MOLECULAR PROBES FOR STEROID RECEPTORS

类固醇受体分子探针

• 批准号: 3483107
• 负责人: JOHN A. KATZENELLENBOGEN
• 金额: $ 18.71万
• 依托单位: UNIVERSITY OF ILLINOIS AT URBANA-CHAMPAIGN
• 依托单位国家: 美国
• 财政年份: 1992
• 资助国家: 美国
• 起止时间: 1992-09-01 至 1995-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/3483107
• 关键词: affinity chromatography; breast neoplasms; drug metabolism; endocrine pharmacology; estrogen inhibitor; estrogen receptors; estrogens; hormone binding protein; laboratory rat; organ culture; photoactivation; uterus

We are developing affinity labeling agents and fluorescent ligands as molecular probes for steroid receptors. I. In order to study the structure of the estrogen receptor and to probe for the gene sites with which it interacts, we will prepare new aziridine- substituted ligands for the estrogen receptor that will be estrogenic (agonistic) and are labeled with iodine-125 at high specific activity. Covalently-labeled receptor will be subjected to proteolysis to generate a 6kDa-peptide fragment that will be sequenced. This should enable the site of ligand binding and convalent attachment to be localized within the known amino acid sequence of the receptor. The gene labeling studies will be done as a collaboration and will involve receptor labeling with the I-125 labeled agonistic aziridine and then protein DNA cross- linking. II. Two approaches will be taken to improve the efficiency of steroid receptor photoaffinity labeling. Since the low efficiency of progesterone receptor photolabeling by R5020 (ca. 5-10%) may be due to the non-reactivity of the excited state, we will prepare R5020 analogs that are fluorine- substituted in a way that will switch the excited state to the more reactive state, thereby increasing the efficiency of radical pair generation and ligand-protein coupling. An acyl azide with a built-in triplet sensitizer will be prepared as an efficient photoaffinity label for the estrogen receptor. III. Three types of fluorescent estrogens will be prepared to enable receptor assay by ligand binding in individual viable breast cancer cells by flow cytometry or image-intensified fluorescence microscopy: Inherently fluorescent ligands based on 2-phenylindenes substituted with appropriate donors and acceptors; photofluorogenic estrogens based on cis-stilbazoles, and ligand conjugates with chelates of europium (III) and terbium (III) ions. The very long luminescence lifetimes of these lanthanide ion complexes may permit the use of the powerful technique of time resolution in receptor assays and microscopic imaging.

我们正在开发亲和力标签剂和荧光灯 配体作为类固醇受体的分子探针。 I.为了 研究雌激素受体的结构，并探测 与之相互作用的基因位点，我们将准备新的Aziridine- 取代的配体用于雌激素受体 雌激素（激动剂），并在高中标记为碘125 具体活动。 共价标记的受体将受到 蛋白水解产生一个6KDA肽片段，将是 测序。 这应该使配体约束的部位和 疗养的附件将位于已知氨基中 受体的酸序列。 基因标记研究将是 作为协作完成，将涉及受体标签 I-125标记为激动剂副氨酸，然后蛋白DNA交叉 链接。 ii。 将采用两种方法来改善 类固醇受体光感标记的效率。 自从 R5020的孕酮受体光标记的低效率 （约5-10％）可能是由于激发 州，我们将准备氟-5020类似物 以将激发态转换为 更具反应性状态，从而提高了自由基的效率 配对生成和配体 - 蛋白耦合。 带有的酰基叠 内置的三重态敏化剂将作为有效的 雌激素受体的光性标签。 iii。 三种类型 荧光雌激素将准备好启用受体分析 通过流动在单个可行乳腺癌细胞中的配体结合 细胞仪或图像密集型荧光显微镜： 固有的基于2-苯基烯的荧光配体 用适当的捐助者和受体代替； 基于顺式 - 斯蒂尔巴唑和配体的光氟化雌激素 与Europium（III）和Terbium（III）离子的螯合物相结合。 这些灯笼离子的发光寿命非常长 复合物可以允许使用强大的时间技术 在受体分析和微观成像中的分辨率。