MOLECULAR PROBES FOR STEROID RECEPTORS

类固醇受体分子探针

• 批准号: 2136925
• 负责人: JOHN A. KATZENELLENBOGEN
• 金额: $ 17.93万
• 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/2136925
• 关键词: affinity chromatography; breast neoplasms; drug metabolism; endocrine pharmacology; estrogen inhibitor; estrogen receptors; estrogens; hormone binding protein; hormone therapy; laboratory rat; neoplasm /cancer chemotherapy; 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.

我们正在开发亲和标记剂和荧光 配体作为类固醇受体的分子探针。一、为了 研究雌激素受体的结构，探索雌激素受体 它与之相互作用的基因位置，我们将制备新的氮杂环丙烷- 雌激素受体的取代配体将是 雌激素(激动剂)，高剂量碘-125标记 特定活动。共价标记的受体将受到 以产生6 kDa的多肽片段，该片段将被 已排序。这应该能够使配体结合的位置和 共价键定位于已知的氨基 受体的酸序列。基因标记研究将是 作为协作完成，将涉及到受体标记 I-125标记激动剂氮杂环丙氨酸与蛋白质DNA交叉反应 链接。II.将采取两个方法来改善 类固醇受体光亲和标记的效率。自.以来 R5020光标记孕激素受体效率低 (约5-10%)可能是由于被激发的 国家，我们将准备R5020类似物，是氟- 替换的方式会将激发态切换到 更多的反应状态，从而提高自由基的效率 配对生成和配体-蛋白质偶联。一种酰基叠氮化物 一种内置的三重态增敏剂将作为一种有效的 雌激素受体的光亲和标记。三、三类 将制备荧光雌激素以使受体分析成为可能 通过Flow在单个存活的乳腺癌细胞中通过配体结合 细胞学或图像增强荧光显微镜： 基于2-苯基取代基的固有荧光配体 取而代之的是适当的供体和受体； 基于顺式二苯乙烯类化合物的光致荧光雌激素及其配体 与Eu(III)和Tb(III)离子的络合物偶联。 这些稀土离子的超长发光寿命 复合体可能允许使用强大的时间技术 在受体分析和显微成像中的分辨率。