Structure and function of human cytochromes P450 enzymes: Xenobiotic metabolism by CYP2A and steroid biosynthesis by CYP17A1

Abstract

Cytochrome P450 (CYP) is a superfamily of heme-containing enzymes that have roles in the breakdown or synthesis of steroids, fatty acids, vitamins, and eicosanoids. They are also the main family of enzymes involved in the Phase I metabolism of xenobiotic compounds, including drugs. Several isoforms of cytochromes P450 of both types are targets for the prevention or treatment of cancers. The xenobiotic metabolizing enzyme CYP2A13 and the steroid synthesis enzyme CYP17A1 are two such enzymes. Understanding the mechanisms of substrate recognition and inhibitor interaction of these enzymes is important for the development of clinical inhibitors and is the focus of this dissertation. Human CYP2A13 is a lung-specific cytochrome P450 that metabolizes the tobacco procarcinogen 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK) into two carcinogenic compounds and thus is a target for prevention of lung cancer. However, CYP2A13 is one of three human CYP2A enzymes, sharing 94% sequence identity with the human hepatic CYP2A6. Despite this very high sequence identity, CYP2A6 metabolizes the procarcinogen NNK at much lower levels. The similarities and differences between these two human CYP2A enzymes with the substrate nicotine and the inhibitor pilocarpine were investigated by determining co-crystal structures. Additionally, a structure was also determined of CYP2A13 with NNK. These structures provide insight into substrate binding by CYP2A13 and CYP2A6 and indicate that CYP2A enzyme selectivity is primarily guided by statics. The third human CYP2A enzyme, CYP2A7, was reported to be an inactive enzyme ten years ago. We utilized the same techniques used to express and purify large quantities of CYP2A6 and CYP2A13 to determine if CYP2A7 was a stable, active enzyme. CYP2A7 was not as stable as CYP2A6 and CYP2A13, and only a very small amount of 7-hydroxycoumarin was detected. That any metabolite was produced does indicate that in some individuals CYP2A7 may contribute to xenobiotic metabolism. Human CYP17A1 catalyzes two steps in the synthesis of androgens. CYP17A1 first catalyzes the 17α-hydroxylation of progesterone and pregnenolone to 17α-hydroxyprogesterone and 17α-hydroxypregnenolone, respectively. Then the 17,20-lyase activity converts these 17α-hydroxyderivatives to androstenedione and dehydroepiandrosterone, respectively. Since the majority of prostate cancers are dependent on androgens for growth, inhibition of CYP17A1 provides a means to inhibit the production of androgens throughout the body. Several inhibitors are currently in clinical trials including abiraterone, TOK-001, and TAK-700. In order to understand the interactions between the inhibitors and target enzyme CYP17A1, we determined the first crystal structure of CYP17A1, which had the inhibitor abiraterone bound. This structure indicates an active site complementary to the steroid core of abiraterone. Some excess volume and polar amino acids not currently interacting with inhibitor that give direction to possible modifications for an inhibitor with increased interactions with the CYP17A1 protein. Future directions include the determination of structure with TOK-001, TAK-700, and at least one substrate. These structures will also aid in the rational design of inhibitors for CYP17A1.