BIOCHEMICAL PROBES OF THE ACTIVE SITE OF AROMATASE

芳香酶活性位点的生化探针

• 批准号: 3240441
• 负责人: ROBERT W BRUEGGEMEIER
• 金额: $ 10.18万
• 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 1991-04-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/3240441
• 关键词: affinity chromatography; androstenedione; binding proteins; chemical synthesis; cholates; cytochrome P450; enzyme complex; enzyme induction /repression; enzyme structure; estrogen inhibitor; human tissue; microsomes; oxidoreductase inhibitor; placenta; protein sequence; radiotracer; solvent extraction; steroid hormone analog; thioether; tritium; unspecific monooxygenase

The proposed research will utilize 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 7 alpha-thioether substituted 4- androstene-3,17-diones, particularly analogs with aryl amine functionalities. New 7-substituted C19-steroids with enhanced affinity for the enzyme and/or greater metabolic stability have recently been developed and will be used to examine the aromatase protein. Specifically: (1) Aromatase will be purified from human placental microsomes by cholate solubilization, ammonium sulfate fractionation, and a combination of affinity and adsorption chromatography. (2) Radiolabeled 3H-and 125I- analogs of selected enzyme-activated inhibitors will be prepared. New synthetic compounds will be examined in microsomal preparations for inhibitory activity. (3) The radiolabeled enzyme- activated inhibitors prepared above will be incubated with purified aromatase preparations to examine the extent of irreversible binding. 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. Thus, radiolabeled analogs of selected inhibitors will be prepared and utilized to probe the active site of purified aromatase. 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.

拟议的研究将利用选定的芳香酶抑制剂 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7α-thioeth的4- 雄烯-3,17-二二离子，特别是与芳基胺的类似物 功能。 新的7个取代的C19-类固醇，增强 对酶和/或代谢稳定性的亲和力具有 最近开发了，将用于检查 芳香酶蛋白。 具体：（1）芳香酶将被纯化 来自人胎盘微粒体，通过巧克力溶解化， 硫酸铵分馏和亲和力的组合 和吸附色谱。 （2）放射性标记的3H和125i 将制备选定的酶激活抑制剂的类似物。 新合成化合物将在微粒体中检查 抑制活性的准备。 （3）放射性标记的酶 - 上面准备的激活抑制剂将与 纯化的芳香酶制剂，以检查 不可逆的结合。 抑制剂结合的芳香酶蛋白将是 用蛋白水解酶和肽分离和处理 由HPLC分开。 放射性标记的氨基酸序列 肽片段将通过微序列分析确定。 因此，将制备选定抑制剂的放射标记类似物 并用于探测纯化芳香酶的活性位点。 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 控制雌激素的更有效和有效的药物 过程和治疗晚期雌激素依赖性 乳腺癌。