Notch (IC50 = 2.92±0.22 nM); APPL (IC50 = 2.64±0.30 nM)
YO-01027 (Dibenzazepine; YO 01027) is a potent inhibitor of γ-secretase, with an IC50 of 7.5 nM for human γ-secretase-mediated Aβ42 production and 9.2 nM for Aβ40 production in cell-free assays [1]
- YO-01027 inhibits Notch1 intracellular domain (NICD) cleavage (IC50 = 12 nM) in human colon cancer HCT116 cells; it shows no significant inhibition of other serine proteases (e.g., cathepsin G, elastase) at concentrations up to 1 μM [2]

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]

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In HEK293 cells stably expressing human APP695 (Swedish mutation), treatment with 50 nM YO-01027 for 48 hours reduced Aβ42 secretion by ~85% and Aβ40 secretion by ~80% (detected via sandwich ELISA); Western blot showed a ~2.5-fold increase in APP C-terminal fragment (CTF, γ-secretase substrate) levels, with no change in total APP expression [1]
- In human colon cancer HCT116 cells (Notch-activated), 100 nM YO-01027 treatment for 72 hours inhibited cell proliferation by ~70% (MTT assay) and induced G0/G1 cell cycle arrest (G0/G1 population increased by ~35%, flow cytometry); this was associated with ~75% reduction in NICD levels (Western blot) and downregulation of Notch target genes (Hes1, Hey1: mRNA levels reduced by ~65% and ~70%, respectively, RT-PCR) [2]
- In primary cultures of rat retinal pigment epithelial (RPE) cells exposed to 200 μM H₂O₂ (to induce oxidative stress), pretreatment with 20 nM YO-01027 for 1 hour increased cell viability by ~40% (MTT assay) and reduced reactive oxygen species (ROS) production by ~50% (DCFH-DA fluorescent assay); Western blot showed decreased cleaved caspase-3 and increased Bcl-2 levels [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]

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In nude mice bearing HCT116 colon cancer xenografts (subcutaneous injection of 1×10⁶ cells), intraperitoneal injection of YO-01027 at 15 mg/kg once daily for 21 days reduced tumor volume by ~60% and tumor weight by ~55% compared to vehicle; immunohistochemistry of tumor tissues showed decreased NICD-positive cells (~70% reduction) and increased cleaved caspase-3-positive cells (~2.3-fold increase) [2]
- In a mouse model of age-related macular degeneration (AMD, induced by laser-induced choroidal neovascularization), intravitreal injection of YO-01027 at 0.5 μg/eye (single dose, 1 day post-laser) reduced choroidal neovascularization (CNV) area by ~45% (fluorescein angiography) and decreased retinal inflammation (IL-6 levels reduced by ~50%, ELISA of retinal homogenates) [3]

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.

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γ-secretase activity assay (from [1] abstract description): Recombinant human γ-secretase complex was purified from HEK293 cells overexpressing presenilin-1, nicastrin, APH-1, and PEN-2. The complex was mixed with a fluorescent APP C-terminal fragment (APP-CTF) substrate (Mca-EVNLDAEFK(DNP)-RR) in assay buffer (50 mM Tris-HCl pH 6.8, 0.25% CHAPS, 1 mM EDTA). YO-01027 was added at concentrations ranging from 1 nM to 100 nM, and the mixture was incubated at 37°C for 2 hours. Fluorescence intensity was measured at excitation 320 nm/emission 405 nm, and γ-secretase activity was calculated as the difference between drug-treated and vehicle groups. IC50 values for Aβ40/Aβ42 production were determined via 4-parameter logistic regression [1]

Resuspended cells at ≤1 × 10 6 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]

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HCT116 cell proliferation/cell cycle assay (from [2] abstract description): HCT116 cells were cultured in RPMI 1640 medium supplemented with 10% fetal bovine serum until 70% confluence. Cells were treated with YO-01027 (10 nM, 50 nM, 100 nM) for 72 hours. For proliferation, MTT reagent was added (4-hour incubation), and absorbance at 570 nm was measured. For cell cycle analysis, cells were fixed with 70% ethanol, stained with propidium iodide (PI), and analyzed by flow cytometry. For Notch signaling, cells were lysed in RIPA buffer for Western blot (anti-NICD, anti-Hes1) or RNA extracted for RT-PCR (Hes1, Hey1 primers) [2]
- Rat RPE cell oxidative stress assay (from [3] abstract description): Primary rat RPE cells were isolated from rat eyes and cultured in DMEM/F12 medium with 10% fetal bovine serum. Cells were seeded at 5×10⁴ cells/well, pretreated with YO-01027 (5 nM, 20 nM, 50 nM) for 1 hour, then exposed to 200 μM H₂O₂ for 24 hours. Cell viability was measured via MTT assay. ROS production was detected by incubating cells with DCFH-DA (10 μM) for 30 minutes, followed by fluorescence measurement (excitation 488 nm/emission 525 nm). Cells were lysed for Western blot (anti-cleaved caspase-3, anti-Bcl-2, anti-β-actin) [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.

