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

• 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
• 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

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.

摘要 细胞色素b5（b5）深刻地影响许多细胞色素P450催化的 然而，b5对P450酶的这种作用的机制是有争议的。其中，B5- 受调节的活性是P450 17 A1的17，20-裂解酶活性（CYP 17 A1，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性疾病， 包括多囊卵巢综合症和前列腺癌，都非常常见，而P450 17 A1抑制剂 醋酸阿比特龙（AA）用于治疗前列腺癌，证明了P450 17 A1在人类疾病中的相关性。 通过不加区别地抑制P450 17 A1的17-羟化酶活性以及其17，20-裂解酶活性， 然而，除非同时给予有效的糖皮质激素，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，其将安全地降低17，20-裂解酶反应的活性。 睾丸激素的产生我们假设，一种药物，破坏了b5与P450 17 A1的相互作用， 选择性阻断17，20-裂解酶反应，降低睾酮的产生，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 17 A1复合物的生物化学和物理性质 能增强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 17 A1和结合的底物，这减少了17，20-裂解酶反应的解偶联。因此，目标 本更新申请的目的是确定b5-P450 17 A1相互作用的生物化学和生物物理学性质， 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。 在目的1中，我们将探索P450 17 A1的构象动力学和结合后诱导的变化 的底物和/或B5。在Aim 2中，Scott 实验室，我们将使用定点诱变，严格的酶学研究，和X射线晶体学， 探索P450 17 A1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 在17，20-裂解酶反应的各个步骤上。在目标3中，与Waskell实验室一起，我们将采用预- 稳态动力学实验，以剖析催化循环中的各个步骤的速率， 没有B5。以这种方式，我们将系统地定义b5对17，20-裂解酶的作用机制 P450 17 A1的活性，并为开发更好的药物安全地抑制雄激素（和雌激素）铺平道路 生产用于治疗人类疾病。