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 are controversial. Among these b5- regulated activities is the 17,20-lyase activity of P450 17A1 (CYP17A1, steroid 17-hydroxylase/17,20-lyase), which is a key step in the biosynthesis of androgens. Diseases of androgen excess and androgen dependence, including polycystic ovary syndrome and prostate cancer, are extremely common, and the P450 17A1 inhibitor abiraterone acetate (AA) is used to treat prostate cancer, proving the relevance of P450 17A1 in human disease. By indiscriminately inhibiting the 17-hydroxylase activity of P450 17A1 in addition to its 17,20-lyase activity, however, AA causes hypertension and potassium loss unless a potent glucocorticoid is co-administered. 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 P450 17A1, 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 biochemical and physical properties of the b5-P450 17A1 complex that enhance the 17,20-lyase reaction. Our central hypothesis is that b5 binding restricts the dynamics of P450 17A1 and bound substrate, which reduces uncoupling of the 17,20-lyase reaction. Consequently, the objectives of this renewal application are to define the biochemical and biophysical nature of the b5-P450 17A1 interaction, determine the rate-limiting step(s) of the 17,20-lyase reaction, and to probe allosteric sites on the complex.
In Aim 1, we will probe the conformational dynamics of P450 17A1 and the changes induced upon binding of substrate and/or b5 using hydrogen-deuterium exchange and mass spectrometry.
In Aim 2, with the Scott laboratory, we will use site-directed mutagenesis, rigorous enzymology studies, and x-ray crystallography to probe the functional properties of a second non-active-site steroid-binding site on P450 17A1 and its influence on the individual steps of the 17,20-lyase reaction.
In Aim 3, with the Waskell laboratory, we will employ pre- steady state kinetic experiments to dissect the rates of individual steps in the catalytic cycle in the presence and absence of b5. In this manner, we will systematically define the mechanism of action of b5 on the 17,20-lyase activity of P450 17A1 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 men?s health, but too much testosterone causes or worsens common diseases, such as prostate cancer in 10% of men or polycystic ovary syndrome in 5-8% of reproductive- aged women. Testosterone production requires the enzyme cytochrome P450 17A1, and inhibitors of P450 17A1 are now used to treat prostate cancer?but?cause side effects including high blood pressure and low potassium. The purpose of this grant application is to understand the details about how P450 17A1 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.
| Peng, Hwei-Ming; Barlow, Chase; Auchus, Richard J (2018) Catalytic modulation of human cytochromes P450 17A1 and P450 11B2 by phospholipid. J Steroid Biochem Mol Biol 181:63-72 |
| Capper, C P; Liu, J; McIntosh, L R et al. (2018) Functional characterization of the G162R and D216H genetic variants of human CYP17A1. J Steroid Biochem Mol Biol 178:159-166 |
| Turcu, Adina F; Nanba, Aya T; Auchus, Richard J (2018) The Rise, Fall, and Resurrection of 11-Oxygenated Androgens in Human Physiology and Disease. Horm Res Paediatr 89:284-291 |
| Rege, Juilee; Turcu, Adina F; Kasa-Vubu, Josephine Z et al. (2018) 11-Ketotestosterone Is the Dominant Circulating Bioactive Androgen During Normal and Premature Adrenarche. J Clin Endocrinol Metab 103:4589-4598 |
| Turcu, Adina F; Auchus, Richard J (2017) Clinical significance of 11-oxygenated androgens. Curr Opin Endocrinol Diabetes Obes 24:252-259 |
| Peng, Hwei-Ming; Auchus, Richard J (2017) Molecular Recognition in Mitochondrial Cytochromes P450 That Catalyze the Terminal Steps of Corticosteroid Biosynthesis. Biochemistry 56:2282-2293 |
| Auchus, Richard J (2017) Steroid 17-hydroxylase and 17,20-lyase deficiencies, genetic and pharmacologic. J Steroid Biochem Mol Biol 165:71-78 |
| Alzahrani, Ali S; Alswailem, Meshael M; Murugan, Avaniyapuram Kannan et al. (2017) A high rate of novel CYP11B1 mutations in Saudi Arabia. J Steroid Biochem Mol Biol 174:217-224 |
| Borges, Beatriz C; Garcia-Galiano, David; da Silveira Cruz-Machado, Sanseray et al. (2017) Obesity-Induced Infertility in Male Mice Is Associated With Disruption of Crisp4 Expression and Sperm Fertilization Capacity. Endocrinology 158:2930-2943 |
| Turcu, Adina F; Mallappa, Ashwini; Elman, Meredith S et al. (2017) 11-Oxygenated Androgens Are Biomarkers of Adrenal Volume and Testicular Adrenal Rest Tumors in 21-Hydroxylase Deficiency. J Clin Endocrinol Metab 102:2701-2710 |
Showing the most recent 10 out of 36 publications
