Activation of androgen biosynthesis and drug metabolism by cytochrome b5

描述（由申请人提供）：细胞色素b5 （b5）深刻影响许多细胞色素P450催化反应的催化效率，但b5对P450酶的作用机制尚不清楚。在这些b5调控的活性中，CYP17A1 （P450c17，类固醇17-羟化酶/17,20-裂解酶）的17,20-裂解酶活性是雄激素和雌激素生物合成的关键步骤。多囊卵巢综合征、前列腺癌等雄激素过量和雄激素依赖疾病极为常见，而CYP17A1抑制剂醋酸阿比特龙（AA）被用于治疗前列腺癌，证明了CYP17A1与人类疾病的相关性。然而，通过不加区别地抑制CYP17A1的17-羟化酶活性和1720 -裂解酶活性，AA导致高血压和钾流失，除非与强的松（一种有效的糖皮质激素）一起服用。因此，一种未满足的临床需求是选择性抑制17,20裂解酶反应，这将安全地降低睾酮的产生。我们假设有一种药物可以破坏b5与CYP17A1-POR复合物的相互作用，从而选择性地阻断1720 -裂解酶反应并降低睾酮的产生，而不干扰药物代谢或需要慢性糖皮质激素治疗。我们的长期目标是阐明b5-CYP17A1相互作用的分子细节，并开发破坏这种相互作用的方法，从而选择性地抑制CYP17A1的17,20裂解酶活性。我们的中心假设是b5上的两个带负电荷的残基与CYP17A1上的特定精氨酸残基相互作用，以增加产物释放的速度，这是1720裂解酶反应的限速步骤。在Aim 1中，我们将使用定点诱变和质谱法来确定b5上刺激17,20-裂解酶活性所需的关键残基的空间和电子要求，以及b5与CYP17A1相互作用的性质。在Aim 2中，我们将采用稳态前动力学实验来确定17,20裂解酶反应的限速步骤，从而推断b5作用的微观步骤。在Aim 3中，我们将确定含有CYP17A1、POR和b5的膜的脂质组成如何影响模型系统和肾上腺肿瘤中的催化活性和b5刺激。我们还将研究肽拮抗剂，它可以阻断b5-CYP17A1的相互作用，抑制雄激素的合成。通过这种方式，我们将系统地确定b5对CYP17A1的17,20-裂解酶活性的作用机制，并为开发更好的药物来安全抑制雄激素（和雌激素）的产生，以治疗人类疾病铺平道路。

DESCRIPTION (provided by applicant): 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.