GSK-3 inhibitor 1

GSK-3
The target of GSK-3 inhibitor 1 is glycogen synthase kinase 3 (GSK-3), including two isoforms: GSK-3α and GSK-3β. For human GSK-3α, the half-maximal inhibitory concentration (IC₅₀) is 0.08 μM [2]
; for human GSK-3β, the IC₅₀ is 0.06 μM [2]
. It shows no significant inhibition of other related kinases (e.g., CDK2, ERK1, JNK2) with IC₅₀ > 10 μM, demonstrating high isoform and kinase selectivity [2]

GSK-3 抑制剂 1 可用于诱导、促进或增强内耳组织（如内耳支持细胞或内耳毛细胞）的生长、增殖或再生[2]。

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1. GSK-3激酶活性抑制：GSK-3抑制剂1以浓度依赖的方式抑制重组人类GSK-3α和GSK-3β的催化活性。0.1 μM浓度下，较溶媒对照组抑制GSK-3α活性85%，抑制GSK-3β活性90% [2]
2. 诱导内耳支持细胞增殖：体外培养分离的小鼠耳蜗支持细胞，用0.1、0.5、1.0 μM的GSK-3抑制剂1处理7天。0.5 μM浓度使支持细胞增殖率较溶媒组增加2.3倍（BrdU掺入实验）；1.0 μM浓度增加3.1倍，且未检测到细胞毒性 [1]
3. 诱导支持细胞向内耳毛细胞分化：用0.5 μM的GSK-3抑制剂1处理小鼠耳蜗支持细胞14天，可诱导其转分化为毛细胞样细胞，免疫荧光染色显示45%的细胞表达毛细胞特异性标志物（Myo7a、钙视网膜蛋白），而溶媒对照组仅3% [1]
4. 促进干细胞/祖细胞自我更新：用0.1–0.5 μM的GSK-3抑制剂1处理人类牙髓干细胞（hDPSCs）5天，自我更新能力增强，克隆形成效率提高1.8–2.5倍（克隆形成实验）。流式细胞术分析证实，干细胞标志物（CD105、CD90）阳性比例保持>95%，表明干细胞表型得以保留 [2]
5. 激活Wnt/β-连环蛋白信号通路：在支持细胞和hDPSCs中，0.5 μM的GSK-3抑制剂1可增加β-连环蛋白的核积累（免疫荧光检测），并使Wnt靶基因（c-Myc、Cyclin D1）的表达上调2.8–3.5倍（实时荧光定量PCR） [1,2]

1. 噪声诱导听力损失（NIHL）小鼠的毛细胞再生：将C57BL/6小鼠暴露于110 dB SPL宽带噪声中2小时，诱导耳蜗毛细胞损失。噪声暴露后第3天开始，将GSK-3抑制剂1（溶于10%二甲基亚砜+90%无菌生理盐水）通过耳蜗内注射给药，剂量为0.5 μM（每耳1 μL），每周1次，持续3周。治疗后4周，组织学分析显示，耳蜗基底回外毛细胞数量较溶媒处理的NIHL小鼠增加60%；听性脑干反应（ABR）检测显示，8 kHz频率下听力阈值降低25 dB，表明功能恢复 [1]
2. 小鼠牙髓干细胞增殖：向C57BL/6小鼠牙髓局部注射0.1 μM的GSK-3抑制剂1（5 μL），7天后BrdU标记显示，增殖的牙髓干细胞数量较溶媒对照组增加2.2倍。免疫组织化学染色证实，增殖细胞表达干细胞标志物（CD105），且保留分化潜能 [2]

1. 重组GSK-3α/β激酶活性实验：
- 将重组人类GSK-3α或GSK-3β蛋白溶于激酶实验缓冲液（20 mM Tris-HCl，pH 7.5，10 mM MgCl₂，1 mM DTT），终浓度为10 ng/μL [2]
- 向激酶溶液中加入系列浓度的GSK-3抑制剂1（0.001–10 μM）或溶媒，随后加入荧光标记的GSK-3特异性肽底物和ATP（终浓度100 μM） [2]
- 反应混合物在30°C孵育60分钟，加入含EDTA的终止缓冲液终止反应 [2]
- 用酶标仪检测荧光强度（激发光485 nm，发射光520 nm），反映底物的磷酸化水平。计算相对于溶媒对照组的激酶活性抑制百分比，从剂量-反应曲线推导IC₅₀值 [2]

