Van Rymenant Yentl, Tanc Muhammet, Van Elzen Roos, Bracke An, De Wever Olivier, Augustyns Koen, Lambeir Anne-Marie, Kockx Mark, De Meester Ingrid, Van Der Veken Pieter
Laboratory of Medical Biochemistry, Department of Pharmaceutical Sciences, University of Antwerp, Antwerp, Belgium.
Laboratory of Medicinal Chemistry, Department of Pharmaceutical Sciences, University of Antwerp, Antwerp, Belgium.
Front Chem. 2021 Apr 14;9:640566. doi: 10.3389/fchem.2021.640566. eCollection 2021.
Fibroblast activation protein (FAP) is a proline-selective protease that belongs to the S9 family of serine proteases. It is typically highly expressed in the tumor microenvironment (TME) and especially in cancer-associated fibroblasts, the main cell components of the tumor stroma. The exact role of its enzymatic activity in the TME remains largely unknown. Hence, tools that enable selective, activity-based visualization of FAP within the TME can help to unravel FAP's function. We describe the synthesis, biochemical characterization, and application of three different activity-based probes (biotin-, Cy3-, and Cy5-labeled) based on the FAP-inhibitor UAMC1110, an in-house developed molecule considered to be the most potent and selective FAP inhibitor available. We demonstrate that the three probes have subnanomolar FAP affinity and pronounced selectivity with respect to the related S9 family members. Furthermore, we report that the fluorescent Cy3- and Cy5-labeled probes are capable of selectively detecting FAP in a cellular context, making these chemical probes highly suitable for further biological studies. Moreover, proof of concept is provided for FAP activity staining in patient-derived cryosections of urothelial tumors.
成纤维细胞活化蛋白（FAP）是一种脯氨酸选择性蛋白酶，属于丝氨酸蛋白酶的S9家族。它通常在肿瘤微环境（TME）中高度表达，尤其是在癌症相关成纤维细胞中，而癌症相关成纤维细胞是肿瘤基质的主要细胞成分。其酶活性在肿瘤微环境中的具体作用在很大程度上仍不清楚。因此，能够在肿瘤微环境中对FAP进行基于活性的选择性可视化的工具有助于揭示FAP的功能。我们描述了基于FAP抑制剂UAMC1110（一种内部开发的分子，被认为是目前可用的最有效和选择性最强的FAP抑制剂）的三种不同的基于活性的探针（生物素标记、Cy3标记和Cy5标记）的合成、生化特性及应用。我们证明这三种探针具有亚纳摩尔级的FAP亲和力，并且对相关S9家族成员具有显著的选择性。此外，我们报告荧光Cy3标记和Cy5标记的探针能够在细胞环境中选择性地检测FAP，这使得这些化学探针非常适合进一步的生物学研究。此外，还提供了在患者来源的尿路上皮肿瘤冷冻切片中进行FAP活性染色的概念验证。
