Limited Chemical Structural Diversity Found to Modulate Thyroid Hormone Receptor in the Tox21 Chemical Library

Abstract

Background:Thyroid hormone receptors (TRs) are critical endocrine receptors that regulate a multitude of processes in adult and developing organisms, and thyroid hormone disruption is of high concern for neurodevelopmental and reproductive toxicities in particular. To date, only a small number of chemical classes have been identified as possible TR modulators, and the receptors appear highly selective with respect to the ligand structural diversity. Thus, the question of whether TRs are an important screening target for protection of human and wildlife health remains.Objective:Our goal was to evaluate the hypothesis that there is limited structural diversity among environmentally relevant chemicals capable of modulating TR activity via the collaborative interagency Tox21 project.Methods:We screened the Tox21 chemical library (8,305 unique structures) in a quantitative high-throughput, cell-based reporter gene assay for TR agonist or antagonist activity. Active compounds were further characterized using additional orthogonal assays, including mammalian one-hybrid assays, coactivator recruitment assays, and a high-throughput, fluorescent imaging, nuclear receptor translocation assay.Results:Known agonist reference chemicals were readily identified in the TR transactivation assay, but only a single novel, direct agonist was found, the pharmaceutical betamipron. Indirect activation of TR through activation of its heterodimer partner, the retinoid-X-receptor (RXR), was also readily detected by confirmation in an RXR agonist assay. Identifying antagonists with high confidence was a challenge with the presence of significant confounding cytotoxicity and other, non-TR-specific mechanisms common to the transactivation assays. Only three pharmaceuticals—mefenamic acid, diclazuril, and risarestat—were confirmed as antagonists.Discussion:The results support limited structural diversity for direct ligand effects on TR and imply that other potential target sites in the thyroid hormone axis should be a greater priority for bioactivity screening for thyroid axis disruptors. https://doi.org/10.1289/EHP5314

In vitro assessment of the capacity of pesticides to act as agonists/antagonists of the thyroid hormone nuclear receptors

“…TRα1 agonist or antagonist. This reinforces the conclusion of the Tox21 screen, performed on 8305 compounds, according to which many environmental chemicals do not act as TRα1 ligand at non-toxic concentrations, either as agonists or antagonists(Paul-Friedman et al 2019). This contrasts with other nuclear receptors, for which a number of environmental ligands have been identified(Toporova et al 2020, Liu et al 2019.…”

“…However the presence of halogens in the chemical structure (fluoride, iodine, chloride, or bromide) is not an indication for a possible binding to TRs, as halogens are frequent is pesticides, and absent from the NH-3 synthetic TRα1 ligand. This study also illustrates the importance of combining several in vitro assays to draw firm conclusions on the mode of action of THD, as previously recommended by others(Paul-Friedman et al 2019), as single endpoints screens produce a high rate of false positives.Our data illustrate the benefit provided by transcriptome analysis for in vitro toxicology: first it is unbiased, and able to capture any unexpected alteration in the cellular physiology. Second, if performed on relevant cellular models, it helps to prioritize some chemicals for in vivo assessment, and limit the use of animals, as recommended by ethical guidelines.…”

Molecular Image-Based Prediction Models of Nuclear Receptor Agonists and Antagonists Using the DeepSnap-Deep Learning Approach with the Tox21 10K Library

“…There are several conflicting reports regarding the in vitro receptor-mediated activity. Chemicals such as bisphenol A (BSA) and its halogenated analogs (tetrabromo-BSA and tetrachloro-BSA) show weak TR antagonist activity but have a potential agonist-like effect at lower concentrations [60,61]. Thus, competitive agonists and antagonists of the steroids have long been known [62][63][64].…”

Single-Cell Distribution Analysis of AR Levels by High-Throughput Microscopy in Cell Models: Application for Testing Endocrine-Disrupting Chemicals

“…Recently, computational frameworks obtained from the ToxCast datasets have been built for all three receptors and the in vitro to in vivo data revealed, at least for ER, that a set of in vitro assays can serve as a substitute for more expensive and time-taking in vivo assays. 2,4,20 Perhaps one of the drawbacks of most current efforts is that the HT assays are largely out of context, meaning they are performed in test tubes (i.e., ligand binding assays) or using engineered cell models (i.e., luciferase assays or GFP-tagged proteins) that, inarguably, do not fully recapitulate the inherent complexity of endogenous systems. One such complexity, often ignored, is the intrinsic cell-to-cell variation in genetically homogeneous populations (phenotypic heterogeneity), a metric that is rarely used in HT assays [17][18][19] and, to our knowledge, has never been used to assess EDC effects on nuclear receptors.…”

“…One of the largest efforts to date has been the U.S. Environmental Protection Agency's (EPA) Endocrine Disruptor Screening Program (EDSP), which aimed to combine HTS with computational analyses to characterize chemicals that interfere with endocrine hormones, focusing on three nuclear receptors pathways: estrogen (ER), androgen (AR), and thyroid (TR) receptors. 1 Through the extensive efforts of the Tox21/ToxCast initiatives, via testing hundreds of chemicals in more than a hundred high-throughput (HT) assays, the National Institute of Environmental Health Sciences (NIEHS) and EPA curated sets of reference chemicals for the ER, AR, and TR pathways [2][3][4] that are relevant to in vivo animal studies, which have included the development of computational models for quantifying activity on ER and AR. 3,5,6 Many studies, including several from our group, 7-11 have described the development of HT high-content assays (HCAs) using the AR as a model system (reviewed in Campana et al 12 ).…”