IWR-1-endo

Tankyrase; Wnt (IC50 = 180 nM)

IWR-1 和 XAV939 在体外具有相似的药理作用，并且作为 Wnt 通路的可逆抑制剂发挥作用。 IWR-1 与 Axin 相互作用以发挥作用，而 XAV939 直接与 TNKS 结合[1]。 IWR-1 (10 μM) 可使 β-连环蛋白破坏复合物稳定。当将 IWR-1 (10 μM) 与 MIF 一起加入培养基时，细胞集落的大小急剧减小，表明在所有 MIF 浓度组中，IWR-1 抑制了 MIF 对 NSPC 增殖的刺激作用。 NSPC 的增殖受到 2、5 和 10 μM IWR-1 的强烈抑制，呈剂量依赖性。 MIF 对 NSPC 发育成神经元谱系的刺激作用被 IWR-1 抑制[2]。在 FSH 最大刺激剂量 (0.5 ng/mL) 存在的情况下，IWR-1 给药会剂量依赖性地抑制 FSH 的刺激作用，在 IWR-1 最大抑制剂量 (1 µM) 下观察到 > 75% 的抑制）。 IWR-1 治疗可部分逆转 FSH 诱导的颗粒细胞 CARTPT mRNA 表达抑制[3]。

研究人员之前曾报道，外显IWR 51仅在高浓度下具有活性。5从数量上讲，51的活性比内显IWR-1低25倍（图2）。有趣的是，烯烃的饱和不影响活性。Sat IWR 52和1在体外试验中同样有效（图2）。这些结果表明，1的降冰片基区域只能容忍微妙的空间扰动。 研究人员还测试了IWR的体内活性，发现1有效地抑制了斑马鱼尾鳍的再生。我们在这里表明，1的最小抑制浓度为0.5μM（图3）。他们进一步证明，IWR的体内活性与其体外活性相关。例如，使用中度抑制剂13和43仅观察到鳍再生的部分抑制。弱抑制剂17仅延缓了尾鳍的生长（图片未显示）[1]。

对于NSPC增殖实验，将单个解离细胞以1×105/ml的密度接种到96孔板中，不同MIF浓度（0、1、2、4、8、16、32ng/ml），有或没有IWR-1（10μM；Sigma，圣路易斯，密苏里州）。四天后，观察神经球，并用倒置显微镜拍摄显微照片。在单一培养中使用六只幼崽，并重复实验3次。通过Image Pro Plus 5.0软件计算神经球的数量和测量神经球的直径来分析图像。将部分细胞接种在NSPC培养基中24孔板上涂有聚-D-赖氨酸盐酸盐（PLL，分子量为70000-150000）的10mm玻璃盖玻片上，四天后用Ki67和Hoechst抗体进行免疫染色。对于Ki67免疫染色细胞，MIF刺激组的MIF浓度为16ng/ml。[2]

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对于NSPC分化研究，将神经球接种在24孔板或烧瓶中的聚-D-赖氨酸盐酸盐（PLL，分子量为70000-150000）涂层的10 mm玻璃盖玻片上，培养基含有DMEM/F-12，补充有2%B27和2%胎牛血清（FBS；Invitrogen），有或没有MIF（16ng/ml）或IWR-1（1或10μM）。7至10天后，停止分化，用4%多聚甲醛固定细胞进行染色，或将细胞收集在RIPA缓冲液中进行蛋白质印迹[2]。

