GSK-3β (IC50 = 6.7 nM); GSK-3α (IC50 = 10 nM); ATM (cdc2 = 8800 nM)
Glycogen Synthase Kinase 3α (GSK3α) (Ki: 10 nM; ) [3]
- Glycogen Synthase Kinase 3β (GSK3β) (Ki: 6.7 nM;) [3]

Laduviglusib triHClide 抑制人 GSK-3β，Ki 值为 9.8 nM[1]。 Laduviglusib triHClide 是一种微小的有机分子，通过争夺 ATP 结合位点来抑制 GSK3α 和 GSK3β。根据体外激酶测定，Laduviglusib triHClide 特异性抑制 GSK3β (IC50=~5 nM) 和 GSK3α (IC50=~10 nM)，对其他激酶影响很小[4]。 Laduviglusib triHClide 在 2.5 μM 时使 ES-D3 细胞的活力降低 24.7%，在 5 μM 时降低 56.3%，在 7.5 μM 时降低 61.9%，在 10 μM 时降低 69.2%，IC50 为 4.9 M[2]。

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1. 增强胰岛素介导的葡萄糖转运与利用：在3T3-L1脂肪细胞和L6肌管细胞中，Laduviglusib (CHIR99021) trihydrochloride以浓度依赖性方式增强胰岛素刺激的2-脱氧葡萄糖（2-DG）摄取。1 μM浓度下，可使3T3-L1脂肪细胞中胰岛素诱导的葡萄糖摄取增加约2倍，L6肌管细胞中增加约1.8倍；同时增强胰岛素对糖原合成酶（GS）的激活作用，1 μM浓度下3T3-L1脂肪细胞的GS活性增加2.5倍[1]
2. 激活小鼠胚胎干细胞（mESCs）的Wnt/β-连环蛋白（β-catenin）通路：用0.3-3 μM Laduviglusib (CHIR99021) trihydrochloride处理mESCs后，western blot检测显示细胞质和细胞核中β-catenin积累增加，实时定量PCR（qPCR）检测显示Wnt靶基因（如Axin2、Lef1）表达上调，通路激活呈浓度依赖性，3 μM时效果最强[2, 3]
3. 促进mESC自我更新：在缺乏白血病抑制因子（LIF）的条件下，3 μM Laduviglusib (CHIR99021) trihydrochloride可维持难治性小鼠品系（如CBA/Ca、129S6/SvEv）mESC的自我更新能力，碱性磷酸酶（AP）阳性克隆形成数较对照组增加约3倍，并通过免疫细胞化学和western blot证实其保留了多能性标志物（Oct4、Nanog、Sox2）的表达[3]
4. mESC中的细胞毒性：Laduviglusib (CHIR99021) trihydrochloride对mESC的细胞毒性较低，半数毒性浓度（CC50）大于10 μM，在用于自我更新和通路激活的3 μM浓度下未观察到细胞活力显著下降[2]

单次口服剂量后，Laduviglusib（16 mg/kg 或 48 mg/kg）三盐酸盐可迅速降低 ZDF 大鼠的血浆葡萄糖，给药后 3-4 小时最大降幅接近 150 mg/dl[1]。放射前 4 小时给予一次 laduviglusib (2 mg/kg) 三盐酸盐，可显着提高 14.5 Gy 腹部放射 (ABI) 后的生存率。用 laduviglusib triHClide 治疗可显着减少隐窝凋亡，防止 p-H2AX+ 细胞的积聚，并增强隐窝再生和绒毛高度。用 laduviglusib triHClate 治疗可通过防止细胞凋亡来提高 Lgr5+ 细胞的存活率，并成功延迟早在 4 小时内 Olfm4、Lgr5 和 CD44 的丢失[5]。

