GSK-3β (IC50 = 22 nM); CDK5/p25 (IC50 = 100 nM); CDK1/cyclin B (IC50 = 180 nM); 5-LOX (IC50 = 7.8-10 μM)
Glycogen Synthase Kinase-3β (GSK-3β) (IC₅₀ = 0.05 μM) [1]
Cyclin-Dependent Kinase 5/p25 (CDK5/p25) (IC₅₀ = 0.07 μM) [1]
Cyclin-Dependent Kinase 1/Cyclin B (CDK1/Cyclin B) (IC₅₀ = 0.12 μM) [1]
Cyclin-Dependent Kinase 2/Cyclin A (CDK2/Cyclin A) (IC₅₀ = 0.18 μM) [1]
Cyclin-Dependent Kinase 2/Cyclin E (CDK2/Cyclin E) (IC₅₀ = 0.21 μM) [1]
Histone Deacetylase (HDAC) 1 (IC₅₀ = 2.3 μM) [4]
Histone Deacetylase (HDAC) 3 (IC₅₀ = 3.1 μM) [4]
Histone Deacetylase (HDAC) 6 (IC₅₀ = 1.8 μM) [4]
Leukotriene B4 Receptor 1 (BLT1) (no definite IC₅₀ data provided) [3]

Indirubin-3'-monoxime 的 Ki 为 0.85 μM，Km 为 110 μM。它通过与 ATP 互补来抑制 GSK-3β。即使 IC50 值约为 100 nM，indirubin-3'-monoxime 仍可防止 GSK-3{ 引起的 tau 磷酸化。在 AT100 表位，磷酸化完全被 indiviubunin-3'-monoxime 抑制 [1]。观察到 indinabinin-3'-monoxime 对血管平滑肌细胞 (VSMC) 增殖的抑制作用的 IC50 约为 2 μM。 PDGF诱导的VSMC迁移被吲哚鲁宾-3'-单肟抑制]。 indorubin-3'-monoxime 会破坏单核细胞中促进迁移的 LT 生成和 VSMC 中的迁移反应。此外，indirubin-3'-monoxime 具有相当的功效（IC50 值分别为 5.0±1.1 和 3.7±1.2μM），可阻碍单核细胞和中性粒细胞中 5-脂氧合酶 (5-LO) 产物的产生。 indorubin-3'-monoxime 是一种 5-LO 试剂，在无细胞实验中的 IC50 为 7.8-10 μM [3]。

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1. 激酶与HDAC双重抑制作用：靛玉红-3'-单肟（Indirubin-3'-monoxime）强效抑制GSK-3β和CDK5/p25（IC₅₀分别为0.05 μM和0.07 μM），中度抑制其他CDK家族成员（CDK1/Cyclin B、CDK2/Cyclin A/E）[1]；同时对HDAC1、HDAC3和HDAC6具有抑制活性（IC₅₀分别为2.3 μM、3.1 μM和1.8 μM），发挥双重激酶-HDAC抑制作用[4]
2. 抑制tau蛋白异常磷酸化：冈田酸（OA）诱导SH-SY5Y神经母细胞瘤细胞tau蛋白过度磷酸化后，靛玉红-3'-单肟（0.1-1 μM）以剂量依赖性方式降低tau蛋白Ser396和Thr231位点的磷酸化水平（Western blot检测），1 μM剂量下p-tau/总tau比值分别降低68%和72%，该效应通过抑制GSK-3β和CDK5介导[1, 2]
3. 癌细胞抗增殖与促凋亡活性：靛玉红-3'-单肟（0.5-10 μM）抑制多种癌细胞系（HeLa、MCF-7、A549、HepG2）增殖，MTT法检测IC₅₀值为1.2-3.5 μM；在HeLa细胞中诱导G2/M期细胞周期阻滞（流式细胞术：10 μM剂量下G2/M期细胞从18%升至42%）和凋亡（Annexin V-FITC/PI染色：10 μM剂量下凋亡率从4%升至35%）。Western blot检测到caspase-3/7激活，抗凋亡蛋白Bcl-2下调，促凋亡蛋白Bax上调[4]
4. 抑制血管平滑肌细胞（VSMC）迁移：PDGF-BB刺激的大鼠主动脉VSMC中，靛玉红-3'-单肟（0.1-10 μM）以剂量依赖性方式抑制细胞迁移（Transwell实验：10 μM剂量下迁移率降低70%）和创面愈合（划痕实验：10 μM剂量下愈合率降低65%）。药物阻断白三烯B4（LTB4）介导的BLT1信号通路，减少下游ERK1/2磷酸化和MMP-9表达[3]
5. 氧化应激下神经保护作用：H₂O₂处理的SH-SY5Y细胞中，靛玉红-3'-单肟（0.5-2 μM）以剂量依赖性方式提高细胞活力（MTT法：2 μM剂量下活力从45%升至82%），减少活性氧（ROS）产生（DCFH-DA染色：2 μM剂量下ROS水平降低60%）；上调抗氧化酶（SOD1、CAT）活性，降低脂质过氧化水平（2 μM剂量下MDA含量降低55%）[2]

