Nonsteroidal anti-inflammatory drugs (NSAIDs) have been known to reduce risk for Alzheimer's disease. In addition to the anti-inflammatory effects of NSAIDs to block cylooxygenase, it has been shown recently that a subset of NSAIDs selectively inhibits the secretion of highly amyloidogenic A␤42 from cultured cells, although the molecular target(s) of NSAIDs in reducing the activity of ␥-secretase for A␤42 generation (␥ 42 -secretase) still remain unknown. Here we show that sulindac sulfide (SSide) directly acts on ␥-secretase and preferentially inhibits the ␥ 42 -secretase activity derived from the 3-[(3-cholamidopropyl)dimethylammonio]-2-hydroxy-1-propanesulfonate-solubilized membrane fractions of HeLa cells, in an in vitro ␥-secretase assay using recombinant amyloid ␤ precursor protein C100 as a substrate. SSide also inhibits activities for the generation of A␤40 as well as for Notch intracellular domain at higher concentrations. Notably, SSide displayed linear noncompetitive inhibition profiles for ␥ 42 -secretase in vitro. Our data suggest that SSide is a direct inhibitor of ␥-secretase that preferentially affects the ␥ 42 -secretase activity. Alzheimer's disease (AD)1 is a dementing neurodegenerative disorder of the elderly characterized pathologically by neuronal loss in the cerebral cortex accompanied by massive deposition of amyloid ␤ peptides (A␤) as senile plaques (1). A␤ is produced by sequential proteolytic cleavages of the amyloid ␤ precursor protein (␤APP) by a set of membrane-bound proteases termed ␤-and ␥-secretases. The C-terminal length of A␤ generated by ␥-secretase is heterogeneous; A␤42 is a relatively minor molecular species of the A␤ secreted from cells, but it has a much higher propensity to aggregate and form amyloid compared with other A␤ species. These findings provide strong support for the hypothesis that the deposition of A␤42 is closely related to the pathogenesis of AD, implicating ␥-secretase as an important therapeutic target.Mutations in PS1 or PS2 genes account for the majority of early onset familial AD, and these mutations cause an increase in the ratio or levels of production of A␤42 (1). It is known that PS is essential for the ␥-secretase-mediated intramembranous cleavage not only for ␤APP but for other type I transmembrane proteins (e.g. Notch, ErbB4, E-cadherin, low density lipoprotein receptor-related protein, and CD44) (2). PS proteins undergo endoproteolysis to generate N-and C-terminal fragments and interact with other proteins (i.e. nicastrin, APH-1, and PEN-2) to form a high molecular weight (HMW) protein complex (3). The functional role of PS complex in ␥-secretase activity still remains unknown. However, aspartyl protease transition state analogue inhibitors of ␥-secretase, which harbors a hydroxyl ethylene isostere or a difluoro alcohol moiety, directly label PS fragments (4 -6). In addition, a systematic analysis using a variety of PS mutants revealed that HMW complex formation of PS as well as conserved aspartyl residues within the transmembrane domain are es...

␥-Secretase is a multimeric membrane protein complex composed of presenilin (PS), nicastrin, Aph-1 and, Pen-2 that is responsible for the intramembrane proteolysis of various type I transmembrane proteins, including amyloid ␤-precursor protein and Notch. The direct labeling of PS polypeptides by transition-state analogue ␥-secretase inhibitors suggested that PS represents the catalytic center of ␥-secretase. Here we show that one of the major ␥-secre- tase inhibitors of dipeptidic type, N-[N-(3,5-difluorophenacetyl)-Lalanyl]-S-phenylglycine t-butyl ester (DAPT), targets the C-terminal fragment of PS, especially the transmembrane domain 7 or more C-terminal region, by designing and synthesizing DAPBpB (N-[N-(3,5-difluorophenacetyl)-L-alanyl]-(S)-phenylglycine-4-(4-(8-biotinamido)octylamino)benzoyl)benzyl)methylamide), a photoactivable DAPT derivative. We also found that DAP-BpB selectively binds to the high molecular weight ␥-secretase complex in an activity-dependent manner. Photolabeling of PS by DAP-BpB is completely blocked by DAPT or its structural relatives (e.g. Compound E) as well as by arylsulfonamides. In contrast, transitionstate analogue inhibitor L-685,458 or ␣-helical peptidic inhibitor attenuated the photolabeling of PS1 only at higher concentrations. These data illustrate the DAPT binding site as a novel functional domain within the PS C-terminal fragment that is distinct from the catalytic site or the substrate binding site.

Notch signaling affects a variety of mammalian stem cells, but there has been limited evidence that a specific Notch molecule regulates adult stem cells. Recently, it was reported that the reduced Notch signaling initiated at the embryonic stage results in a gradual hair graying phenotype after birth. Here we demonstrate that the oral administration of a gamma-secretase inhibitor (GSI) to wild-type adult C57/Bl6 mice led to a gradual increase in gray spots, which remained unchanged for at least 20 weeks after discontinuing the GSI. In GSI-treated mice, there was a severe decrease in unpigmented melanocytes in the bulge/subbulge region where melanocyte stem cells are located. While we confirmed that Notch1+/-Notch2+/- double heterozygous mice with a C57/Bl6 background were born with a normal hair color phenotype and gradually turned gray after the second hair cycle, in the c-kit mutant Wv background, Notch1+/- and Notch2+/- mice had larger white spots on the first appearance of hair than did the Wv/+ mice, which did not change throughout life. Notch1+/-Notch2+/-Wv/+ mice had white hair virtually all over the body at the first appearance of hair and the depigmentation continued to progress thereafter. Using a neural crest organ culture system, GSI blocked the generation of pigmented melanocytes when added to the culture during the period of melanoblast proliferation, but not during the period of differentiation. These observations imply roles of Notch signaling in both development of melanocyte during embryogenesis and maintenance of melanocyte stem cells in adulthood, while the degree of requirement is distinct in these settings: the latter is more sensitive than the former to the reduced Notch signaling. Furthermore, Notch1 and Notch2 cooperates with c-kit signaling during embryogenesis, and they cooperate with each other to regulate melanocyte homeostasis after birth.

Divergent synthesis of multifunctional molecular probes based on caprolactam-derived dipeptidic gamma-secretase inhibitors (GSIs), Compound E (CE) and LY411575 analogue (DBZ), was efficiently accomplished by means of Cu(I)-catalyzed azide/alkyne fusion reaction. Photoaffinity labeling experiments using these derivatives coupled to photoactivatable and biotin moieties provided direct evidence that the molecular targets of CE and DBZ are the N-terminal fragment of presenilin 1 within the gamma-secretase complex. Moreover, these photoprobes directly targeted signal peptide peptidase. These data suggest that the divergent synthesis of molecular probes has been successfully applied to characterize the interaction of GSIs with their molecular targets and define the structural requirements for inhibitor binding to intramembrane-cleaving proteases.