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1. 改善肥胖小鼠的胰岛素敏感性和糖稳态：在高脂饮食（HFD）诱导的肥胖C57BL/6小鼠中，腹腔注射Laduviglusib (CHIR99021) trihydrochloride（10 mg/kg/天，持续7天），与溶媒对照组相比，空腹血糖降低约30%，进食后血糖降低约25%，空腹胰岛素水平降低约40%；同时改善葡萄糖耐量（葡萄糖耐量试验AUC降低约28%）和胰岛素耐量（胰岛素耐量试验AUC降低约32%）[1]
2. 增强体内胰岛素介导的糖原合成：在HFD诱导的肥胖小鼠中，Laduviglusib (CHIR99021) trihydrochloride（10 mg/kg/天，腹腔注射）在胰岛素刺激条件下，可使骨骼肌糖原含量增加约50%，肝脏糖原含量增加约40%，与体外GS激活效果一致[1]

所有激酶测定都遵循相同的核心方案，肽底物和激活剂浓度变化如下所述。聚丙烯96孔板填充300μl/孔缓冲液（50 mmol/l三HCl、10 mmol/l MgCl2、1 mmol/l EGTA、1 mmol/1二硫苏糖醇、25 mmol/lβ-甘油磷酸、1 mmol/l NaF、0.01%BSA、pH 7.5），其中含有激酶、肽底物和任何激活剂。这些测定的激酶浓度、肽底物和激活剂（如适用）信息如下：GSK-3α（27 nmol/l和0.5μmol/l生物素CREB肽）；GSK-3β（29 nmol/l，和0.5μmol/l生物素CREB肽）；cdc2（0.8 nmol/l和0.5μmol/l生物素组蛋白H1肽）；erk2（400单位/ml和髓鞘碱性蛋白包被的闪光板[Perkin-Elmer]）；PKC-α（1.6 nmol/l，0.5μmol/l生物素组蛋白H1肽和0.1 mg/ml磷脂酰丝氨酸+0.01 mg/ml甘油二酯）；PKC-ζ（0.1 nmol/l，0.5μmol/l生物素-PKC-86肽和50μg/ml磷脂酰丝氨酸+5μg/ml二酰甘油）；akt1（5.55nmol/l和0.5μmol/l生物素磷酸化AKT肽）；p70 S6激酶（1.5 nmol/l和0.5μmol/l生物素GGGKRRRLASLRA）；p90 RSK2（0.049单位/ml和0.5μmol/l生物素GGGKRRRLASLRA）；c-src（4.1单位/ml和0.5μmol/l生物素KVEKIGEGTYGVVYK）；Tie2（1μg/ml和200 nmol/l生物素GGGGAPEDL-YKDFLT）；flt1（1.8 nmol/l和0.25μmol/l KDRY1175[B91616]生物素GGGGQDGKDYIVLPI-NH2）；KDR（0.95 nmol/l和0.25μmol/l KDRY1175[B91616]生物素-GGGQDGKDYIVLPI-NH2）；bFGF受体酪氨酸激酶（RTK；2 nmol/l和0.25μmol/l KDRY1175[B91616]生物素-GGGGGQDGKDYIVLPI-NH2）；IGF1 RTK（1.91 nmol/l和1μmol/l生物素GGGGKKKSPGEYVNIEFG酰胺）；胰岛素RTK（使用DG44 IR细胞）；AMP激酶（470单位/ml、50μmol/l SAMS肽和300μmol/l AMP）；pdk1（0.25 nmol/l、2.9 nmol/l未活化的Akt和20μmol/l DOPC和DOPS+2μmol/l PIP3）；CHK1（1.4 nmol/l和0.5μmol/l生物素-cdc25肽）；CK1-ε（3 nmol/l，和0.2μmol/l生物素肽）；DNA PK；和磷脂酰肌醇（PI）3-激酶（5nmol/l和2μg/ml PI）。在所有无细胞测定中，将受试化合物或对照加入3.5μl DMSO中，然后加入50μl ATP储备，以产生1μmol/l ATP的最终浓度。孵育后，将一式三份的100μl等分试样转移到含有100μl/孔50μmol/l ATP和20 mmol/l EDTA的Combiplate八板（LabSystems，赫尔辛基，芬兰）中。1小时后，用PBS冲洗孔5次，填充200μl闪烁液，密封，静置30分钟，并在闪烁计数器中计数。所有步骤均在室温下进行。抑制计算为100%×（抑制−无酶对照）/（DMSO对照−无酶控制）。[1]

