Structure of Microsomal Enzymes in Estrogen Biosynthesis

描述(由申请人提供)：我们建议通过X光确定 两种膜结合酶的结晶学研究 来自人胎盘：细胞色素P450芳香酶(P450arom)和 雌酮/脱氢表雄酮硫酸酯酶(ES)。P450arom和ES，以及 17β-羟基类固醇脱氢酶1，构成三酶系 负责活性雌激素的生物合成。高水平的雌激素会使 与乳腺肿瘤的发生和增殖有关。当前 他莫昔芬及其类似物等治疗药物是雌激素受体 药效有限且产生不良副作用的拮抗剂。 参与雌激素生物合成的酶提供了替代和 诱人的抗乳腺癌药物靶点，以及这一目标的最终目标 项目就是设计这样的药物。活性雌激素的战略性降低 通过同时抑制两种或全部三种药物，在乳腺肿瘤或其附近的水平 这个酶系统的成员，可能被证明是最务实的方式 防止乳腺肿瘤细胞的增殖。P450arom和Es的鉴定 晶体结构将有助于更好地理解分子 这些酶的作用机理和合理的设计 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级的晶体 ES和P450arom的小单晶。我们将继续使用这两种方式 常规和自动化方法系统地筛查大量的 P450arom的结晶条件。我们已经制备了几种抗体 结合P450arom的片段具有高亲和力和特异性。我们将使用 这些碎片作为工具将P450arom结晶为可溶的 络合物，增加了获得该化合物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个 研究免疫特异性相互作用的分子基础的机会 在P450arom和抗体的互补决定区域之间， 这一信息可用于活动抑制的设计 具有疫苗含义的药效团或免疫反应性多肽。

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.