Notch (IC50 = 2.92±0.22 nM); APPL (IC50 = 2.64±0.30 nM)

YO-01027 targets the N-terminal Presenilin fragment and directly interacts with the γ-secretase complex. When APPL- or Notch-expressing cells are exposed to increasing concentrations of YO-01027, APPL CTF fragment accumulation progresses and NICD production declines in a strictly dose-dependent manner. (Source: ) YO-01027 at 10 μM decreases the quantity and activity of breast cancer stem cells (BCSCs).[2] According to a recent study, YO-01027 inhibits the production of the mucin protein MUC16 in undifferentiated cells via Notch inhibition at both the preconfluent and confluent stages, but not in postmitotic stratified cells. This effect is concentration-dependent.[3]

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Here researchers demonstrate that Notch3 is highly expressed in undifferentiated and differentiated HCLE and HCjE cells, and that Notch1 and Notch2 biosynthesis is enhanced by induction of differentiation with serum-containing media. Inhibition of Notch signaling with DBZ impaired MUC16 biosynthesis in a concentration-dependent manner in undifferentiated cells at both preconfluent and confluent stages, but not in postmitotic stratified cells. In contrast to protein levels, the amount of MUC16 transcripts were not significantly reduced after DBZ treatment, suggesting that Notch regulates MUC16 posttranscriptionally. Immunoblots of DBZ-treated epithelial cells grown at different stages of differentiation revealed no differences in the levels of MUC1 and MUC4. Conclusions: These results indicate that MUC16 biosynthesis is posttranscriptionally regulated by Notch signaling at early stages of epithelial cell differentiation, and suggest that Notch activation contributes to maintaining a mucosal phenotype at the ocular surface.[3]

YO-01027 increases latency compared to control mice (18-28 days) and significantly reduces MCF7 tumors but not MDA-MB-231 tumors when administered intraperitoneally (1 mg/mL) on the day of cell injection and every 3 days after that. When MCF7 tumors did develop, they were considerably smaller thanks to YO-01027 treatment.[2] In intestine adenomas, YO-01027 treatment in C57BL/6 mice dose-dependently reduces the proliferation of epithelial cells and promotes goblet cell generation.[4]

To ascertain the effective linear range and maximal inhibitory dose of YO-01027, pilot studies are conducted utilizing varying drug concentrations spanning from 0.1 nM to 250 nM. When Notch or APPL expression is induced, six hours prior to protein harvesting, YO-01027 is added at the appropriate concentrations to the S2 cell medium. In the lysis buffer for protein extraction and immunoblot analysis, YO-01027 is additionally added for every sample at the appropriate concentration.

Resuspended cells at ≤1 × 10⁶ are incubated with preconjugated primary antibodies BEREP4-FITC (1:10), CD44-APC (1:20), and CD24-PE (1:10) for 10 minutes at 4 °C in 100 μL sorting buffer (PBS containing 0.5% bovine serum albumin, 2 mM EDTA). After being cleaned with PBS, the cells are centrifuged for two minutes at 800 × g. Cells are resuspended in 500 μL of sorting buffer for analysis, and FACSCalibur is used to measure fluorescence and WinMIDI 2.8 is used for analysis. Following primary antibody incubation, cells are resuspended in 1× HBSS for sorting. Using FACSAria, cells are sorted at 16 p.s.i. with HBSS serving as the sheath fluid. The lowest quintile of CD24-positive cells plus all CD24-negative cells make up the CD24low cell population, which is gated by FACS.

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HCLE and HCjE cells were grown at different stages of differentiation, representing nondifferentiated (preconfluent and confluent) and differentiated (stratified) epithelial cultures. Notch signaling was blocked with the γ-secretase inhibitor dibenzazepine (DBZ). The presence of Notch intracellular domains (Notch1 to Notch3) and mucin protein (MUC1, -4, -16) was evaluated by electrophoresis and Western blot analysis. Mucin gene expression was determined by TaqMan real-time polymerase chain reaction.[3]

Mice: In this study, male C57BL/6J wild-type (WT) and Apo E^-/- mice are used. Four weeks of daily treatment are administered to Ang II-treated mice via intraperitoneal injection, starting the day before mini-pump implantation and continuing every day thereafter with either a saline vehicle or the γ-secretase inhibitor dibenzazepine (DBZ) (1 mg/kg/d, dissolved in saline). Using an automated tail-cuff system, blood pressure is measured in conscious mice. Every rodent is sedated. To facilitate additional histological and molecular analysis, the aortic tissues are removed.

