Shanghai Key Laboratory of Bioactive Small Molecules & Department of Pharmaceutical Analysis, School of Pharmacy, Fudan University, Shanghai 201203, P. R. China.
China State Institute of Pharmaceutical Industry, Shanghai 201203, P. R. China.
Anal Chem. 2021 Apr 27;93(16):6501-6507. doi: 10.1021/acs.analchem.1c00413. Epub 2021 Apr 18.
Fibroblast activation protein-alpha (FAPα) is a key modulator of the microenvironment in multiple pathologies and is becoming the next pan-cancer target for cancer diagnostics and therapeutics. Chemiluminescence (CL) luminophores are considered as one of the most sensitive families of probes for detection and imaging applications due to their high signal-to-noise ratio. Until now, however, no such effective CL probe was reported for FAPα detection. Herein, we developed a novel CL probe for the detection of endogenous FAPα activity by incorporating FAPα-specific dipeptide substrates (glycine-proline) to the improved Schaap's adamantylidene-dioxetane. In this manner, we designed three CL probes (, , and ) with the dipeptide substrate blocked by N-terminal benzyloxycarbonyl, --butoxycarbonyl or -quinoline-4-carboxylic acid, respectively, which was used as the masking group to restrain the chemiexcitation energy. Probe exhibited the optimal specificity for the discrimination of FAPα from dipeptidase IV and prolyl oligopeptidase, which was elucidated by molecular docking simulation. Upon FAPα cleavage, was turned on for the highly selective and sensitive detection of FAPα with a limit of detection of 0.785 ng/mL. Furthermore, the ability of to image FAPα was effectively demonstrated in vitro, including various biological samples (plasma and tissue preparations), and in living systems (tumor cells and tumor-bearing mice). Furthermore, this newly established probe could be easily extended to evaluate FAPα inhibitors. Overall, we anticipate that probe will offer a facile and cost-effective alternative in the early detection of pathologies, individual tailoring of drug therapy, and drug screening.
成纤维细胞激活蛋白-α（FAPα）是多种病理微环境的关键调节因子，正在成为癌症诊断和治疗的下一个泛癌靶点。化学发光（CL）荧光团因其高信噪比而被认为是检测和成像应用中最灵敏的探针家族之一。然而，到目前为止，还没有报道用于 FAPα 检测的这种有效的 CL 探针。在此，我们通过将 FAPα 特异性二肽底物（甘氨酸-脯氨酸）引入改进的 Schaap 的金刚烷亚胺-二氧杂环丁烷，开发了一种用于检测内源性 FAPα 活性的新型 CL 探针。通过这种方式，我们设计了三个 CL 探针（、和），其中二肽底物分别被 N-端苄氧羰基、叔丁氧羰基或 -喹啉-4-羧酸封闭，用作掩蔽基团以抑制化学激发能。探针显示出对 FAPα 与二肽酶 IV 和脯氨酰寡肽酶的区分的最佳特异性，这通过分子对接模拟得到了阐明。在 FAPα 切割后，探针被打开，可高度选择性和灵敏地检测 FAPα，检测限为 0.785ng/mL。此外，在体外，包括各种生物样品（血浆和组织制剂）和活体系统（肿瘤细胞和荷瘤小鼠）中，成功证明了 对 FAPα 成像的能力。此外，这种新建立的探针可以很容易地扩展到评估 FAPα 抑制剂。总体而言，我们预计探针将为疾病的早期检测、药物治疗的个体化定制和药物筛选提供一种简便且经济有效的替代方案。
