Structure-based subsite specificity mapping of human cathepsin D using statine-based inhibitors.

Human cathepsin D is a lysosomal aspartic protease that has been implicated in breast cancer metastasis and Alzheimer's disease. Based on a crystal structure of a human cathepsin D-pepstatin A complex, a series of statine-containing inhibitors was designed, synthesized, and tested for inhibitory activity toward the enzyme in vitro. The compounds were modified systematically at individual positions (P4, P3, P2, P1, and P2t) with the aim of mapping the cathepsin D subsite preferences. The experimentally obtained SAR data were correlated on the basis of molecular modeling. Side-chain preferences for the peptidomimetic inhibitors differed from those found previously using peptide substrates (Scarborough PE et al., 1993, Protein Sci 2:264-276). In addition, the effects of single side-chain modifications were often nonadditive. Structure-activity relationships, modeling, and thermodynamic analysis indicated that entropy plays a major stabilizing role in inhibitor binding to cathepsin D.