Screening e identificazione di nuovi interferenti endocrini dell'attività dell'aromatasi

Human aromatase (CYP19A1) belongs to the cytochrome P450 superfamily and is responsible for the last step of steroidogenesis catalyzing the conversion of androgens into estrogens. The enzyme is part of the endocrine system and it is essential for the regulation of the circulating sexual hormones. As such, it is a known target of the so-called endocrine disrupting chemicals (EDCs), a series of dangerous molecules from different environmental sources that can be present in soil and groundwater and can enter the food chain. Nowadays, screening methods and biological assays are required in order to test new and emerging pollutants and identify the ones potentially acting as EDCs with toxic effects on human health. Here, computational methods, biophysical and biological assays were used to screen and identify molecules interfering with aromatase activity. The compounds were chosen based on a watch-list of chemicals from the EU Directive on Environmental Quality Standards 2008/105/EC. First, molecular docking simulations were applied to establish the compounds able to fit in human aromatase active site (30 out of 33). Second, spectroscopic assay were performed using the purified recombinant enzyme (rArom) and allowed the identification of the ones binding in the active site of the protein (6 out of 30). Last, the catalytic activity of rArom and full-length aromatase transfected in a prostate cancer cell line (PC3) was evaluated in the presence of these potential EDCs. Out of the 33 starting compounds, the herbicide glyphosate and the neonicotinoid insecticide thiacloprid were identified to inhibit aromatase activity by 66% and 40%, respectively, when used in a concentration of 1 μM. The data show that the integrated approach applied here, allowed to restrict, step-by-step, the number of molecules to be tested and granted the identification of two compounds acting as aromatase inhibitors at ppm concentrations. Such an approach will be extended to screen and identify other EDCs acting on aromatase activity.