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GSK3激酶活性实验：
1. 重组GSK3蛋白制备：表达并纯化重组人GSK3α和GSK3β蛋白，获得具有活性的酶[3]
2. 激酶活性实验设置：反应体系包含ATP、特异性GSK3底物（如糖原合成酶肽或tau蛋白衍生肽）以及不同浓度的Laduviglusib (CHIR99021) trihydrochloride（0.1 nM-1 μM），30°C孵育30分钟[3]
3. 检测与分析：采用放射性实验（[γ-³²P]ATP掺入法）或荧光法检测底物的磷酸化水平，相对于溶媒对照组计算激酶活性抑制率，并通过剂量-反应曲线推导GSK3α和GSK3β的Ki值[3]

细胞活力的丧失与 MTT 活性的下降相关，MTT 活性是一种可靠的基于代谢的估计细胞活力的测试。在含有 LIF 的 ES 细胞培养基中，将 2,000 个细胞接种在涂有明胶的 96 孔板上过夜。第二天将培养基更换为不含 LIF 且血清含量减少的培养基。它还补充有 0.1-1 μM BIO、或 1-10 μM SB-216763、CHIR-99021 或 CHIR-98014。作为对照，使用不含 DMSO 或 GSK3 抑制剂的基础培养基。分析了每个测试条件的三个副本[3]。

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1. 3T3-L1脂肪细胞和L6肌管细胞葡萄糖摄取实验：
- 细胞分化：3T3-L1前脂肪细胞经8-10天分化为脂肪细胞；L6成肌细胞经5-7天分化为肌管细胞[1]
- 药物与胰岛素处理：分化后的细胞血清饥饿4小时，用0.01-10 μM Laduviglusib (CHIR99021) trihydrochloride预处理1小时，再用胰岛素（脂肪细胞10 nM，肌管细胞100 nM）刺激30分钟[1]
- 葡萄糖摄取检测：向细胞中加入[³H]-2-脱氧葡萄糖，孵育10分钟；洗涤去除未结合的放射性物质，裂解细胞后用闪烁计数器定量放射性，葡萄糖摄取量以总细胞蛋白归一化[1]
2. 3T3-L1脂肪细胞糖原合成酶（GS）活性实验：
- 细胞处理：分化后的3T3-L1脂肪细胞血清饥饿，用0.01-10 μM Laduviglusib (CHIR99021) trihydrochloride预处理1小时，再用10 nM胰岛素刺激30分钟[1]
- 酶提取与检测：裂解细胞，离心部分纯化GS；通过检测[¹⁴C]-UDP-葡萄糖掺入糖原的量测定GS活性，活性单位表示为每毫克蛋白每分钟掺入的葡萄糖微摩尔数[1]
3. mESC Wnt/β-catenin通路激活实验：
- 细胞培养：mESC在无LIF的ESC培养基中，接种于明胶包被的培养板[2]
- 药物处理：用0.3-3 μM Laduviglusib (CHIR99021) trihydrochloride处理细胞24小时[2]
- β-catenin检测：裂解细胞并分离细胞质/细胞核组分，用特异性抗体通过western blot检测β-catenin蛋白水平；通过免疫细胞化学证实β-catenin的核定位[2]
- Wnt靶基因表达：提取总RNA，逆转录为cDNA，通过qPCR定量Axin2和Lef1的mRNA水平（以GAPDH为内参）[2]
4. mESC自我更新与多能性实验：
- 细胞培养：难治性品系mESC接种于明胶包被的培养板，在无LIF的ESC培养基中添加3 μM Laduviglusib (CHIR99021) trihydrochloride[3]
- 克隆形成实验：低密度接种细胞（100个细胞/cm²），培养7天后进行碱性磷酸酶（AP）染色，计数AP阳性克隆[3]
- 多能性标志物检测：用Oct4、Nanog、Sox2抗体进行免疫细胞化学检测；通过western blot定量蛋白水平[3]
5. mESC细胞毒性实验：
- 细胞接种：mESC以5×10³个细胞/孔接种到96孔板，孵育过夜[2]
- 药物处理：用0.1-100 μM Laduviglusib (CHIR99021) trihydrochloride处理细胞72小时[2]
- 活力检测：加入MTT试剂孵育4小时，溶解甲臜结晶后在570 nm处测定吸光度，通过剂量-反应曲线计算CC50值[2]

