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

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Abstract Background Cysteine cathepsin L (CatL) is emerging as a key biomarker of cancer progression, making it an attractive candidate for non-invasive molecular imaging. To monitor enzymatic activity directly, rather than merely protein expression, activity-based probes (ABPs) enable selective targeting of enzymes through covalent binding at their active sites. Despite vinyl sulfones are well-established irreversible inhibitors of cathepsins, their application as positron emission tomography (PET) ABPs remains unexplored. In this study we describe the design, synthesis, and evaluation of a novel class of 68 Ga-labeled vinyl sulfone-based ABPs targeting CatL. Results A series of ABPs were synthesized based on the established vinyl sulfone scaffold K11777 (K777). Structure-activity relationship (SAR) analysis revealed that retaining a homophenylalanine (hPhe) residue at the P1 position resulted in greater CatL potency compared to its corresponding leucine (Leu) analogue. Evaluation of different linkers at the P3 position further revealed that polyethylene glycol (PEG) linkers preserved high CatL selectivity, whereas a piperazine linker increased off-target binding to cathepsin B (CatB). Based on these findings, two representative radiotracers were selected for further biological evaluation: the PEG2-linked [ 68 Ga]CREANT-101 and the piperazine-linked [ 68 Ga]CREANT-102. In vivo pharmacokinetic studies in healthy mice demonstrated that [ 68 Ga]CREANT-102 exhibited prolonged blood circulation and high non-specific tissue uptake, whereas [ 68 Ga]CREANT-101 displayed rapid systemic clearance and low background signal, supporting its selection for tumor imaging. In both U-87 MG and HT-29 xenograft models, [ 68 Ga]CREANT-101 showed modest absolute tumor uptake in vivo. However, subsequent ex vivo analysis of tumor homogenates confirmed successful and specific engagement of active cathepsins within the target tissue. Conclusions A novel class of 68 Ga-labeled vinyl sulfone ABPs targeting CatL was successfully developed and preclinically evaluated. The lead tracer, [ 68 Ga]CREANT-101, demonstrated high selectivity and a favorable background profile, but showed limited tumor accumulation in vivo. Ex vivo findings indicated that this limited tumor uptake was not attributable to insufficient target affinity, but rather to suboptimal cellular internalization and restricted tumor bioavailability, likely resulting from rapid systemic clearance and in vivo metabolic instability. Therefore, future optimization of this tracer class should focus on improving these pharmacokinetic properties to enhance in vivo tumor targeting.

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