Structure of Microsomal Enzymes in Estrogen Biosynthesis

描述（由申请人提供）：我们建议通过X射线测定 晶体学两种膜结合酶的三维结构 来自人胎盘：细胞色素P450芳香酶（P450 arom）和 雌酮/脱氢表雄酮硫酸酯酶（ES）。P450 arom和ES，沿着与 17 β-羟基类固醇脱氢酶1型，构成三酶系统 负责活性雌激素的生物合成。高水平的雌激素 与乳腺肿瘤的发生和增殖有关。电流 治疗剂如他莫昔芬及其类似物是雌激素受体 具有有限功效并产生不希望的副作用的拮抗剂。 参与雌激素生物合成的酶提供了替代和 有吸引力的抗乳腺癌药物靶点，这一最终目标是 该项目旨在设计此类药物。策略性降低活性雌激素 在乳腺肿瘤处或附近的水平，通过同时抑制两种或所有三种 这种酶系统的成员，可能是最实用的方法， 防止乳腺肿瘤细胞增殖。P450 arom和ES的解析 晶体结构将导致更好地了解分子 这些酶的作用机制和合理的设计， 过渡态类似物作为抑制剂，从而最小化或消除 不良副作用。我们已经生长出了衍射质量的晶体， ES和P450 arom的小单晶。我们将继续使用两者 传统的和自动化的方法来系统地筛选大量的 P450芳族化合物的结晶条件。我们已经制备了几种抗体 以高亲和力和特异性结合P450 arom的片段。我们将使用 这些片段作为以可溶性形式结晶P450芳族化合物的工具， 络合物，提高获得这种良好晶体的总体机会 难以结晶的膜蛋白。后一种方法提供了一种 研究免疫特异性相互作用的分子基础的机会 在P450 arom和抗体的互补决定区之间， 这些信息可以用来设计抑制活性的 药效团或具有疫苗意义的免疫应答肽。

DESCRIPTION (provided by applicant): We propose to determine by X-ray crystallography the three-dimensional structures of two membrane-bound enzymes from human placenta: cytochrome P450 aromatase (P450arom) and estrone/dehydroepiandrosterone sulfatase (ES). P450arom and ES, along with 17beta-hydroxysteroid dehydrogenase type 1, constitute the three-enzyme system responsible for biosynthesis of active estrogens. High levels of estrogen have been linked to the initiation and proliferation of breast tumors. Current therapeutic agents, such as Tamoxifen and its analogs, are estrogen-receptor antagonists that have limited efficacy and produce undesirable side effects. The enzymes involved in estrogen biosynthesis provide alternative and attractive anti-breast cancer drug targets, and the ultimate objective of this project is to design such drugs. The strategic lowering of active estrogen levels at or near breast tumors, by simultaneous inhibition of two or all three members of this enzyme system, could turn out to be the most pragmatic way to prevent proliferation of breast-tumor cells. Elucidation of the P450arom and ES crystal structures will lead to better understanding of the molecular mechanisms of action of these enzymes and the rational design of transition-state analogs as inhibitors, thereby minimizing or eliminating undesirable side effects. We have already grown diffraction-quality crystals of ES and small single crystals of P450arom. We will continue to use both conventional and automated methods to screen systematically a large number of crystallization conditions for P450arom. We have prepared several antibody fragments that bind P450arom with high affinity and specificity. We will use these fragments as tools for crystallizing P450arom in the form of soluble complexes, enhancing the overall chances of obtaining good crystals of this difficult-to-crystallize membrane protein. The latter approach provides an opportunity to investigate the molecular basis of immune-specific interactions between P450arom and the complementarity-determining regions of the antibody, and this information could be exploited for the design of activity-suppressing pharmacophores or immune-responsive peptides with vaccine implications.