- Supervisors: Prof. Rui Moreira, Prof. Maria M. M. Santos, and Prof. Filipe Elvas
- Institution: Faculty of Pharmacy of the University of Lisbon
- Defended: 22 July 2026
- Download the PhD thesis
Abstract: The main objective of this work was the development and evaluation of novel activity-based PET probes for imaging tumor-associated cathepsins. Using the dipeptidyl vinyl sulfone scaffold of K11777 (K777) as a starting point, a series of selective cathepsin probes was designed, synthesized, and functionalized with the NODA-GA chelator. The inhibitory activities of the probe precursors and non-radioactive complexes were assessed through enzymatic assays, and two lead compounds were subsequently radiolabeled with gallium-68 (68Ga) resulting in two PET tracers, [68Ga]CREANT-101 and [68Ga]CREANT-102, obtained with high radiochemical purity and yield. Following radiolabeling optimization, extensive in vitro and in vivo pre-clinical evaluation was performed. Biochemical experiments showed selective binding to CatL in recombinant enzymes and tumor cell lysates. Subsequent pharmacokinetic studies demonstrated a more favorable biodistribution profile for [68Ga]CREANT-101 compared to [68Ga]CREANT-102, including faster blood clearance and decreased non-specific uptake. Therefore, the probe [68Ga]CREANT-101 was selected for further investigation due to its potential to achieve higher tumor-to-background ratios. Nevertheless, µPET/CT imaging and biodistribution studies in tumor-bearing mice highlighted low tumor uptake of the probe. To investigate this discrepancy, an ex vivo radiolabeling of tumor homogenates was subsequently performed showing that the probe was able to bind active cathepsins confirming engagement of the probe within the tissue environment.
Additionally, clickable vinyl sulfone-based activity-based probes were developed as chemoproteomic tools to enable future proteome-wide identification of on- and off-target interactions. This complementary strategy establishes a platform for integrating molecular imaging and chemoproteomics, providing a powerful framework for the rational optimization of cathepsin-targeted imaging agents.