Identifying androgen receptor antagonists using a metabolically competent high-throughput screening assay

Area of Science:

• Endocrinology
• Pharmacology
• Toxicology

Background:

• Androgen receptor (AR) plays a crucial role in sexual development and function.
• AR dysregulation is linked to endocrine disruption, cancer, and other diseases.
• Existing screening methods often overlook the role of metabolism in identifying AR modulators.

Purpose of the Study:

• To optimize a high-throughput screening assay that incorporates metabolic processes.
• To identify novel AR antagonists, particularly those requiring metabolic activation.
• To accurately classify compounds that modulate AR activity following metabolic transformation.

Main Methods:

• Developed and validated a high-throughput screening assay using rat liver microsomes (RLM) for metabolic activation.
• Screened the LOPAC library + 88 Tox21 compounds (1365 unique compounds) for AR antagonists.
• Conducted assays with heat-inactivated RLM and metabolic stability assays to confirm activity and identify bioactivated compounds.

Main Results:

• Identified 55 potential AR antagonists, with 9 showing significant potency shifts in the presence of RLM.
• Discovered four novel parent compounds requiring metabolism for enhanced AR antagonism.
• 4,5-dianilinophthalimide (DAPH) was identified as a true AR antagonist requiring metabolism, exhibiting increased potency with RLM inclusion.

Conclusions:

• Metabolism is critical for accurately identifying certain AR modulators.
• The developed assay effectively detects AR antagonists that require metabolic activation.
• DAPH represents a significant finding as a novel, metabolism-dependent AR antagonist.