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Cathepsin B-Activatable Cell-Penetrating Peptides
Kuhne, K.; Behring, L.; Belter, B.; Wodtke, R.; Pietzsch, J.; Löser, R.;
Protease activity is increasingly drawn into the spotlight as a crucial modulator in cancer angiogenesis, invasion, and metastasis [1]. Elevated activity of multiple members of the family of cysteine cathepsins has been shown to correlate with increased metastasis and therapy resistance [2, 3]. Especially high expression levels of extracellular cathepsin B (CatB) indicate poor prognosis in neoplastic diseases, making CatB an interesting target for functional characterization of cancers by activity-based molecular imaging. It is our aim to develop such an imaging probe for CatB by combination of a polyarginine-based, activatable cell-penetrating peptide [4] (ACPP) and an optimised endopeptidase substrate for CatB. 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 CatB. We have generated a CatB-endoprotease substrate by C-terminally elongating the CatB carboxydipeptidase substrate Abz GIVR*AK(Dnp) OH [5] (Abz – amino-benzoyl, Dnp – dinitrophenyl, * – cleavage site) to the octapeptide Abz GIVR*AK(Dnp)GX CONH2, which could be used as activation site in the final ACPP. Introduction of any amino acid other than glycine at the P4’ position resulted in hysteretic kinetics for the CatB-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-ESI-MS-based analysis of serum-incubated substrates, the positions P1 and P3’ where 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 > 24 h under concomitant improvement of kinetic substrate efficiency towards CatB. Based on this optimised CatB-endopeptidase substrate, we have synthesised a fluorescently labelled ACPP with which we were able to demonstrate CatB-dependent uptake and subsequent nucleolar accumulation of the activated peptide in human U87 MG glioma cells. Radiolabelling of the probe with copper-64 was enabled by conjugating the ACPP to NODAGA as chelating moiety. Its evaluation in vivo using PET imaging is under current investigation.

[1] Yang et al., Cancer Growth Metastasis 2009, 2, 13
[2] Aggarwal and Sloane, Proteomics Clin. Appl. 2014, 8, 427
[3] Löser and Pietzsch, Front. Chem. 2015, 3, article 37
[4] Jiang et al., PNAS, 2004, 101, 17867
[5] Cotrin et al., Anal. Biochem. 2004, 335, 244
  • Poster
    Frontiers in Medicinal Chemistry, 24.-27.03.2019, Würzburg, Deutschland

Publ.-Id: 29159 - Permalink