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第一个实验检查了WNT信号抑制剂IWR-1对基础和FSH诱导的雌二醇产生和细胞数量的影响。WNT抑制剂稳定AXIN2与CTNNB1的相互作用，导致CTNNB1降解并抑制典型的WNT途径。处理包括含有DMSO的培养基（稀释剂对照组）或含有0.1、1.0或10µMIWR-1的培养基，可添加或不添加最大刺激剂量的FSH（0.5 ng/ml；NHPP），持续6天，每次重复实验中每个处理12个孔。媒体每两天更换一次。在培养的第6天，取出培养基并储存在-20°C下，直至分析雌二醇浓度，然后洗涤细胞，用库尔特计数器（Beckman Coulter）进行胰蛋白酶处理和计数，该计数器设置为计数5至20µm大小的细胞，如前所述[15]。使用不同日期获得的卵巢重复该实验4次。[3]
在第二个实验中，确定了FSH和最大抑制剂量IWR-1对选定WNT通路成员和其他FSH作用调节剂的mRNA丰度的影响。在这个实验中，在有或没有FSH（0.5 ng/ml）的情况下，用DMSO（载体对照）或最大有效剂量的IWR-1（1µM）处理颗粒细胞（每次处理24孔）。在培养的第6天，取出培养基并在-20°C下储存，直至分析雌二醇浓度，然后将细胞裂解并保存在-80°C下，直至进行总RNA分离。使用不同日期获得的卵巢重复该实验4次。[3]
在第三个实验中，确定了IWR-1处理对CTNB1和AXIN2蛋白丰度的影响。对于该实验，按照实验2所述分离和培养颗粒细胞。在培养的第6天，取出培养基并储存在-20°C下，直至分析雌二醇浓度。然后清洗细胞，从孔中吸出，在3000 g下离心5分钟。然后将细胞沉淀在液氮中快速冷冻，并在-80°C下保存，直至蛋白质印迹分析[3]。

[1]. Structure-activity relationship studies of small-molecule inhibitors of Wnt response. Bioorg Med Chem Lett. 2009 Jul 15;19(14):3825-7.

[2]. Macrophage migration inhibitory factor promotes proliferation and neuronal differentiation of neural stem/precursor cells through Wnt/β-catenin signal pathway. Int J Biol Sci. 2013 Nov 28;9(10):1108-20.

[3]. Regulation and Regulatory Role of WNT Signaling in Potentiating FSH Action during Bovine Dominant Follicle Selection. PLoS One. 2014 Jun 17;9(6):e100201.

IWR-1-endo is a dicarboximide having an endo bridged phthalimide structure, substituted at nitrogen by a 4-(quinolin-8-ylcarbamoyl)benzoyl group. It has a role as an axin stabilizer and a Wnt signalling inhibitor. It is a dicarboximide, a bridged compound, a member of quinolines and a member of benzamides.

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Suppression of oncogenic Wnt-mediated signaling holds promise as an anti-cancer therapeutic strategy. We previously reported a novel class of small molecules (IWR-1/2, inhibitors of Wnt response) that antagonize Wnt signaling by stabilizing the Axin destruction complex. Herein, we present the results of structure-activity relationship studies of these compounds.[1]

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Macrophage migration inhibitory factor (MIF) is a highly conserved and evolutionarily ancient mediator with pleiotropic effects. Recent studies demonstrated that the receptors of MIF, including CD44, CXCR2, CXCR4 and CD74, are expressed in the neural stem/progenitor cells (NSPCs). The potential regulatory effect of MIF on NSPCs proliferation and neuronal differentiation, however, is largely unknown. Here, we investigated the effect of MIF on NSPC proliferation and neuronal differentiation, and further examined the signal pathway by which MIF transduced these signal effects in mouse NSPCs in vitro. The results showed that both Ki67-positive cells and neurosphere volumes were increased in a dose-dependent manner following MIF treatment. Furthermore, the expression of nuclear β-catenin was significantly stronger in MIF-stimulated groups than that in control groups. Conversely, administration of IWR-1, the inhibitor of Wnt/β-catenin pathway, significantly inhibited the proliferative effect of MIF on NSPCs. Immunostaining and Western blot further indicated that doublecortin (DCX) and Tuj 1, two neuronal markers, were evidently increased with MIF stimulation during NSPC differentiation, and there were more Tuj1-positive cells migrated out from neurospheres in MIF-stimulated groups than those in control groups. During NSPC differentiation, MIF increased the activity of β-galactosidase that responds to Wnt/β-catenin signaling. Wnt1 and β-catenin proteins were also up-regulated with MIF stimulation. Moreover, the expression of DCX and Tuj 1 was inhibited significantly by IWR-1. Taken together, the present study indicated that MIF enhances NSPC proliferation and promotes the neuronal differentiation, by activating Wnt/β-catenin signal pathway. The interaction between MIF and Wnt/β-catenin signal pathway may play an important role in modulating NSPC renewal and fate during brain development.[2]

