Structure and Function of Integral Membrane Enzyme Human Aromatase

描述(申请人提供)：人细胞色素P450芳香酶(P450arom)是内质网的一种完整的膜半蛋白，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，P450arom进行了大量的生化和生物物理研究，但其结构与功能的关系及其催化机制仍然知之甚少。用P450arom抑制剂抑制雌激素的生物合成是治疗激素依赖性乳腺癌的有效方法。到目前为止，获得人类P450arom的衍射级晶体的尝试还没有成功。我们生长了从人足月胎盘中提纯的全长、高活性P450arom的雄烯二酮配合物的单晶，收集了完整的衍射数据到2.90E分辨率，并获得了结构的解决方案。在这里，我们建议开展一项研究，以确定人类P450arom的结构与功能的关系。我们的假设是，从功能性质的角度分析人P450芳香配体络合物的原子结构将有助于阐明底物和抑制剂的起源、催化重要残基的作用、反应中间产物的性质以及作用机理，并且根据这些结构基础进行配体设计和优化将导致新的高亲和力抑制剂。检验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-羟基睾酮以及(2)抑制剂依西美坦、来曲唑、福美斯坦、阿那曲唑、法洛唑和氨基谷氨酸亚胺的络合物的晶体结构。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，在特定的目标3中，我们将结合目标1和2的数据与反应中间体的结构数据来研究酶的机理。通过在衍射过程中用X射线光电子引发酶-底物络合物晶体中的芳构化反应，并用分光光度计原位监测Soret带的跃迁，我们计划捕捉反应中间产物的结构快照。完成这些目标将导致具体目标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。作为该项目未来的一个方向，我们计划结晶P450arom与P450-还原酶的络合物，以研究电子转移氧化还原反应的机理。与公众健康相关：芳香酶是一种独特的酶，可以在人体内制造所有雌激素。我们提出了一项研究计划，以揭开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的乳腺癌药物奠定基础，这些药物对芳香酶高度特异，但副作用最小。

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.