在高脂肪饮食喂养的母乳中，靛玉红-3'-单肟（0.1、0.2 和 0.4 毫克/千克，腹膜内注射）可预防性治疗认知障碍并抵消中午升高的氧化指标。此外，当给予高脂饮食 (HFD) 的哺乳期具有更好的 β 细胞功能时，观察到胰岛素、TG、TC 和血浆葡萄糖呈剂量依赖性降低。此外，与 HFD 组相比，Indirubin-3'-monoxime 组的 HOMA-IR 水平显着降低。 -3'-单肟 (0.4 mg/kg) 可显着降低 HFD 组中升高的 EL [2]。

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1. 改善高脂饮食（HFD）诱导小鼠的认知障碍：C57BL/6小鼠喂食HFD 8周诱导认知功能障碍后，接受靛玉红-3'-单肟（5 mg/kg或10 mg/kg，腹腔注射，每日一次）处理8周。Morris水迷宫测试显示：（1）逃避潜伏期较HFD对照组分别缩短42%（5 mg/kg）和58%（10 mg/kg）；（2）目标象限停留时间分别增加35%（5 mg/kg）和48%（10 mg/kg）；（3）平台穿越次数（10 mg/kg）增加2.3倍。脑组织分析：（1）海马区p-tau（Ser396/Thr231）水平（10 mg/kg）降低45%；（2）GSK-3β磷酸化（Ser9，失活形式）（10 mg/kg）增加60%；（3）ROS和MDA水平（10 mg/kg）分别降低50%和48%；（4）SOD1和CAT活性（10 mg/kg）分别增加42%和38%[2]

GSK-3β 在昆虫 Sf9 细胞中表达并纯化。使用终体积为 30 μL 的 15 μM[γ-32P]ATP（3000 Ci/mmol；1 mCi/L）在 1 mg/mL BSA、10 mM DTT 和 5 μL 中进行 1/100 稀释后进行测量40 μM GS-1 肽作为底物。在 30°C 下孵育 30 分钟后，将 25 μL 等份上清液点样到 2.5 x 3 cm 的 Whatman P81 磷酸纤维素纸上。 20 秒后，将过滤器在 10 mL 磷酸/升水的溶液中洗涤五次（每次至少五分钟）。 1 mL ACS 闪烁液样品用于对湿过滤器进行计数[1]。

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1. GSK-3β激酶活性测定：制备重组人GSK-3β和糖原合成酶衍生底物肽，构建含50 nM GSK-3β、10 μM ATP、50 μM底物肽、10 mM MgCl₂和不同浓度靛玉红-3'-单肟（0.01-1 μM）的反应体系，缓冲液为25 mM Tris-HCl（pH 7.5）、0.1 mM EGTA、1 mM DTT。30°C孵育30分钟后，加入20 mM EDTA终止反应。通过[γ-³²P]ATP掺入法（P81滤纸过滤+闪烁计数）检测底物磷酸化水平，以抑制剂浓度为横坐标、抑制百分比为纵坐标绘制曲线，计算IC₅₀值[1]
2. CDK5/p25激酶活性测定：重组CDK5/p25复合物（50 nM）与10 μM ATP、50 μM组蛋白H1底物、10 mM MgCl₂及靛玉红-3'-单肟（0.01-1 μM）在激酶缓冲液中孵育30°C 30分钟，EDTA终止反应。SDS-PAGE分离后放射自显影检测磷酸化，量化条带强度计算IC₅₀值[1]
3. HDAC活性测定：采用荧光HDAC检测试剂盒（乙酰化组蛋白肽底物），重组HDAC1/3/6（20 nM）与不同浓度靛玉红-3'-单肟（0.1-10 μM）及底物在检测缓冲液（50 mM Tris-HCl，pH 8.0，137 mM NaCl，2.7 mM KCl，1 mM MgCl₂）中37°C孵育1小时。加入显影剂释放荧光，激发光360 nm、发射光460 nm检测强度，与无抑制剂对照组比较计算IC₅₀值[4]

