BIOCHEMICAL PROBES OF THE ACTIVE SITE OF AROMATASE

芳香酶活性位点的生化探针

• 批准号: 3240443
• 负责人: ROBERT W BRUEGGEMEIER
• 金额: $ 12.42万
• 依托单位: OHIO STATE UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 1988
• 资助国家: 美国
• 起止时间: 1988-05-01 至 1994-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/3240443
• 关键词: active sites; affinity chromatography; androstenedione; aromatase; binding proteins; carbon; chemical models; chemical synthesis; computer simulation; conformation; covalent bond; cytochrome P450; high performance liquid chromatography; human tissue; microsomes; oxidoreductase inhibitor; placenta; protein purification; protein sequence; radionuclides; radiotracer; solvent extraction; steroid hormone analog; stoichiometry; thioether; tritium

The research proposed in this competing renewal application will continue and extend investigations on the utilization of selected aromatase inhibitors as biochemical tools in studying the active site of aromatase. Investigations by the P.I. have demonstrated enhanced affinity of microsomal aromatase for several 7alpha-thioether-substituted 4-androstene- 3,17-diones and 1,4-androstadiene-3,17-diones. New 7-sub-stituted C19- steroids with enhanced affinity for the enzyme and/or greater metabolic stability have been developed and used in structure-activity studies of aromatase inhibition. The overall goal of our research is to use specific aromatase inhibitors to map the steroid binding site and study the structure of the aromatase cytochrome P450 protein. Investigations of the interactions of these 7-substituted steroidal inhibitors with the aromatase enzyme complex have recently focused on employing purified cytochrome P450arom. Isolation and purification of cytochrome P450arom from human placental microsomes is accomplished employing sodium cholate solubilization, ammonium sulfate fractionation, and gel chromatography. Two radiolabeled enzyme-activated inhibitors are being examined, and biochemical studies recently demonstrated direct evidence of covalent binding of these inhibitors to cytochrome P450arom. The specific aims of this renewal application are: (1) Further examination of the extent of covalent binding of radiolabeled enzyme-activated inhibitors with purified aromatase preparations will be performed. The stoichiometry of the covalent binding will be determined for the radiolabeled inhibitors. (2) Inhibitor-bound aromatase protein will be isolated and treated with proteolytic enzymes, and peptides will be separated by HPLC. The amino acid sequence of radiolabeled peptide fragments will be determined by microsequence analysis. (3) New radiolabeled [14C]- and [3H]- analogs of selected enzyme-activated or affinity inhibitors will be prepared. These inhibitors will be utilized in the examination of various regions in the steroid binding site. Also, certain radiolabeled inhibitors will be employed to study the mechanism of the enzyme-activated irreversible binding. (4) Molecular modeling and computational chemistry will be utilized in determination of possible conformations of the active site of cytochrome P450arom and possible interactions with inhibitors. Thus, radiolabeled analogs of selected inhibitors will be prepared and utilized to probe the active site of purified aromatase and the mechanism of inactivation. Knowledge of the chemistry of the enzymatic "pocket" of aromatase is critical in the further design and development of more effective and potent agents for the control of estrogenic processes and for the treatment of advanced estrogen-dependent breast cancer.

在此竞争更新申请中提出的研究将继续 并扩展对选定芳香酶使用的研究 抑制剂作为研究芳香酶的活性位点的生化工具。 P.I.的调查已经表现出增强的亲和力 微粒体芳香酶，用于几个7Alpha-硫骨取代的4-丁香烯 - 3,17-二离子和1,4-androstadiene-3,17-Diones。 新的7-Sub-Situed C19-- 类固醇对酶和/或代谢更大的亲和力增强 稳定性已开发并用于结构活性研究 芳香酶抑制。 我们研究的总体目标是使用特定 芳香酶抑制剂以绘制类固醇结合位点并研究 芳香酶细胞色素P450蛋白的结构。 调查 这些7个取代的类固醇抑制剂与芳香酶的相互作用 酶复合物最近专注于采用纯化的细胞色素 P450AROM。 从人类的细胞色素P450arom隔离和纯化 胎盘微粒体采用巧克力钠完成 溶解度，硫酸铵分馏和凝胶色谱法。 正在检查两个放射性标记的酶激活抑制剂，并 生化研究最近证明了共价的直接证据 这些抑制剂与细胞色素P450arom的结合。 具体目的 此更新应用是：（1）进一步检查 放射性标记的酶激活抑制剂与纯化的抑制剂的共价结合 将执行芳香酶制剂。 化学计量的 将确定用于放射性标记抑制剂的共价结合。 （2） 抑制剂结合的芳香酶蛋白将被分离并用 蛋白水解酶和肽将通过HPLC分离。 氨基 放射性标记肽片段的酸序列将由 Microsequence分析。 （3）新的放射性标记[14C]和[3H] - 类似物的类似物 将准备选定的酶激活或亲和力抑制剂。 这些 抑制剂将用于检查 类固醇结合位点。 另外，某些放射标记的抑制剂将是 用于研究酶激活不可逆的机制 结合。 （4）分子建模和计算化学将是 用于确定活性位点的可能构象 细胞色素P450AROM和可能与抑制剂相互作用。 因此， 将准备并利用选定抑制剂的放射性标签类似物 探测纯化的芳香酶的活性位点和机制 失活。 了解化学的酶“口袋”的化学知识 芳香酶在进一步的设计和开发中至关重要 有效且有效的药物，用于控制雌激素过程和 治疗晚期雌激素依赖性乳腺癌。