Screening a combinatorial peptide library to develop a human glandular kallikrein 2-activated prodrug as targeted therapy for prostate cancer
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Screening a combinatorial peptide library to develop a human glandular kallikrein 2-activated prodrug as targeted therapy for prostate cancer. / Janssen, Samuel; Jakobsen, Carsten M.; Rosen, D. Marc; Ricklis, Rebecca M.; Reineke, Ulrich; Christensen, Soeren B.; Lilja, Hans; Denmeade, Samuel R.
In: Molecular Cancer Therapeutics, Vol. 3, No. 11, 01.11.2004, p. 1439-1450.Research output: Contribution to journal › Journal article › Research › peer-review
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TY - JOUR
T1 - Screening a combinatorial peptide library to develop a human glandular kallikrein 2-activated prodrug as targeted therapy for prostate cancer
AU - Janssen, Samuel
AU - Jakobsen, Carsten M.
AU - Rosen, D. Marc
AU - Ricklis, Rebecca M.
AU - Reineke, Ulrich
AU - Christensen, Soeren B.
AU - Lilja, Hans
AU - Denmeade, Samuel R.
PY - 2004/11/1
Y1 - 2004/11/1
N2 - 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.
AB - 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.
UR - http://www.scopus.com/inward/record.url?scp=9444268199&partnerID=8YFLogxK
M3 - Journal article
C2 - 15542783
AN - SCOPUS:9444268199
VL - 3
SP - 1439
EP - 1450
JO - Molecular Cancer Therapeutics
JF - Molecular Cancer Therapeutics
SN - 1535-7163
IS - 11
ER -
ID: 232596005