Improving Stability of Cathepsin B Endopeptidase Substrates as Potential Cleavage Sites in Activatable Cell-Penetrating Peptides

Objectives: The cysteine protease cathepsin B, whose expression in tumors correlates with increased metastasis, therapy resistance, and a generally poor prognosis, represents an excellent target for molecular imaging using radiotracers [1]. We aim to develop a cathepsin B specific, substrate-based radiotracer derived from poly-d-arginine-based activatable cell penetrating peptides [2]. With in vivo application of peptides being often limited by short biological half-life, stabilization against proteolytic degradation is a key aspect in the development of this agent.
Methods: Octapeptide substrates containing the FRET pair aminobenzoyl/dinitrophenyl (Abz/Dnp) were synthesized by solid phase peptide synthesis in high purities and good yields, using non-proteinogenic and N-methylated amino acids (AA) for stabilization. All substrates were evaluated for cleavage efficiency by cathepsin B in orientation to [3]. In vitro stability studies were performed in human serum, with analysis by UPLC-ESI-MS, using the UV absorbance of Dnp (λ = 365 nm) for quantification and subsequent ESI-MS analysis for identification of degradation products.
Results: Rapid degradation has been observed for the endopeptidase substrate Abz-Gly-Ile-Val-Arg-Ala-Lys(Dnp)-Gly-Ser-NH2 in the in vitro serum stability assay (T_1/2 = 3.7 min), which was due to cleavage at the P1-P1’ cleavage site (Arg-Ala) as indicated by LC-MS analysis. In a first step, arginine was substituted by citrulline to decrease susceptibility to trypsin-like serum proteases, which increased serum stability (T_1/2 = 8.9 min). The non-proteinogenic AA homoarginine, homocitrulline and O-carboxybenzylserine are being tested as further potential substitutes for arginine. Secondary cleavage sites, identified at P4-P3 (Gly-Ile) and P2’-P3’ (Lys-Gly), were suppressed by insertion of N^α-methyl-isoleucine and N^α-methyl-glycine.
Conclusions: After the optimization of the endopeptidase substrate with regards to cathepsin B-specific cleavage, substrate stabilization against other proteases is a crucial step to a peptide-based radiotracer. We have demonstrated the potential for stabilization by introduction of citrulline, with further stabilization by insertion of N-methylated and non-proteinogenic amino acids ongoing, which will pave the way to the envisaged substrate-based imaging probes.
References:
1. Löser & Pietzsch, Front. Chem. 2015, 3, 37
2. Jiang et al., PNAS 2004, 101, 17867
3. Cotrin et al., Anal. Biochem. 2004, 335, 244