Cell-Based High-Throughput Screening for Aromatase Inhibitors in the Tox21 10K Library.

Area of Science:

• Endocrinology
• Biochemistry
• Toxicology

Background:

• Endocrine disruption can occur through nonreceptor-mediated mechanisms, including inhibition of aromatase, an enzyme crucial for regulating androgen and estrogen balance.
• Aromatase inhibitors (AIs) are important for understanding and potentially treating hormone-dependent conditions.

Purpose of the Study:

• To adapt and validate the AroER tri-screen assay for high-throughput screening (HTS) to identify novel aromatase inhibitors.
• To screen the U.S. Tox21 10K compound library for potential AIs.
• To characterize the identified AIs for selectivity and novel structures.

Main Methods:

• Adapted the AroER tri-screen assay from 96-well to 1536-well format for HTS.
• Screened the Tox21 library using the tri-screen assay in the presence of testosterone to identify potential AIs.
• Rescreened identified compounds and known actives/inactives across all three assay formats (ERα agonist, AI/ERα antagonist, ERα antagonist).
• Confirmed potential AIs using a biochemical tritiated water-release aromatase assay and investigated binding characteristics.

Main Results:

• Screening identified 302 potential AIs from the Tox21 library.
• Of 333 compounds retested, 113 (34%) were identified as potential AIs, independent of cytotoxicity and ER antagonism.
• Structure-activity analysis revealed both conventional and novel AI structures.
• Ten novel AI compounds were confirmed active in the biochemical assay, with four showing low efficacy in HTS only.

Conclusions:

• The high-throughput AroER tri-screen assay is an accurate and efficient method for identifying selective aromatase interactors in large chemical libraries.
• The study successfully identified novel potential aromatase inhibitors, including drugs and fungicides.
• These findings provide a foundation for further investigation into the mechanisms and applications of these novel AIs.