Department of Nuclear Medicine, Peking University First Hospital, Beijing 100034, China.
Department of Nuclear Medicine, Peking University International Hospital, Beijing 102206, China.
Mol Pharm. 2021 Oct 4;18(10):3750-3762. doi: 10.1021/acs.molpharmaceut.1c00273. Epub 2021 Sep 7.
Arg-Arg-Leu (RRL) is a potent tumor-homing tripeptide. However, the binding target is unclear. In this study, we intended to identify the binding target of RRL and evaluate the tumor targeting of Tc-MAG-RRL . Biotin-RRL, 5-TAMRA-RRL, and Tc-MAG-RRL were designed to trace the binding target and tumor lesion. Immunoprecipitation-mass spectrometry was conducted to identify the candidate proteins and determination of the subcellular localization was also performed. A pull-down assay was performed to demonstrate the immunoprecipitate. Fluorescence colocalization and cell uptake assays were performed to elucidate the correlation between the selected binding protein and RRL, and the internalization mechanism of RRL. Biodistribution and imaging were performed to evaluate the tumor accumulation and targeting of Tc-MAG-RRL. The target for RRL was screened to be heat shock protein 70 (HSP70). The prominent uptake distribution of RRL was concentrated in the membrane and cytoplasm. A pull-down assay demonstrated the existence of HSP70 in the biotin-RRL captured complex. Regarding fluorescence colocalization and cell uptake assays, RRL may interact with HSP70 at the nucleotide-binding domain (NBD). Clathrin-dependent endocytosis and macropinocytosis could be a vital internalization mechanism of RRL. imaging and biodistribution both demonstrated that Tc-MAG-RRL can trace tumors with satisfactory accumulation in hepatoma xenograft mice. The radioactive signals accumulated in tumor lesions can be blocked by VER-155008, which can bind to the NBD of HSP70. Our findings revealed that RRL may interact with HSP70 and that Tc-MAG-RRL could be a prospective probe for visualizing overexpressed HSP70 tumor sections.
精氨酸-精氨酸-亮氨酸（RRL）是一种有效的肿瘤归巢三肽。然而，其结合靶标尚不清楚。在本研究中，我们旨在鉴定 RRL 的结合靶标，并评估 Tc-MAG-RRL 的肿瘤靶向性。设计了生物素-RRL、5-TAMRA-RRL 和 Tc-MAG-RRL 来追踪结合靶标和肿瘤病变。进行免疫沉淀-质谱分析以鉴定候选蛋白，并进行亚细胞定位测定。进行下拉实验以证明免疫沉淀的存在。荧光共定位和细胞摄取实验阐明了所选结合蛋白与 RRL 之间的相关性以及 RRL 的内化机制。进行生物分布和成像实验以评估 Tc-MAG-RRL 的肿瘤积累和靶向性。筛选出 RRL 的靶标为热休克蛋白 70（HSP70）。RRL 的摄取分布主要集中在膜和细胞质中。下拉实验证明 HSP70 存在于生物素-RRL 捕获的复合物中。关于荧光共定位和细胞摄取实验，RRL 可能与 HSP70 在核苷酸结合域（NBD）相互作用。网格蛋白依赖的内吞作用和巨胞饮作用可能是 RRL 内化的重要机制。成像和生物分布实验均表明，Tc-MAG-RRL 可追踪荷瘤异种移植小鼠的肿瘤，具有令人满意的积累。放射性信号在肿瘤病变中的积累可被 VER-155008 阻断，该化合物可与 HSP70 的 NBD 结合。我们的研究结果表明，RRL 可能与 HSP70 相互作用，Tc-MAG-RRL 可能是可视化 HSP70 过表达肿瘤切片的有前途的探针。
