SPECIFIC AND EFFICIENT PEPTIDE-SUBSTRATES FOR ASSAYING THE PROTEOLYTIC ACTIVITY OF PROSTATE-SPECIFIC ANTIGEN

Prostate-specific antigen (PSA) is a serine protease secreted by both normal prostate glandular cells and prostate cancer cells. The major p roteolytic substrates for PSA are the gel-forming proteins in semen, s emenogelin (Sg) I and II. On the basis of the PSA cleavage map for Sg I and II, a series of small peptides (i.e., less than or equal to 7 am ino acids) was synthesized and coupled at the COOH terminus to 7-amino -4-methyl coumarin. Using these fluorescently tagged substrates, K(m)s and k(cat)s were determined for PSA hydrolysis, and the substrates we re also tested for activity against a panel of purified proteases. Pre viously, a variety of chymotrypsin substrates have been used to assay the enzymatic activity of PSA. The present studies have identified a p eptide sequence with a high degree of specificity for PSA (i.e., no de tectable hydrolysis by chymotrypsin) and improved K(m)s and k(cat)s ov er previously used substrates. On the basis of these parameters, the b est peptide substrate for PSA has the amino acid sequence HSSKLQ. Usin g PC-82 human prostate cancer xenografts and human prostate tissues, t his PSA substrate was used to document that prostate cancer cells secr ete enzymatically active PSA into the extracellular fluid but that onc e in the blood, PSA is not enzymatically active. On the basis of this information, it should be possible to use the HSSKLQ peptide as a carr ier to target peptide-coupled prodrugs for selective activation within sites of PSA-secreting, metastatic prostate cancer cells and not with in the blood or other nonprostatic normal tissues.