AUTHOR=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 

TITLE=In Vitro and In Situ Activity-Based Labeling of Fibroblast Activation Protein with UAMC1110-Derived Probes

JOURNAL=Frontiers in Chemistry

VOLUME=Volume 9 - 2021

YEAR=2021

URL=https://www.frontiersin.org/journals/chemistry/articles/10.3389/fchem.2021.640566

DOI=10.3389/fchem.2021.640566

ISSN=2296-2646

ABSTRACT=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 sub-nanomolar 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 in situ FAP activity staining in patient-derived cryosections of urothelial tumors.