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-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.
  
  
  CONCLUSIONS
  The hK2-activated thapsigargin prodrug represents potential novel targeted therapy for prostate cancer.
authors.affiliation: Sidney Kimmel Comprehensive Cancer Center at Johns Hopkins, Bunting Blaustein Cancer Research Building, 1650 Orleans Street, Baltimore, MD 21231, USA.
authors.forename: Samuel
authors.initials: S
authors.lastname: Janssen
authors.forename: Carsten M
authors.initials: CM
authors.lastname: Jakobsen
authors.forename: D Marc
authors.initials: DM
authors.lastname: Rosen
authors.forename: Rebecca M
authors.initials: RM
authors.lastname: Ricklis
authors.forename: Ulrich
authors.initials: U
authors.lastname: Reineke
authors.forename: Soeren B
authors.initials: SB
authors.lastname: Christensen
authors.forename: Hans
authors.initials: H
authors.lastname: Lilja
authors.forename: Samuel R
authors.initials: SR
authors.lastname: Denmeade
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D000970:Antineoplastic Agents / Q000737:chemistry / Q000378:metabolism* / Q000494:pharmacology*; D002401:Cathepsin B / Q000037:antagonists & inhibitors / Q000378:metabolism; D045744:Cell Line, Tumor; D004353:Drug Evaluation, Preclinical; D006801:Humans; D006868:Hydrolysis; D008297:Male; D051379:Mice; D015394:Molecular Structure; D019151:Peptide Library; D010455:Peptides / Q000737:chemistry / Q000378:metabolism; D011355:Prodrugs / Q000737:chemistry / Q000378:metabolism* / Q000494:pharmacology*; D011471:Prostatic Neoplasms / Q000201:enzymology / Q000378:metabolism / Q000473:pathology*; D013379:Substrate Specificity; D019284:Thapsigargin / Q000031:analogs & derivatives / Q000737:chemistry / Q000378:metabolism / Q000494:pharmacology; D020840:Tissue Kallikreins / Q000097:blood / Q000378:metabolism*; D014357:Trypsin / Q000378:metabolism
chemical_list: D000970:Antineoplastic Agents; D019151:Peptide Library; D010455:Peptides; D011355:Prodrugs; D019284:Thapsigargin; D020840:Tissue Kallikreins; D014357:Trypsin; D002401:Cathepsin B