Prolidase, a Potential Enzyme Target for Melanoma:  Design of Proline-Containing Dipeptide-like Prodrugs

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-<SCP>l</SCP>-proline (GLY-PRO), a standard substrate of prolidase, determined in tumor cell lines, exhibited a high correlation (r² = 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 <SCP>l</SCP>-phenylalanine moiety of melphalan to the N-terminus of <SCP>l</SCP> and <SCP>d</SCP> 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 <SCP>l</SCP>- and <SCP>d</SCP>-proline prodrugs of melphalan, designated as prophalan-<SCP>l</SCP> and prophalan-<SCP>d</SCP>, respectively, indicated a ~7-fold higher rate of activation of prophalan-<SCP>l</SCP> compared to prophalan-<SCP>d</SCP> in SK-MEL-5 cell homogenates. Prophalan-<SCP>l</SCP> exhibited cytotoxicity (GI<INF>50</INF> = 74.8 μM) comparable to that of melphalan (GI<INF>50</INF> = 57.0 μM) in SK-MEL-5 cells while prophalan-<SCP>d</SCP> was ineffective, suggesting that prolidase-specific activation to the parent drug may be essential for cytotoxic action. Thus, melphalan prodrugs such as prophalan-<SCP>l</SCP> that are cleavable by prolidase offer the potential for enhanced selectivity by facilitating cytotoxic activity only in cells overexpressing prolidase. Keywords: Bioinformatics; prolidase; enzyme targeting; melanoma; prodrugs; melphalan