1. 耳蜗支持细胞分离及增殖实验：
- 从P3-P5 C57BL/6小鼠幼崽中分离耳蜗，无菌条件下取出柯蒂氏器，通过酶消化和机械吹打分散支持细胞，以5×10³个/孔接种于胶原包被的96孔板 [1]
- 细胞在含生长因子的DMEM/F12培养基中培养，用0.1–1.0 μM的GSK-3抑制剂1或溶媒处理，7天培养期的最后24小时向培养基中加入BrdU [1]
- 多聚甲醛固定细胞，曲拉通X-100透化后，用抗BrdU抗体进行免疫染色，荧光显微镜下计数BrdU阳性细胞数，计算相对于溶媒对照组的增殖率 [1]
2. 毛细胞转分化实验：
- 分离的小鼠耳蜗支持细胞接种于含盖玻片的24孔板，用0.5 μM的GSK-3抑制剂1处理14天，每3天更换一次培养基 [1]
- 固定细胞后，用抗Myo7a（毛细胞标志物）和Sox2（支持细胞标志物）一抗进行免疫染色，再加入荧光二抗 [1]
- 用图像分析软件量化Myo7a阳性细胞比例，共聚焦显微镜下观察转分化细胞的形态 [1]
3. 干细胞自我更新（克隆形成）实验：
- 分离人类牙髓干细胞（hDPSCs），以100个/孔接种于6孔板，用0.1–0.5 μM的GSK-3抑制剂1或溶媒处理 [2]
- 培养14天后，甲醇固定克隆，结晶紫染色，计数含>50个细胞的克隆，克隆形成效率计算为（克隆数/接种细胞数）×100% [2]
4. Wnt通路激活实验（实时荧光定量PCR）：
- 支持细胞或hDPSCs用0.5 μM的GSK-3抑制剂1处理24小时后，提取总RNA [1,2]
- 以1 μg总RNA合成互补DNA（cDNA），用c-Myc、Cyclin D1和内参基因GAPDH的特异性引物进行实时荧光定量PCR [1,2]
- 采用2^(-ΔΔCt)法计算相对基因表达水平，比较GSK-3抑制剂1处理组与溶媒对照组的差异 [1,2]

1. Noise-induced hearing loss (NIHL) mouse model for hair cell regeneration:
- Male C57BL/6 mice (8–10 weeks old) were randomly divided into three groups (n=8 per group): normal control (no noise exposure), vehicle-treated NIHL, and GSK-3 inhibitor 1-treated NIHL [1]
- NIHL was induced by exposing mice to 110 dB SPL broadband noise for 2 hours in a sound-attenuated chamber. ABR thresholds were measured 3 days post-noise exposure to confirm hearing loss [1]
- GSK-3 inhibitor 1 was formulated as a 0.5 μM solution in 10% DMSO + 90% sterile saline. Intracochlear injection (1 μL per cochlea) was performed under anesthesia using a microinjector. Injections were administered once weekly for 3 weeks, starting 3 days post-noise exposure [1]
- At 4 weeks post-treatment, ABR testing was repeated to assess hearing function. Mice were euthanized, and cochleae were dissected, fixed, and stained with phalloidin (to label hair cell stereocilia) for histological analysis of hair cell number [1]
2. Mouse dental pulp stem cell proliferation model:
- Female C57BL/6 mice (6–8 weeks old) were randomly divided into vehicle and GSK-3 inhibitor 1 groups (n=6 per group) [2]
- GSK-3 inhibitor 1 was prepared as a 0.1 μM solution in sterile phosphate-buffered saline (PBS). Under anesthesia, a small hole was drilled in the maxillary first molar, and 5 μL of the test solution or vehicle (PBS) was injected into the dental pulp using a microsyringe [2]
- BrdU (50 mg/kg) was administered intraperitoneally to mice 24 hours before euthanasia (7 days post-injection). Dental pulp tissues were dissected, fixed, embedded in paraffin, and sectioned. Immunohistochemical staining for BrdU and CD105 was performed to identify proliferating stem cells [2]

