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A highly efficient and versatile synthetic method for amines was established using nitrobenzenesulfonamides (Ns-amides) as both a protecting and activating group. The alkylation of N-monosubstituted Ns-amides either proceeded conventionally or under Mitsunobu conditions to provide the N,N-disubstituted sulfonamides, and the Ns group was removed easily with soft nucleophiles via Meisenheimer complexes to give the corresponding secondary amines. The major advantage of this protocol is that both alkylation and deprotection proceed under mild conditions. Thus, with this methodology, the total synthesis of linear and/or macrocyclic natural polyamines can be accomplished efficiently.

Neuroligin (NLG), a postsynaptic adhesion molecule, is involved in the formation of synapses by binding to a cognate presynaptic ligand, neurexin. Here we report that neuroligin-1 (NLG1) undergoes ectodomain shedding at the juxtamembrane stalk region to generate a secreted form of NLG1 and a membrane-tethered C-terminal fragment (CTF) in adult rat brains in vivo as well as in neuronal cultures. Pharmacological and genetic studies identified ADAM10 as the major protease responsible for NLG1 shedding, the latter being augmented by synaptic NMDA receptor activation or interaction with soluble neurexin ligands. NLG1-CTF was subsequently cleaved by presenilin/γ-secretase. Secretion of soluble NLG1 was significantly upregulated under a prolonged epileptic seizure condition, and inhibition of NLG1 shedding led to an increase in numbers of dendritic spines in neuronal cultures. Collectively, neuronal activity-dependent proteolytic processing of NLG1 may negatively regulate the remodeling of spines at excitatory synapses.

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...

The total synthesis of ecteinascidin 743 (1), an extremely potent antitumor agent, has been accomplished. The synthesis features Ugi's 4CC reaction, intramolecular Heck reaction, phenol-aldehyde cyclization, and acid-induced intramolecular sulfide formation.

Sphingosine kinases (SphK) 1 and 2 phosphorylate sphingosine to generate sphingosine-1-phosphate (S1P), a pluripotent lipophilic mediator implicated in a variety of cellular events. Here we show that the activity of β-site APP cleaving enzyme-1 (BACE1), the rate limiting enzyme for amyloid-β peptide (Aβ) production, is modulated by S1P in neurons. Treatment by SphK inhibitor, RNAi knockdown of SphK or overexpression of S1P degrading enzymes decreased BACE1 activity to reduce Aβ production. S1P specifically bound to full-length BACE1 and increased its proteolytic activity, suggesting that the cellular S1P directly modulates BACE1 activity. Notably, the relative activity of SphK2 was upregulated in the brains of patients with Alzheimer disease. The unique modulatory effect of cellular S1P on BACE1 activity is a novel potential therapeutic target for Alzheimer disease.

␥-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.

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