Human Cytochrome P450 Enzymes in Steroidogenesis and Drug Metabolism
Creators: name: Burris-Hiday, Sarah
Publisher: name: My University
Year: 2023
Subjects: subject: Cytochrome P450
subject: NADPH cytochrome P450 reductase
subject: castration resistant prostate cancer
subject: protein-protein interaction
subject: allosteric modulation
subject: drug metabolism
subject: Biological Chemistry
subject: Molecular, Cellular and Developmental Biology
subject: Pharmacy and Pharmacology
subject: Chemistry
subject: Science (General)
subject: Health Sciences
subject: Science

description: Cytochrome P450 enzymes are a class of heme-containing monooxygenases involved in multiple critical pathways in the human body. They are most commonly known for their role in drug metabolism, but they are also involved in production of steroid hormones, fatty acids, eicosanoids, and vitamins. They perform catalysis on this wide variety of substrates through the use of their heme prosthetic group. Of the 57 human P450 enzymes, most require a redox partner protein to deliver electrons to the catalytic heme iron, and 48 of those enzymes use the redox partner NADPH-cytochrome P450 reductase. This dissertation investigates how reductase modulates various drug metabolizing and steroidogenic P450 enzymes, and then focuses on inhibition of a specific steroidogenic P450, CYP17A1, for the treatment of prostate cancer. For electron transfer to the P450 heme, the reductase FMN-containing domain directly binds to the proximal side of the P450, yet little is known about the details of this P450/reductase interaction. To stabilize these highly transient interactions, artificial fusion proteins of the FMN-containing domain of reductase with either drug metabolizing or steroidogenic P450 enzymes were designed, expressed, and purified. The functions of these fusion proteins were investigated in parallel with their non-fusion counterparts in order to interrogate the effect that localization of the FMN domain of reductase has on P450 activity. P450 enzymes were characterized by their ability to metabolize substrate and bind both substrate and inhibitor. However, all experiments were performed with the oxidized form of the FMN domain of reductase, thus the resulting modulation of P450 activity was not due to electron transfer to the P450 heme. Intriguingly, the FMN domain of reductase was found to increase, decrease, or have no effect on P450 activity dependent on the ligand and the P450 isoform. This suggests that the FMN domain allosterically modulates P450 activity differentially for various P450 isoforms, revealing an intricate communication between the P450 active site and the P450/FMN domain interaction site on the P450 surface 15-20 angstroms away. One specific steroidogenic P450 enzyme, CYP17A1, is an important drug target for the treatment of prostate cancer. Androgen-driven prostate cancer is typically treated with androgen deprivation therapy by chemical castration. However, prostate cancer cells often adapt to survive under even castration levels of androgens, resulting in castration resistant prostate cancer. Abiraterone acetate is an FDA approved CYP17A1 inhibitor designed to treat this advanced and fatal stage of prostate cancer. Abiraterone beneficially inhibits CYP17A1, and metabolites of abiraterone have been found to disrupt CYP17A1 and androgen receptor activity. However, abiraterone as well as its metabolites typically also inhibit another P450, CYP21A2, leading to decreased corticosteroid production and unwanted side effects. Therefore, the work herein aimed to design abiraterone metabolite analogs with modifications on the A and B rings of the steroid core as well as on the 3-pyridine ring. Analogs were analyzed for their selectivity for CYP17A1 inhibition over CYP21A2. In general, modifications on the pyridine ring retained more inhibition of CYP17A1 than modifications on the steroid core. One inhibitor of CYP17A1 was found to be selective for CYP17A1 over CYP21A2, showing promise for prostate cancer treatment. In conclusion, this dissertation aims to investigate the role of P450 allosteric modulation by redox partner proteins, as well as abiraterone’s modulation of CYP17A1 activity for the treatment of prostate cancer.