1. In vitro cytotoxicity: Treatment of cochlear supporting cells and hDPSCs with GSK-3 inhibitor 1 at concentrations up to 5 μM for 14 days did not affect cell viability (MTT assay), with viability >90% compared to vehicle control [1,2]
2. In vivo local toxicity: In the NIHL mouse model, intracochlear injection of GSK-3 inhibitor 1 (0.5 μM) did not induce inflammation (hematoxylin-eosin staining) or damage to adjacent cochlear structures (e.g., spiral ganglion neurons) [1]

[1]. SOLUBILIZED COMPOSITIONS FOR CONTROLLED PROLIFERATION OF STEM CELLS / GENERATING INNER EAR HAIR CELLS USING GSK3 INHIBITORS: III. 20170252449 A1

[2]. 1h-pyrrole-2,5-dione compounds and methods of using them to induce self-renewal of stem/progenitor supporting cells. Patent WO2018125746A1.

1. GSK-3 inhibitor 1 is a small-molecule inhibitor of glycogen synthase kinase 3 (GSK-3α/β), developed for applications in regenerative medicine, including inner ear hair cell regeneration (for treating hearing loss) and stem cell self-renewal regulation [1,2]
2. Mechanism of action: GSK-3 inhibitor 1 selectively binds to the ATP-binding pocket of GSK-3α/β, inhibiting their kinase activity. This leads to stabilization and nuclear translocation of β-catenin, activating the canonical Wnt/β-catenin signaling pathway, which promotes proliferation of supporting cells/stem cells and induces transdifferentiation of cochlear supporting cells into hair cells [1,2]
3. Formulation characteristics: The specified patents disclose solubilized compositions of GSK-3 inhibitor 1, including aqueous formulations with DMSO, polyethylene glycol, or cyclodextrin as solubilizers, designed to improve solubility and bioavailability for local administration (intracochlear, dental pulp injection) [1,2]
4. Therapeutic potential: It has potential utility in treating sensorineural hearing loss (caused by hair cell damage/loss) and in regenerative therapies requiring enhanced stem cell self-renewal (e.g., dental pulp regeneration, tissue repair) [1,2]
5. Chemical class: GSK-3 inhibitor 1 belongs to the 1H-pyrrole-2,5-dione compound class, with a chemical structure optimized for high GSK-3 selectivity and low off-target kinase inhibition [2]

C22H17CLFN5O2

437.8541

337.10223

603272-51-1

603272-51-1

78357782

Yellow to orange solid

8.592

80.12

3

5

2

31

788

0

Cl[H].FC1=C([H])C2C([H])([H])N([H])C([H])([H])C([H])([H])N3C([H])=C(C4C(N([H])C(C=4C4=C([H])N=C5C([H])=C([H])C([H])=C([H])N45)=O)=O)C(=C1[H])C3=2

CXXAOCQHGIGIBJ-UHFFFAOYSA-N

InChI=1S/C22H16FN5O2.ClH/c23-13-7-12-9-24-4-6-27-11-15(14(8-13)20(12)27)18-19(22(30)26-21(18)29)16-10-25-17-3-1-2-5-28(16)17;/h1-3,5,7-8,10-11,24H,4,6,9H2,(H,26,29,30);1H

3-(6-fluoro-1,10-diazatricyclo[6.4.1.04,13]trideca-2,4,6,8(13)-tetraen-3-yl)-4-imidazo[1,2-a]pyridin-3-ylpyrrole-2,5-dione;hydrochloride

GSK-3 inhibitor 1

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，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)

DMSO: ~12.5 mg/mL (~28.55 mM)

配方 1 中的溶解度: ≥ 1.25 mg/mL (2.85 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 12.5 mg/mL澄清的DMSO储备液加入到400 μL PEG300中，混匀；再向上述溶液中加入50 μL Tween-80，混匀；然后加入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: ≥ 1.25 mg/mL (2.85 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 12.5 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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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。