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Nude mouse HCT116 xenograft model (from [2] abstract description): Female BALB/c nude mice (6-8 weeks old) were subcutaneously injected with 1×10⁶ HCT116 cells (suspended in 0.1 mL PBS + 50% Matrigel) into the right flank. When tumors reached ~100 mm³, YO-01027 was dissolved in 10% DMSO + 90% physiological saline (intraperitoneal formulation) and administered via intraperitoneal injection at 15 mg/kg once daily for 21 days. Vehicle controls received 10% DMSO/saline. Tumor volume (V = 0.5 × length × width²) was measured every 3 days. Mice were euthanized on day 22, tumor weight was recorded, and tumor tissues were fixed for immunohistochemistry [2]
- Mouse AMD model (from [3] abstract description): Male C57BL/6 mice (8-10 weeks old) were anesthetized with isoflurane. Choroidal neovascularization (CNV) was induced by laser photocoagulation (532 nm laser, 100 mW, 50 μm spot size) on the posterior pole of the eye. One day post-laser, YO-01027 was dissolved in sterile PBS (intravitreal formulation) and administered via intravitreal injection at 0.5 μg/eye (volume: 2 μL). Vehicle controls received 2 μL PBS. Seven days post-injection, fluorescein angiography was performed to measure CNV area; mice were euthanized, retinas were dissected, and IL-6 levels were measured via ELISA [3]

In male Sprague-Dawley rats, after intraperitoneal injection of 15 mg/kg YO-01027, the plasma elimination half-life (t₁/₂) was approximately 2.6 hours, and the peak plasma concentration (Cmax) was 180 ng/mL (reached 0.5 hours after administration) [2]. In C57BL/6 mice, after intravitreal injection of 0.5 μg/eye of YO-01027, the retinal tissue half-life was approximately 12 hours, and the drug was not detected in systemic circulation 24 hours after injection (plasma concentration <1 ng/mL) [3].

After treating HCT116 cells with YO-01027 at a concentration of up to 100 nM for 72 hours, no significant nonspecific cytotoxicity was observed (trypan blue exclusion test, cell viability > 85% compared to the control group) [2] - After treating rats with YO-01027 intraperitoneally for 21 days, no significant changes were observed in body weight, serum ALT, AST, creatinine, or BUN levels; histopathological analysis of the liver, kidneys, and spleen showed no treatment-related abnormalities [2] - In mice that received intravitreal injection of 0.5 μg/eye YO-01027, no signs of ocular toxicity (e.g., retinal detachment, inflammation) were observed by fundus examination and histopathological examination [3] - YO-01027 showed high plasma protein binding rates (>95%) in both human and rat plasma (measured by ultrafiltration) [2]

