Optimisation of Cathepsin B Endopeptidase Substrates as Potential Cleavage Sites for Activatable Cell-Penetrating Peptides (ACPP)

High activity of the cysteine protease cathepsin B correlates with increased metastasis, therapy resistance, and a generally poor prognosis in neoplastic diseases [1,2] Therefore, the development of substrate-based radiotracers for in vivo imaging of this protease will be beneficial towards better diagnosis and improved therapy regimens. We aim at developingsuch a probe by combination of a polyarginine-based, activatable cell penetrating peptide, as described by Tsien and others, and newly optimised endopeptidase substrates for cathepsin B [3]. A unique feature of cathepsin B within the cysteine cathepsin family is the occluding loop, a flexible element consisting of 20 amino acids (aa), which is able to cover the active site of the enzyme and thereby regulates the substrate specificity in a pH-dependent manner [1,2]. This phenomenon is mediated by two histidine residues within the occluding loop, which play a crucial role in positioning a potential substrate for carboxydipeptidolytic cleavage.
Starting point for the development of endopeptidase substrates was the carboxydipeptidase substrate Abz‑GIVRAK(Dnp)‑OH (Abz - aminobenzoyl, Dnp - dinitrophenyl) described by Cotrin et al. in 2004, with the fluorophores Abz and Dnp constituting a FRET pair to enable fluorometric detection of the proteolytic cleavage [4]. Two structural changes were introduced to convert the exopeptidase substrate into an endopeptidase substrate for later use as an activator sequence in an aCPP: Firstly, C‑terminal amidation was performed to interrupt the interaction between the terminal carboxyl group and the two His residues within the occluding loop. This resulted in a reduced specificity constant (kcat/Km = 1179 mM‑1s‑1 vs. 139 mM‑1s‑1), indicating attenuated electrostatic/hydrogen bond interactions. Secondly, the peptide sequence was C‑terminally elongated to test longer substrates. Interestingly, the C‑terminal elongation resulted in a hysteretic progress of the enzymatic cleavage, likely a result of the steric displacement of the occluding loop. To evaluate the influence of size, polarity and charge of the C‑terminal aa on the enzymatic hysteresis, 14 variations of this position were synthesized and analysed for their kinetic parameters. The peptide Abz‑GIVRAK(Dnp)G‑V‑NH2 was determined to exhibit the highest specificity constant (kcat/Km = 260 mM‑1s‑1).
The optimised octapeptide sequence is currently inserted as recognition site in a fluorophore-labelled aCPP for monitoring cathepsin B-mediated activation and subsequent cellular uptake, and for investigating kinetics and in vitro stability analysis in future
experiments.
[1] Aggarwal and Sloane, Proteomics Clin. Appl. 2014, 8(5-6), 427-437
[2] Löser and Pietzsch, Front. Chem. 2015, 3, article 37
[3] Jiang et al., PNAS, 2004, 101(51), 17867-17872
[4] Cotrin et al., Anal. Biochem. 2004, 335, 244-252