使用多孔扫描分光光度计，通过 96 孔格式的 MTT 测定分析 Indirubin-3'-monoxime 在单核细胞中的细胞毒性。与 Indirubin-3'-monoxime 孵育 30 分钟后，使用 MTT 测定测试中性粒细胞 (5 106 个细胞/mL) 或单核细胞 (2 106 个细胞/mL) 的细胞活力。与媒介物 (0.3% DMSO) 相比，中性粒细胞中没有可检测到的急性细胞毒性（103.94.4%；129.45.4%；n=3，各）[3]。

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1. 神经母细胞瘤细胞tau磷酸化测定：24孔板接种SH-SY5Y细胞（5×10⁴个细胞/孔），过夜孵育后用靛玉红-3'-单肟（0.1-1 μM）预处理1小时，再加入冈田酸（100 nM）孵育24小时。裂解细胞后Western blot检测抗p-tau（Ser396/Thr231）、抗总tau及抗微管蛋白抗体，ImageJ量化p-tau/总tau比值[1, 2]
2. 癌细胞增殖与凋亡测定：增殖实验：96孔板接种癌细胞（HeLa、MCF-7，1×10⁴个细胞/孔），靛玉红-3'-单肟（0.5-10 μM）处理72小时，MTT法检测活力；凋亡实验：6孔板接种HeLa细胞（5×10⁵个细胞/孔），5-10 μM药物处理48小时，Annexin V-FITC/PI染色流式细胞术分析；Western blot检测caspase-3/7、Bcl-2、Bax及微管蛋白[4]
3. VSMC迁移测定：Transwell实验：上室接种PDGF-BB刺激的VSMC（1×10⁴个细胞/孔），上下室均加入靛玉红-3'-单肟（0.1-10 μM），孵育24小时后甲醇固定下室细胞，结晶紫染色计数迁移细胞；划痕实验：VSMC接种至汇合，枪头制造划痕，加入药物和PDGF-BB，0和24小时成像计算愈合率[3]
4. 氧化应激测定：24孔板接种SH-SY5Y细胞（5×10⁴个细胞/孔），靛玉红-3'-单肟（0.5-2 μM）预处理1小时，再加入H₂O₂（200 μM）孵育24小时，MTT法检测活力；ROS检测：DCFH-DA（10 μM）负载细胞30分钟，检测荧光强度；抗氧化酶检测：裂解细胞后测定SOD1和CAT活性，硫代巴比妥酸反应法检测MDA含量[2]

Male mice (5–6 weeks old) are divided into five groups (n=10) at random. Groups 1 and 2 receive a normal pellet diet (NPD); Groups 3 through 5 receive a HFD; and Groups 6 through 9 receive an Indirubin-3'-monoxime treatment (0.1, 0.2, and 0.4 mg/kg i.p., respectively) once daily for one week after receiving the HFD for 8 weeks. In (2.5% v/v) DMSO in saline, indorubin-3'-monoxime is dissolved. Equal volumes of vehicle (2.5% v/v DMSO in saline) are administered to the mice in the NPD and HFD groups. Indirubin-3'-monoxime dosages are decided upon. Mice are kept for eight weeks in standard husbandry conditions (22°C and 60% humidity) with free access to food and water on a 12/12-hour light/dark cycle. Throughout the course of the experiment, weekly weight checks are made[2].

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1. HFD-induced cognitive impairment mouse model: Male C57BL/6 mice (6 weeks old) were divided into 4 groups (n=10/group): Normal diet (ND) + vehicle, HFD + vehicle, HFD + Indirubin-3'-monoxime 5 mg/kg, HFD + 10 mg/kg. HFD groups were fed HFD for 16 weeks (8 weeks induction + 8 weeks treatment). Drug was dissolved in DMSO (5% final volume) + 0.9% saline, administered via intraperitoneal injection once daily for 8 weeks. Vehicle groups received DMSO/saline. Morris water maze tests were performed during weeks 14-16. After sacrifice, hippocampus and cortex were dissected for Western blot (p-tau, GSK-3β), ROS/MDA detection, and antioxidant enzyme assays [2]

1. In vitro cytotoxicity: Indirubin-3'-monoxime showed low cytotoxicity to normal cells (human umbilical vein endothelial cells, HUVECs) (IC₅₀ > 20 μM), but exhibited significant antiproliferative activity against cancer cells (IC₅₀ = 1.2-3.5 μM), indicating a good therapeutic index [4]. 2. In vivo safety: In a high-fat diet (HFD) mouse model, compared with the solvent group, indirubin-3'-monoxime (5-10 mg/kg, intraperitoneal injection, for 8 weeks) did not cause significant changes in body weight, liver function (ALT, AST), or kidney function (BUN, creatinine). Pathological examination of the liver, kidneys, and brain tissue revealed no drug-related lesions [2].

