Structure and Function of Integral Membrane Enzyme Human Aromatase

人芳香酶整合膜酶的结构与功能

• 批准号: 7743555
• 负责人: DEBASHIS GHOSH
• 金额: $ 27.29万
• 依托单位: HAUPTMAN-WOODWARD MEDICAL RESEARCH INST
• 依托单位国家: 美国
• 财政年份: 2009
• 资助国家: 美国
• 起止时间: 2009-01-01 至 2010-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7743555
• 关键词: 15q21.1; Active Sites; Adverse effects; Affinity; Amino Acids; Aminoglutethimide; Anabolism; Androgens; Androstenedione; Antineoplastic Agents; Aromatase; Aromatase Inhibitors; Binding; Biochemical; CYP19A1 gene; Catalysis; Chromosomes; Collaborations; Complex; Crystallization; Cytochrome P450; Cytochromes; Data; Data Set; Databases; Docking; Dose; Electron Transport; Endoplasmic Reticulum; Enzymes; Estradiol; Estriol; Estrogens; Estrone; Exemestane; Fadrozole; Flavoproteins; Formestane; Future; Genes; Goals; Hemeproteins; Hormones; Human; Human body; Hydrophobicity; In Situ; Investigation; Laboratories; Lead; Length; Letrozole; Ligands; Lipid Bilayers; Membrane; Methods; Molecular; Molecular Structure; Monitor; NADP; NADPH-Ferrihemoprotein Reductase; Nature; Oxidation-Reduction; Oxidoreductase; Pathway interactions; Placenta; Predisposition; Process; Property; Proteins; Reaction; Recombinants; Reporting; Research; Resolution; Roentgen Rays; Role; Solutions; Specificity; Structure; Structure-Activity Relationship; Substrate Specificity; Temperature; Testing; Testosterone; Work; anastrozole; base; design; effective therapy; enzyme mechanism; enzyme substrate complex; hormone therapy; improved; inhibitor/antagonist; malignant breast neoplasm; member; methyl group; novel; prevent; public health relevance; research study

DESCRIPTION (provided by applicant): Human cytochrome P450 aromatase (P450arom), an integral membrane hemeprotein of the endoplasmic reticulum, catalyzes the synthesis of estrogens from androgens in the presence of P450-reductase. Despite intense biochemical and biophysical investigations for the past 35 years, the structure-function relationships of P450arom and its catalytic mechanism remain poorly understood. Inhibition of estrogen biosynthesis by P450arom inhibitors is an effective therapy for hormone-dependent breast cancers. Attempts to obtain diffraction-quality crystals of human P450arom have so far been unsuccessful. We have grown single crystals of the androstenedione-complex of the full-length, highly active P450arom purified from human term placenta, gathered complete diffraction data to 2.90E resolution and obtained a solution for the structure. Here, we propose to launch an investigation in order to determine of the structure-function relationships of human P450arom. Our hypothesis is that analysis of the atomic structures of human P450arom-ligand complexes in terms of their functional properties will lead to the elucidation of the origin of substrate and inhibitor specificities, roles of the catalytically important residues, nature of the reaction intermediates, as well as the mechanism of action, and that ligand design and optimization guided by these structural basis will lead to novel high-affinity inhibitors that are exclusive for the target. The specific aims to test the hypothesis are to determine the crystal structures of the complexes of P450arom with its (1) natural substrates androstenedione, testosterone, and 161-hydroxy-testosterone, as well as with (2) the inhibitors exemestane, letrozole, formestane, anastrozole, fadrozole and aminoglutethimide. These findings will reveal the molecular mechanism for substrate and inhibitor specificities and help build a structure-activity database based on the atomic level descriptions of the enzyme-ligand interactions. Next, in specific aim 3 we will investigate the enzyme mechanism by combining the data from aims 1 and 2 with structural data on reaction intermediates. By initiating the aromatization reaction in an enzyme-substrate complex crystal with X-ray photoelectrons during diffraction and in situ monitoring of the Soret band transition with a spectrophotometer, we plan to capture structural snap shots of the reaction intermediates. Completion of these goals will lead to the implementation of specific aim 4 - discovery of new inhibitors through docking, design, synthesis, testing, and optimization, in collaboration with chemists. As a future direction of the project, we plan to crystallize a complex of P450arom with P450- reductase for investigating the mechanism of redox reactions by electron transfer. PUBLIC HEALTH RELEVANCE: Aromatase is a unique enzyme that makes all estrogens in the human body. We propose a research plan to unravel the molecular details of how aromatase works and how aromatase inhibitors prevent it from making estrogens. Results from this investigation will form the basis for future discovery of novel breast cancer drugs that are highly specific for aromatase but cause minimal side effects.

描述（由申请人提供）：人细胞色素P450芳香酶（P450arom）是内质网的整合膜血红素蛋白，在P450还原酶存在下催化雄激素合成雌激素。尽管过去 35 年进行了大量的生化和生物物理研究，但 P450arom 的结构-功能关系及其催化机制仍然知之甚少。 P450arom 抑制剂抑制雌激素生物合成是治疗激素依赖性乳腺癌的有效方法。迄今为止，获得人 P450arom 衍射质量晶体的尝试尚未成功。我们生长了从人足月胎盘中纯化的全长、高活性 P450arom 的雄烯二酮复合物的单晶，收集了分辨率为 2.90E 的完整衍射数据，并获得了该结构的解决方案。在这里，我们建议开展一项研究，以确定人类 P450arom 的结构与功能关系。我们的假设是，根据功能特性对人 P450arom-配体复合物的原子结构进行分析，将有助于阐明底物和抑制剂特异性的起源、催化重要残基的作用、反应中间体的性质以及作用机制，并且以这些结构基础为指导的配体设计和优化将产生独特的新型高亲和力抑制剂。 为目标。检验该假设的具体目的是确定 P450arom 与其 (1) 天然底物雄烯二酮、睾酮和 161-羟基睾酮，以及 (2) 抑制剂依西美坦、来曲唑、福美坦、阿那曲唑、法屈唑和氨基鲁米特的复合物的晶体结构。这些发现将揭示底物和抑制剂特异性的分子机制，并有助于建立基于酶-配体相互作用的原子水平描述的结构-活性数据库。接下来，在具体目标 3 中，我们将通过将目标 1 和 2 的数据与反应中间体的结构数据相结合来研究酶机制。通过在衍射过程中用 X 射线光电子引发酶-底物复合晶体中的芳构化反应，并用分光光度计原位监测索雷带跃迁，我们计划捕获反应中间体的结构快照。这些目标的完成将导致具体目标4的实施——与化学家合作，通过对接、设计、合成、测试和优化发现新的抑制剂。作为该项目未来的方向，我们计划结晶 P450arom 与 P450-还原酶的复合物，以研究电子转移氧化还原反应的机制。公共健康相关性：芳香酶是一种独特的酶，可产生人体内的所有雌激素。我们提出了一项研究计划，以揭示芳香酶如何发挥作用以及芳香酶抑制剂如何阻止其产生雌激素的分子细节。这项研究的结果将为未来发现新型乳腺癌药物奠定基础，这些药物对芳香酶具有高度特异性，但副作用最小。