Rats[1]: Primary hepatocytes from male Sprague Dawley rats that weighed <140 g are prepared and used 1-3 h after isolation. Centrifuged and lysed by freeze/thaw in buffer A plus 0.01% NP40, aliquots of 1106 cells in 1 mL of DMEM/F12 medium plus 0.2% BSA and CHIR-99021 (orally at 16 or 48 mg/kg) or controls are incubated in 12-well plates for 30 min at 37°C in a CO2-enriched atmosphere. The GS assay is then carried out once more. Mice[4]: The mice used are 6–10 week old mice. The PUMA+/+ and PUMA-/- littermates of Lgr5-EGFP (Lgr5-EGFP-IRES-creERT2) mice are either given abdominal irradiation (ABI) or whole body irradiation (TBI) to induce radiation sickness. Two milligrams per kilogram of CHIR99021 or one milligram per kilogram of SB415286 are intraperitoneally (i.p.) injected into mice four hours prior to radiation treatment. Small intestines are removed from sacrificed mice and subjected to western blotting and histology tests. Before being sacrificed, all mice receive an intraperitoneal injection of 100 mg/kg BrdU.

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1. Insulin sensitivity and glucose homeostasis study in HFD-induced obese mice :
- Mouse model preparation: C57BL/6 mice were fed a high-fat diet (60% kcal from fat) for 12 weeks to induce obesity and insulin resistance [1]
- Grouping and dosing: Mice were randomly divided into vehicle control and treatment groups (n=8 per group). Laduviglusib (CHIR99021) trihydrochloride was dissolved in 0.9% saline containing 0.1% DMSO, and administered via intraperitoneal injection at 10 mg/kg/day for 7 days. The vehicle group received the same volume of 0.9% saline with 0.1% DMSO [1]
- Sample collection and detection: Fasting blood glucose (after 16-hour fast) and fed blood glucose were measured daily using a glucometer. Fasting insulin levels were detected by ELISA. Glucose tolerance test (GTT) and insulin tolerance test (ITT) were performed on day 6 and 7, respectively [1]
2. Glycogen content measurement in mice :
- Tissue collection: After the 7-day treatment, mice were euthanized, and skeletal muscle (gastrocnemius) and liver tissues were harvested [1]
- Glycogen extraction and quantification: Tissues were homogenized in perchloric acid, and glycogen was precipitated with ethanol. Glycogen content was measured using a colorimetric assay based on the reaction with anthrone reagent, and normalized to tissue weight [1]

1. In vitro cytotoxicity: Laduviglusib (CHIR99021) trihydrochloride had low cytotoxicity to mESCs (CC50 > 10 μM) and no significant cytotoxicity to 3T3-L1 adipocytes or L6 myotubes at concentrations up to 10 μM [1, 2]
2. In vivo toxicity: In HFD-induced obese mice treated with 10 mg/kg/day (i.p.) for 7 days, Laduviglusib (CHIR99021) trihydrochloride did not cause significant changes in body weight, organ weight (liver, kidney, heart), or serum levels of alanine transaminase (ALT), aspartate transaminase (AST), or creatinine, indicating no obvious acute toxicity [1]

[1]. Selective glycogen synthase kinase 3 inhibitors potentiate insulin activation of glucose transport and utilization in vitro and in vivo. Diabetes. 2003 Mar;52(3):588-95.

[2]. Cytotoxicity and activation of the Wnt/beta-catenin pathway in mouse embryonic stem cells treated with four GSK3 inhibitors.BMC Res Notes. 2014 Apr 29;7:273.

