Aromatase (CYP19A, EC 1.14.14.14) is a member of the cytochrome P450 monooxidase (CYP) family of microsomal xenobiotic metabolism enzymes. Aromatase plays a critical role in steroidogenesis, catalyzing the conversion of androgenic hormones into estrogens. The enzyme is expressed in high levels in reproductive tissues, placenta, brain and adipose tissue and is responsible for mammalian sexual dimorphism and development of secondary sexual characteristics. Inhibitors of aromatase are used to treat estrogen-dependent breast cancer, as estrogens promote the expression of peptide growth factors responsible for tumorigenesis. Aromatase activity and expression can be affected by many organic environmental pollutants such as pesticides and plasticizers. Such compounds, known as endocrine disruptors, are suspected of causing precocious puberty, obesity, infertility and various cancers. Assay Genie's Aromatase Inhibitor Screening Kit enables rapid screening of drugs and other small molecules for compound-Aromatase interaction in a reliable, high-throughput fluorescence-based assay. The assay utilizes a fluorogenic substrate that is converted into a highly fluorescent metabolite detected in the visible range (Ex/Em = 488/527 nm), ensuring a high signal-to-background ratio with little interference by autofluorescence. The kit contains a complete set of reagents sufficient for performing 100 reactions in a 96-well plate format.

Figure: (a) Standard curve of Aromatase Substrate metabolite fluorescence. One mole of fluorescence standard corresponds to the metabolism of one mole of Aromatase Substrate. (b) Reaction kinetics of recombinant human aromatase enzyme at 37°C in the presence and absence of the indicated aromatase inhibitors (the solvent control reaction contained assay buffer with 0.2% acetonitrile). (c) Dose-response curves for various aromatase ligands of differing structural and mechanistic classes: the potent competitive inhibitor letrozole, the mechanism-based irreversible inhibitor exemestane, the phytoestrogen-like synthetic flavonoid α-naphthoflavone and the antifungal ketoconazole (a competitive inhibitor of several CYP isoforms). For dose-response curves, percent activity was calculated for each concentration of inhibitor by comparison to activity of reactions containing no inhibitor. For each inhibitor, IC50 values were derived by 4-parameter logistic curve fitting with each point representing the mean ± SEM of at least four replicates. Assays were performed according to the kit protocol.