Activation of androgen biosynthesis and drug metabolism by cytochrome b5

细胞色素 b5 激活雄激素生物合成和药物代谢

基本信息

批准号：
8691516
负责人：
RICHARD J. AUCHUS
金额：
$ 34.21万
依托单位：
UNIVERSITY OF MICHIGAN AT ANN ARBOR
依托单位国家：
美国
项目类别：
财政年份：
2009
资助国家：
美国
起止时间：
2009-09-30 至 2018-03-31
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/8691516
关键词：
Acetates Adrenal Gland Neoplasms Adverse effects Anabolism Androgen Therapy Androgens Animals Applications Grants Arginine Biochemical Biochemistry Biological Models CYP17A1 gene CYP2C19 gene CYP2E1 gene CYP3A4 gene Cancer Etiology Charge Chemicals Chronic Clinical Complex Cytochrome P450 Cytochromes b5 Dependence Development Disease Dissociation Drug Metabolic Detoxication Drug usage Electronics Enzymes Estrogens Fruit Gene Expression Regulation Genetic Polymorphism Glucocorticoids Goals Grant Health Hormones Human Hypertension In Vitro Kinetics Knowledge Laboratories Ligands Lipids Malignant neoplasm of prostate Mass Spectrum Analysis Medical Membrane Microscopic Mixed Function Oxygenases Molecular Mus Names Nature Patients Peptides Pharmaceutical Preparations Phospholipids Pigments Plants Polycystic Ovary Syndrome Potassium Prednisone Process Production Property Prostatic Diseases Prostatic hypertrophy Reaction Regulation Sexual Maturation Site-Directed Mutagenesis Sodium Chloride Staging Steroid 17-alpha-monooxygenase Steroid 21-Monooxygenase Testosterone Woman Work Xenobiotics abiraterone catalyst cost defined contribution drug metabolism human disease in vivo inhibitor/antagonist leydig interstitial cell male men novel strategies public health relevance research study success

项目摘要

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.

描述（由申请人提供）：细胞色素b5（b5）深刻地影响许多细胞色素P450催化反应的催化效率，但b5对P450酶的这种作用的机制尚不清楚。在这些 b5 调节的活性中，CYP17A1 的 17,20-裂解酶活性（P450c17，类固醇 17-羟化酶/17,20-裂解酶）是雄激素和雌激素生物合成的关键步骤。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极为常见，CYP17A1抑制剂醋酸阿比特龙（AA）用于治疗前列腺癌，证明了CYP17A1在人类疾病中的相关性。然而，除了 17,20-裂解酶活性之外，AA 还会不加区别地抑制 CYP17A1 的 17-羟化酶活性，从而导致高血压和钾丢失，除非与强的松（一种强效糖皮质激素）一起使用。因此，未满足的临床需求是17,20-裂解酶反应的选择性抑制剂，它将安全地降低睾酮的产生。我们假设一种破坏 b5 与 CYP17A1-POR 复合物相互作用的药物将选择性地阻断 17,20-裂解酶反应并降低睾酮产生，而不会干扰药物代谢或需要长期糖皮质激素治疗。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，以提高产物释放速率，这是 17,20-裂解酶反应的限速步骤。在目标 1 中，我们将使用定点诱变和质谱法来确定刺激 17,20-裂解酶活性所需的 b5 上关键残基的空间和电子需求以及 b5 与 CYP17A1 相互作用的性质。在目标2中，我们将采用前稳态动力学实验来确定17,20-裂解酶反应的限速步骤，从而推导出b5作用的微观步骤。在目标 3 中，我们将确定含有 CYP17A1、POR 和 b5 的膜的脂质成分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 相互作用并抑制雄激素合成。通过这种方式，我们将系统地确定b5对CYP17A1 17,20-裂合酶活性的作用机制，并为开发更好的药物以安全地抑制雄激素（和雌激素）产生以治疗人类疾病铺平道路。