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Identifying Environmental Chemicals as Agonists of the Androgen Receptor by using a Quantitative High-Throughput Screening Platform

Lynch C, Sakamuru S, Huang R, Stavreva DA, Varticovski L, Hager GL, Judson RS, Houck KA, Kleinstreuer NC, Casey W, Paules RS, Simeonov A, Xia M.
Toxicology (2017) DOI: PMID: 28478275



The androgen receptor (AR, NR3C4) is a nuclear receptor whose main function is acting as a transcription factor regulating gene expression for male sexual development and maintaining accessory sexual organ function. It is also a necessary component of female fertility by affecting the functionality of ovarian follicles and ovulation. Pathological processes involving AR include Kennedy’s disease and Klinefelter’s syndrome, as well as prostate, ovarian, and testicular cancer. Strict regulation of sex hormone signaling is required for normal reproductive organ development and function. Therefore, testing small molecules for their ability to modulate AR is a first step in identifying potential endocrine disruptors. We screened the Tox21 10K compound library in a quantitative high-throughput format to identify activators of AR using two reporter gene cell lines, AR β-lactamase (AR-bla) and AR-luciferase (AR-luc). Seventy-five compounds identified through the primary assay were characterized as potential agonists or inactives through confirmation screens and secondary assays. Biochemical binding and AR nuclear translocation assays were performed to confirm direct binding and activation of AR from these compounds. The top seventeen compounds identified were found to bind to AR, and sixteen of them translocated AR from the cytoplasm into the nucleus. Five potentially novel or not well-characterized AR agonists were discovered through primary and follow-up studies. We have identified multiple AR activators, including known AR agonists such as testosterone, as well as novel/not well-known compounds such as prulifloxacin. The information gained from the current study can be directly used to prioritize compounds for further in-depth toxicological evaluations, as well as their potential to disrupt the endocrine system via AR activation.


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

Every hexagon represents a class of structurally similar compounds. The color gradient is indicative of the enrichment of AR 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 AR in the AR-bla and AR-luc assays. Clusters with multiple actives in their class are closer to maroon, while clusters with no activity are a light grey color. Empty clusters with no available (N/A) compounds in them are darker grey in color.

Figure 2. Concentration-response curves alongside binding data of potentially novel AR agonists.

Fifteen point dilutions and receptor binding studies of, GSK232420A (A), Norethisterone enanthate (B), and prulifloxacin (C) were performed. Data were collected from follow-up studies, including the AR-bla confirmation assay, the AR-luc confirmation assay, and the receptor binding screen. They were expressed as mean ± SD from triplicate experiments for each assay.

Figure 3. Translocation of GFP-AR protein of top 17 compounds.

A mouse mammary adenocarcinoma cell line that expresses GFP-AR was treated with the vehicle control (DMSO), the positive control (Testosterone), or each of the top 17 potential and known agonists for 30 min, at concentrations indicated in Table 3. An automated image analysis of localization was performed and the mean GFP-AR intensity for both the nucleus and the cytoplasm was identified. The ratio of both intensities was calculated and the triplicate values were then normalized to the corresponding control (DMSO) values (A). Data represent the mean ± SD (n = 3).
**, P < 0.01; ***, P < 0.001. Representative micrographs of the GFP-AR, nucleus, and combined channels of the cells treated with 62.5 nM of the representative three potential novel compounds, as well as the positive and negative controls, are shown in B.


Table 1. Assay performance using Tox21-88 duplicates and 10K triplicate runs.

For each assay, the reproducibility was calculated for the Tox21-88 compounds (duplicates in each plate) and for the 10K compound library (three copies) with compounds plated in different well locations.

Table 2. Previously identified AR reference compounds and their present activity.

Purity Rating: A = MW Confirmed, Purity > 90%.
a % Efficacy is based on the maximal efficacy produced by R1881 (positive control).
b Compound was identified as inactive if the efficacy was below 30% of R1881’s activity.
c Values are in mean ± SD format, where n = 3.

Table 3. Potential novel and representative AR modulators.

Potential novel and representative AR modulators identified through qHTS, clusters, binding, and translocation.
Purity Rating: A = MW Confirmed, Purity > 90%; Ac = Purity > 90%, Low concentration of sample.
a % Efficacy is based on the maximal efficacy produced by R1881.
b These compounds have documented direct connections to AR as modulators.
c Values are in mean ± SD format, where n = 3.
d Compound was identified as inactive if the efficacy was below 30% of R1881’s activity.

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