In Vivo Visualization of γ-Glutamyl Transpeptidase Activity with an Activatable Self-Immobilizing Near-Infrared Probe

Abstract

γ-Glutamyl transpeptidase (GGT), a type of cell membrane-bound enzyme, is closely involved in a wide range of physiological and pathological processes, and a large number of fluorogenic probes have been developed to detect the activity of GGT. However, the use of these imaging reagents to visualize GGT activity in vivo is largely limited because of rapid diffusion and clearance of activated fluorophores. Herein, by merging quinone methide and a fluorogenic enzyme substrate, we report an activatable self-immobilizing near-infrared probe for the in vitro and in vivo imaging of GGT activity. This probe is initially fluorescently silent, but the selective activation by GGT is able to significantly increase its fluorescence intensity at 714 nm and covalently anchor activated fluorophores at the site of interest. We have shown that this probe induced a much stronger fluorescence on live GGT-overexpressing cells compared to regular fluorogenic probes and allowed wash-free and real-time imaging of enzyme activity. More importantly, the use of this probe in the imaging of GGT activity in U87MG tumor-bearing mice by i.v. administration indicates that this self-immobilizing reagent is capable of efficiently enhancing its retention at the detection target and thus leads to much improved detection sensitivity compared to regular fluorogenic probes. This study demonstrates the advantage of fluorogenic probes with activatable anchors in the noninvasive imaging of enzyme activity in highly dynamic in vivo systems.

Progress on Multifunction Enzyme-Activated Organic Fluorescent Probes for Bioimaging

“…An activatable self-immobilizing fluorophore (GGTIN-1) was designed by merging quinone methide and a fluorogenic enzyme substrate ( Figure 2A ), activated by γ-glutamyl transpeptidase (GGT) for the in vitro and in vivo imaging ( Li et al, 2020b ). GGT is a type of cell membrane-bound enzyme, overexpressed specifically in cancer cells.…”

“… Design strategy and application of multifunction enzyme-activated probes with single enzyme. (A) Design of GGT-activated self-immobilizing fluorophore and its real-time imaging in mice [adapted with permission from Li et al (2020b ) Copyright © 2020, American Chemical Society]. (B) Design of Casp3-activatable cell-permeable fluorophore and its application on apoptosis imaging in HeLa cells [adapted with permission from Jin et al (2021 ) Copyright © 2021, American Chemical Society].…”

Near-Infrared Fluorescent Probes for the Detection of Cancer-Associated Proteases

“…This minimal crosstalk due to the large Stokes shifts could avoid signal interference between the dye pair and successfully detected small cancerous lesions in colon cancer. Meanwhile, Xie and co-workers recently developed the self-immobilizing NIR probe GGTIN-1, which undergoes enzymatic glutamyl cleavage followed by self-immolative elimination of a PABA linker and finally covalent conjugation to either GGT or nearby proteins via an exposed electrophilic trap . GGTIN-1 was administered intravenously in mice to successfully image GGT activity in tumors from up to 1 h post injection and up to 24 h in total.…”

Precipitated Fluorophore-Based Molecular Probe for In Situ Imaging of Aminopeptidase N in Living Cells and Tumors

“…Many enzymes are found to cluster in microdomains with particular subcellular localization, and their levels in various organelles can be dynamically changed, which is related to their function in biological processes, occurrence, and development of diseases. − Thus, comprehensive analysis of subcellular enzyme expression and distribution holds great importance for the discovery of biomarkers, early diagnosis of diseases, and rational formulation of personalized treatment projects . Fluorescence imaging is now a powerful tool for the real-time study of the dynamics of enzymatic activity and intracellular localization in their native environments, which is significant both for fundamental research and biomedical applications. ,− Small-molecular enzymatic fluorescent probes are one of the most flexible contrast agents in fluorescence imaging and exploring biological processes. ,, However, a common problem of these probes is the rapid diffusion of the liberated fluorophores after enzymatic activation and the potential accumulation at intracellular locations rather than the site they generate, which may result in unreliable detection results .…”