Structural Characterization of Aromatase

Institution: Beckman Research Institute of the City of Hope
Investigator(s): Yanyan Hong, M.S. -
Award Cycle: 2005 (Cycle 11) Grant #: 11GB-0125 Award: $67,543
Award Type: Dissertation Award
Research Priorities
Etiology and Prevention>Etiology: the role of environment and lifestyle

Initial Award Abstract (2005)
Introduction: Studies have shown that breast cancer growth often depends on the presence of estrogens, the female sex hormones, which attach to their breast canceer cell receptors to induce these abnormal cells to divide without control. Tamoxifen is a drug that is extremely effective in blocking the action of estrogens, but, in many patients, it stops working effectively after about five years.

Aromatase is the enzyme that produces estrogen. From several clinical trials, aromatase inhibitors, including anastrozole (Arimidex), letrozole (Femara), and exemestane (Aromasin), were found to be effectively challenging tamoxifen as an estrogen blocker. They are a new line of estrogen inhibiting drugs, acting through the aromatase pathway, to treat estrogen-sensitive breast cancers by preventing estrogen production in the first place.

Hypotheses: 1. How do these inhibitors interact with aromatase? 2. What are the structural features needed to design new aromatase inhibitors with greater potency and selectivity?

Methodology: One biophysical technique, i.e., protein X-ray crystallography, will be used to understand the structural basis of the drug recognition of aromatase. Meanwhile, the active site peptides of aromatase that interact with an anticancer drug, exemestane, will be identified by proteomic and mass spectral analysis.

Innovative elements: During the last several years, aromatase inhibitors have been shown to be novel and better types of drugs than tamoxifen in treating hormone-responsive breast cancer in postmenopausal women. The availability of a three-dimensional structure of aromatase would be valuable in understanding how these inhibitors interact with the enzyme and in providing critical information for the design of inhibitors with greater potency and selectivity.

Advocacy involvement: This project will provide important information for the understanding of the molecular action of drug recognition by aromatase and for the development of its fourth-generation inhibitors with potentially greater clinical efficacy for the prevention and the treatment of hormone-responsive breast cancer.

Final Report (2007)
Aromatase converts androgen to estrogen. Aromatase inhibitors (AIs) block the synthesis of estrogen and provide effective therapies for hormone-dependent breast cancer. The structure-function characterization of aromatase is crucial in understanding the structural basis of aromatase-drug interaction and designing new potent AIs for the treatment of hormone-dependent breast cancer.

Human recombinant aromatase, with the removal of the membrane anchor domain, was purified from E. coli. Purified recombinant aromatase was shown to be structurally stable and functionally active from the aromatase activity assay and the ultraviolet (UV)-visible spectral analysis. Using this recombinant protein, we determined the interaction of different ligands with aromatase by UV-visible spectral analysis. Crystallization of purified recombinant aromatase was carried out by the vapor diffusion method, and yielded needle clusters. Alternatives, including microseeding, to obtain crystals suitable for X-ray diffraction analysis are in the process.

Proteolysis using pure and active human recombinant aromatase, combined with MALDI mass spectrometry analysis, provided useful information about the 3-D fold of aromatase and ligand-induced conformational changes of aromatase. A clamping mechanism of steroid binding to aromatase was proposed based on site-directed mutagenesis, reaction intermediate study, proteomic analysis, and computer-assisted molecular modeling. A molecular mechanism of the mysterious aromatization reaction was also proposed. The molecular basis for exemestane-mediated irreversible inhibition of aromatase were discussed. Finally, we investigated the interaction of aromatase with different classes of substrates and inhibitors to evaluate the predicted active site of aromatase and the hypothetical clamping mechanism.

The crystal structure of aromatase remains unsolved because it has been difficult to obtain adequate amounts of purified active enzyme due to its membrane-bound character and heme-binding instability. This stable, active and purified recombinant aromatase give us a unique opportunity to study its structure-function relationship. Furthermore, needle crystal clusters of human recombinant aromatase provide a promising perspective for x-ray crystallography. These comprehensive structure-function studies of aromatase provide valuable information for understanding the mechanism of aromatization reaction and the molecular action of drug recognition by this enzyme, and designing new AIs with greater potency and selectivity for the treatment of hormone-dependent breast cancer.

Symposium Abstract (2007)
The third-generation aromatase inhibitors (AIs), including anastrozole, letrozole, and exemestane, are widely used as the first-line drugs in the endocrine treatment of estrogen-dependent breast cancer in postmenopausal women. Although these AIs are potent and specific, resistance to them does occur. Therefore, new AIs could provide alternative treatments for breast cancer patients who are not responsive to current AIs. However, the structural basis of drug binding to aromatase is not well understood because the three-dimensional (3-D) structure of aromatase is unknown.

In our laboratory, a structurally stable and functionally active human aromatase has been expressed in E. coli and has been successfully purified. Using this purified aromatase, molecular features of the interaction of different substrates and inhibitors with aromatase were studied by UV-visible spectral analysis. Proteomic studies of this purified aromatase combined with MOLDI-TOF MS revealed a 3-D fold of human aromatase, and indicated that helices B and C and the B-C loop of aromatase might undergo dramatic conformational changes when the enzyme binds to steroidal substrate or inhibitor. Moreover, efforts to crystallize aromatase have generated needle cluster crystals.

Based on site-directed mutagenesis, proteomic analysis, and computer modeling, we proposed a clamping mechanism of androstenedione and exemestane binding to the active site of aromatase. We also investigated the mechanism basis for the aromatization reaction and exemestane-mediated irreversible inhibition of human aromatase. To examine the accuracy of the predicted active site pocket of aromatase and the hypothetical clamping mechanism of steroid binding to aromatase, we recently investigated the interaction of aromatase with four different classes of substrates and inhibitors, each having its own unique structural features. We also compared the interaction of aromatase with two synthetic A-ring modified steroids. This study demonstrates the importance of the A ring of steroidal inhibitors for their binding to aromatase, and provides useful information in the development of new AIs for the treatment of hormone-dependent breast cancer.

Molecular Basis for the Aromatization Reaction and Exemestame-Mediated Irreversible Inhibition of Human Aromatase. Mol Endocrinol 21 (2): 401-414. relevant
Periodical:Molecular Endocrinology
Index Medicus: Mol Endocrinol
Authors: Hong, Y., Yu, B., Sherman, M., Yuan, Y.-C., Zhou, D. and Chen, S.
Yr: 2007 Vol: 21 Nbr: Abs: Pg:401-14

Aromatase inhibitors: Structure Features and Biochemical Characterization. (Ann NY Acad Sci 1089: 237-251.
Periodical:New York Academy of Sciences
Index Medicus:
Authors: Hong, Y. and Chen, S.
Yr: 2006 Vol: 1089 Nbr: Abs: Pg:237-51

New experimental models for aromatase inhibitor resistance. J Steroid Biochem Mol Biol 106(1-5):8-15.
Periodical:Journal of Steroid Biochemistry and Molecular Biology
Index Medicus: J Steroid Biochem Mol Biol
Authors: Chen S, Masri S, Hong Y, Wang X, Phung S, Yuan YC, Wu X 2007
Yr: 2007 Vol: 106 Nbr: Abs: Pg:8-15