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¹ Sidney Kimmel Comprehensive Cancer Center at Johns Hopkins, Baltimore, Maryland; ² Danish University of Pharmaceutical Sciences, Copenhagen, Denmark; ³ Jerini AG, Berlin, Germany; and ⁴ Memorial Sloan Kettering Cancer Center, New York, New York

Requests for reprints: Samuel Denmeade, Sidney Kimmel Comprehensive Cancer Center at Johns Hopkins, Bunting Blaustein Cancer Research Building, 1650 Orleans Street, Baltimore, MD 21231. Phone: 410-502-3941; Fax: 410-614-8397. E-mail: denmesa{at}jhmi.edu

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-leucinoylamino]dodecanoyl)-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 K_m of 26.5 µmol/L, k_cat of 1.09 s^–1, and a k_cat/K_m 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.

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Received 6/ 9/04; revised 8/12/04; accepted 8/27/04.