[1]. Mol Pharmacol . 2010 Apr;77(4):567-74.

[2]. Cancer Res . 2010 Jan 15;70(2):709-18

[3]. Invest Ophthalmol Vis Sci . 2011 Jul 29;52(8):5641-6.

[4]. Nature . 2005 Jun 16;435(7044):959-63.

CLE and HCjE cells were grown at different stages of differentiation, representing nondifferentiated (preconfluent and confluent) and differentiated (stratified) epithelial cultures. Notch signaling was blocked with the γ-secretase inhibitor dibenzazepine (DBZ). The presence of Notch intracellular domains (Notch1 to Notch3) and mucin protein (MUC1, -4, -16) was evaluated by electrophoresis and Western blot analysis. Mucin gene expression was determined by TaqMan real-time polymerase chain reaction. Results: Here we demonstrate that Notch3 is highly expressed in undifferentiated and differentiated HCLE and HCjE cells, and that Notch1 and Notch2 biosynthesis is enhanced by induction of differentiation with serum-containing media. Inhibition of Notch signaling with DBZ impaired MUC16 biosynthesis in a concentration-dependent manner in undifferentiated cells at both preconfluent and confluent stages, but not in postmitotic stratified cells. In contrast to protein levels, the amount of MUC16 transcripts were not significantly reduced after DBZ treatment, suggesting that Notch regulates MUC16 posttranscriptionally. Immunoblots of DBZ-treated epithelial cells grown at different stages of differentiation revealed no differences in the levels of MUC1 and MUC4. Conclusions: These results indicate that MUC16 biosynthesis is posttranscriptionally regulated by Notch signaling at early stages of epithelial cell differentiation, and suggest that Notch activation contributes to maintaining a mucosal phenotype at the ocular surface.[3]

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he gamma-secretase aspartyl protease is responsible for the cleavage of numerous type I integral membrane proteins, including amyloid precursor protein (APP) and Notch. APP cleavage contributes to the generation of toxic amyloid beta peptides in Alzheimer's disease, whereas cleavage of the Notch receptor is required for normal physiological signaling between differentiating cells. Mutagenesis studies as well as in vivo analyses of Notch and APP activity in the presence of pharmacological inhibitors indicate that these substrates can be differentially modulated by inhibition of mammalian gamma-secretase, although some biochemical studies instead show nearly identical dose-response inhibitor effects on Notch and APP cleavages. Here, we examine the dose-response effects of several inhibitors on Notch and APP in Drosophila melanogaster cells, which possess a homogeneous form of gamma-secretase. Four different inhibitors that target different domains of gamma-secretase exhibit similar dose-response effects for both substrates, including rank order of inhibitor potencies and effective concentration ranges. For two inhibitors, modest differences in inhibitor dose responses toward Notch and APP were detected, suggesting that inhibitors might be identified that possess some discrimination in their ability to target alternative gamma-secretase substrates. These findings also indicate that despite an overall conservation in inhibitor potencies toward different gamma-secretase substrates, quantitative differences might exist that could be relevant for the development of therapeutically valuable substrate-specific inhibitors.[1]

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Notch receptor signaling pathways play an important role not only in normal breast development but also in breast cancer development and progression. We assessed the role of Notch receptors in stem cell activity in breast cancer cell lines and nine primary human tumor samples. Stem cells were enriched by selection of anoikis-resistant cells or cells expressing the membrane phenotype ESA(+)/CD44(+)/CD24(low). Using these breast cancer stem cell populations, we compared the activation status of Notch receptors with the status in luminally differentiated cells, and we evaluated the consequences of pathway inhibition in vitro and in vivo. We found that Notch4 signaling activity was 8-fold higher in stem cell-enriched cell populations compared with differentiated cells, whereas Notch1 signaling activity was 4-fold lower in the stem cell-enriched cell populations. Pharmacologic or genetic inhibition of Notch1 or Notch4 reduced stem cell activity in vitro and reduced tumor formation in vivo, but Notch4 inhibition produced a more robust effect with a complete inhibition of tumor initiation observed. Our findings suggest that Notch4-targeted therapies will be more effective than targeting Notch1 in suppressing breast cancer recurrence, as it is initiated by breast cancer stem cells.[2]

