Department of Cell Biology and Physiology, University of New Mexico Health Science Center, Albuquerque, New Mexico 87131, USA.
ACS Chem Biol. 2010 Jul 16;5(7):681-90. doi: 10.1021/cb1000636.
Recent clinical studies implicate the role of G protein-coupled estrogen receptor, GPR30, in aggressive forms of breast, ovarian, and endometrial cancers. However, the functional role of GPR30 at cellular and molecular levels remains less clear and controversial, particularly its subcellular location. The primary objective of this study was to develop radiolabeled neutral and charged GPR30-targeted nonsteroidal analogues to understand the influence of ligand charge on cell binding, cellular permeability, and in vivo tumor imaging. Therefore, we developed a series of GPR30-targeted (111/113)In(III)-labeled analogues using macrocyclic and acyclic polyamino-polycarboxylate chelate designs that would render either a net negative or neutral charge. In vitro biological evaluations were performed to determine the role of negatively charged analogues on receptor binding and activation using calcium mobilization and phosphoinositide 3-kinase assays. In vivo evaluations were performed on GPR30-expressing human endometrial Hec50 tumor-bearing mice to characterize the biodistribution and potential application of GPR30-targeted imaging agents for translational research. In vitro functional assays revealed an effect of charge, such that only the neutral analogue activated GPR30-mediated rapid signaling pathways. These observations are consistent with expectations for initial rates of membrane permeability and suggest an intracellular rather than the cell surface location of functional receptor. In vivo studies revealed receptor-mediated uptake of the radiotracer in target organs and tumors; however, further structural modifications will be required for the development of future generations of GPR30-targeted imaging agents with enhanced metabolic properties and decreased nonspecific localization to the intestines.
最近的临床研究表明，G 蛋白偶联雌激素受体 GPR30 在侵袭性乳腺癌、卵巢癌和子宫内膜癌中发挥作用。然而，GPR30 在细胞和分子水平上的功能作用仍不明确且存在争议，尤其是其亚细胞定位。本研究的主要目的是开发放射性标记的中性和带电 GPR30 靶向非甾体类似物，以了解配体电荷对细胞结合、细胞通透性和体内肿瘤成像的影响。因此，我们使用大环和无环多氨基多羧酸盐螯合物设计开发了一系列 GPR30 靶向 (111/113)In(III)-标记的类似物，这些类似物带有净负电荷或中性电荷。进行了体外生物学评估，以使用钙动员和磷酸肌醇 3-激酶测定来确定带负电荷的类似物对受体结合和激活的作用。在表达 GPR30 的人子宫内膜 Hec50 荷瘤小鼠中进行了体内评估，以表征 GPR30 靶向成像剂用于转化研究的生物分布和潜在应用。体外功能测定表明电荷有影响，只有中性类似物激活了 GPR30 介导的快速信号通路。这些观察结果与膜通透性初始速率的预期一致，并表明功能性受体位于细胞内而不是细胞表面。体内研究表明放射性示踪剂在靶器官和肿瘤中通过受体介导摄取；然而，为了开发具有增强代谢特性和减少对肠道非特异性定位的新一代 GPR30 靶向成像剂，还需要进一步的结构修饰。
