Activation of androgen biosynthesis and drug metabolism by cytochrome b5

摘要 细胞色素b5(B5)深刻影响多种细胞色素p450的催化效率 然而，b5对P450酶的这种作用机制(S)是有争议的。其中b5- 调节活性是P450 17A1的17，20-裂解酶活性(CyP17A1，类固醇17-羟基酶/17，20-裂解酶)， 这是雄激素生物合成的关键一步。雄激素过多和雄激素依赖疾病， 包括多囊卵巢综合征和前列腺癌，以及P450 17A1抑制物 阿比特龙(AA)被用于治疗前列腺癌，证明了P450 17A1与人类疾病的相关性。 通过不分青红皂白地抑制P450 17A1的17-羟基酶活性以及其17，20-裂解酶活性， 然而，除非联合使用有效的糖皮质激素，否则AA会导致高血压和钾丢失。 因此，一个未得到满足的临床需要是一种选择性的17，20-裂解酶反应抑制剂，它将安全地降低 睾丸素的产生。我们假设，一种药物，破坏b5与P450 17A1的相互作用，将 在不干扰药物代谢的情况下选择性地阻断17，20-裂解酶反应并降低睾酮的产生 或需要慢性糖皮质激素治疗。 我们的长期目标是阐明b5-p450 17a1复合体的生化和物理性质。 增强了17，20-裂解酶反应。我们的中心假设是b5结合限制了p450的动力学。 17A1和结合底物，减少了17，20-裂解酶反应的解偶联。因此，目标是 该更新申请的目的是定义B5-P450 17A1相互作用的生化和生物物理性质， 确定17，20-裂解酶反应的限速步骤(S)，并探索络合物上的变构位点。 在目标1中，我们将探讨P45017A1的构象动力学以及结合后引起的变化 底物和/或b5使用氢-氚交换和质谱学。在《目标2》中，斯科特 实验室，我们将使用定点突变、严格的酶学研究和x射线结晶学来 探讨P450 17A1上第二个非活性部位类固醇结合位点的功能特性及其影响 在17，20-裂解酶反应的各个步骤上。在Aim 3中，我们将与Waskell实验室一起，采用预 在存在和存在的情况下，分析催化循环中各个步骤的速率的稳态动力学实验 没有b5。通过这种方式，我们将系统地确定b5对17，20-裂解酶的作用机制。 P450 17A1的活性，为开发更好的安全抑制雄激素(和雌激素)的药物铺平道路 用于治疗人类疾病的生产。

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 (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.