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STRUCTURE OF MICROSOMAL ENZYMES--ESTROGEN BIOSYN

微生物酶的结构--雌激素生物合成酶

基本信息

批准号：
6182203
负责人：
DEBASHIS GHOSH
金额：
$ 11.39万
依托单位：
HAUPTMAN-WOODWARD MEDICAL RESEARCH INST
依托单位国家：
美国
项目类别：
财政年份：
1999
资助国家：
美国
起止时间：
1999-04-01 至 2001-03-31
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/6182203
关键词：
aromatase crystallization enzyme inhibitors enzyme structure estradiol estrogen receptors estrogens molecular cloning monoclonal antibody protein purification protein sequence steroid hormone biosynthesis sulfatases unspecific monooxygenase

项目摘要

Estrogens play essential roles in physiology and pathology of human development and reproduction. High-levels of the most active estrogen, 17beta-estradiol, have been linked to hormone-dependent breast cancer. Three enzymes primarily responsible for intracrine biosynthesis of nearly all of 17beta-estradiol in postmenopausal women and also in breast tumor cells are Cytochrome P450 Aromatase (P450arom), Type 1 17beta- Hydroxysteroid Dehydrogenase (17beta-HSD1) and Estrone/DHEA Sulfatase (ES). Although significant advances have been made in our laboratory towards understanding the structure-function relationship of 17beta-HSD1, three-dimensional structures of P450arom (55kDa) and ES (Subunit: 64kDa) are not yet available. The major objective of this proposed research is crystallization of two membrane-bound naturally-occurring, full-length, fully-active human enzymes P450arom and ES, in order to proceed with their high-resolution crystal structure analysis. No mammalian cytochrome P450 or steroid sulfatase has been crystallized to date. We have optimized the purification processes for both enzymes from microsomal fractions of human placenta and are able to routinely obtain milligram quantities of the highly purified, homogeneous enzymes suitable for crystallization. We have completed the initial biochemical characterization and substrate-specificity studies of purified ES. We have prepared an activity-suppressing monoclonal antibody to P450arom. Single microcrystals of P450arom, showing the characteristic red coloration and active enzyme, have been obtained. We have initiated cloning of the variable domain Fv of the anti-P450arom antibody. The plan is to use the Fv fragment in P450arom crystallization and crystal structure analysis of the P450arom-Fv complex. Affinity-optimized recombinant anti- P450arom Fv could have therapeutic as well as diagnostic applications. Most of the anti-breast cancer agents in use to-day, such as tamoxifen or its analogs, work by blocking the estrogen receptor and have been shown to have serious side-effects. The enzymes pose attractive alternative targets for lowering estrogen levels in breast tumor tissues. Simultaneous inhibition of two or all three enzymes could not only be one of the most effective treatments of breast cancer, but also provide means for its prevention in high-risk populations. The long-term objective is structure-based design of inhibitors with high specificities, but mutually exclusive affinities for their respective targets, and no affinity for the estrogen receptor.

雌激素在人类发育和生殖的生理和病理中起着重要作用。高水平的最活跃的雌激素，17 β-雌二醇，与乳腺癌有关。在绝经后妇女和乳腺肿瘤细胞中，主要负责几乎所有17 β-雌二醇的内分泌生物合成的三种酶是细胞色素P450芳香酶（P450 arom）、1型17 β-羟基类固醇脱氢酶（17 β-HSD 1）和雌酮/DHEA硫酸酯酶（ES）。虽然我们的实验室在理解17 β-HSD 1的结构-功能关系方面取得了重大进展，但P450 arom（55 kDa）和ES（亚基：64 kDa）的三维结构尚未获得。这项研究的主要目的是结晶两种膜结合的天然存在的全长完全活性的人类酶P450 arom和ES，以进行高分辨率的晶体结构分析。迄今为止，没有哺乳动物细胞色素P450或类固醇硫酸酯酶被结晶。我们已经优化了人胎盘微粒体组分中两种酶的纯化工艺，并且能够常规获得毫克量的高度纯化的、均质的、适合于结晶的酶。我们已经完成了初步的生化特性和底物特异性研究纯化ES。我们制备了抗P450 arom活性抑制单克隆抗体。获得了P450 arom的单晶，显示出特征性的红色和活性酶。我们已经开始克隆抗P450 arom抗体的可变结构域Fv。计划将Fv片段用于P450 arom结晶和P450 arom-Fv复合物的晶体结构分析。亲和力优化的重组抗P450 arom Fv可以具有治疗以及诊断应用。目前使用的大多数抗乳腺癌药物，如他莫昔芬或其类似物，通过阻断雌激素受体起作用，并已被证明具有严重的副作用。这些酶为降低乳腺肿瘤组织中的雌激素水平提供了有吸引力的替代靶点。同时抑制两种或所有三种酶不仅是乳腺癌最有效的治疗方法之一，而且还为高危人群的预防提供了手段。长期目标是基于结构设计具有高特异性的抑制剂，但对各自靶点的亲和力相互排斥，并且对雌激素受体没有亲和力。