MOLECULAR PROBES FOR STEROID RECEPTORS

我们正在开发亲和标记剂和荧光标记剂。 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， 与之相互作用的基因位点，我们将制备新的氮丙啶- 雌激素受体的取代配体， 雌激素（激动剂）并用高碘-125标记 具体活动。 共价标记的受体将受到 蛋白水解产生一个6 kDa的肽片段， 测序 这将使配体结合位点和 共价连接位于已知的氨基内， 受体的酸性序列。 基因标记研究将是 作为一个合作完成，将涉及受体标记与 I-125标记的激动剂氮丙啶，然后蛋白质DNA交叉， 链接。 二. 将采取两种方法来改善 类固醇受体光亲和标记的效率。 以来 R5020光标记孕酮受体效率低 （大约。5-10%）可能是由于激发的非反应性 如前所述，我们将制备R5020类似物， 以一种将激发态切换到 更多的反应状态，从而提高自由基的效率 配对产生和配体-蛋白质偶联。 一种酰基叠氮化物， 一种内置的三重态敏化剂将被制备成一种有效的 雌激素受体的光亲和标记。 三. 三种类型的 将制备荧光雌激素以进行受体测定 通过流式细胞术检测单个活乳腺癌细胞中的配体结合 流式细胞术或图像增强荧光显微术： 基于2-苯基茚的固有荧光配体 以适当的供体和受体取代; 基于顺式-芪唑的光致荧光雌激素和配体 与铕（III）和铽（III）离子的螯合物的缀合物。 这些镧系离子的发光寿命很长， 复合体可能允许使用强大的时间技术 在受体测定和显微成像中的分辨率。

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.