RECEPTOR MEMBRANE ANCHORING WITH SYNTHETIC ANTIESTROGENS

雌激素受体（hER）是配体诱导的转录因子，对乳腺癌的进展至关重要。 雌激素拮抗剂他莫昔芬通过阻断激素与hER结合来治疗乳腺癌。 虽然他莫昔芬化疗是有益的，这种药物会导致子宫内膜癌。 这种严重的副作用是由于他莫昔芬不能完全抑制细胞核中的hER功能。本研究的长期目标集中于开发一种完全阻断hER功能的新模式。 该范例基于利用合成配体来抑制hER核定位。 拟议的研究将测试以下假设：由于hER影响细胞核中的转录，因此将hER募集到质膜的新型合成配体将完全抑制hER介导的转录激活。 将通过评价和化学合成具有不同hER结合和膜结合结构域的嵌合配体来检验这一假设。 将利用膜锚定功能、嵌合hER配体和hER-配体复合物的表面等离子体共振测量来研究体外膜亲和力。此外，嵌合配体将通过转录报告基因测定和亚细胞定位的绿色荧光蛋白测定进行体内分析。初步工作已经证明，（a）可以有效地合成7-α-取代的β-雌二醇hER配体，和（B）通过将Ras膜靶向序列与hER基因融合，消除了hER促进的报告基因在体内的表达。 因此，Ras膜靶向提供比hER核定位信号强得多的导向力。 这些数据表明，靶向hER质膜的合成配体可能会导致完全的激素阻断，从而提供更有效的抗雌激素治疗。 本文提出的策略有可能提供一种新的核激素受体拮抗剂的方法。

Estrogen Receptors (hERs) are ligand-inducible transcription factors critical for the progression of breast cancers. The estrogen antagonist tamoxifen is used to treat breast carcinomas by blocking hormone binding to hERs. Although tamoxifen chemotherapy is beneficial, this drug causes endometrial (uterine) cancer. This serious side effect results from the inability of tamoxifen to fully inhibit hER function in the cell nucleus. The long-term objective of this research focuses on the development of a novel paradigm for total blockage of hER function. This paradigm is based on utilizing synthetic ligands to prohibit hER nuclear localization. The proposed research will test the following hypothesis: Since hERs affect transcription in the cell nucleus, novel synthetic ligands that recruit hERs to the plasma membrane will fully inhibit hER-mediated transcriptional activation. This hypothesis will be tested through evaluation and chemical synthesis of chimeric ligands with distinct hER-binding and membrane-binding domains. Surface plasmon resonance measurements of membrane anchoring functionality, chimeric hER ligands and hER-ligand complexes will be utilized to investigate membrane affinities in vitro. Furthermore, chimeric ligands will be analyzed in vivo through transcriptional reporter gene assays and green fluorescent protein assays of subcellular localization. Preliminary work has already demonstrated that (a) 7-alpha-substituted beta-estradiol hER ligands can be efficiently synthesized and (b) that hER-promoted reporter gene expression in vivo is abolished by genetically fusing the Ras membrane targeting sequence to hERs. Thus, Ras membrane targeting provides a much stronger directing force than hER nuclear localization signals. These data suggest that synthetic ligands that target hERs to the plasma membrane may confer total hormonal blockage, thereby providing more effective antiestrogen therapeutics. The strategy proposed herein has the potential to provide a novel approach to nuclear hormone receptor antagonism.