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：
9913550
负责人：
RICHARD J. AUCHUS
金额：
$ 47.81万
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 2022-04-30
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/9913550
关键词：
21-hydroxylase deficiency Acetates Active Sites Adverse drug effect Affect Allosteric Site Anabolism Androgen Therapy Androgens Animals Antineoplastic Agents Applications Grants Binding Binding Sites Biochemical Biochemistry Biological Biophysics CYP17A1 gene Chronic Clinical Clinical Trials Complex Consumption Coupling Crystallography Cytochrome P450 Cytochromes b5 Data Dependence Deuterium Development Disease Dose Drug Metabolic Detoxication Drug usage Enzymatic Biochemistry Enzymes Estrogens Fruit Gene Expression Genetic Polymorphism Glucocorticoids Goals Grant Hormones Hydrocortisone Hydrogen Hydrophobicity Hydroxylation Hypertension Impairment In Vitro Individual Kinetics Laboratories Malignant neoplasm of prostate Mass Spectrum Analysis Medical Michigan Mixed Function Oxygenases Molecular Conformation Mus Mutation NADP Nature Patients Pharmaceutical Preparations Pharmacology Pigments Plants Polycystic Ovary Syndrome Potassium Prednisone Process Production Property Prostatic hypertrophy Reaction Regulation Reproduction Risk Side Site-Directed Mutagenesis Steroid 17-alpha-monooxygenase Steroids Structure Testosterone Universities Woman Work X-Ray Crystallography Xenobiotics abiraterone aged androgen sensitive catalyst drug metabolism experimental study human disease hydrophilicity in vivo inhibitor/antagonist leydig interstitial cell male male health men physical property reproductive side effect wasting

项目摘要

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.

抽象的细胞色素 b5 (b5) 深刻影响许多细胞色素 P450 催化的催化效率反应，但 b5 对 P450 酶的这种作用机制尚存在争议。其中b5- 受调节的活性是 P450 17A1 的 17,20-裂解酶活性（CYP17A1，类固醇 17-羟化酶/17,20-裂解酶），这是雄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，非常常见，P450 17A1 抑制剂醋酸阿比特龙 (AA) 用于治疗前列腺癌，证明了 P450 17A1 在人类疾病中的相关性。通过不加选择地抑制 P450 17A1 的 17,20-裂解酶活性以及 17-羟化酶活性，然而，除非同时使用强效糖皮质激素，否则 AA 会导致高血压和钾流失。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。我们假设一种药物可以破坏 b5 与 P450 17A1 的相互作用，选择性阻断 17,20-裂解酶反应并降低睾酮产生，而不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-P450 17A1 复合物的生化和物理特性增强 17,20-裂合酶反应。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 17,20-裂解酶反应的解偶联。因此，目标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，并探测复合物上的变构位点。在目标 1 中，我们将探讨 P450 17A1 的构象动力学以及结合后诱导的变化使用氢-氘交换和质谱分析底物和/或b5。在目标 2 中，与 Scott 在实验室中，我们将使用定点诱变、严格的酶学研究和 X 射线晶体学来探究 P450 17A1 上第二个非活性位点类固醇结合位点的功能特性及其影响 17,20-裂解酶反应的各个步骤。在目标 3 中，我们将与 Waskell 实验室合作，采用预稳态动力学实验剖析催化循环中各个步骤的速率缺乏b5。通过这种方式，我们将系统地定义b5对17,20-裂合酶的作用机制 P450 17A1 的活性，并为开发更好的药物来安全抑制雄激素（和雌激素）铺平道路用于治疗人类疾病的生产。