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Follicular development occurs in wave like patterns in monotocous species such as cattle and humans and is regulated by a complex interaction of gonadotropins with local intrafollicular regulatory molecules. To further elucidate potential mechanisms controlling dominant follicle selection, granulosa cell RNA harvested from F1 (largest) and F2 (second largest) follicles isolated at predeviation (PD) and onset of diameter deviation (OD) stages of the first follicular wave was subjected to preliminary RNA transcriptome analysis. Expression of numerous WNT system components was observed. Hence experiments were performed to test the hypothesis that WNT signaling modulates FSH action on granulosa cells during follicular waves. Abundance of mRNA for WNT pathway members was evaluated in granulosa cells harvested from follicles at emergence (EM), PD, OD and early dominance (ED) stages of the first follicular wave. In F1 follicles, abundance of CTNNB1 and DVL1 mRNAs was higher and AXIN2 mRNA was lower at ED versus EM stages and DVL1 and FZD6 mRNAs were higher and AXIN2 mRNA was lower in F1 versus F2 follicle at the ED stage. Bovine granulosa cells were treated in vitro with increasing doses of the WNT inhibitor IWR-1+/- maximal stimulatory dose of FSH. IWR-1 treatment blocked the FSH-induced increase in granulosa cell numbers and reduced the FSH-induced increase in estradiol. Granulosa cells were also cultured in the presence or absence of FSH +/- IWR-1 and hormonal regulation of mRNA for WNT pathway members and known FSH targets determined. FSH treatment increased CYP19A1, CCND2, CTNNB1, AXIN2 and FZD6 mRNAs and the stimulatory effect on CYP19A1 mRNA was reduced by IWR-1. In contrast, FSH reduced CARTPT mRNA and IWR-1 partially reversed the inhibitory effect of FSH. Results support temporal and hormonal regulation and a potential role for WNT signaling in potentiating FSH action during dominant follicle selection.[3]

C25H19N3O3

409.44

409.142

, 73.34; H, 4.68; N, 10.26; O, 11.72

1127442-82-3

44483163

Off-white to yellow solid powder

1.4±0.1 g/cm3

643.9±55.0 °C at 760 mmHg

343.2±31.5 °C

0.0±1.9 mmHg at 25°C

1.741

2.65

79.37

1

4

3

31

772

4

ZGSXEXBYLJIOGF-UHFFFAOYSA-N

InChI=1S/C25H19N3O3/c29-23(27-19-5-1-3-14-4-2-12-26-22(14)19)15-8-10-18(11-9-15)28-24(30)20-16-6-7-17(13-16)21(20)25(28)31/h1-12,16-17,20-21H,13H2,(H,27,29)

4-[(3aR,4S,7R,7aS)-1,3,3a,4,7,7a-hexahydro-1,3-dioxo-4,7-methano-2H-isoindol-2-yl]-N-8-quinolinyl-benzamide

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)

配方 1 中的溶解度: ≥ 2.5 mg/mL (6.11 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 (6.11 mM) (饱和度未知) in 10% DMSO + 90% (20% SBE-β-CD in 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900 μL 20% SBE-β-CD生理盐水溶液中，混匀。
*20% SBE-β-CD 生理盐水溶液的制备（4°C，1 周）：将 2 g SBE-β-CD 溶解于 10 mL 生理盐水中，得到澄清溶液。

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配方 3 中的溶解度: ≥ 2.5 mg/mL (6.11 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 储备液加入到 900 μL 玉米油中并混合均匀。

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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;
4、 如产品在配制过程中出现沉淀/析出，可通过加热(≤50℃)或超声的方式助溶;
5、为保证最佳实验结果，工作液请现配现用！
6、如不确定怎么将母液配置成体内动物实验的工作液，请查看说明书或联系我们;
7、 以上所有助溶剂都可在 Invivochem.cn网站购买。