RO4929097 (RO4929097; R4733; RO04929097)

γ secretase (IC50 = 4 nM); Notch (IC50 = 5 nM); Aβ40 (IC50 = 14 nM)

体外活性：RO4929097 减少 HEK293 细胞中 Aβ 肽分泌到培养基中的量，EC50 为 14 nM。在基于 Notch 细胞的报告基因检测中，RO4929097 强烈抑制 Notch 加工，EC50 为 5 nM。 RO4929097 在无细胞和细胞测定中的效力处于低纳摩尔范围，相对于 75 种其他各种类型的蛋白质（包括受体、离子通道和酶）观察到 >100 倍的选择性（CEREP 小组）。治疗 5 天后，RO4929097 减少了人 NSCLC A549 细胞中 ICN 的产生，从而在组织培养中诱导扁平化且转化较少的肿瘤细胞表型。 RO4929097 阻断人非小细胞肺癌细胞中的 Notch 加工并降低 Notch 转录靶基因 Hes1 的表达。 RO4929097 处理显示 SUM149 中直接 Notch 靶基因 Hes1、Hey1 和 Heyl 的表达减少了 2 至 3 倍，SUM190 细胞中的表达减少了 3.5 至 8 倍。 RO4929097 以剂量依赖性方式适度抑制 SUM149 细胞的生长。相对于媒介物处理的对照，RO4929097 浓度为 1 μM 时，SUM149 细胞的生长抑制为 20%，SUM190 细胞的生长抑制为 10%。 RO4929097 减少 T 细胞产生炎症细胞因子。此外，通过 RO4929097 治疗，TH2 细胞因子优于 TH1 细胞因子。此外，RO4929097 会增加 T 细胞激活诱导的 IL-6 产生。 RO4929097 治疗后，选定的黑色素瘤细胞系显示出 NOTCH 下游效应子 HES1 的下调。检测到原代黑色素瘤细胞系经 RO4929097 处理后形成的黑色素球数量减少。激酶测定：使用 RO4929097 后，使用多种抗 Aβ 抗体和 Origen 1.5 分析仪通过 ECL 测定来测量 Aβ 肽。 4G8 鼠 mAb 结合 Aβ 肽（氨基酸 18-21 内）中紧邻 α-分泌酶裂解位点远端的表位。 G2–10 鼠 mAb 与 γ-分泌酶介导的切割后暴露的 C 末端结合，生成 Aβ40 肽的氨基酸 40。 FCA3542 兔抗体与 γ-分泌酶介导的切割后暴露的 C 末端结合，生成 Aβ42 肽的氨基酸 42。 4G8 mAb 采用生物素-LC-磺基-N-羟基琥珀酰亚胺-酯进行生物素化。 G2–10 和 FCA3542 抗体用 TAG-N-羟基琥珀酰亚胺酯进行钌酰化。 Aβ(x-40) 可使用生物素化 4G8 和钌酰化 G2–10 进行检测。使用生物素化 4G8 和钌基化 FCA3542 检测 Aβ(x-42)。细胞测定：将原代黑色素瘤细胞系（包括 WM35 和 WM98.1）按每孔 2.5 × 103 个细胞接种在 12 孔培养皿中，一式三份。第二天（第 0 天）更换培养基，添加 DMSO 或 10 μM RO4929097，每 3-4 天更换一次。在指定的时间点，将细胞固定在 10% 福尔马林溶液中，并在 4°C 下储存在 PBS 中。第18-24天，所有板都用结晶紫染色。用 10% 乙酸洗脱颜色后，在 590 nm 处读取光密度。

与载体治疗的动物相比，在 21 天的方案中，每天一次或每天两次向携带 A549 NSCLC 异种移植物的裸鼠口服注射 3 至 60 mg/kg RO4929097，持续 7、14 或 21 天，结果显着抑制肿瘤生长。肿瘤生长抑制值范围为66%至91%。当小鼠按照 7+/14- 方案每天两次用 60 mg/kg RO4929097 治疗时，治疗最初会引起已建立的 A549 肿瘤的消退。在 21 天周期结束时（第 47 天），与载体对照小鼠相比，肿瘤生长预防率仍为 91%。肿瘤生长的抑制作用持续时间较长，可持续长达治疗后 34 天（第 67 天）。第 67 天，这些小鼠再次接受相同剂量的 RO4929097 治疗，进行第二个周期（7 天），直到第 74 天。重要的是，给药完成后，抗肿瘤作用仍然持续。 RO4929097 导致 A549 异种移植模型中与血管生成相关的基因表达减少。相比之下，RO4929097抗性H460a异种移植物在这些基因的表达方面几乎没有变化，强调了RO4929097的体内抗血管生成作用机制。对于 IL6 和 IL8 过表达的肿瘤，RO4929097 不再影响血管生成或肿瘤相关成纤维细胞的浸润。

