Optimisation and stabilisation of cathepsin B-endopeptidase substrates: Towards a Cathepsin B-activated cell-penetrating peptide

Cathepsin B (CTB), whose expression in tumours correlates with increased metastasis, therapy resistance, and generally poor prognosis, represents an excellent target for molecular imaging using radiotracers [1]. It is our aim to develop a CTB-specifc, substrate-based radiotracer based on activatable, poly-D-arginine-derived cell pentrating peptides [2]. Central prerequisite for such a probe is an endopeptidase substrate for CTB used as an activator sequence that shows efficient cleavage kinetics towards CTB and is stable in circulation and against other relevant cathepsins. After newly identifying the P4' position as major determinant of CTB endopeptidase specificity, we determined Val (k_cat/K_M=245 mM^-1s^-1, 16xGly) as the ideal amino acid at this position. In terms of stabilisation, we were able to increase serum half-life from 3.6 min to >1440 min by amino acid exchange at P1 and N²-methylation of a secondary cleavage site, while still retaining good cleavability by CTB. Analysis of cleavage by other relevant ctahepsins is currently ongoing.

Literature
[1] Löser & Pietzsch Front. Chem. 2015, 3, 37
[2] Jang et al. PNAS 2004, 101, 17867