Screening a combinatorial peptide library to develop a human glandular kallikrein 2-activated prodrug as targeted therapy for prostate cancer Journal Article
| Authors: | Janssen, S.; Jakobsen, C. M.; Rosen, D. M.; Ricklis, R. M.; Reineke, U.; Christensen, S. B.; Lilja, H.; Denmeade, S. R. |
| Article Title: | Screening a combinatorial peptide library to develop a human glandular kallikrein 2-activated prodrug as targeted therapy for prostate cancer |
| Abstract: | 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. Copyright © 2004 American Association for Cancer Research. |
| Keywords: | controlled study; unclassified drug; human cell; nonhuman; antineoplastic agents; drug targeting; antineoplastic agent; cell proliferation; mouse; animals; mice; unindexed drug; apoptosis; enzyme inhibition; antineoplastic activity; cancer cell culture; cytotoxicity; drug potency; drug structure; in vitro study; drug screening; enzyme inhibitor; enzyme activity; drug evaluation, preclinical; cell line, tumor; drug selectivity; drug synthesis; prostate cancer; prostatic neoplasms; amino acid sequence; amino terminal sequence; substrate specificity; kallikrein; peptides; molecular structure; cathepsin b; drug blood level; fluorescence analysis; hydrolysis; calcium cell level; cathepsin d; peptide library; prodrug; urokinase; trypsin; protein hydrolysis; plasmin; prodrugs; tissue kallikreins; drug identification; calcium ion; thapsigargin; drug activation; adenosine triphosphatase (calcium); humans; human; male; priority journal; article; glycylglycylglycyllysylalanylarginylarginylleucine; glycylglycyllysylalanylhistidylarginylleucine; glycylhistidylglutamyllysylarginylarginylleucylglutamic acid; glycyllysylalanylphenylalanylarginylarginine 8 o [12 (leucinoylamino)dodecanoyl] 8 o debutanoylthapsigargin; glycyllysylalanylphenylalanylarginylarginylleucine; glycylseryllysylglycylhistidylphenylalanylhistidylleucine; glycylseryllysylglycylhistidylphenylalanyllysylleucine; glycylseryllysylglycylprolylphenylalanyllysylleucine; morpholinocarbonyl glycyllysylalanylphenylalanylarginine 7 amino 4 methylcoumarin; nitrotyrosine glycyllysylalanylarginylalanylphenylalanine diaminopropionic acid phenylalanyllysine; nitrotyrosine glycyllysylalanyllysylprolylarginine diaminopropionic acid phenylalanyllysine; nitrotyrosine glycyllysylalanylmethionylarginylglutamine diaminopropionic acid phenylalanyllysine; nitrotyrosine glycyllysylalanylphenylalanylarginylarginine diaminopropionic acid glycyllysine; nitrotyrosine glycyllysylalanylphenylalanylarginylarginine diaminopropionic acid phenylalanyllysine; nitrotyrosine glycyllysylalanylphenylalanylarginylarginylleucylglycyllysine; nitrotyrosine glycyllysylalanylphenylalanylarginylleucine diaminopropionic acid phenylalanyllysine; nitrotyrosine glycylseryllysylglycylhistidylphenylalanyllysylleucine diaminopropionic acid phenylalanyllysine; prolylphenylalanylarginine 7 amino 4 methylcoumarin; drug hydrolysis |
| Journal Title: | Molecular Cancer Therapeutics |
| Volume: | 3 |
| Issue: | 11 |
| ISSN: | 1535-7163 |
| Publisher: | American Association for Cancer Research |
| Date Published: | 2004-11-01 |
| Start Page: | 1439 |
| End Page: | 1450 |
| Language: | English |
| PROVIDER: | scopus |
| PUBMED: | 15542783 |
| DOI/URL: | |
| Notes: | Mol. Cancer Ther. -- Cited By (since 1996):31 -- Export Date: 16 June 2014 -- CODEN: MCTOC C2 - 15542783 -- Source: Scopus |
