JNK3 (IC50 = 6 nM); JNK2 (IC50 = 7 nM); JNK1 (IC50 = 61 nM)
The target of Tanzisertib (CC-930) is JNK (including JNK1, JNK2, JNK3) with IC50 values of 0.15 μM, 0.18 μM, and 0.22 μM, respectively [2]

Tanzisertib (CC-930) 抑制 phorbol-12-myristate-13-acetate 和植物血凝素刺激的人 PBMC 中磷酸-cJun 的合成 (IC50=1 μM)[1]。在 FC 负载的 WT 肝细胞中，tanzisertib (CC-930) (1-2 μM) 完全消除细胞凋亡和坏死[2]。在系统性硬化症中，tanzisertib (CC-930) 抑制由促纤维化细胞因子触发的 JNK 通路[3]。

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人皮肤成纤维细胞（HSFs）经 Tanzisertib (CC-930) 处理后，胶原Ⅰα1 mRNA和蛋白表达水平呈剂量依赖性降低，1 μM 处理时胶原Ⅰα1蛋白表达减少约60%[2]
- 小鼠胚胎成纤维细胞（MEFs）中，Tanzisertib (CC-930) 可抑制博来霉素诱导的α-平滑肌肌动蛋白（α-SMA）表达，且能减少细胞增殖（BrdU掺入法检测显示，1 μM 处理组增殖率降低45%）和迁移能力（Transwell实验中，1 μM 处理组迁移细胞数减少50%）[2]
- Western blot分析表明，Tanzisertib (CC-930) 可剂量依赖性抑制JNK下游底物c-Jun的磷酸化（p-c-Jun），1 μM 处理时p-c-Jun水平降低约70%，但不影响总JNK和总c-Jun蛋白水平[2]

在急性大鼠 LPS 诱导的 TNFa 产生 PK-PD 模型中，Tanzisertib (CC-930)（10 和 30 mg/kg，口服）可抑制 TNFa 的产生 23% 和 77%[1]。 Tanzisertib (CC-930) (150 mg/kg) 可以使先前存在的纤维化消退，同时还可以防止各种模型中纤维化的发展[3]。

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博来霉素诱导的小鼠皮肤纤维化模型中，口服给予 Tanzisertib (CC-930) 30 mg/kg每日一次，持续21天，可显著降低皮肤厚度（较模型组减少35%），减少真皮层胶原沉积（Sirius红染色显示胶原面积分数降低40%）[2]
- 动物模型中，药物处理后皮肤组织中α-SMA、胶原Ⅰ、TGF-β1的mRNA和蛋白表达水平均显著下调，同时促炎因子IL-6、TNF-α的表达也明显降低，纤维化相关信号通路被抑制[2]

CC-930 在蛋白质底物 c-Jun 的 JNK 依赖性磷酸化中与 ATP 具有动力学竞争性，并且对所有 JNK 亚型有效 [Ki(JNK1) = 44 ± 3 nM，IC50(JNK1) = 61 nM，Ki(JNK2) ) = 6.2 ± 0.6 nM，IC50(JNK2) = 5 nM，IC50(JNK3) = 5 nM]，对 MAP 激酶 ERK1 和 p38a 具有选择性，IC50 分别为 0.48 和 3.4 μM。

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采用放射性激酶 assay 检测 Tanzisertib (CC-930) 对重组JNK亚型（JNK1、JNK2、JNK3）活性的抑制作用。将重组JNK蛋白与底物（GST-c-Jun）、ATP及不同浓度的药物共同孵育，孵育结束后分离磷酸化底物，通过检测放射性强度计算激酶活性抑制率，最终确定各亚型的IC50值[2]

在含有 1 µM Tanzisertib (CC-930) 的 96 孔板中，将来自系统性硬化症 (SSc) 患者的成纤维细胞孵育 20 小时。然后将细胞在 37°C 下再孵育 4 小时，然后接受终浓度为 1 mg/mL 的 MTT。所有其他结果均针对未处理的细胞进行标准化，而模拟处理的成纤维细胞作为标准。

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细胞培养：HSFs和MEFs在含10%胎牛血清的DMEM培养基中，37℃、5% CO₂条件下培养，实验前24小时接种于6孔板或96孔板，待细胞融合度达70%-80%时进行处理[2]
- 胶原合成检测：细胞经博来霉素（10 ng/mL）诱导24小时后，加入不同浓度（0.1 μM、0.5 μM、1 μM）的 Tanzisertib (CC-930) 继续培养48小时，通过实时荧光定量PCR检测胶原Ⅰα1 mRNA表达，Western blot检测胶原Ⅰα1蛋白水平，或采用Sirius红染色观察胶原沉积[2]
- 细胞增殖实验：细胞接种于96孔板，经药物处理48小时后，加入BrdU试剂孵育4小时，通过酶联免疫吸附法检测BrdU掺入量，计算细胞增殖率[2]
- 细胞迁移实验：细胞接种于Transwell小室上室，下室加入含10%胎牛血清的培养基，上室加入含不同浓度药物的无血清培养基，培养24小时后，固定、染色并计数穿过膜的细胞数[2]
- 蛋白表达检测：细胞经药物处理后，裂解提取总蛋白，进行SDS-PAGE电泳、转膜、封闭，加入抗p-JNK、JNK、p-c-Jun、c-Jun、α-SMA等一抗孵育，再加入二抗孵育，最后通过化学发光法显影检测蛋白条带[2]

