Structure and Function of Integral Membrane Enzyme Human Aromatase

描述（由申请人提供）：人细胞色素P450芳香化酶（P450arom）是内质网的一种完整的膜血红蛋白，在P450还原酶存在的情况下催化雄激素合成雌激素。尽管在过去的35年里进行了大量的生物化学和生物物理研究，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激素依赖性乳腺癌的有效治疗方法。迄今为止，获得人体p450 - rom衍射质量晶体的尝试尚未成功。我们培养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2.90E分辨率的完整衍射数据，并获得了该结构的溶液。在此，我们建议开展一项调查，以确定人类P450arom的结构-功能关系。我们的假设是，对人类p450芳香配体复合物的原子结构进行功能特性分析，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，并在这些结构基础的指导下进行配体设计和优化，从而产生针对靶标的新型高亲和力抑制剂。验证这一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-羟基睾酮配合物的晶体结构，以及与(2)抑制剂依西美坦、来曲唑、福美斯坦、阿那曲唑、法唑和氨酰硫胺配合物的晶体结构。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，在具体的目标3中，我们将通过结合目标1和目标2的数据和反应中间体的结构数据来研究酶的机制。通过在衍射过程中使用x射线光电子在酶-底物复合晶体中启动芳构化反应，并使用分光光度计对Soret带跃迁进行原位监测，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的实现-通过对接，设计，合成，测试和优化，与化学家合作，发现新的抑制剂。作为该项目的未来方向，我们计划结晶P450arom与P450-还原酶的配合物，以研究电子转移氧化还原反应的机制。公共卫生相关性：芳香化酶是一种独特的酶，在人体内产生所有雌激素。我们提出了一项研究计划，以揭示芳香化酶如何工作的分子细节，以及芳香化酶抑制剂如何阻止它产生雌激素。这项研究的结果将为未来发现对芳香化酶具有高度特异性但副作用最小的新型乳腺癌药物奠定基础。

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.