Development of a specific inhibitor for the placental protease, cathepsin P

Gene duplications in rodents have given rise to a family of proteases that are expressed exclusively in placenta. To define the biological role of these enzymes specific inhibitors are needed to differentiate their activities from other more ubiquitously expressed proteases, such as cathepsins B and L. Libraries of peptidyl inhibitors based upon a 4-cyclohexanone pharmacophore were screened for inhibition of cathepsins P, L, and B. The tightest binding dipeptidyl inhibitor for cathepsin P contained Tyr in P2 and Trp in P2′, consistent with the specificity of this enzyme for hydrophobic amino acids at these sites in synthetic substrates. An inhibitor containing Trp in both P2 and P2′ provided better discrimination between cathepsin P and cathepsins B and L. Extension of the inhibitors to include P3, and P3′ amino acids identified an inhibitor with Trp in P2, P2′, and P3, and Phe in P3′ that bound to cathepsin P with a Ki of 32 nM. This specificity for inhibitors with hydrophobic aromatic amino acids in these four positions is unique among the lysosomal cysteine proteases. This inhibitor bound to cathepsin P an order of magnitude tighter than to mouse and human cathepsin L and two orders of magnitude tighter than to human cathepsin B. Cbz-Trp-Trp-4-cyclohexanone-Trp-Phe-OMe can discriminate cathepsin P from cathepsins B and L and consequently can be used to specifically inhibit and identify cathepsin P in cellular systems.