Dual-Targeting PET Tracers Enable Enzyme-Mediated Self-Assembly for the PET Imaging of Legumain Activity

Abstract

Legumain, a lysosomal cysteine protease overexpressed in a variety of tumors, has been considered a promising biomarker for various cancers. Precise detection of legumain activity in the lysosome represents an important strategy for early diagnosis and prognosis of tumors. Small-molecule probes with the property of target-enabled self-assembly hold great potential for molecular imaging. In this study, we reported two dual-targeting radiotracers ([18F]SF-AAN-M and [18F]SF-AAN-HEM) with a property of legumain-mediated self-assembly for positron emission tomography (PET) imaging. Both the radiotracers were synthesized with high labeling yield (>50%) and the radiochemical purity was over 99% via one-step straightforward 18F-labeling. Both tracers were efficiently activated by the reducing agent and legumain to self-assemble into aggregates and showed enhanced retention in legumain-overexpressed MDA-MB-468 cells and tumors, indicating that the introduction of lysosome-targeting morpholine increased the tumor uptake and extended the retention of radiotracers in legumain-overexpressed tumors. In addition, [18F]SF-AAN-HEM with a hydrophilic (histidine-glutamate)3 tag displayed significantly reduced liver uptake with no conspicuous reduction in tumor uptake, affording high signal-to-noise ratios (tumor/liver and tumor/muscle). All of these results suggest that dual-targeting tracer [18F]SF-AAN-HEM could provide a promising tool for in vivo monitoring legumain activity in tumors.

Legumain-Triggered Macrocyclization of Radiofluorinated Tracer for Enhanced PET Imaging

“…These suggested that cyclized [ 18 F]AlF-RS-C cannot accumulate in the tumor site due to the absence of the legumain recognition sequence and the lysosome-targeting moiety morpholine. In comparison with previous results, the uptake of [ 18 F]AlF-RSM in MDA-MB-468 tumors was comparable to that of [ 18 F]SF-AAN-M, 10 demonstrating that the change of labeling group does not affect the specificity of the tracer.…”

“…The molar activity was calculated to be 33.69 ± 1.50 GBq/μmol (n = 3), which was significantly higher than [ 18 F]SF-AAN-M (13.21 ± 0.36 GBq/ μmol) reported in our previous study. 10 To evaluate the stability of [ 18 F]AlF-RSM, it was incubated in mouse serum for 4 h and continuously monitored the RCP by radio-HPLC. As shown in Figure 2f, the RCP of [ 18 F]AlF-RSM was maintained above 95% after 4 h of incubation, demonstrating the good stability of [ 18 F]AlF-RSM.…”

“…The k cat /K m value was 11214.2 M −1 s −1 , which was a little lower than our previously reported [ 19 F]SF-AAN-M (k cat /K m = 20,413.41 M −1 s −1 ). 10 This may be attributed to the steric hindrance of the [ 18 F]AlF-RESCA group.…”

“…Meanwhile, it is overexpressed in the majority of solid tumors, such as breast cancer, colon cancer, and prostate cancer. , Thus, it is critical to sensitively detect legumain expression levels in different tumors for early cancer diagnosis and evaluation . A number of probes targeting legumain based on imaging techniques have been developed for tumor diagnosis, such as fluorescence imaging, − magnetic resonance imaging, and positron emission tomography (PET) imaging. − In particular, PET is widely used in clinics with the advantages of real-time detection, deep tissue penetration, and high sensitivity. − Up to now, the PET tracers targeting legumain mainly are small molecules based on good tissue permeability. However, the probes with small size have the characteristics of rapid clearance and washout from the target tissues. , Hence, it is of significant value to develop an efficient mean to enhance the accumulation of radiotracers to amplify the imaging signals …”

Cathepsin B-Activated PET Tracer for In Vivo Tumor Imaging

“…The SF scaffold containing a 2-cyano-benzothiazole and a masked cysteine can trigger a click condensation reaction to self-assemble in situ . Tracers were designed by combining the SF scaffold with various enzyme substrates, while attaching the labeling groups for PET imaging. , Based on the above strategies, we report a novel small-molecule tracer [ 68 Ga]­NOTA-SF-CV for the detection of cathepsin B activity via PET imaging, which is composed of a recognition sequence Cit-Val , for cathepsin B-specific cleavage, a 1,4,7-triaazacyclononane-1,4,7-triacetic acid chelator NOTA for 68 Ga-labeling, and an SF scaffold (Scheme A). When the tracer enters into cells, it undergoes reduction, and the Cit-Val substrate is cleaved by cathepsin B.…”