Screening a combinatorial peptide library to develop a human glandular kallikrein 2-activated prodrug as targeted therapy for prostate cancer.
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 th...
| Main Authors: | , , , , , , , |
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| Format: | Journal article |
| Language: | English |
| Published: |
2004
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| _version_ | 1797076959746850816 |
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| author | Janssen, S Jakobsen, C Rosen, D Ricklis, R Reineke, U Christensen, S Lilja, H Denmeade, SR |
| author_facet | Janssen, S Jakobsen, C Rosen, D Ricklis, R Reineke, U Christensen, S Lilja, H Denmeade, SR |
| author_sort | Janssen, S |
| collection | OXFORD |
| description | 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 Km of 26.5 micromol/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. |
| first_indexed | 2024-03-07T00:10:53Z |
| format | Journal article |
| id | oxford-uuid:793237d4-f79d-44ad-9f48-489cf7b21541 |
| institution | University of Oxford |
| language | English |
| last_indexed | 2024-03-07T00:10:53Z |
| publishDate | 2004 |
| record_format | dspace |
| spelling | oxford-uuid:793237d4-f79d-44ad-9f48-489cf7b215412022-03-26T20:35:49ZScreening a combinatorial peptide library to develop a human glandular kallikrein 2-activated prodrug as targeted therapy for prostate cancer.Journal articlehttp://purl.org/coar/resource_type/c_dcae04bcuuid:793237d4-f79d-44ad-9f48-489cf7b21541EnglishSymplectic Elements at Oxford2004Janssen, SJakobsen, CRosen, DRicklis, RReineke, UChristensen, SLilja, HDenmeade, SR 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 Km of 26.5 micromol/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. |
| spellingShingle | Janssen, S Jakobsen, C Rosen, D Ricklis, R Reineke, U Christensen, S Lilja, H Denmeade, SR Screening a combinatorial peptide library to develop a human glandular kallikrein 2-activated prodrug as targeted therapy for prostate cancer. |
| title | Screening a combinatorial peptide library to develop a human glandular kallikrein 2-activated prodrug as targeted therapy for prostate cancer. |
| title_full | Screening a combinatorial peptide library to develop a human glandular kallikrein 2-activated prodrug as targeted therapy for prostate cancer. |
| title_fullStr | Screening a combinatorial peptide library to develop a human glandular kallikrein 2-activated prodrug as targeted therapy for prostate cancer. |
| title_full_unstemmed | Screening a combinatorial peptide library to develop a human glandular kallikrein 2-activated prodrug as targeted therapy for prostate cancer. |
| title_short | Screening a combinatorial peptide library to develop a human glandular kallikrein 2-activated prodrug as targeted therapy for prostate cancer. |
| title_sort | screening a combinatorial peptide library to develop a human glandular kallikrein 2 activated prodrug as targeted therapy for prostate cancer |
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