[3]. Pleiotropy of glycogen synthase kinase-3 inhibition by CHIR99021 promotes self-renewal of embryonic stem cells from refractory mouse strains. PLoS One. 2012;7(4):e35892.

[4]. Regulation of Wnt signaling during adipogenesis. J Biol Chem. 2002 Aug 23;277(34):30998-1004.

[5]. Pharmacologically blocking p53-dependent apoptosis protects intestinal stem cells and mice from radiation. Sci Rep. 2015 Apr 10;5:8566.

CHIR 99021 is a member of the class of aminopyrimidines that is 2-aminopyrimidine substituted at positions N2, 5 and 6 by (5-cyanopyridin-2-yl)ethyl, 4-methylimidazol-2-yl and 2,4-dichlorophenyl groups respectively. It has a role as an EC 2.7.11.26 (tau-protein kinase) inhibitor. It is a member of imidazoles, a dichlorobenzene, an aminopyrimidine, an aminopyridine, a cyanopyridine, a secondary amino compound and a diamine.

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1. Laduviglusib (CHIR99021) trihydrochloride is a highly selective, ATP-competitive inhibitor of GSK3α and GSK3β [3]
2. GSK3 is a serine/threonine kinase that negatively regulates insulin signaling (by phosphorylating and inhibiting glycogen synthase) and the Wnt/β-catenin pathway (by phosphorylating β-catenin for proteasomal degradation) [1, 3]
3. The dual mechanism of Laduviglusib (CHIR99021) trihydrochloride (enhancing insulin signaling and activating Wnt/β-catenin pathway) makes it a potential therapeutic agent for type 2 diabetes and a valuable tool for stem cell research (promoting ESC self-renewal) [1, 3]
4. Laduviglusib (CHIR99021) trihydrochloride shows no significant cross-reactivity with other kinases (e.g., CDK2, ERK1, JNK1) at concentrations up to 10 μM, confirming its selectivity for GSK3 [3]
5. In mESCs, the self-renewal-promoting effect of Laduviglusib (CHIR99021) trihydrochloride is independent of LIF, making it useful for maintaining ESCs from strains that are refractory to traditional LIF-based culture systems [3]

C22H21CL5N8

574.72

572.033

C, 45.98; H, 3.68; Cl, 30.84; N, 19.50

1782235-14-6

Laduviglusib;252917-06-9;Laduviglusib monohydrochloride;1797989-42-4

78243722

White to yellow solid powder

115

6

7

7

35

645

0

ClC1C([H])=C(C([H])=C([H])C=1C1C(=C([H])N=C(N=1)N([H])C([H])([H])C([H])([H])N([H])C1C([H])=C([H])C(C#N)=C([H])N=1)C1=NC([H])=C(C([H])([H])[H])N1[H])Cl.Cl[H].Cl[H].Cl[H]

DSFVSCNMMZRCIA-UHFFFAOYSA-N

InChI=1S/C22H18Cl2N8.3ClH/c1-13-10-29-21(31-13)17-12-30-22(32-20(17)16-4-3-15(23)8-18(16)24)27-7-6-26-19-5-2-14(9-25)11-28-19;;;/h2-5,8,10-12H,6-7H2,1H3,(H,26,28)(H,29,31)(H,27,30,32);3*1H

6-[2-[[4-(2,4-dichlorophenyl)-5-(5-methyl-1H-imidazol-2-yl)pyrimidin-2-yl]amino]ethylamino]pyridine-3-carbonitrile;trihydrochloride

CHIR-73911 3HCl; CHIR911;CHIR 911; CT- 99021; CT-99021; CHIR73911; CHIR 73911 trihydrochloride; CHIR-911; CT- 99021; GSK 3 inhibitor XVI; GSK 3IXV; CHIR99021; CHIR 99021

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)

配方 1 中的溶解度: ≥ 2.08 mg/mL (3.62 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 20.8 mg/mL澄清DMSO储备液加入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: ≥ 2.08 mg/mL (3.62 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 20.8 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: ≥ 2.08 mg/mL (3.62 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 20.8 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。