Gallium-68 Vinyl Sulfone Probes Target Tumor Cathepsins in Preclinical PET Study

Researchers have designed and tested a new family of gallium-68 labeled activity-based probes that covalently latch onto active cathepsin L, a protease increasingly linked to cancer progression, and evaluated them for positron emission tomography imaging of tumors. The work, published in EJNMMI Radiopharmacy and Chemistry, describes a proof-of-concept study exploring vinyl sulfone chemistry as a scaffold for PET activity-based probes targeting cathepsins, an application the authors note remains unexplored, according to the study team led by Oliviero Cini and Filipe Elvas.

The study’s authors note that during malignant transformation these enzymes are often upregulated and secreted into the acidic tumor microenvironment, where they contribute to extracellular matrix degradation, invasion, angiogenesis and metastasis. Cathepsin L activity in particular has been strongly associated with advanced tumor grade and unfavorable prognosis across a range of solid malignancies, which is why the group chose it as the imaging target. Because protein expression levels do not necessarily reflect enzymatic activity, the team pursued activity-based probes, small molecules that bind covalently to the catalytic cysteine residue of the active enzyme, allowing direct visualization of function rather than abundance.

The chemical starting point was K777, a well-validated dipeptidyl vinyl sulfone inhibitor with high potency toward human cathepsins L and B. Vinyl sulfones act as Michael acceptors, reacting with catalytic cysteines while offering better metabolic stability than alternative electrophilic warheads such as acrylamides or epoxides. The researchers modified the scaffold at two positions: the solvent-exposed P3 site, where different linker chemistries were attached to carry the NODA-GA chelator for gallium-68 complexation, and the P1 position, where a leucine-containing analogue was synthesized for comparison with the native homophenylalanine residue.

Structure-activity analysis using fluorogenic enzyme assays revealed clear preferences. Retaining homophenylalanine at P1 produced markedly greater cathepsin L potency than the leucine analogue: the gallium-complexed reference compound with hPhe showed a cathepsin L inactivation constant of 0.32 micromolar, while the leucine version was roughly fourfold weaker at 1.3 micromolar. Linker choice proved equally consequential. Polyethylene glycol linkers of varying lengths preserved high selectivity, with cathepsin B inhibition remaining above 100 micromolar and selectivity indices exceeding 313. A piperazine linker, by contrast, substantially increased off-target cathepsin B binding, dropping the selectivity index to 30 despite achieving the strongest cathepsin L potency in the series at 0.11 micromolar.

Importantly, the team demonstrated that complexation with gallium did not alter enzymatic affinity or selectivity, comparing non-chelated precursors with their metal-bound reference compounds and finding essentially identical kinetic profiles. From these data, two representative radiotracers advanced to biological evaluation: the PEG2-linked [68Ga]CREANT-101 and the piperazine-linked [68Ga]CREANT-102. Both tracers showed slightly hydrophilic partition coefficients, with logD values of -0.18 and -0.15 respectively, indicating that the linker architecture had minimal impact on lipophilicity.

In vitro binding assays on recombinant cathepsins B, L and S confirmed that both radiotracers selectively labeled cathepsin L, with efficient labeling visible within ten minutes of incubation. Pre-incubation with the pan-cathepsin inhibitor K777 abolished binding, verifying target specificity. Competitive co-incubation experiments, in which cathepsin L was mixed with either cathepsin B or cathepsin S before probe addition, produced a dominant cathepsin L signal with limited cross-reactivity, consistent with the kinetic data.

Stability testing revealed a divergence between the two tracers. In mouse plasma, [68Ga]CREANT-101 degraded gradually, falling from 86 percent intact at 30 minutes to 73 percent at 60 minutes, while [68Ga]CREANT-102 remained above 90 percent intact at both timepoints. In vivo metabolite analysis in healthy Balb/C mice showed more rapid degradation for both compounds: [68Ga]CREANT-101 retained 45.0 percent intact tracer at 30 minutes and 24.5 percent at 60 minutes, whereas [68Ga]CREANT-102 fared better with 60.6 percent and 50.6 percent respectively.

Pharmacokinetic imaging in healthy mice exposed stark differences attributable to linker chemistry. [68Ga]CREANT-101 cleared rapidly from blood, dropping from 2.71 percent injected dose per gram at 30 minutes to 0.36 percent at three hours, with low uptake in lungs, heart and kidneys. [68Ga]CREANT-102 persisted far longer, with blood activity of 9.04 percent injected dose per gram at 30 minutes and 1.71 percent at three hours, accompanied by elevated non-specific uptake across peripheral tissues. The researchers linked this prolonged circulation to higher plasma protein binding: [68Ga]CREANT-101 maintained a free fraction of 5.7 to 9.5 percent, while [68Ga]CREANT-102 showed markedly lower values of 1.21 to 2.95 percent. Both tracers cleared through mixed renal and hepatobiliary pathways, with substantial bladder and intestinal activity.

Based on its favorable clearance and low background profile, [68Ga]CREANT-101 was selected for tumor imaging in two xenograft models: U-87 MG glioblastoma as a positive model and HT-29 colorectal carcinoma as a low-expressing comparator. Peak tumor uptake on dynamic PET reached 3.05 percent injected dose per milliliter in U-87 MG tumors and 3.12 percent in HT-29 tumors, but by three hours ex vivo biodistribution confirmed only modest absolute uptake of 0.19 and 0.36 percent injected dose per gram respectively. Time-activity curve analysis showed a steep decline from early to late phases, consistent with perfusion-driven uptake and limited tracer retention. Notably, despite clear differences in cathepsin L expression between the two cell lines in vitro, no corresponding advantage emerged in vivo for the U-87 MG model.

The critical finding came from ex vivo analysis of tumor homogenates. When [68Ga]CREANT-101 was incubated with lysates prepared from the excised tumors, distinct radioactive bands appeared in both U-87 MG and HT-29 samples, consistent with the molecular weights of mature cathepsins L and B. Pre-treatment with K777 eliminated the signal in U-87 MG homogenates, confirming specific covalent engagement of active cathepsins within the tissue. This result indicated that the limited tumor uptake was not caused by insufficient target affinity but rather by suboptimal cellular internalization and restricted tumor bioavailability, likely driven by rapid systemic clearance and in vivo metabolic instability. The authors suggest that future optimization of this vinyl sulfone probe class should focus on improving these pharmacokinetic properties to enhance in vivo tumor targeting, providing a rational foundation for developing selective cathepsin L-targeted activity-based probes for precision oncology.

Subject of Research: Development and preclinical evaluation of gallium-68 labeled vinyl sulfone activity-based probes for PET imaging of tumor-associated cathepsin L

Article Title: Development and preclinical evaluation of selective vinyl sulfone-based probes for PET imaging of tumor-associated cathepsins

Article References: Cini, O., Miranda, A., Willocx, D., Karakasidi, A., Groot, A. D., Moreira, T. D. V., Leandro, A. P., Santos, M. M. M., Moreira, R., & Elvas, F. (2026). Development and preclinical evaluation of selective vinyl sulfone-based probes for PET imaging of tumor-associated cathepsins. EJNMMI Radiopharmacy and Chemistry, 11(1), Article 64. https://doi.org/10.1186/s41181-026-00493-5

Image Credits: AI Generated

DOI: 10.1186/s41181-026-00493-5

Keywords: PET imaging, cathepsin L, vinyl sulfone, activity-based probes, gallium-68, tumor imaging, preclinical evaluation, radiotracers, K777 scaffold, cysteine cathepsins, xenograft models, molecular imaging