Profiling Drug Metabolism (P450) Proteins in Breast Cancer

Institution: Scripps Research Institute
Investigator(s): Aaron Wright, Ph.D. -
Award Cycle: 2006 (Cycle 12) Grant #: 12FB-0027 Award: $105,147
Award Type: Postdoctoral Fellowship
Research Priorities
Biology of the Breast Cell>Pathogenesis: understanding the disease

Initial Award Abstract (2006)
Normal breast cell regulation and function is modulated by multiple enzyme families. One enzyme family, the cytochrome P450s, is responsible for both metabolism of foreign compounds that enter the cell as well as the synthesis of key regulatory compounds such as estrogen. Many currently used breast cancer therapeutics target aromatase, a P450 enzyme important to the synthesis of estrogen, including anastrozole (Arimidex®) and letrozole (Femara®) for the treatment of estrogen (hormone)-dependent breast cancer. These enzymes are also known to transform foreign compounds that enter the cell to carcinogens which enhance cancer cell proliferation. Additional ways to inhibit and regulate the P450 enzyme family will provide new alternatives for cancer treatment and prevention. What is needed is a way of determining cytochrome P450 enzyme activity in both normal and cancerous breast tissues. An inventory of P450 enzyme activity will support the development of novel therapeutics to treat breast cancer. Current methods for assessing enzyme activity measure the relative protein abundance. However, enzymes in cells can be in inactive forms, inhibited forms, or modulated forms that are not detected by quantitative measurements.

In this research, we will develop enzyme probes that have a chemical group reacting with active cytochrome P450 enzymes in normal and cancerous breast cells. The probes also are developed with a chemical “handle” that allows them to “light up” for easy identification. The probes will be added to a particular cell line (cell lines will differ in their state of invasiveness, presence of estrogen receptors which are key to hormone-dependent breast cancer, and their potential for forming breast tumors) and given time to react with P450 enzymes. After treatment the second compound is added and the amount of enzymes that have reacted with the probes can be measured. Thus, a profile of active cytochrome P450 enzymes in various breast cell lines will be generated. Furthermore, the probes can be added to the breast cells in the presence of key breast cancer drugs such as tamoxifen and anastrozole to determine how they effect or are affected by these drugs. These types of activity studies may permit more directed pharmaceutical development of new breast cancer therapeutics.

This research will use newly developed chemical probes to determine which cytochrome P450 enzymes are active in normal and cancerous breast cells. This research will also assess the effects on cytochrome P450 enzymes by two common breast cancer therapeutics, tamoxifen and anastrozole. It is likely that these studies may also explain the effects of P450 enzymes on the degradation of these important cancer drugs.

Final Report (2008)
Cytochrome P450 (P450) enzymes are critical to the regulation and synthesis of endogenous signaling molecules and the metabolism of xenobiotics, including drugs. Due to their significant role in the oxidative metabolism and clearance of breast cancer chemotherapeutics, the catalytic activity of P450s is critical to the pharmaceutical industry. We recently developed an activity-based chemical probe for evaluating mouse liver microsomal P450 activity in vitro and in vivo. We discovered broad promiscuity of our probe among P450s, labeling multiple P450 subfamilies.

However, there are fairly significant differences between murine and human P450s which cloud organism comparisons. As a final project under the CBCRP funding we have developed a novel activity-based probe suite for analyzing human P450 activity, and evaluating fluctuations in those activities due to breast cancer drugs. The probes contain varied chemical architectures validated as mechanism-based inhibitors of the P450 enzyme family. A panel of human P450s with differing substrate selectivity and biological catalytic activity were selected for analysis. We tested the selectivity and sensitivity of the probes, and used them to profile the effects of aromatase inhibitors, anastrozole and formestane, drugs used for treating post-menopausal breast cancer, on human P450 activity. We observed fascinating results, including inhibitory profiles of aromatase (P450 19A1), and dynamic inhibition and induction of other P450s by drugs that are supposed to be specific for aromatase.

This work provides a novel platform for testing new breast cancer therapeutics and analyzing their effect on P450 activities.

Symposium Abstract (2007)
The cytochrome P450 superfamily of enzymes oxidizes several exogenous and endogenous compounds including drugs, carcinogens, fatty acids, and steroids. Additionally, mutations in many P450 genes cause inborn errors of metabolism and contribute to increased risk of cancer. Variations in expression level and activity of several P450 enzymes between individuals can also lead to differences in metabolism and carcinogen formation.

We have created derivatives of 2-ethynylnaphthalene and phenylisothiocyanate as “click chemistry”-compatible activity-based probes for monitoring NADPH-dependent P450 activity in liver microsomal proteomes. These probes were found to label numerous P450s in an activity-based manner both in vitro and in living animals, where the impact of chemical inducers of P450 expression in liver could be detected. Furthermore, in experiments synonymous with drug-drug interactions, the impact on P450 activity due to multiple xenobiotics could be monitored by alterations in probe labeling.

Further work to develop an in vivo chemical probe for monitoring P450 activity may facilitate the determination of those P450 enzymes responsible for carcinogen formation or drug metabolism which in turn could be used for developing more efficacious therapeutics. An activity-based chemical probe approach may help facilitate the determination of those compounds being metabolized by P450 enzymes to carcinogenic compounds that are known inducers of breast cancer pathology, as well as the P450 enzymes responsible for metabolism of breast cancer therapeutics.

Chemical proteomic probes for profiling cytochrome p450 activities and drug interactions in vivo.
Periodical:Chemistry and Biology
Index Medicus: Chem Biol
Authors: Wright AT, Cravatt BF
Yr: 2007 Vol: 14 Nbr: 9 Abs: Pg:1043-51

Activity-based protein profiling: from enzyme chemistry to proteomic chemistry.
Periodical:Annual Review of Biochemistry
Index Medicus: Annu Rev Biochem
Authors: Cravatt BF, Wright AT, Kozarich JW
Yr: 2008 Vol: 77 Nbr: Abs: Pg:383-414