An Activity-Based Near-Infrared Glucuronide Trapping Probe for Imaging β-Glucuronidase Expression in Deep Tissues
An Activity-Based Near-Infrared Glucuronide Trapping Probe for Imaging β-Glucuronidase Expression in Deep Tissues
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DOI：
10.1021/ja209335z
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发表时间：
2012-02-15
影响因子：
15
通讯作者：
Leu, Yu-Ling
中科院分区：
文献类型：
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作者：
Cheng, Ta-Chun;Roffler, Steve R.;Leu, Yu-Ling
β-葡糖醛酸糖苷酶是一种有吸引力的报道基因和前药转化酶。开发用于β-葡萄糖醛酸苷酶活性成像的近红外（NIR）探针将是理想的，可以在临床前研究中估计报告基因表达和个性化葡萄糖醛酸苷前药癌症治疗。然而，NIR葡萄糖醛酸探针尚未上市。在这项工作中，我们开发了两种荧光探针用于检测β-葡萄糖醛酸酶活性，一种用于NIR范围（含有IR-820染料），另一种用于可见光范围[含有异硫氰酸荧光素（FITC）]，通过利用二氟甲基苯酚-葡萄糖醛酸苷部分（TrapG）捕获活性酶附近的荧光染料。β-葡糖醛酸糖苷酶介导的TrapG的葡糖醛酸键的水解产生高度反应性的烷基化基团，其促进荧光染料与位于β-葡糖醛酸糖苷酶表达位点附近的亲核部分的连接。FITC-TrapG被选择性地捕获在纯化的β-葡萄糖醛酸苷酶或表达β-葡萄糖醛酸苷酶的CT 26细胞（CT 26/m β G）上，但不被捕获在牛血清白蛋白或用作对照的非β-葡萄糖醛酸苷酶表达的CT 26细胞上。β-葡萄糖醛酸酶激活的FITC-TrapG不干扰β-葡萄糖醛酸酶活性，并且可以标记β-葡萄糖醛酸酶附近的旁观者蛋白。FITC-TrapG和NIR-TrapG均特异性成像皮下CT 26/m β G肿瘤，但只有NIR-TrapG可以成像移植到肝脏深处的CT 26/m β G肿瘤。因此，NIR-TrapG可以提供用于可视化体内β-葡萄糖醛酸酶活性的有价值的工具。
beta-glucuronidase is an attractive reporter and prodrug-converting enzyme. The development of near-IR (NIR) probes for imaging of beta-glucuronidase activity would be ideal to allow estimation of reporter expression and for personalized glucuronide prodrug cancer therapy in preclinical studies. However, NIR glucuronide probes are not yet available. In this work, we developed two fluorescent probes for detection of beta-glucuronidase activity, one for the NIR range (containing IR-820 dye) and the other for the visible range [containing fluorescein isothiocyanate (FITC)], by utilizing a difluoromethylphenol-glucuronide moiety (TrapG) to trap the fluorochromes in the vicinity of the active enzyme. beta-glucuronidase-mediated hydrolysis of the glucuronyl bond of TrapG generates a highly reactive alkylating group that facilitates the attachment of the fluorochrome to nucleophilic moieties located near beta-glucuronidase-expressing sites. FITC-TrapG was selectively trapped on purified beta-glucuronidase or beta-glucuronidase-expressing CT26 cells (CT26/m beta G) but not on bovine serum albumin or non-beta-glucuronidase-expressing CT26 cells used as controls. beta-glucuronidase-activated FITC-TrapG did not interfere with beta-glucuronidase activity and could label bystander proteins near beta-glucuronidase. Both FITC-TrapG and NIR-TrapG specifically imaged subcutaneous CT26/m beta G tumors, but only NIR-TrapG could image CT26/m beta G tumors transplanted deep in the liver. Thus NIR-TrapG may provide a valuable tool for visualizing beta-glucuronidase activity in vivo.