Molecular imaging of drug-modulated protein-protein interactions in living subjects
Molecular imaging of drug-modulated protein-protein interactions in living subjects
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DOI：
10.1158/0008-5472.can-03-2972
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发表时间：
2004-03-15
期刊：
影响因子：
11.2
通讯作者：
Gambhir, SS
中科院分区：
文献类型：
--
作者：
Paulmurugan, R;Massoud, TF;Gambhir, SS
蛋白质相互作用网络介导细胞对环境刺激的反应，并指导许多不同细胞功能途径的执行。在细胞内合成或从外部环境募集的小分子介导许多蛋白质相互作用。小分子介导的蛋白质相互作用的研究对于了解癌症中的异常信号转导途径以及药物开发和验证非常重要。在这项研究中，我们使用分裂合成海肾荧光素酶（hRLUC）蛋白片段辅助互补来评估由小分子雷帕霉素介导的人蛋白FRB和FKBP 12的异源二聚化。在细胞系中研究了有效二聚化所需的雷帕霉素浓度及其抑制二聚化所需的竞争性结合剂子囊霉素浓度。该系统在细胞培养物中在转录水平上通过使用肿瘤坏死因子a控制核因子kappaB启动子/增强子元件，以及在相互作用水平上通过控制二聚化剂雷帕霉素的浓度来双重调节。雷帕霉素介导的FRB和FKBP 12的二聚化也在活小鼠中通过使用冷却的电荷耦合器件照相机定位、定量和定时基于hRLUC互补的生物发光成像信号来研究。这种分裂报告系统可用于有效筛选调节蛋白质-蛋白质相互作用的小分子药物，也可用于评估活体动物中的药物。两者都是靶向蛋白质-蛋白质相互作用（包括癌细胞中的信号传导通路）的候选药物的临床前评价中的重要步骤。
Networks of protein interactions mediate cellular responses to environmental stimuli and direct the execution of many different cellular functional pathways. Small molecules synthesized within cells or recruited from the external environment mediate many protein interactions. The study of small molecule-mediated interactions of proteins is important to understand abnormal signal transduction pathways in cancer and in drug development and validation. In this study, we used split synthetic renilla luciferase (hRLUC) protein fragment-assisted complementation to evaluate heterodimerization of the human proteins FRB and FKBP12 mediated by the small molecule rapamycin. The concentration of rapamycin required for efficient dimerization and that of its competitive binder ascomycin required for dimerization inhibition were studied in cell lines. The system was dually modulated in cell culture at the transcription level, by controlling nuclear factor kappaB promoter/enhancer elements using tumor necrosis factor a, and at the interaction level, by controlling the concentration of the dimerizer rapamycin. The rapamycin-mediated dimerization of FRB and FKBP12 also was studied in living mice by locating, quantifying, and timing the hRLUC complementation-based bioluminescence imaging signal using a cooled charged coupled device camera. This split reporter system can be used to efficiently screen small molecule drugs that modulate protein-protein interactions and also to assess drugs in living animals. Both are essential steps in the preclinical evaluation of candidate pharmaceutical agents targeting protein-protein interactions, including signaling pathways in cancer cells.