Molecular basis of azole fungicide interactions with human estrogen receptor alpha (ERα): an integrated in silico and in vitro toxicological assessment.

The extensive agricultural use of azole fungicides, while essential for crop protection, has raised growing concern regarding their potential to disrupt endocrine signaling in humans. This study examined the molecular basis of interactions between selected azole compounds and human estrogen receptor alpha (ERα) to determine their capacity to interfere with receptor function. An integrated in silico and in vitro workflow was employed. Molecular docking was first performed for the azoles alongside the endogenous agonist estradiol (E2) and the antagonist 4-hydroxytamoxifen (OHT) as reference ligands, followed by binding free energy estimation using MM/GBSA analysis. The five top-ranked azoles were subsequently characterized through ADMET profiling and density functional theory (DFT) calculations to assess their physicochemical and electronic properties. Molecular dynamics simulations and post-simulation MM/GBSA analysis were then conducted to evaluate the stability and dynamic behavior of the azole-ERα complexes. Experimental validation was performed using a bioluminescence resonance energy transfer (BRET)-based assay. Computational analyses identified five azoles with notable binding affinity toward ERα, and molecular dynamics simulations confirmed the structural stability of the corresponding complexes, supported by favorable MM/GBSA binding energies. Experimentally, three azoles (flusilazole, myclobutanil, and tebuconazole) intrinsically induced cytosolic ERα dimerization, consistent with agonist-like activity, whereas the remaining two azoles (fenbuconazole and paclobutrazol) significantly suppressed E2-induced ERα dimerization, consistent with antagonist-like activity comparable to that produced by OHT. Together, these findings provide mechanistic insight into ERα modulation by azole fungicides and highlight their relevance for human endocrine toxicity and health risk assessment.