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Aspartic proteases have been the subject of intensive physicochemical research in the areas of catalytic mechanism, three-dimensional structure, zymogen activation and substrate specificity.1,2 The important roles of various members of this family in human diseases, including hypertension,3 cancer,4 malaria5 and AIDS6 have made these enzymes the target of rational drug design efforts. Thermodynamic characterization of inhibitor binding is important for our understanding of the mechanism and energetics of enzyme-drug interactions. Recent developments in high sensitivity isothermal titration calorimetry (ITC) have made it possible to obtain extensive thermodynamic information for molecular interactions.9,10 The binding of pepstatin to three aspartic proteases, endothiapepsin from Endothia parasitica, plasmepsin II from Plasmodium falciparum and cathepsin D from human liver have been characterized using this technique.7,8

Cathepsin D (EC 3.4.23.5) (CatD) is an intracellular aspartic protease (AP) that is normally found in the lysosomes of higher eukaryotes. CatD shares two structural features with lysosomal enzymes that distinguish it from most secreted, extracellular APs. First, the mature enzyme from humans is found as a two-chain enzyme as a result of post-translational cleavage and removal of an insertion loop in the N-domain. Second, CatD contains phosphorylated, N-linked oligosaccharides that target the enzyme to lysosomes via man-nose-6-phosphate receptors (MPR). Interest in CatD as a target for drug design results from its association with several biological processes of therapeutic significance, particularly breast cancer and Alzheimer’s disease (Table 1). Recent studies have implicated CatD in the processing of (β-amyloid precursor protein to promote amyloid plaque formation in Alzheimer’s brain. Numerous studies of primary breast cancers have demonstrated that elevated levels of CatD were correlated with an increased risk of metastasis and shorter relapse-free survival. High levels of CatD produced in the vicinity of the growing tumor may degrade the extracellular matrix, either directly or indirectly by potentiating the activity of other tissue proteases, such as collagenase, and thereby promote tumor growth and metastasis. While a role for CatD or other APs in metastasis has been questioned by recent results that show a negative effect of pepstatin A in an in vitro invasion assay using breast cancer-derived MCF7 cells, new studies have revealed that elevated levels of CatD secretion in breast cancers correlate with tumor aggressiveness.

The structures of three aspartic proteases, penicillopepsin, endothiapepsin and rhizopuspepsin, have been determined in complexes with difluorostatone (DFS)-containing peptide-based inhibitors [1–3]. In all three structures, the hydrated core of the inhibitors display similar conformations and interactions with the active site aspartate groups. One of the gem-hydroxyls is positioned between both aspartates, the other one interacts with a single aspartate, and the fluorines do not interact with either of them. Since one of the hydroxyl groups is located near the position of the active site water observed in the native structures, these inhibitors have been proposed as analogs of transition state intermediates of peptide bond hydrolysis.