An Activity-Based Near-Infrared Glucuronide Trapping Probe for Imaging β-Glucuronidase Expression in Deep Tissues

Abstract

β-glucuronidase is an attractive reporter and prodrug-converting enzyme. The development of near-IR (NIR) probes for imaging of β-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 β-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. β-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 β-glucuronidase-expressing sites. FITC-TrapG was selectively trapped on purified β-glucuronidase or β-glucuronidase-expressing CT26 cells (CT26/mβG) but not on bovine serum albumin or non-β-glucuronidase-expressing CT26 cells used as controls. β-glucuronidase-activated FITC-TrapG did not interfere with β-glucuronidase activity and could label bystander proteins near β-glucuronidase. Both FITC-TrapG and NIR-TrapG specifically imaged subcutaneous CT26/mβG tumors, but only NIR-TrapG could image CT26/mβG tumors transplanted deep in the liver. Thus NIR-TrapG may provide a valuable tool for visualizing β-glucuronidase activity in vivo.

PET Imaging of β-Glucuronidase Activity by an Activity-Based 124I-Trapping Probe for the Personalized Glucuronide Prodrug Targeted Therapy

“…We previously demonstrated that a near infrared glucuronide trapping probe (NIR-TrapG) is an activity-based probe that allows specific and direct detection of bG activity in preclinical models (21). In addition, the glucuronide trapping probe did not inhibit bG enzyme activity, which can improve substrate activation, imaging intensity, and might not affect the conversion of bG-based prodrugs (43,44).…”

“…45). Moreover, the glucuronide trapping moiety (difluoromethylphenol) can be conjugated with various probes for multiimaging systems, such as fluorescent dyes (FITC and IR-820) for optical imaging (21) and radioactive isotopes ( 124 I) for micro-PET imaging. In the future, the glucuronide trapping probe could also be linked to clinical used diethylenetriamine pentaacetic acid (DTPA; ref.…”

“…at À80 C, and sectioned into 5-mm slices. Consecutive sections were either directly placed onto a phosphor imaging plate for autoradiography and detected by Typhoon 9410 phosphorimager (GE Healthcare Life Sciences) or stained with the b-glucuronidase Reporter Gene Staining Kit (Sigma-Aldrich) as previously described (21).…”

“…1A. (1) was prepared as described previously (21). To prepare of Tyramine-TrapG, tyramine was coupled with compound 1 via an amide linkage to obtain compound 3.…”

“…Only the highly signal penetrating NIR-TrapG with near infrared spectrum properties can be used in long-term tracking (for about 72 hours) of bG activity in deep liver tissues; the FITCTrapG cannot be. NIR-TrapG is a good probe to monitor the bG activity in small animals (21) but the near infrared fluorescent probes are still not feasible for human use as the large thickness of the human body causes a decrease in fluorescence emission and optical imaging systems for medical usage are not available. Development of a positron emission tomography (PET) glucuronide probe to image bG activity will provide a novel method to allow optimization of the protocols for bG-based personalized cancer therapy.…”