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he self-renewing epithelium of the small intestine is ordered into stem/progenitor crypt compartments and differentiated villus compartments. Recent evidence indicates that the Wnt cascade is the dominant force in controlling cell fate along the crypt-villus axis. Here we show a rapid, massive conversion of proliferative crypt cells into post-mitotic goblet cells after conditional removal of the common Notch pathway transcription factor CSL/RBP-J. We obtained a similar phenotype by blocking the Notch cascade with a gamma-secretase inhibitor. The inhibitor also induced goblet cell differentiation in adenomas in mice carrying a mutation of the Apc tumour suppressor gene. Thus, maintenance of undifferentiated, proliferative cells in crypts and adenomas requires the concerted activation of the Notch and Wnt cascades. Our data indicate that gamma-secretase inhibitors, developed for Alzheimer's disease, might be of therapeutic benefit in colorectal neoplastic disease.[4]

C26H23F2N3O3

463.48

463.17

C, 67.38; H, 5.00; F, 8.20; N, 9.07; O, 10.36

209984-56-5

YO-01027;209984-56-5

11454028

Yellow to orange solid powder.

1.4±0.1 g/cm3

801.3±65.0 °C at 760 mmHg

257-259ºC

438.4±34.3 °C

0.0±2.8 mmHg at 25°C

1.637

4.6

2

5

5

34

756

2

FC1C([H])=C(C([H])=C(C=1[H])C([H])([H])C(N([H])[C@@]([H])(C([H])([H])[H])C(N([H])[C@]1([H])C(N(C([H])([H])[H])C2=C([H])C([H])=C([H])C([H])=C2C2=C([H])C([H])=C([H])C([H])=C12)=O)=O)=O)F

QSHGISMANBKLQL-OWJWWREXSA-N

InChI=1S/C26H23F2N3O3/c1-15(29-23(32)13-16-11-17(27)14-18(28)12-16)25(33)30-24-21-9-4-3-7-19(21)20-8-5-6-10-22(20)31(2)26(24)34/h3-12,14-15,24H,13H2,1-2H3,(H,29,32)(H,30,33)/t15-,24-/m0/s1

(2S)-2-[[2-(3,5-difluorophenyl)acetyl]amino]-N-[(7S)-5-methyl-6-oxo-7H-benzo[d][1]benzazepin-7-yl]propanamide

Dibenzazepine;  YO01027; Iminostilbene; YO 01027; DBZ; 209984-56-5; (S)-2-(2-(3,5-Difluorophenyl)acetamido)-N-((S)-5-methyl-6-oxo-6,7-dihydro-5H-dibenzo[b,d]azepin-7-yl)propanamide; Dibenzazepine (Deshydroxy LY 411575); Deshydroxy LY-411575; DBZ; C26H23F2N3O3; YO01027; YO-01027; Deshydroxy LY-411575

2934.99.9001

Powder      -20°C    3 years

                     4°C     2 years

In solvent   -80°C    6 months

                  -20°C    1 month

Room temperature (This product is stable at ambient temperature for a few days during ordinary shipping and time spent in Customs)

Solubility in Formulation 1: ≥ 2.5 mg/mL (5.39 mM) (saturation unknown) in 10% DMSO + 90% Corn Oil (add these co-solvents sequentially from left to right, and one by one), clear solution.
For example, if 1 mL of working solution is to be prepared, you can add 100 μL of 25.0 mg/mL clear DMSO stock solution to 900 μL of corn oil and mix evenly.

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Solubility in Formulation 2: 0.5% hydroxyethyl cellulose: 6 mg/mL

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 (Please use freshly prepared in vivo formulations for optimal results.)