Targeting Presenilin-type Aspartic Protease Signal Peptide Peptidase with γ-Secretase Inhibitors

Abstract

Presenilin is implicated in the pathogenesis of Alzheimer's disease. It is thought to constitute the catalytic subunit of the ␥-secretase complex that catalyzes intramembrane cleavage of ␤-amyloid precursor protein, the last step in the generation of amyloidogenic A␤ peptides. The latter are major constituents of amyloid plaques in the brain of Alzheimer's disease patients. Inhibitors of ␥-secretase are considered potential therapeutics for the treatment of this disease because they prevent production of A␤ peptides. Recently, we discovered a family of presenilin-type aspartic proteases. The founding member, signal peptide peptidase, catalyzes intramembrane cleavage of distinct signal peptides in the endoplasmic reticulum membrane of animals. In humans, the protease plays a crucial role in the immune system. Moreover, it is exploited by the hepatitis C virus for the processing of the structural components of the virion and hence is an attractive target for anti-infective intervention. Signal peptide peptidase and presenilin share identical active site motifs and both catalyze intramembrane proteolysis. These common features let us speculate that ␥-secretase inhibitors directed against presenilin may also inhibit signal peptide peptidase. Here we demonstrate that some of the most potent known ␥-secretase inhibitors efficiently inhibit signal peptide peptidase. However, we found compounds that showed higher specificity for one or the other protease. Our findings highlight the possibility of developing selective inhibitors aimed at reducing A␤ generation without affecting other intramembranecleaving aspartic proteases. Alzheimer's disease (AD)1 is characterized by the formation of senile plaques in the brain. Major constituents of these plaques are the amyloidogenic 40-and 42-residue-long A␤ peptides A␤40 and A␤42, respectively (1). The amyloid cascade hypothesis casually links the generation of amyloid plaques with the neuropathological changes accompanying the symptoms typical of this disease (2). A␤ peptides are generated from the type I transmembrane protein ␤-APP (␤-amyloid precursor protein) by sequential proteolysis (3). The protein is first cleaved in the exoplasmic domain by the ␤-site APP-cleaving enzyme (BACE) to release the ectodomain (4, 5). The residual membrane-anchored stub of 99 residues (C99) is subsequently cleaved in the center of the transmembrane region by ␥-secretase (6). The resulting cleavage products, an A␤ peptide and the amyloid intracellular domain (AICD), are liberated from the lipid bilayer toward the exoplasm and cytosol, respectively (7-9).To date, the majority of characterized familial AD mutations are clustered along the presenilin-1 (PS1) gene (10, 11). They are thought to accelerate disease onset by increasing the A␤42/ A␤40 ratio (12). It is not well understood how these mutations, which are essentially scattered along the entire PS1 gene, can lead to a specific increase in the production of the 42-residuelong peptide that corresponds to the most amyloidogenic form of A␤ (13). ...

Signal Peptide Peptidase and γ-Secretase Share Equivalent Inhibitor Binding Pharmacology

“…3). The binding affinities (K d ) were 1.2 Ϯ 0.26 and 2.2 Ϯ 0.36 nM, respectively, consistent with the potencies of these compounds for inhibition of ␥-secretase and/or SPP (13,15,31 To evaluate the contribution of the presenilins to binding of each radioligand, homogenates were prepared from mouse embryonic fibroblast cell lines lacking expression of presenilin-1, presenilin-2, or both (43,44 (Fig. 4) 2 -ketone, and the NSAID sulindac sulfide.…”

“…Additional experiments indicated IC 50 values of ϳ2 and 10 M for the inhibitors (Z-LL) 2 -ketone and L-685,458, respectively, whereas no inhibition was observed in the presence of 10 M DAPT, BMS-299897, BMS-433796, or IN973 (data not shown). These potencies are lower than those reported previously (31,34). Although we do not understand the exact cause, it seems likely that differences in the assay conditions would provide the explanation.…”

“…prot, protein. (31,33). Furthermore, we showed that one of these non-transition state inhibitors, BMS-433796, exhibited noncompetitive displacement of [ 3 H]L-685,458 from SPP, suggesting a distinct binding site, which is potentially equivalent to the allosteric inhibitor binding site proposed for ␥-secretase (17,18).…”

“…Its activity is required for histocompatibility antigen E (HLA E) epitope formation (28); maturation of hepatitis C virus (29); and, in zebrafish, neuronal cell survival (30). Like presenilin, SPP can be photolabeled by a ␥-secretase transition state analog inhibitor, indicating conservation of active-site structure within the two enzymes (31,32). Furthermore, some of the non-transition state inhibitors also inhibit SPP (31,33), and even A␤ modulators affect SPP (34).…”

“…Like presenilin, SPP can be photolabeled by a ␥-secretase transition state analog inhibitor, indicating conservation of active-site structure within the two enzymes (31,32). Furthermore, some of the non-transition state inhibitors also inhibit SPP (31,33), and even A␤ modulators affect SPP (34). In contrast to presenilin, which requires additional subunits for enzyme activity, the active SPP enzyme does not require additional subunits for activity (32).…”