Bioinformatics tools such as Perl, Visual Basic, Cluster, and TreeView were used to analyze public gene expression databases in order to identify potential
enzyme targets for
prodrug strategies. The analyses indicated that
prolidase might be a desirable
enzyme target based on its differential expression in
melanoma cancer cell lines and its high substrate specificity for
dipeptides containing
proline at the carboxy terminus. RT-PCR expression of
prolidase and hydrolytic activity against N-
glycyl-l-proline (
GLY-PRO), a standard substrate of
prolidase, determined in tumor cell lines, exhibited a high correlation (r(2) = 0.95). These results suggest the possibility of targeting
prolidase with
prodrugs of
anticancer agents for enhanced selectivity. The feasibility of such a scenario was tested by (a) synthesizing
prodrugs of
melphalan that comprised linkage of the carboxy terminus of the
l-phenylalanine moiety of
melphalan to the N-terminus of l and d stereoisomers of
proline and (b) determining their bioconversion and antiproliferative activities in SK-MEL-5 cells, a
melanoma cancer cell line with high expression levels of
prolidase. The results of hydrolysis studies of the l- and d-
proline prodrugs of
melphalan, designated as
prophalan-l and
prophalan-d, respectively, indicated a approximately 7-fold higher rate of activation of
prophalan-l compared to
prophalan-d in SK-MEL-5 cell homogenates.
Prophalan-l exhibited cytotoxicity (GI(50) = 74.8 microM) comparable to that of
melphalan (GI(50) = 57.0 microM) in SK-MEL-5 cells while
prophalan-d was ineffective, suggesting that
prolidase-specific activation to the parent drug may be essential for cytotoxic action. Thus,
melphalan prodrugs such as
prophalan-l that are cleavable by
prolidase offer the potential for enhanced selectivity by facilitating cytotoxic activity only in cells overexpressing
prolidase.