使用 RO4929097 后，使用 Origen 1.5 分析仪和一系列抗 Aβ 抗体通过 ECL 测定对 Aβ 肽进行定量。 α-分泌酶裂解位点紧邻 4G8 鼠 mAb 结合的 Aβ 肽（氨基酸 18-21 内）表位的远端。 C 末端在 γ-分泌酶介导的裂解产生 Aβ40 肽的氨基酸 40 后暴露，与 G2–10 鼠 mAb 结合。 Aβ42 肽的 C 末端通过 γ-分泌酶介导的裂解产生氨基酸 42，与兔抗体 FCA3542 结合。生物素-LC-磺基-N-羟基琥珀酰亚胺-酯用于对 4G8 mAb 进行生物素化。使用 TAG-N-羟基琥珀酰亚胺酯，G2-10 和 FCA3542 抗体被钌酰化。生物素化 4G8 和钌基化 G2–10 用于检测 Aβ(x–40)。生物素化 4G8 和钌基化 FCA3542 用于检测 Aβ(x-42)。

以 5 × 10⁴ 细胞的密度接种 IBC 细胞系 SUM149 和 SUM190。 0.1 nM 至 10 μM 的 RO4929097 作为媒介物施用或在第二天逐渐增加剂量。 72小时后对细胞进行胰蛋白酶消化，并使用血细胞计数器对活细胞进行计数。

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癌症基底细胞癌在炎症性癌症（IBC）患者中很常见，已被证明对辐射有抵抗力，并富含癌症干细胞。Notch通路在乳腺癌症干细胞的自我更新中起着重要作用，并促进炎症信号传导，从而促进乳腺癌症干细胞表型。在此，我们在富集癌症起始细胞的体外模型中使用γ分泌酶抑制剂RO4929097抑制Notch信号传导（3D克隆发生测定）和常规2D克隆发生测定，以比较SUM149和SUM190 IBC细胞系对放射增敏的影响。RO4929097下调了Notch靶基因Hes1、Hey1和HeyL，并显示出SUM190和SUM149中锚定非依赖性生长的显著减少。然而，该药物提高了假定的自我更新测定乳腺球形成效率。为了评估推定的癌症干细胞的放射增敏作用，在其标准（2D）和自我更新富集（3D）培养条件下，将细胞暴露于增加剂量的辐射，无论是否使用1μM RO4929097。在常规的2D克隆形成试验中，RO4929097显著地使SUM190细胞对电离辐射敏感，并在SUM149细胞中具有适度的放射增敏作用。然而，在3D克隆试验中，在较高剂量下，SUM149和SUM190细胞都显示出辐射防护作用。这两种细胞系都表达IL-6和IL-8细胞因子，已知它们介导Notch抑制的功效并促进干细胞的自我更新。我们进一步表明，RO429097抑制了一些炎性细胞因子的正常T细胞合成，包括TNF-α，TNF-α是微环境中IL-6和IL-8产生的潜在介质。这些数据表明，可能需要额外的靶向剂通过Notch抑制选择性靶向IBC干细胞，对微环境影响的评估可能会进一步揭示这种抑制剂的潜在作用[2]。

Mice: Mice treated with RO4929097 are given oral doses of suspensions ranging from 3 to 60 mg/kg RO4929097 in accordance with the prescribed regimens. RO4929097 is dosed at 60 mg/kg/d every other week for 4 weeks (7+/7- × 2 cycles) in the Calu-6 xenograft model. RO4929097 is dosed once daily at 10 mg/kg for 21 days for all other xenograft models. One-way ANOVA, the post hoc Bonferroni t test, and the Mann-Whitney rank-sum test are used to determine statistical analysis. When P ≤ 0.05, differences between groups are deemed significant. A549 tumors from both the vehicle-treated and specific RO4929097-treated groups are taken, processed, and embedded in paraffin for an overnight fix. They are then sectioned at 5 μM and stained with H&E for histopathology evaluation. The histology images were obtained with an Olympus BX51 microscope (×40 objective) mounted on a Nikon DS-Fi1 equipped with the NIS-Elements F2.20 software. Three A549 tumours are flash-frozen for Western blot analysis, one for each of the two groups (7 (60 mg/kg) or 21 days (3 and 30 mg/kg). H-200 antibody is used at a dilution of 1:1,000 to detect collagen type V, and at a dilution of 1:5,000 to detect MFAP5.

