Inhibition of cathepsin D by synthetic oligopeptides.

A number of synthetic oligopeptides with the COOH terminus D-amino acid situated third from the potentially cleavable phenylalanyl-phenylalanyl bond, typified by < Glu-D-Phe-Pro-Phe-Phe-Val-D-Trp (Peptide VI) were shown to be potent competitive inhibitors of cathepsin D and pepsin. Peptide VI, which forms an equimolar nonproductive enzyme l inhibitor complex, inhibited the hydrolysis of methyl[‘4C]glycinated hemoglobin by porcine pepsin with a Ki of 0.4 pM, by rabbit liver cathepsin D with a Ki of 0.01 PM, and the cleavage of the COOH terminus L-tryptophanyl analog at the Phe-Phe bond by cathepsin D with a Ki of 0.06 M. The pH profile of the inhibition of cathepsin D by the peptide followed that of the enzyme reaction. Derivatization of the carboxyl group at the COOH terminus into a nondissociable form diminished the inhibitory activity. The minimum unit to display significant inhibition is probably the segment of Pro-Phe-Phe-Val-DTrp. The interaction contributed by the additional 2 NH2 terminus residues facilitated binding. Enantiomerit substitution at the Phe-Phe unit or the valyl residue reduced the inhibitory capacity. The COOH terminus residue is preferably a large hydrophobic Damino acid. This class of inhibitors was effective only on carboxyl proteinases such as cathepsin D and pepsin. Bovine (Ychymotrypsin and subtilisin inactivated the inhibitors by cleaving the Phe-Phe bond. In the presence of Peptide VI at a molar ratio of peptide to enzyme of lOO:l, the rapid inactivation of cathepsin D by acetyl-DL-norleucine methyl ester and Cu2+ was retarded by about 20%, and the slow inactivation by 1,2-epoxy-3-(p-nitrophenoxy)propane was reduced by about 65%. It is proposed that the potent inhibitory activity of the peptide may be attributable, in part, to the perturbation of the function of a catalytically critical carboxylate group in the active site of the enzyme by the COOH terminus Damino acid carboxylate group of the peptide, which binds along the primary and secondary binding sites.