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Identification of Estrogen-Related Receptor Alpha Agonists in the Tox21 Compound Library

Caitlin Lynch, Jinghua Zhao, Ruili Huang, Noriko Kanaya, Lauren Bernal, Jui-Hua Hsieh, Scott S Auerbach, Kristine L Witt, B Alex Merrick, Shiuan Chen, Christina T Teng, and Menghang Xia.
Endocrinology (2018). DOI: PMID: 29216352



The estrogen-related receptor alpha (ERRα) is an orphan nuclear receptor (NR) that plays a role in energy homeostasis and controls mitochondrial oxidative respiration. Increased expression of ERRα in certain ovarian, breast, and colon cancers has a negative prognosis, indicating an important role for ERRα in cancer progression. An interaction between ERRα and peroxisome proliferator-activated receptor-γ coactivator-1α (PGC-1α) has also recently been shown to regulate an enzyme in the β-oxidation of free fatty acids, thereby suggesting that ERRα plays an important role in obesity and type 2 diabetes. Therefore, it would be prudent to identify compounds that can act as activators of ERRα. In this study, we screened ∼10,000 (8,311 unique) compounds, known as the Tox21 10K collection, to identify agonists of ERRα. We performed this screen using two stably transfected HEK 293 cell lines, one with the ERRα-reporter alone and the other with both ERRα-reporter and PGC-1α expression vectors. After the primary screening, we identified more than five agonist clusters based on compound structural similarity analysis (e.g., statins). By examining the activities of the confirmed ERRα modulators in other Tox21 NR assays, eliminating those with promiscuous NR activity, and performing follow-up assays (e.g., siRNA knockdown) we identified compounds that might act as endocrine disrupters through effects on ERRα signaling. This study is the first comprehensive analysis in discovering potential endocrine disrupters which affect the ERRα signaling pathway.


Figure 1. Structural class heat map of ERR agonist activity.

Both the ERR and PGC/ERR cell lines were screened and divided into clusters. Each hexagon represents a class of structurally similar compounds. The color gradient is indicative of the enrichment of ERR actives in that specific cluster [negative logarithmic scale of the P value, −log (P value)]. Each color represents a group of chemicals with similar scaffolds to activate ERR. Clusters with multiple actives in their class are closer to a maroon color, whereas clusters with no activity are a light gray color. Empty clusters with no available (N/A) compounds in them are darker gray in color.

Figure 2. Concentration curves of the statin structural class.

The 11-point dilution of the (a) ERR and (b) PGC/ERR follow-up studies is represented in a concentration curve for four of the statins from structural classes 15.23 and 40.23. The stable cell lines were treated with each respective compound for 18 hours in 1536-well plates. ONE-Glo was then added and the luminescence intensity was calculated. The efficacies were compared with the positive control, genistein. Data are expressed as mean ± standard error of the mean using triplicate assays.

Figure 3. Selectivity of structural clusters and singletons.

Heat maps were generated based on the (a) EC50 and (b) efficacy of each of the chosen compounds against 15 different NR agonist assays.

Figure 4. ERR activity modulation by compounds in the statin cluster.

(a) An ERR-luciferase assay was conducted by cotreating the known ERR inverse agonist, XCT790, at concentrations of 0 μM, 5 μM, or 10 μM with atorvastatin (23 μM), atorvastatin calcium (23 μM), cerivastatin sodium (23 μM), fluvastatin (23 μM), lovastatin (23 μM), or simvastatin (46 μM). The activities were compared with each respective compound without cotreatment of XCT790. (b) A knockdown ERR-luciferase assay was performed by transfecting ERR cells with either control or ERR siRNA. Cells were then treated with DMSO (0.2%), cerivastatin sodium (569 nM), or fluvastatin (15 μM). Each bar represents the mean ± standard deviation (n = 3). *P < 0.05, **P < 0.01, ***P < 0.001. ns, not significant.

Figure 5. ERR, COX8, and IDH3 mRNA expression modulation by cerivastatin sodium and fluvastatin.

The ERR cell line was transfected with control or ERR siRNA and treated with DMSO (0.2%), cerivastatin sodium (569 nM), or fluvastatin (15 μM). The RNA was extracted and quantitative reverse transcription PCR was performed to determine mRNA expression of (a) ERR, (b) COX8, and (c) IDH3. Each gene was expressed relative to the levels of β-actin and normalized to the levels of their respective vehicle control (DMSO). Each bar represents the mean ± standard deviation (n = 3). *P < 0.05, **P < 0.01, ***P < 0.001.


Table 1. Positive Control 10K Screen Statistics.

Table 2. Assay Performance Validation Using the 10K Triplicate Run and Tox21-88 Duplicates.

Supplemental Materials

Supplemental Material

Table S1. qHTS primary and follow-up screen data
Table S2. qHTS primary and follow-up screen data for single actives