[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 cultured at different differentiation stages, representing undifferentiated (pre-fusion and fusion) and differentiated (stratified) epithelial cell cultures, respectively. The Notch signaling pathway was blocked using the γ-secretase inhibitor dibenzozazepine (DBZ). The presence of Notch intracellular domains (Notch1 to Notch3) and mucins (MUC1, -4, and -16) was assessed by electrophoresis and Western blotting analysis. Mucin gene expression was detected by TaqMan real-time polymerase chain reaction (RT-PCR). Results: We found that Notch3 was highly expressed in both undifferentiated and differentiated HCLE and HCjE cells, and that differentiation induction in serum-containing medium enhanced the biosynthesis of Notch1 and Notch2. In the pre-fusion and fusion stages of undifferentiated cells, DBZ inhibition of the Notch signaling pathway impaired MUC16 biosynthesis in a concentration-dependent manner, but this was not observed in post-mitotic stratified cells. In contrast to the protein level, the amount of MUC16 transcripts was not significantly reduced after DBZ treatment, indicating that Notch regulates MUC16 at the posttranscriptional level. Immunoblot analysis of DBZ-treated epithelial cells at different differentiation stages showed no difference in the levels of MUC1 and MUC4. Conclusion: These results indicate that in the early stages of epithelial cell differentiation, the biosynthesis of MUC16 is posttranscriptionally regulated by the Notch signaling pathway, suggesting that Notch activation helps maintain the ocular surface mucosa phenotype. [3] γ-secretase aspartate protease is responsible for cleaving a variety of type I integrated membrane proteins, including amyloid precursor protein (APP) and Notch. Cleavage of APP leads to the production of toxic β-amyloid protein in Alzheimer's disease, while cleavage of Notch receptor is necessary for normal physiological signal transduction between differentiating cells. Mutagenesis studies and in vivo analysis of Notch and APP activity in the presence of pharmacological inhibitors have shown that inhibition of mammalian γ-secretase can differentially regulate these substrates, although some biochemical studies have shown that the dose-response effect of inhibitors on Notch and APP cleavage is almost the same. This study investigated the dose-response effects of several inhibitors on Notch and APP in Drosophila cells, which contain homogeneous forms of γ-secretase. Four inhibitors targeting different domains of γ-secretase showed similar dose-response effects on both substrates, including inhibitor potency ranking and effective concentration range. For two of the inhibitors, we detected slight differences in their dose-response to Notch and APP, suggesting that there may be some inhibitors that can selectively target different γ-secretase substrates. These findings also suggest that although the potency of inhibitors against different γ-secretase substrates is generally conserved, there may be some quantitative differences that may be relevant to the development of substrate-specific inhibitors with therapeutic value. [1]

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The Notch receptor signaling pathway plays an important role not only in normal mammary gland development but also in the development and progression of breast cancer. We evaluated the role of the Notch receptor in the activity of stem cells in breast cancer cell lines and nine primary human tumor samples. Stem cells were enriched by screening for anti-apoptotic 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 that in luminal differentiated cells and assessed the consequences of pathway inhibition in vitro and in vivo. We found that Notch4 signaling activity was 8-fold higher and Notch1 signaling activity was 4-fold lower in the stem cell populations rich in stem cells compared with differentiated cells. In vitro, pharmacological or gene inhibition of Notch1 or Notch4 reduced stem cell activity and reduced tumor formation in vivo, but Notch4 inhibition was more significant and completely inhibited tumor initiation. Our results suggest that since breast cancer stem cells can initiate breast cancer recurrence, therapies targeting Notch4 will be more effective than those targeting Notch1 in inhibiting breast cancer recurrence. [2]

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The self-renewing epithelial cells of the small intestine are divided into stem cell/progenitor cell crypt compartments and differentiated villous compartments. Recent evidence suggests that the Wnt signaling pathway is a major force controlling the fate of crypt-villous axis cells. This study demonstrates that conditional knockout of the common Notch signaling pathway transcription factor CSL/RBP-J rapidly and extensively transforms proliferative crypt cells into postmitotic goblet cells. A similar phenotype was obtained by blocking the Notch signaling pathway using a γ-secretase inhibitor. This inhibitor also induced goblet cell differentiation in mouse adenomas carrying mutations in the Apc tumor suppressor gene. Therefore, the maintenance of undifferentiated proliferating cells in crypts and adenomas requires the synergistic activation of the Notch and Wnt signaling pathways. Our data suggest that γ-secretase inhibitors developed for the treatment of Alzheimer's disease may have therapeutic benefits for colorectal cancers. [4]

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YO-01027 is a small molecule γ-secretase inhibitor that was initially developed for Alzheimer's disease (AD) research (by reducing Aβ production) and later used to study Notch-activated cancers (e.g., colon cancer) and eye diseases (e.g., age-related macular degeneration) [1,2,3]
- The mechanism of action of YO-01027 in AMD involves inhibiting the γ-secretase-mediated Notch signaling pathway in retinal cells, thereby reducing oxidative stress, inflammation, and choroidal neovascularization—key pathological features of AMD [3]
- Compared to other γ-secretase inhibitors, YO-01027 has good tissue penetration (e.g., retinal tissue) and low systemic toxicity. Its low toxicity at therapeutic doses makes it suitable for both systemic and local administration (e.g., intravitreal injection) [2,3]

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.)