Shiuan Chen

Division of Immunology, Beckman Research Institute of the City of Hope, Duarte, CA 91010

Received 12/18/97 Accepted 7/15/98

6. AROMATASE INHIBITORS AND ENZYME STRUCTURE-FUNCTION STUDIES

Aromatase inhibitors are thought to be of value in treating estrogen-dependent breast cancer, especially in postmenopausal patients. As described above, estrogens in postmenopausal patients are mostly produced in peripheral adipose tissues and in cancer cells, and the peripheral aromatase is not under gonadotropin regulation (39). Therefore, in postmenopausal patients, complications due to a feedback regulatory mechanism which increases luteinizing hormone (LH) and follicle-stimulating hormone (FSH) after aromatase inhibitor treatment does not occur. In premenopausal women, LH and FSH stimulate the synthesis of aromatase in ovaries and may counteract the effects of some aromatase inhibitors, as has been observed for aminoglutethimide (AG, structure see figure 4) (40). In view of this, the inhibition of the enzyme has been considered as a potential therapy for breast cancer in postmenopausal women. Throughout the years, a number of very potent and highly selective aromatase inhibitors have been synthesized and tested as drugs for the treatment of breast cancer (structures of aromatase inhibitors are shown in figure 4).

Figure 4. Structures of seven aromatase inhibitors and two phytoestrogens

AG was initially introduced as an anticonvulsant agent and was the first aromatase inhibitor approved for use by FDA for breast cancer treatment. It was also shown to inhibit cytochrome P450 cholesterol SCC (side-chain cleavage) (41). In addition to problems caused by a lack of specificity, it has also been shown that in some patients the aromatase activity in breast tumors is significantly increased after AG treatment (5). In recent years, several potent and specific aromatase inhibitors (e.g., Lentaron, Arimidex, and Letrozole) have been developed and are being used in the treatment of breast cancer. The application of these inhibitors in treating breast cancer was recently reviewed by Brodie and Njar (42).

Aromatase inhibitor development has been based primarily on inhibitor structure-activity relationship studies. Aromatase inhibitors can be categorized into two types: steroidal and nonsteroidal inhibitors (see figure 4). In general, steroidal aromatase inhibitors are analogues of androgen substrates and nonsteroidal inhibitors perturb the catalytic properties of the heme prosthetic group of aromatase. While a number of the inhibitors have been shown to be very potent and specific inhibitors of aromatase, the exact nature of their interactions with aromatase are not known. This is especially true for nonsteroidal inhibitors since these compounds have very diverse structures. Although the structures of these compounds are different, it is thought that they bind to the active site of aromatase, as indicated by competitive inhibition of the enzyme.

We have investigated the interaction of a number of aromatase inhibitors with different aromatase mutants [using a mammalian cell expression method (15)] for evaluating the accuracy of our computer model as well as for determining the binding characteristics of different steroidal and nonsteroidal inhibitors. We have published extensively in this area (43-51). The aim of our aromatase structure-function studies has been to use results generated with steroidal inhibitors to refine our computer model and then explain results generated with nonsteroidal inhibitors using the refined model. We feel that useful information has been generated from these studies.

Our enzyme structure-function 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 make significant contributions to the binding of the substrate and conversion of androgen to estrogen. Mutations in these regions reduce the binding of the substrate and inhibitors. Characterization of two mutants, H480K and H480Q, further suggests that His-480 is hydrogen bonded to the 3-keto group of the androgen substrate (52). The molecular basis of the interaction of various inhibitors with aromatase has been discussed in details and computer models have been presented in two recent publications from our laboratory (50,51). While several important regions of aromatase have been recognized by computer modeling from several laboratories (49,50, 53-55), a few disagreements still exist among models from different groups.

Our laboratory recently succeeded in expressing aromatase using the insect cell expression method and has purified the enzyme to homogeneity (unpublished results). It is our hope that we will be able to better study the aromatase structure-function relationship using purified enzyme preparations.