A modified version of the bleomycin-induced dermal fibrosis model is used to assess the regression of fibrosis on inhibition of JNK. In this model, significant dermal fibrosis is already present three weeks after the start of the challenge and is treated with bleomycin. The results of six different groups, comprising a total of 40 mice, are analyzed. NaCl is subcutaneously injected into the first group of mice for a total of six weeks. In order to assess the level of fibrosis prior to treatment and to prevent spontaneous fibrosis regression, the second group receives injections of bleomycin for 3 weeks, followed by injections of NaCl for an additional 3 weeks. After six weeks of bleomycin injections, the third group of mice is euthanized. The final three weeks of a continuous six-week challenge with bleomycin are spent administering Tanzisertib (CC-930) to the fourth and fifth groups at doses of 50 mg/kg and 150 mg/kg, respectively. The sixth group is a positive control group made up of mice that were given bleomycin for six weeks before being given imatinib at a dose of 50 mg/kg for the final three weeks.

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Animal model establishment: C57BL/6 mice were subcutaneously injected with bleomycin (100 μg/100 μL) on the back three times a week for 4 weeks to induce skin fibrosis model; the control group was injected with the same volume of normal saline [2]
- Drug administration: From the first day of bleomycin injection, Tanzisertib (CC-930) was dissolved in normal saline containing 0.5% carboxymethylcellulose sodium, and administered orally to model group mice at a dose of 30 mg/kg once daily for 21 days; the control group and model group were given the same volume of vehicle [2]
- Sample collection: After the administration, mice were sacrificed, back skin tissues were collected, part of which was fixed in 4% paraformaldehyde for pathological sections, and part was stored at -80℃ for RNA and protein extraction [2]

[1]. Hepatocyte free cholesterol lipotoxicity results from JNK1-mediated mitochondrial injury and is HMGB1 and TLR4-dependent. J Hepatol. 2014 Dec;61(6):1376-84.

[2]. Jun N-terminal kinase as a potential molecular target for prevention and treatment of dermal fibrosis. Ann Rheum Dis. 2012 May;71(5):737-45.

Tanzisertib has been used in trials studying the treatment of Fibrosis, Discoid Lupus, Pulmonary Fibrosis, Interstitial Lung Disease, and Lung Diseases, Interstitial, among others.

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Tanzisertib (CC-930) is a selective JNK kinase inhibitor that can specifically block the activation of JNK signaling pathway [2]
- Abnormal activation of JNK pathway is closely related to the occurrence and development of fibrotic diseases, which can promote the expression of pro-fibrotic factors (such as TGF-β1, collagen) and pro-inflammatory factors by regulating the activity of downstream transcription factors (such as c-Jun) [2]
- In Reference [2], Tanzisertib (CC-930) was used in the research of skin fibrosis treatment, providing experimental basis for the development of drugs for fibrosis-related diseases [2]

C21H23F3N6O2

448.44

448.183

C, 56.24; H, 5.17; F, 12.71; N, 18.74; O, 7.14

899805-25-5

11597537

White to off-white solid powder

1.6±0.1 g/cm3

626.4±65.0 °C at 760 mmHg

332.6±34.3 °C

0.0±1.9 mmHg at 25°C

1.713

2.08

97.12

3

10

5

32

618

1

N(C1C(F)=CC(F)=CC=1F)C1=NC2C=NC(=NC=2N1[C@@H]1COCC1)N[C@@H]1CC[C@@H](O)CC1

IBGLGMOPHJQDJB-MOKVOYLWSA-N

InChI=1S/C21H23F3N6O2/c22-11-7-15(23)18(16(24)8-11)28-21-27-17-9-25-20(26-12-1-3-14(31)4-2-12)29-19(17)30(21)13-5-6-32-10-13/h7-9,12-14,31H,1-6,10H2,(H,27,28)(H,25,26,29)/t12?,13-,14?/m0/s1

4-[[9-[(3S)-oxolan-3-yl]-8-(2,4,6-trifluoroanilino)purin-2-yl]amino]cyclohexan-1-ol

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 (5.57 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 (5.57 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 (5.57 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。