Extracellular cathepsin B is a driver of tumor progression and metastasis, and its potential as diagnostic and prognostic marker is increasingly recognized. To harness its activity for triggering the uptake of activatable cell-penetrating peptides (ACPP) in vivo, kinetically suitable and stable endopeptidase substrates for this cysteine protease, which mainly acts as carboxydipeptidase, are required. This challenge was tackled by C-terminal elongation of the previously identified GIVRAK sequence to octapeptides and systematic structural variation, which has revealed that endopeptidase activity of cathepsin B is associated with kinetic hysteresis and the P4’ residue plays a key role in this regard, as further investigated by enzyme-substrate docking in silico. By replacing the N-terminal motif with GFLG and focused N-methylation of the backbone, substrate serum half-life was extended from 3.7 min to 23.4 h. Integrating this sequence into the fluorophore-conjugated ACPP and fluorescence microscopy in U87MG cells confirmed cathepsin B-mediated uptake on the basis of selective inhibitors and control probes. PET imaging and biodistribution studies ex vivo with a NODAGA-conjugated ACPP analogue radiolabeled with copper-64 in a murine U87MG-derived xenograft model together with radiopharmacological investigations in normal Wistar rats demonstrated more favorable pharmacokinetics compared to the corresponding CPP. Although tumor-associated proteolytic activation in vivo is indicated, this does not contribute to tumor retention as judged from control experiments under pharmacological blockade of cathepsin B and with non-functional analogues. The obtained results are discussed in context of previous data for radiolabeled ACPPs and implications for the general use of ACPPs for radiotheranostic approaches are highlighted.

Extracellular cathepsin B is a driver of tumor progression and metastasis, and its potential as diagnostic and prognostic marker is increasingly recognized. To harness its activity for triggering the uptake of activatable cell-penetrating peptides (ACPP) in vivo, kinetically suitable and stable endopeptidase substrates for this cysteine protease, which mainly acts as carboxydipeptidase, are required. This challenge was tackled by C-terminal elongation of the previously identified GIVRAK sequence to octapeptides and systematic structural variation, which has revealed that endopeptidase activity of cathepsin B is associated with kinetic hysteresis and the P4’ residue plays a key role in this regard, as further investigated by enzyme-substrate docking in silico. By replacing the N-terminal motif with GFLG and focused N-methylation of the backbone, substrate serum half-life was extended from 3.7 min to 23.4 h. Integrating this sequence into the fluorophore-conjugated ACPP and fluorescence microscopy in U87MG cells confirmed cathepsin B-mediated uptake on the basis of selective inhibitors and control probes. PET imaging and biodistribution studies ex vivo with a NODAGA-conjugated ACPP analogue radiolabeled with copper-64 in a murine U87MG-derived xenograft model together with radiopharmacological investigations in normal Wistar rats demonstrated more favorable pharmacokinetics compared to the corresponding CPP. Although tumor-associated proteolytic activation in vivo is indicated, this does not contribute to tumor retention as judged from control experiments under pharmacological blockade of cathepsin B and with non-functional analogues. The obtained results are discussed in context of previous data for radiolabeled ACPPs and implications for the general use of ACPPs for radiotheranostic approaches are highlighted.