity of 85% or greater and were further tested in 12-point concentration-response with target concentrations ranging from 0.015 to 100 μM. An EC50 was obtained for 276 of these 301 chemicals. To date, this is the largest set of chemicals screened for binding to TTR. Utilization of this assay is a significant contribution toward expanding the suite of assays used to identify chemicals with the potential to disrupt thyroid hormone homeostasis.
转甲状腺素（TTR）是一种血清结合蛋白，负责将甲状腺激素（TH）转运至靶组织，并维持可用 TH 的平衡。化学结合 TTR 并随后置换 TH 已被确定为筛选潜在扰乱甲状腺系统的化学物质的终点。为了解决有关化学物质与 TTR 结合的数据缺乏的问题，我们优化了一种利用荧光探针 8-苯胺-1-萘磺酸（ANSA）和人蛋白 TTR 的测定方法，利用分层方法筛选了来自美国环保署 ToxCast ph1_v2、ph2 和 e1k 文库的 1500 多种化学物质。对单一高浓度（目标 100 μM）的测试导致 888 种化学物质具有 20%或更高的活性，基于 ANSA 从 TTR 中的置换。其中，282 种化学物质的活性为 85%或更高，并在 0.015 至 100 μM 的目标浓度范围内进行了 12 点浓度反应测试。其中 276 种化学物质获得了 EC50。迄今为止，这是筛选与 TTR 结合的最大一组化学物质。利用这种测定方法是朝着扩大用于识别具有潜在扰乱甲状腺激素动态平衡能力的化学物质的测定方法套件迈出的重要一步。
