RECEPTOR MEMBRANE ANCHORING WITH SYNTHETIC ANTIESTROGENS

雌激素受体(HERS)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HERS的结合来治疗乳腺癌。尽管他莫昔芬化疗是有益的，但这种药物会导致子宫内膜(子宫)癌。这种严重的副作用是因为他莫昔芬无法完全抑制她在细胞核中的功能。这项研究的长期目标是开发一种新的范例来完全阻断她的功能。这种模式是基于利用合成配体来禁止她的核定位。这项拟议的研究将检验以下假设：由于HERS影响细胞核内的转录，因此将HERS聚集到质膜上的新型合成配体将完全抑制HER介导的转录激活。这一假设将通过评估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验证。膜锚定官能度、嵌合HER配体和HER-配体复合体的表面等离子体共振测量将被用于研究膜的体外亲和力。此外，嵌合配体将通过转录报告基因分析和绿色荧光蛋白亚细胞定位分析在体内进行分析。初步的工作已经证明：(A)7-α-取代的β-雌二醇HER配体可以被有效地合成；(B)通过将RAS膜靶向序列与HER的基因融合，HER促进的报告基因在体内的表达被取消。因此，RAS膜靶向提供了比她的核定位信号更强的定向力。这些数据表明，针对质膜HER的合成配体可能会完全阻断激素，从而提供更有效的抗雌激素治疗药物。本文提出的策略有可能为核激素受体拮抗提供一种新的方法。

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.