Cytochrome b5 (b5) profoundly influences the catalytic efficiency of many cytochrome P450-catalyzed reactions, yet the mechanism(s) of this action of b5 on P450 enzymes is not known. Among these b5- regulated activities is the 17,20-lyase activity of CYP17A1 (P450c17, steroid 17-hydroxylase/17,20-lyase), which is a key step in the biosynthesis of androgens and estrogens. Diseases of androgen excess and androgen dependence, including polycystic ovary syndrome and prostate cancer, are extremely common, and the CYP17A1 inhibitor abiraterone acetate (AA) is used to treat prostate cancer, proving the relevance of CYP17A1 in human disease. By indiscriminately inhibiting CYP17A1's 17-hydroxylase activity in addition to the 17,20-lyase activity, however, AA causes hypertension and potassium loss unless given with prednisone, a potent glucocorticoid. Consequently, an unmet clinical need is a selective inhibitor of the 17,20-lyase reaction, which will safely lower testosterone production. We hypothesize that a drug, which disrupts the interaction of b5 with the CYP17A1-POR complex, will selectively block the 17,20-lyase reaction and lower testosterone production without disturbing drug metabolism or requiring chronic glucocorticoid therapy. Our long-term goal is to elucidate the molecular details of the b5-CYP17A1 interaction and to develop approaches to disrupt this interaction and thus selectively inhibit the 17,20-lyase activity of CYP17A1. Our central hypothesis is that two negatively-charged residues on b5 interact with specific arginine residues on CYP17A1 to increase the rate of product release, the rate-limiting step of the 17,20-lyase reaction.
In Aim 1, we will use site-directed mutagenesis and mass spectrometry to determine the steric and electronic requirements of key residues on b5 necessary to stimulate 17,20-lyase activity and the nature of b5 interaction with CYP17A1.
In Aim 2, we will employ pre-steady state kinetic experiments to determine the rate-limiting step of the 17,20-lyase reaction and thus deduce the microscopic step of b5 action.
In Aim 3, we will determine how the lipid composition of membranes containing CYP17A1, POR, and b5 influences catalytic activity and b5 stimulation in model systems and adrenal tumors. We will also being work with peptide antagonists, which block the b5-CYP17A1 interaction and inhibit androgen synthesis. In this manner, we will systematically define the mechanism of action of b5 on the 17,20-lyase activity of CYP17A1 and pave the way for development of better drugs to safely inhibit androgen (and estrogen) production for the treatment of human diseases.
Testosterone, the male hormone, is important for health, but too much testosterone causes or worsens common diseases, such as polycystic ovary syndrome in 5-8% of women and prostate cancer in 10% of men. Testosterone production requires a key enzyme, named CYP17A1, and inhibitors of CYP17A1 are now used to treat prostate cancer-but-cause side effects, most commonly high blood pressure and low potassium. The purpose of this grant application is to understand how CYP17A1 makes testosterone, so that we can develop better inhibitors, which eliminate testosterone production but do not have the side effects of the drugs used currently.
|Yoshimoto, Francis K; Auchus, Richard J (2016) Rapid kinetic methods to dissect steroidogenic cytochrome P450 reaction mechanisms. J Steroid Biochem Mol Biol 161:13-23|
|Liu, Teresa T; Grubisha, Melanie J; Frahm, Krystle A et al. (2016) Opposing Effects of Cyclooxygenase-2 (COX-2) on Estrogen Receptor Î² (ERÎ²) Response to 5Î±-Reductase Inhibition in Prostate Epithelial Cells. J Biol Chem 291:14747-60|
|Turcu, Adina F; Auchus, Richard J (2016) Novel treatment strategies in congenital adrenal hyperplasia. Curr Opin Endocrinol Diabetes Obes 23:225-32|
|Sondhi, Varun; Owen, Bryn M; Liu, Jiayan et al. (2016) Impaired 17,20-Lyase Activity in Male Mice Lacking Cytochrome b5 in Leydig Cells. Mol Endocrinol 30:469-78|
|Capper, Cameron P; Rae, James M; Auchus, Richard J (2016) The Metabolism, Analysis, and Targeting of Steroid Hormones in Breast and Prostate Cancer. Horm Cancer 7:149-64|
|Turcu, Adina F; Nanba, Aya T; Chomic, Robert et al. (2016) Adrenal-derived 11-oxygenated 19-carbon steroids are the dominant androgens in classic 21-hydroxylase deficiency. Eur J Endocrinol 174:601-9|
|Mizrachi, Dario; Chen, Yujie; Liu, Jiayan et al. (2015) Making water-soluble integral membrane proteins in vivo using an amphipathic protein fusion strategy. Nat Commun 6:6826|
|Simonov, Alexandr N; Holien, Jessica K; Yeung, Joyee Chun In et al. (2015) Mechanistic Scrutiny Identifies a Kinetic Role for Cytochrome b5 Regulation of Human Cytochrome P450c17 (CYP17A1, P450 17A1). PLoS One 10:e0141252|
|Pallan, Pradeep S; Wang, Chunxue; Lei, Li et al. (2015) Human Cytochrome P450 21A2, the Major Steroid 21-Hydroxylase: STRUCTURE OF THE ENZYMEÂ·PROGESTERONE SUBSTRATE COMPLEX AND RATE-LIMITING C-H BOND CLEAVAGE. J Biol Chem 290:13128-43|
|Turcu, Adina F; Rege, Juilee; Chomic, Robert et al. (2015) Profiles of 21-Carbon Steroids in 21-hydroxylase Deficiency. J Clin Endocrinol Metab 100:2283-90|
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