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Screening the 10K Tox21 chemical library for thyroid hormone receptor modulators
Houck, K., A. Bone, K. Paul-Friedman, C. Hsu, S. Sakamuru, M. Xia, R. Huang, V. Soni, D. Stavreva, AND K. Crofton. Screening the 10K Tox21 chemical library for thyroid hormone receptor modulators. Society of Toxicology, San Antonio, TX, March 11 - 15, 2018.
We evaluated the hypothesis that TR is highly selective, and that modulation of TR activity is not likely to be a key mechanism of action for thyroid hormone disruption by environmentally-relevant chemicals. Poster presentation at Society of Toxicology meeting March 2018
Few ligands for the thyroid hormone receptor (TR) have been identified outside of endogenous ligands and pharmaceuticals, which suggests that TR is a very selective nuclear receptor (NR). However, large and diverse chemical libraries, particularly of environmental chemicals, have not been tested for TR activity. We evaluated the hypothesis that TR is highly selective, and that modulation of TR activity is not likely to be a key mechanism of action for thyroid hormone disruption by environmentally-relevant chemicals. A TR luciferase reporter gene assay in the rat pituitary-derived GH3 cell line was used in a screen against the 10K Tox21 chemical library using a quantitative high-throughput screening approach in both agonist and antagonist format. In the agonist mode, a total of 28 active compounds were identified with potencies ranging from 0.01-91 uM. To confirm these compounds as TR agonists, a series of additional assays were performed: (1) the GH3 cell line; (2) a second TR reporter gene assay--human TR-beta in a mammalian one-hybrid format; (3) a mammalian one-hybrid assay for the retinoid X receptor (RXR-alpha), a potential contributor to activation through a permissive heterodimer effect; and, (4) a TR:coactivator recruitment assay using the human TR-beta ligand-binding domain and SRC-2 peptide. Results showed all 28 compounds repeated as actives in the GH3 assay, with 22 direct TR ligands and 6 via RXR activity. The antagonist assay mode identified 2352 actives of which 1488 had an AC50 for cytotoxicity 3-fold or less then the corresponding reporter gene AC50 and were considered false positives. We confirmed activity in the 2 reporter gene assays; however, the coactivator recruitment assay failed in antagonist mode. As an alternative, we tested 285 available putative antagonists in a NR translocation assay using GFP-tagged human TR, shown to be responsive to a known TR antagonist, 1-850. Of these, 43 exhibited significantly increased NR translocation activity at the highest concentration tested, 100 uM, but only 17 demonstrated activity at lower concentrations. Fourteen of these were pharmaceutical compounds not previously shown to have TR activity. Overall, this work supports the hypothesis that thyroid hormone disruption via direct TR perturbation by environmental chemicals is a rare event, and that other modes of action are likely of higher importance for screening this chemical space. This abstract does not necessarily represent the views of the US EPA.
URLs/Downloads:SOT_TRQHTS_2018_CLEARANCE.PDF (PDF,NA pp, 995.046 KB, about PDF)
SOT_2018_TR_HOUCK_V2.PDF (PDF,NA pp, 56.621 KB, about PDF)
Record Details:Record Type: DOCUMENT (PRESENTATION/POSTER)
Organization:U.S. ENVIRONMENTAL PROTECTION AGENCY
OFFICE OF RESEARCH AND DEVELOPMENT
NATIONAL CENTER FOR COMPUTATIONAL TOXICOLOGY