GSK-3β (Ki = 38 nM); GSK-3β (Ki = 38 nM)

AR-A014418 抑制表达人四重复 tau 蛋白的 3T3 成纤维细胞中 GSK3 特异性位点 (Ser-396) 的 tau 磷酸化，IC50 为 2.7 μM，并保护培养的 N2A 细胞免遭阻断 PI3K/PKB 通路诱导的死亡。 AR-A014418 可预防海马切片中 β-淀粉样肽引起的神经变性。[1]而在 NGP 和 SH-5Y-SY 细胞中，AR-A014418 降低神经内分泌标志物并抑制神经母细胞瘤细胞的生长。 [2]

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用AR-A014418处理的神经母细胞瘤细胞在所有剂量和时间点的生长都显著降低（P<0.001）。与对照组相比，在第6天，10μM（NGP-53%对0%，SH-5Y-SY-38%对0%，P<0.001）治疗的细胞系生长减少，相应于肿瘤标志物ASCL1和CgA表达的显著降低[2]。 结论：与Tyr216的GSK-3β磷酸化相比，AR-A014418治疗神经母细胞瘤细胞系降低了Tyr279的GSK-3α磷酸化水平，并通过维持凋亡来抑制生长。本研究支持进一步的研究，以阐明GSK-3α抑制下调NE肿瘤标志物表达和神经母细胞瘤生长的机制[2]。

AR-A014418（0–4 mg/kg，腹腔注射）可延迟 G93A 突变型人类 SOD1 的 ALS 小鼠的症状发作、增强运动功能、减缓疾病进展并延迟疾病终点。 [3] 此外，AR-A014418 通过改变 NMDA 和代谢型受体的信号传导以及 TNF-和 IL-1 在脊髓中的传输来抑制小鼠的乙酸和福尔马林诱导的伤害感受。 [4]

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在这项研究中，研究人员研究了小鼠糖原合成酶激酶-3β（GSK-3β）的选择性抑制剂AR-A014418的镇痛作用。用AR-A014418（0.1和1mg/kg，腹膜内[ip]）预处理30分钟可抑制ip注射乙酸引起的伤害性感觉。AR-A014418预处理（0.1和.3 mg/kg，ip）也减少了福尔马林诱导的舔舐的晚期（炎症）阶段，而不影响第一阶段（神经源性）的反应。在另一组实验中，AR-A014418（0.1-10μg/部位）与福尔马林共注射（ipl）抑制了福尔马林诱导的伤害性疼痛的晚期。此外，AR-A014418给药（1和10ng/部位，鞘内[it]）抑制了福尔马林诱导的舔舐的两个阶段。此外，AR-A014418共注射（10 ng/位点，它）分别抑制了谷氨酸、N-甲基-D-天冬氨酸（NMDA）、（±）-1-氨基环戊烷反式-1,3-二羧酸（反式ACPD）、肿瘤坏死因子α（TNF-α）和白细胞介素-1β（IL-1β）诱导的伤害性感觉，抑制率分别为47±12%、48±11%、31±8%、46±13%和44±11%。此外，用另一种GSK-3β抑制剂NP031115（3和10mg/kg，ip）预处理30分钟也减轻了福尔马林诱导的伤害性疼痛的晚期。总的来说，这些结果提供了令人信服的证据，表明AR-A014418通过局部、全身和中枢途径给药，在几种伤害感受的小鼠模型中产生了镇痛作用。AR-A014418依赖的镇痛作用是通过代谢型和离子型（NMDA）受体调节谷氨酸能系统和抑制细胞因子（TNF-α和IL-1β）信号传导来诱导的。

煅烧/碘化丙啶的摄取用于测定细胞活力。钙黄绿素 AM 仅被死细胞吸收，并在被活细胞内的酯酶裂解时发出橙红色荧光。简而言之，N2A 细胞在体外培养两天，然后在 AR-A014418 或载体 (DMSO) 存在下暴露于 50 μM LY-294002 24 小时。然后，将 2 μM PI 和 1 μM calcein-AM 与 N2A 细胞一起孵育 30 分钟。然后用含有 2 mM CaCl2 的 Hanks' 缓冲盐水溶液冲洗培养物 3 次，并使用荧光显微镜和 Zeiss Axiovert 135 显微镜观察细胞。在至少三个不同的实验中，每孔分析三个视野（随机选择）（约 300 个细胞/视野）。 PI 阳性细胞与所有其他细胞相比的百分比用于测量细胞死亡。扣除载体处理的培养物中存在的死亡细胞的数量后，在每个实验中计算特定的细胞死亡。

