Activation of androgen biosynthesis and drug metabolism by cytochrome b5

University Of Michigan At Ann Arbor, Ann Arbor MI

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Abstract

Abstract 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 (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. AA inhibits both the 17-hydroxylase and 17,20-lyase activities, and 17-hydroxylase inhibition causes accumulation of 11- deoxycorticosterone, a mineralocorticoid that 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 more safely lower testosterone than AA. We hypothesize disruption of the allosteric effects of b5 on 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 biophysical properties of the b5-P450 17A1 complex that are required to catalyze the 17,20-lyase reaction. Our central hypothesis is that b5 binding allosterically restricts the conformational dynamics of the P450 17A1-substrate complex, which maximizes the coupling of the 17,20-lyase reaction. Consequently, the objectives of this renewal application are to define the mechanism of b5 action on P450 17A1 during the 17,20-lyase reaction and to characterize the allosteric action of b5 on the conformation dynamics of the b5-P450 17A1 complex on a range of timescales. In Aim 1, we will dissect the mechanism of b5 action on the the 17,20-lyase reaction from the oxyferrous P450 17A1-substrate complex through the second electron transfer and chemical steps, using stop-flow spectroscopy and rapid chemical quench with a set of 17-hydroxysteroid substrates and a series of P450 17A1 and b5 mutations that disrupt 17,20-lyase activity. In Aim 2, we will reposition the cysteine residues P450 17A1 scaffold to insert fluorescent tags on specific residues. We will use these tagged P450 17A1 molecules in phospholipid nanodiscs to study the regional dynamics of P450 17A1. In Aim 3, we will probe these changes in native P450 17A1 conformational dynamics on the msec-sec time scale using hydrogen-deuterium exchange. These experiments will systematically define the mechanism of action of b5 on the 17,20-lyase activity of P450 17A1. This knowledge will inform strategies to target the 17,20-lyase activity and to inhibit androgen (and estrogen) production for the treatment of human diseases, without the limitations of current therapies.