Beyond Competitive Binding: New Biochemical Insights Challenge Endocrine Disrupting Chemical Screening Paradigms.

Endocrine-disrupting chemicals (EDCs) pose significant health risks at environmentally relevant levels. Global screening efforts have predominantly relied on competitive binding assays. Here, using biochemical assays and computational simulations, we identified a crucial but largely overlooked noncompetitive binding mode between EDCs and the androgen receptor (AR). Two underappreciated sites within the AR ligand-binding domain, active function 2 (AF2) and binding function 3 (BF3), were found to influence AR activity and coregulator recruitment. Specifically, AF2-binders sterically obstruct coactivator docking, while BF3-binders indirectly weaken coactivator interactions via allosteric coupling. We show that these newly recognized surfaces hamper AR-driven transcription even in the presence of endogenous hormones. Expanding the regulatory landscape of AR beyond its conventional ligand-binding domain, we find that noncompetitive binding was prevalent among 7841 EDCs. Notably, the AF2 site emerged as a key hotspot, with median preference scores exceeding those of other sites by more than 3-fold among active EDCs. These findings challenge the current paradigm of EDC screening, underscore the need for broadened assay development, and pave the way for innovative methods tailored to noncompetitive mechanisms.