[1]. Luistro L, et al. Preclinical profile of a potent gamma-secretase inhibitor targeting notch signaling with in vivo efficacy and pharmacodynamic properties. Cancer Res. 2009, 69(19), 7672-7680.

[2]. Debeb BG, et al. Pre-clinical studies of Notch signaling inhibitor RO4929097 in inflammatory breast cancer cells. Breast Cancer Res Treat. 2012 Jul;134(2):495-510.

[3]. Huynh C, et al. The novel gamma secretase inhibitor RO4929097 reduces the tumor initiating potential of melanoma. PLoS One. 2011, 6(9), e25264.

RO4929097 is a member of the class of dibenzoazepines that is the amide formed from formal condensation of the carboxy group of 2,2-dimethyl-3-oxo-3-[(2,2,3,3,3-pentafluoropropyl)amino]propanoic acid with the amino group of (7S)-7-amino-5,7-dihydrodibenzo[b,d]azepin-6-one. It has a role as an EC 3.4.23.46 (memapsin 2) inhibitor. It is a dibenzoazepine, a lactam, an organofluorine compound and a dicarboxylic acid diamide.
Ro4929097 has been used in trials studying the treatment of Sarcoma, LYMPHOMA, Neoplasms, Wilm's Tumor, and OSTEOSARCOMA, among others.
Gamma-Secretase Inhibitor RO4929097 is an orally bioavailable, small-molecule gamma secretase (GS) inhibitor with potential antitumor activity. Gamma secretase inhibitor RO4929097 binds to GS and blocks activation of Notch receptors, which may inhibit tumor cell proliferation. The integral membrane protein GS is a multi-subunit protease complex that cleaves single-pass transmembrane proteins, such as Notch receptors, at residues within their transmembrane domains. Overexpression of the Notch signaling pathway has been correlated with increased tumor cell growth.

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Basal breast cancer, common among patients presenting with inflammatory breast cancer (IBC), has been shown to be resistant to radiation and enriched in cancer stem cells. The Notch pathway plays an important role in self-renewal of breast cancer stem cells and contributes to inflammatory signaling which promotes the breast cancer stem cell phenotype. Herein, we inhibited Notch signaling using a gamma secretase inhibitor, RO4929097, in an in vitro model that enriches for cancer initiating cells (3D clonogenic assay) and conventional 2D clonogenic assay to compare the effect on radiosensitization of the SUM149 and SUM190 IBC cell lines. RO4929097 downregulated the Notch target genes Hes1, Hey1, and HeyL, and showed a significant reduction in anchorage independent growth in SUM190 and SUM149. However, the putative self-renewal assay mammosphere formation efficiency was increased with the drug. To assess radiosensitization of putative cancer stem cells, cells were exposed to increasing doses of radiation with or without 1 μM RO4929097 in their standard (2D) and self-renewal enriching (3D) culture conditions. In the conventional 2D clonogenic assay, RO4929097 significantly sensitized SUM190 cells to ionizing radiation and has a modest radiosensitization effect in SUM149 cells. In the 3D clonogenic assays, however, a radioprotective effect was seen in both SUM149 and SUM190 cells at higher doses. Both cell lines express IL-6 and IL-8 cytokines known to mediate the efficacy of Notch inhibition and to promote self-renewal of stem cells. We further showed that RO429097 inhibits normal T-cell synthesis of some inflammatory cytokines, including TNF-α, a potential mediator of IL-6 and IL-8 production in the microenvironment. These data suggest that additional targeting agents may be required to selectively target IBC stem cells through Notch inhibition, and that evaluation of microenvironmental influences may shed further light on the potential effects of this inhibitor.[2]

