Screening a combinatorial peptide library to develop a human glandular kallikrein 2-activated prodrug as targeted therapy for prostate cancer

Samuel Janssen, Carsten M. Jakobsen, D. Marc Rosen, Rebecca M. Ricklis, Ulrich Reineke, Soeren B. Christensen, Hans Lilja, Samuel R. Denmeade

Research output: Contribution to journalArticlepeer-review

42 Scopus citations


Objective: Prostate cancer cells secrete the unique protease human glandular kallikrein 2 (hK2) that represents a target for proteolytic activation of cytotoxic prodrugs. The objective of this study was to identify hK2-selective peptide substrates that could be coupled to a cytotoxic analogue of thapsigargin, a potent inhibitor of the sarcoplasmic/endoplasmic reticulum calcium ATPase pump that induces cell proliferation-independent apoptosis through dysregulation of intracellular calcium levels. Methods: To identify peptide sequence requirements for hK2, a combination of membrane-bound peptides (SPOT analysis) and combinatorial chemistry using fluorescence-quenched peptide substrates was used. Peptide substrates were then coupled to 8-O-(12[L-leucinoylaminoldodecanoyl)-8-O-debutanoylthapsigargin (L12ADT), a potent analogue of thapsigargin, to produce a prodrug that was then characterized for hK2 hydrolysis, plasma stability, and in vitro cytotoxicity. Results: Both techniques indicated that a peptide with two arginines NH2-terminal of the scissile bond produced the highest rates of hydrolysis. A lead peptide substrate with the sequence Gly-Lys-Ala-Phe-Arg-Arg (GKAFRR) was hydrolyzed by hK2 with a Km of 26.5 μmol/L, kcat of 1.09 s-1, and a kcat/Km ratio of 41,132 s-1 mol/L-1. The GKAFRR-L12ADT prodrug was rapidly hydrolyzed by hK2 and was stable in plasma, whereas the GKAFRR-L peptide substrate was unstable in human plasma. The hK2-activated thapsigargin prodrug was not activated by cathepsin B, cathepsin D, and urokinase but was an excellent substrate for plasmin. The GKAFRR-L12ADT was selectively cytotoxic in vitro to cancer cells in the presence of enzymatically active hK2. Conclusion: The hK2-activated thapsigargin prodrug represents potential novel targeted therapy for prostate cancer.

Original languageEnglish (US)
Pages (from-to)1439-1450
Number of pages12
JournalMolecular cancer therapeutics
Issue number11
StatePublished - Nov 2004

ASJC Scopus subject areas

  • Oncology
  • Cancer Research


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