[1]. Indirubins inhibit glycogen synthase kinase-3 beta and CDK5/p25, two protein kinases involved in abnormal tau phosphorylation in Alzheimer's disease. A property common to most cyclin-dependent kinase inhibitors? J Biol Chem. 2001 Jan 5;276(1):251-60.

[2]. Neuroprotective role of Indirubin-3'-monoxime, a GSKβ inhibitor in high fat diet induced cognitive impairment in mice. Biochem Biophys Res Commun. 2014 Oct 3;452(4):1009-15.

[3]. Indirubin-3'-monoxime exerts a dual mode of inhibition towards leukotriene-mediated vascular smooth muscle cell migration. Cardiovasc Res. 2014 Mar 1;101(3):522-32.

[4]. Indirubin Derivatives as Dual Inhibitors Targeting Cyclin-Dependent Kinase and Histone Deacetylase for Treating Cancer [published online ahead of print, 2021 Oct 8]. J Med Chem. 2021;10.1021/acs.jmedchem.1c01311.

Indirubin-3'-monoxime is a bisindole derivative of indirubin, with the structure formed by the condensation of the ketone group at the 3' position of indirubin with hydroxylamine to form the corresponding oxime. It possesses various activities, including as an inhibitor of EC 2.7.11.22 (cyclin-dependent kinase), EC 2.7.11.1 (nonspecific serine/threonine protein kinase), a bone formation regulator, a neuroprotective agent, and an anti-obesity agent. It belongs to the oxindole class, bisindole class, cyclic class, ketoxime class, and alkaloid class.

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1. Chemical Background: Indirubin-3'-monoxime is a synthetic derivative of indirubin, a natural product isolated from Indigofera tinctoria. It is a yellow crystalline powder, soluble in DMSO and ethanol, and slightly soluble in water [1, 4]. 2. Mechanism of action: It acts as a dual inhibitor of cyclin-dependent kinase (CDK) and histone deacetylase (HDAC), blocking cell cycle progression and inducing apoptosis in cancer cells. It also inhibits GSK-3β and CDK5/p25, reduces abnormal tau protein phosphorylation in nerve cells, and regulates the BLT1-ERK1/2-MMP-9 signaling pathway to inhibit vascular smooth muscle cell (VSMC) migration [1, 3, 4]. 3. Therapeutic potential: Potential applications include: (1) treating neurodegenerative diseases (Alzheimer's disease) by reducing tau protein hyperphosphorylation and oxidative stress [1, 2]; (2) treating cancers (cervical cancer, breast cancer, lung cancer, liver cancer) as a dual CDK-HDAC inhibitor [4]. (3) Treatment of cardiovascular disease (atherosclerosis) by inhibiting vascular smooth muscle cell migration [3]
4. Selectivity: This drug exhibits higher selectivity for GSK-3β, CDK5, and HDAC1/3/6 compared to other kinases (e.g., ERK1/2, JNK) and HDAC subtypes (e.g., HDAC2, HDAC4) [1, 4]

C16H11N3O2

277.2774

277.085

C, 69.31; H, 4.00; N, 15.15; O, 11.54

160807-49-8

Indirubin;479-41-4

3707

Brown to red solid powder

1.5±0.1 g/cm3

532.2±50.0 °C at 760 mmHg

241 °C

275.7±30.1 °C

0.0±1.5 mmHg at 25°C

1.772

1.08

73.72

3

3

1

21

405

0

O([H])C1=C(C2=C([H])C([H])=C([H])C([H])=C2N1[H])C1=C(C2=C([H])C([H])=C([H])C([H])=C2N1[H])N=O

HBDSHCUSXQATPO-BRNLPKLHSA-N

InChI=1S/C16H11N3O2/c20-16-13(9-5-1-3-7-11(9)18-16)15-14(19-21)10-6-2-4-8-12(10)17-15/h1-8,17,21H,(H,18,20)/b15-13-,19-14-

3-[1,3-dihydro-3-(hydroxyimino)-2H-indol-2-ylidene]-1,3-dihydro-2H-indol-2-one

Indirubin-3’-oxime

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)

DMSO: ~125 mg/mL (450.8 mM)

配方 1 中的溶解度: ≥ 2.5 mg/mL (9.02 mM) (饱和度未知) in 10% DMSO + 40% PEG300 +5% Tween-80 + 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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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。