Hideaki Natsugari

Sulindac Sulfide Is a Noncompetitive γ-Secretase Inhibitor That Preferentially Reduces Aβ42 Generation

Nonsteroidal anti-inflammatory drugs (NSAIDs) have been known to reduce risk for Alzheimers 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 Aβ42 from cultured cells, although the molecular target(s) of NSAIDs in reducing the activity of γ-secretase for Aβ42 generation (γ42-secretase) still remain unknown. Here we show that sulindac sulfide (SSide) directly acts on γ-secretase and preferentially inhibits the γ42-secretase activity derived from the 3-[(3-cholamidopropyl)dimethylammonio]-2-hydroxy-1-propanesulfonate-solubilized membrane fractions of HeLa cells, in an in vitroγ-secretase assay using recombinant amyloid β precursor protein C100 as a substrate. SSide also inhibits activities for the generation of Aβ40 as well as for Notch intracellular domain at higher concentrations. Notably, SSide displayed linear noncompetitive inhibition profiles for γ42-secretase in vitro. Our data suggest that SSide is a direct inhibitor of γ-secretase that preferentially affects the γ42-secretase activity.

Both Notch1 and Notch2 contribute to the regulation of melanocyte homeostasis.

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 γ‐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.

Atropisomeric Properties of 7-, 8-, and 9-Membered-Ring Dibenzolactams: Conformation, Thermal Stability, and Chemical Reactivity

The atropisomeric enantiomers of 7-, 8-, and 9-membered-ring dibenzolactams were separated by using chiral HPLC, and their stereochemistries were clarified by using X-ray crystallographic analysis. The atropisomers showed high stereochemical stability with the 8-membered ring being the most stable. In 7- and 8-membered dibenzolactams, highly stereoselective C7-methylation proceeded from the lower side of the ring to provide the products with a C7-methyl group in the pseudoaxial orientation, which converted to thermodynamically more stable isomers with the pseudoequatorial C7-methyl group. In 9-membered dibenzolactam, C7-methylation occurred from the opposite (upper) side of the ring to provide a thermodynamically stable product with the pseudoequatorial C7-methyl group.

Dual antitumor mechanisms of Notch signaling inhibitor in a T-cell acute lymphoblastic leukemia xenograft model

Constitutive activation of Notch signaling is required for the proliferation of a subgroup of human T‐cell acute lymphoblastic leukemias (T‐ALL). Previous in vitro studies have demonstrated the therapeutic potential of Notch signaling inhibitors for treating T‐ALL. To further examine this possibility, we applied a γ‐secretase inhibitor (GSI) to T‐ALL xenograft models. Treatment of established subcutaneous tumors with GSI resulted in partial or complete regression of tumors arising from four T‐ALL cell lines that were also sensitive to GSI in vitro. To elucidate the mechanism of action, we transduced DND‐41 cells with the active form of Notch1 (aN1), which conferred resistance to in vitro GSI treatment. Nevertheless, in vivo treatment with GSI induced a partial but significant regression of subcutaneous tumors that developed from aN1‐transduced DND‐41 cells, whereas it induced complete regression of tumors that developed from mock‐transduced DND‐41 cells. These findings indicate that the remarkable efficacy of GSI might be attributable to dual mechanisms, directly via apoptosis of DND‐41 cells through the inhibition of cell‐autonomous Notch signaling, and indirectly via disturbance of tumor angiogenesis through the inhibition of non‐cell‐autonomous Notch signaling. (Cancer Sci 2009; 100: 2444–2450)

Isolation and characterization of atropisomers of seven-membered-ring benzolactams.

The atropisomeric properties of seven-membered-ring benzolactams (7a-c and 8a) [1,5-benzodiazepin-2-one (a), 1,5-benzothiazepin-4-one (b), and 1-benzazepin-2-one (c)] were examined. The atropisomers were isolated as the diastereomers with an (S)-phenethylamide moiety, which were characterized by X-ray crystallography, and the barriers to their interconversion were clarified.

Solid-phase synthesis of photoaffinity probes: highly efficient incorporation of biotin-tag and cross-linking groups

A benzophenone cross-linking group and a biotin-tag hybrid, resin 1a, attached to our novel resin 2 was readily converted to the photoaffnity probe 20 by condensation with the ligand carboxylic acid 19 and cleavage from the resin without purification.

O1-02-07: Different actions of dipeptidic compounds on presenilins define the enzyme specificity of γ-secretase inhibitor

accumulates as full-length protein. Objectives: Three possibilities are being tested that could explain the attenuated A 42-response of PS1Exon9. (i) The attenuated A 42-response is caused by lack of endoproteolytic cleavage, or (ii) is specifically associated with the S290C mutation, or (iii) resides in the 291-319 deletion or its conformational effects on regions flanking exon 9. Methods: CHO cell lines with stable overexpression of WT-PS1 or various PS-1 mutants are being compared in their response to the A 42-lowering NSAID sulindac sulfide. Results: CHO cells overexpressing WT-PS1 or mutants PS1Exon9, S290C, M292D and 291-319 have been created. M292D suppresses endoproteolytic cleavage but is otherwise fully functional in -secretase cleavage. Surprisingly, we found that the S290C mutation alone did not cause increased A 42 production indicating that the pathogenic effect of PS1Exon9 is a consequence of the combined action of the S290C mutation and the 291-319 deletion. Accordingly, the S290C mutation behaved similar to WT-PS1 and was not responsible for the blunted A 42-response of PS1Exon9 after sulindac sulfide treatment. Ongoing studies further aim to clarify whether lack of endoproteolysis or the 291-319 deletion underlie the attenuated A 42-response of PS1Exon9. Conclusions: These studies should resolve the molecular basis for the attenuated A 42-response of the PS1Exon9 mutation. They may further lead to definition of a critical region within PS1 that is required for the A 42-lowering activity of certain NSAIDs.