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Several reports have demonstrated a role for aberrant NOTCH signaling in melanoma genesis and progression, prompting us to explore if targeting this pathway is a valid therapeutic approach against melanoma. We targeted NOTCH signaling using RO4929097, a novel inhibitor of gamma secretase, which is a key component of the enzymatic complex that cleaves and activates NOTCH. The effects of RO4929097 on the oncogenic and stem cell properties of a panel of melanoma cell lines were tested both in vitro and in vivo, using xenograft models. In human primary melanoma cell lines, RO4929097 decreased the levels of NOTCH transcriptional target HES1. This was accompanied by reduced proliferation and impaired ability to form colonies in soft agar and to organize in tridimensional spheres. Moreover, RO4929097 affected the growth of human primary melanoma xenograft in NOD/SCID/IL2gammaR-/- mice and inhibited subsequent tumor formation in a serial xenotransplantation model, suggesting that inhibition of NOTCH signaling suppresses the tumor initiating potential of melanoma cells. In addition, RO4929097 decreased tumor volume and blocked the invasive growth pattern of metastatic melanoma cell lines in vivo. Finally, increased gene expression of NOTCH signaling components correlated with shorter post recurrence survival in metastatic melanoma cases. Our data support NOTCH inhibition as a promising therapeutic strategy against melanoma.[3]

C22H20F5N3O3

469.4

469.142

C, 56.29; H, 4.29; F, 20.24; N, 8.95; O, 10.23

847925-91-1

49867930

White to off-white solid powder

1.4±0.1 g/cm3

696.3±55.0 °C at 760 mmHg

374.9±31.5 °C

0.0±2.2 mmHg at 25°C

1.558

4.81

97.77

3

8

5

33

771

1

N([C@@H]1C(=O)NC2C=CC=CC=2C2C=CC=CC1=2)C(=O)C(C)(C)C(=O)NCC(F)(F)C(F)(F)F

OJPLJFIFUQPSJR-INIZCTEOSA-N

InChI=1S/C22H20F5N3O3/c1-20(2,18(32)28-11-21(23,24)22(25,26)27)19(33)30-16-14-9-4-3-7-12(14)13-8-5-6-10-15(13)29-17(16)31/h3-10,16H,11H2,1-2H3,(H,28,32)(H,29,31)(H,30,33)/t16-/m0/s1

2,2-dimethyl-N-[(7S)-6-oxo-5,7-dihydrobenzo[d][1]benzazepin-7-yl]-N'-(2,2,3,3,3-pentafluoropropyl)propanediamide

2934.99.9001

Powder      -20°C    3 years

                     4°C     2 years

In solvent   -80°C    6 months

                  -20°C    1 month

注意： 请将本产品存放在密封且受保护的环境中（例如氮气保护），避免吸湿/受潮。

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

配方 1 中的溶解度: ≥ 2.5 mg/mL (5.33 mM) (饱和度未知) in 10% DMSO + 40% PEG300 + 5% Tween80 + 45% Saline (这些助溶剂从左到右依次添加，逐一添加), 澄清溶液。
例如，若需制备1 mL的工作液，可将100 μL 25.0 mg/mL澄清DMSO储备液加入到400 μL PEG300中，混匀；然后向上述溶液中加入50 μL Tween-80，混匀；加入450 μL生理盐水定容至1 mL。
*生理盐水的制备：将 0.9 g 氯化钠溶解在 100 mL ddH₂O中，得到澄清溶液。

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配方 2 中的溶解度: ≥ 2.5 mg/mL (5.33 mM) (饱和度未知) in 10% DMSO + 90% Corn Oil (这些助溶剂从左到右依次添加，逐一添加), 澄清溶液。
例如，若需制备1 mL的工作液，可将 100 μL 25.0 mg/mL 澄清 DMSO 储备液添加到 900 μL 玉米油中并混合均匀。

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配方 3 中的溶解度: 2% DMSO+30% PEG 300+5% Tween+ddH2O: 10 mg/mL

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请根据您的实验动物和给药方式选择适当的溶解配方/方案：
1、请先配制澄清的储备液(如：用DMSO配置50 或 100 mg/mL母液(储备液));
2、取适量母液，按从左到右的顺序依次添加助溶剂，澄清后再加入下一助溶剂。以 下列配方为例说明 (注意此配方只用于说明，并不一定代表此产品 的实际溶解配方):
10% DMSO → 40% PEG300 → 5% Tween-80 → 45% ddH2O (或 saline);
假设最终工作液的体积为 1 mL, 浓度为5 mg/mL: 取 100 μL 50 mg/mL 的澄清 DMSO 储备液加到 400 μL PEG300 中，混合均匀/澄清；向上述体系中加入50 μL Tween-80，混合均匀/澄清；然后继续加入450 μL ddH2O (或 saline)定容至 1 mL；
3、溶剂前显示的百分比是指该溶剂在最终溶液/工作液中的体积所占比例;
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