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Development of a 5α-reductase High-throughput Screening Assay for Androgen Steroidogenesis
Citation:
Foley, B., W. Stewart, M. Feshuk, K. Paul-Friedman, R. Thomas, AND C. Deisenroth. Development of a 5α-reductase High-throughput Screening Assay for Androgen Steroidogenesis. 10th Annual Meeting of the American Society for Cellular and Computational Toxicology (ASCCT), Durham, North Carolina, October 12 - 14, 2021. https://doi.org/10.23645/epacomptox.16896796
Impact/Purpose:
Poster presented to the 10th Annual Meeting of the American Society for Cellular and Computational Toxicology (ASCCT) October 2021. Many high-throughut screening assays are utilized by the Endocrine Disruptor Screening Program to evaluate effects on estrogen, androgen, and thyroid endocrine pathways. Reducing the reliance on animal testing requires development of in vitro new approach methods that provide adequate coverage for targets relevant to endocrine screening. The objective of this study was to develop a high-throughput assay for screening inhibition of human 5 alpha-reductase; an enzyme responsible for androgen steroidogenesis that is critical for normal male sexual development and maturation. The target is considered an important in vitro data gap to the guideline in vivo Hershberger assay and will complement a broader battery of assays for androgen steroidogenesis and function.
Description:
The US EPA employs high-throughput screening assays to identify environmental chemicals that may pose a risk to human health. Many assays are utilized by the Endocrine Disruptor Screening Program to evaluate effects on estrogen, androgen, and thyroid endocrine pathways. Altered androgen hormone biosynthesis contribute to endocrine disruption that may result in impaired reproductive and sexual development. Steroid 5α-reductase enzymes catalyze the conversion of testosterone into the more potent androgen 5α-dihydrotestosterone. Type 2 5α-reductase enzyme (SRD5A2) deficiency is associated with decreased virilization in males and presents an important mode-of-action when evaluating environmental chemical exposure. The objective of this study was to develop a high-throughput assay for screening inhibition of human SRD5A2. NanoBRET Target Engagement assay technology was used to evaluate modulation of testosterone binding to SRD5A2 in a 384-well cell-based format. Michaelis-Menten kinetics of the SRD5A2-NanoLuc fusion protein demonstrated normal conversion of testosterone to 5α-dihydrotestosterone (Km: 434 nM). Initial evaluation with 5α-reductase reference inhibitors dutasteride, finasteride, and epristeride revealed significant gain-of-signal. Screening of 1803 blinded ToxCast chemicals identified 91 chemicals with bioactivity hit counts. Additional filtering for assay-specific cytotoxicity and autofluorescence resulted in 24 chemicals with putative bioactivity toward SRD5A2. Overall, the NanoBRET assay technology demonstrated high precision (rCV: 5.2%), modest dynamic range (S/B: 1.41 FC), and marginal assay quality (rZ’: 0.13). Finasteride was the most potent compound identified in the screen, suggesting sufficient sensitivity for identifying potent inhibitors of enzyme function. The views expressed do not reflect the views or policies of the U.S. EPA.
URLs/Downloads:
DOI: Development of a 5α-reductase High-throughput Screening Assay for Androgen Steroidogenesis
FOLEY_ASCCT_2021_POSTER_V7.PDF (PDF, NA pp, 1469.066 KB, about PDF)