Identifying environmental chemicals as agonists of the androgen receptor by using a quantitative high-throughput screening platform
Introduction
The androgen receptor (AR, NR3C4) is a transcription factor which regulates male sexual development, while also maintaining accessory sexual organ function (Culig et al., 2002b). The structure of AR includes an N-terminal region which contains the activation function-1 (AF-1), a DNA-binding domain (DBD), a hinge region, and a ligand binding domain (LBD) which contains the ligand-regulated AF-2 (Culig et al., 2002b, Roy et al., 2001). AR is an evolutionarily conserved receptor and is closely related to the human glucocorticoid and progesterone receptors including even recognizing analogous DNA response elements. However, these receptors have a different hormone ligand specificity (Culig et al., 2002b). AR is the main transcription factor implicated in transmitting hormone signals inside the prostate gland (Culig et al., 2002a). As a key transcription factor regulating male sexual development (Pihlajamaa et al., 2015), altering regulation of this nuclear receptor causes abnormal development of the prostate (Wen et al., 2015).
AR activation can occur through direct or indirect pathways (Culig et al., 2002b, Roy et al., 2001). Direct AR activation occurs through a multi-step process. First, the unliganded receptor sequestered by heat shock proteins and immunophilins in the cytoplasm of the cell binds to a ligand through the LBD. This causes a conformational change allowing for the dissociation from the complex anchoring AR in the cytoplasm. Once free, AR homodimerizes and the nuclear localization signal amino acid sequence becomes exposed. The nuclear localization signal subsequently binds to importins, which then transport AR into the nucleus (DeFranco, 1999, Roy et al., 2001). Once inside the nucleus, the ligand-receptor complex and its co-activators accumulate at sequence-specific nuclear foci (Roy et al., 2001, Tyagi et al., 2000). However, like its other nuclear receptor counterparts, AR can also be activated through multiple other pathways in an indirect manner (Davey and Grossmann, 2016); direct ligand binding is not necessary.
Xenobiotic perturbation of AR has many possible adverse outcomes in humans. This includes multiple types of endocrine disruption such as changes in spermatogenesis and the synthesis of sex hormones (Cook et al., 1999). AR is a key driver of prostate cancer growth and AR expression and sensitivity have also been shown to increase in the androgen-responsive human prostatic carcinoma (LNCaP) cell line when grown in androgen-depleted medium (Culig et al., 2002a, Kokontis et al., 1994). Other studies have shown that AR also has an important role in the modulation of multiple additional cancer types including liver, kidney, and bladder, and is linked to hepatocellular hypertrophy (Chang et al., 2014, Fujimoto et al., 2012). Therefore, recognizing exogenous compounds and environmental chemicals which activate AR is critical in detecting endocrine disrupters and possible cancer modulators.
To quickly evaluate the effect of environmental chemicals on the endocrine system, quantitative high-throughput screening (qHTS) has become a useful way to detect modulation of relevant receptors (Hsu et al., 2014, Huang et al., 2014). As part of the Tox21 program, a federal collaboration among the National Institutes of Health, including the National Center for Advancing Translational Sciences and the National Toxicology Program at the National Institute of Environmental Health Sciences, the Environmental Protection Agency, and the Food and Drug Administration, we screened the Tox21 collection of ∼10,000 environmental chemical and drug (Tox21 10K) samples (∼8300 unique compounds) for their AR agonist potential. From our primary screening assay (Tox21 10K), we conducted follow-up studies including a confirmation screen, binding assay, and translocation assay to identify a number of compounds which activate the androgen receptor.
Section snippets
Compound library
The Tox21 chemical library consists of approximately 10,000 (∼8300 unique) small molecules (NCATS, 2016, PubChem, 2013), including pesticides, drugs, industrial chemicals, and food additives, commercially sourced by the NTP, NCATS, and EPA (Attene-Ramos et al., 2013). These compounds were selected based on multiple criteria, including environmental hazards or exposure concerns, compounds with properties conducive to HTS (molecular weight, volatility, solubility, logP), commercial availability,
qHTS performance and reproducibility
We conducted the primary assay by screening the Tox21 10K compound library in a qHTS platform, using AR-bla (LBD, partial receptor) and AR-luc (full receptor) cells, to identify environmental chemicals and drugs as possible AR agonists (see PubChem assay IDs 743036, 743053, 743040). R1881, the positive control for these assays, showed consistent activity throughout the screen with an EC50 of 1.06 ± 0.10 nM in the AR-bla assay and 0.10 ± 0.02 nM for the AR-luc assay. The assay performance statistics
Discussion
The two qHTS assays, AR-bla and AR-luc, enabled us to identify novel and known AR agonist compounds. The AR is an important receptor in sexual development; dysregulation by endogenous or exogenous factors results in changes in the activity, amount, or polymorphisms of this receptor which may lead to adverse medical conditions including developmental diseases, such as Klinefelter syndrome or Kennedy's disease, or cancer (Ran et al., 2015, Skakkebæk et al., 2014, Tanaka et al., 2012). Therefore,
Conflicts of interest
The authors declare they have no actual or potential competing financial interests.
Funding
This research did not receive any specific grant from funding agencies in the public, commercial, or not-for-profit sectors.
Acknowledgements
This work was supported by the U.S. Environmental Protection Agency (Interagency Agreement #Y3-HG-7026-03) and the interagency agreement IAG #NTR 12003 from the National Institute of Environmental Health Sciences/Division of the National Toxicology Program to the National Center for Advancing Translational Sciences, National Institutes of Health. We also acknowledge the support of the NCI High Throughput Facility.
The views expressed in this paper are those of the authors and do not necessarily
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