Molecular mechanism of exemestane inhibition of aromatase. A site-directed mutagenesis study

5774 Aromatase converts androgen to estrogen. Research from our and other laboratories has confirmed that aromatase is expressed at a higher level in breast cancer tissue than in benign tissue. In situ estrogen biosynthesis in tumor tissue has been shown to play both an autocrine and an endocrine role in promoting tumor growth. Aromatase inhibitors have been demonstrated to be important drugs in the treatment of breast cancer in postmenopausal women. Exemestane is an irreversible (Type 1), steroidal aromatase inhibitor. While it is thought that exemestane achieves its inhibition by binding to the active site of aromatase, the molecular nature for the interaction of this compound with the enzyme is not yet determined. During the past ten years, our laboratory has prepared 51 mutants using the CHO cell expression method. The studies have revealed that two regions, the I helix (Cys-299 to Ser-312) and the “hydrophobic” pocket (Ile-474 to His-480), are important parts of the active site and makes significant contributions to the binding of the substrate and conversion of androgen to estrogen. To better understand how exemestane interacts with aromatase, we first performed inhibitory profile analyses on the wild-type enzyme and 10 mutants. Exemestane was found to be significantly less potent in the inhibition of the mutants, E302D (IC50 = 32 nM), D309A (IC50 = 65 nM) and S478T (IC50 = 68 nM), than the wild-type aromatase (IC50 = 24 nM). A time course study of the inhibition of different aromatase mutants by exemestane (at 20 nM) was then performed. A definitive time-dependent inhibition of the wild-type aromatase was observed, as expected for a suicide inhibitor. However, time-dependent inhibition of three mutants could not be demonstrated. Glu-302, Asp-309 and Ser-478 have been previously suggested to play important roles in the binding of the androgen substrate. These recent results indicate that these amino acid residues may participate in the mechanism of the suicide inhibition of aromatase by exemestane. A computer model to illustrate the interaction of exemestane with aromatase has been generated. This information is important for the understanding of the molecular action of exemestane, a third-generation aromatase inhibitor, and for the development of the fourth-generation inhibitors.