Cathepsin B-Activatable Cell-Penetrating Peptides for Imaging Cancer-related Cathepsin B

In our understanding of the many drivers of malignant progression and cancer metastasis, proteases are increasingly drawn into the spotlight as crucial agents in cancer angiogenesis, invasion, and metastasis [1]. Elevated activities of multiple members of the cathepsin family have been shown to correlate with increased metastasis and high therapy resistance [2, 3]. Especially high expression levels of extracellular cathepsin B (CTB) indicate poor prognosis in neoplastic diseases, making CTB an interesting target for activity-based molecular imaging in diagnostics as well as in therapy monitoring for personalised therapies.
It is our aim to develop such a probe by combination of a polyarginine-based, activatable cell penetrating peptide (ACPP), as first described by R. Tsien, and an optimised endopeptidase substrate for CTB [4]. Substrate optimisation proofed to be challenging as two entirely opposite factors needed to be balanced – high stability against serum proteases to prevent premature cleavage of the activation sequence, while retaining efficient and specific endoproteolytic cleavability by CTB.
We have generated a CTB-endoprotease substrate by C-terminally elongating the CTB carboxydipeptidase substrate Abz GIVR*AK(Dnp) OH (Abz - aminobenzoyl, Dnp – dinitrophenyl, * – cleavage site), described by Cotrin et al. in 2004, to the octapeptide Abz GIVR*AK(Dnp)GX CONH2, which could be used as activation site in the final ACPP [5]. Introduction of any amino acid other than glycine at the P4’ position resulted in hysteretic kinetics for the CTB-catalysed hydrolysis of the octapeptides, which might indicate the displacement of the occluding loop from the active site upon interaction with the substrates. Using LC-ES-MS-based analysis of serum-incubated substrates, the positions P1 and P3’ were determined to be primary determinants of serum stability. After suppression of the P3’ instability by Nα-methylation and optimisation within the positions P1-P3, we were able to increase serum half-life from < 5 min to > 1440 min under concomitant improvement of kinetic substrate efficiency towards CTB.
Currently, the substrate is optimised towards CTB-specificity within the cathepsin protease family. Additionally, cell uptake studies of a fluorescently labelled ACPP using the optimised CTB-endoprotease substrate sequence are ongoing. Using this fluorescent probe, we plan to study cell uptake, in vivo stability and initial biodistribution. Furthermore, an ACPP conjugated to radiometal-chelating entities is currently prepared, which will allow for PET imaging in vivo.

Literature
(1) Yang et al., Cancer Growth Metastasis 2009, 2, 13-27
(2) Aggarwal and Sloane, Proteomics Clin. Appl. 2014, 8(5-6), 427-437
(3) Löser and Pietzsch, Front. Chem. 2015, 3, article 37
(4) Jiang et al., PNAS, 2004, 101(51), 17867-17872
(5) Cotrin et al., Anal. Biochem. 2004, 335, 244-252