通过钙黄绿素/碘化丙啶的摄取来评估细胞活力。钙黄绿素 AM 被活细胞内的酯酶吸收并裂解，产生黄绿色荧光，而 PI 仅被死细胞吸收，产生橙红色荧光。简而言之，N2A 细胞在体外培养 2 天，然后在 AR-A014418 或载体 (DMSO) 存在下用 50 μM LY-294002 处理 24 小时。随后，将 N2A 细胞与 2 μM PI 和 1 μM 钙黄绿素-AM 一起孵育 30 分钟。然后用含有 2 mM CaCl2 的 Hanks 缓冲盐水溶液冲洗培养物 3 次，并使用 Zeiss Axiovert 135 显微镜通过荧光显微镜观察细胞。在至少三个不同的实验中，每孔分析三个视野（随机选择）（~300 个细胞/视野）。细胞死亡表示为 PI 阳性细胞占细胞总数的百分比。在每个实验中，减去载体处理的培养物中存在的死亡细胞的数量后获得特定的细胞死亡。

First, 56 Tg mice are split up into four groups to study the effects of GSK-3 inhibition on the clinical symptoms, lifespan, and motor behavior function of ALS. 14 animals per group are given intraperitoneal injections of 0.5 mL of normal saline five times a week starting 60 days after birth, along with 0 μg (control group), 1 μg (group A), 2 μg (group B), or 4 μg (group C) of AR-A014418 per gram of mouse. At the endpoint outlined below, mice are sacrificed.

[1]. Bhat R, Xue Y, Berg S, Structural insights and biological effects of glycogen synthase kinase 3-specific inhibitor AR-A014418. J Biol Chem. 2003 Nov 14;278(46):45937-45.

[2]. Specific glycogen synthase kinase-3 inhibition reduces neuroendocrine markers and suppresses neuroblastoma cell growth. Cancer Biol Ther. 2014 May;15(5):510-5.

[3]. Inhibition of glycogen synthase kinase-3 suppresses the onset of symptoms and disease progression of G93A-SOD1 mouse model of ALS. Exp Neurol. 2007 Jun;205(2):336-46.

[4]. The antinociceptive effects of AR-A014418, a selective inhibitor of glycogen synthase kinase-3 beta, in mice. J Pain. 2011 Mar;12(3):315-22.

Glycogen synthase kinase 3 (GSK3) is a serine/threonine kinase that has been implicated in pathological conditions such as diabetes and Alzheimer's disease. We report the characterization of a GSK3 inhibitor, AR-A014418, which inhibits GSK3 (IC50 = 104 +/- 27 nM), in an ATP-competitive manner (Ki = 38 nM). AR-A014418 does not significantly inhibit cdk2 or cdk5 (IC50 > 100 microM) or 26 other kinases demonstrating high specificity for GSK3. We report the co-crystallization of AR-A014418 with the GSK3beta protein and provide a description of the interactions within the ATP pocket, as well as an understanding of the structural basis for the selectivity of AR-A014418. AR-A014418 inhibits tau phosphorylation at a GSK3-specific site (Ser-396) in cells stably expressing human four-repeat tau protein. AR-A014418 protects N2A neuroblastoma cells against cell death mediated by inhibition of the phosphatidylinositol 3-kinase/protein kinase B survival pathway. Furthermore, AR-A014418 inhibits neurodegeneration mediated by beta-amyloid peptide in hippocampal slices. AR-A014418 may thus have important applications as a tool to elucidate the role of GSK3 in cellular signaling and possibly in Alzheimer's disease. AR-A014418 is the first compound of a family of specific inhibitors of GSK3 that does not significantly inhibit closely related kinases such as cdk2 or cdk5.[1]

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Glycogen synthase kinase (GSK)-3 has recently been implicated in the pathogenesis of neurodegenerative diseases. Although the neuroprotective effects of GSK-3 inhibitors in Alzheimer's disease have been established, their effects on amyotrophic lateral sclerosis (ALS) have not been well defined. This study was undertaken to evaluate the effects of GSK-3 inhibition in the G93A-SOD1 mouse model of ALS. Groups of G93A-SOD1 mice were treated with varying concentrations of GSK-3 inhibitor VIII, a specific GSK-3 inhibitor that crosses the BBB, intraperitoneally 5 days a week after 60 days of age. The GSK-3 inhibitor VIII treatment significantly delayed the onset of symptoms and prolonged the life span of the animals, and inhibited the activity of GSK-3 in a concentration-dependent manner. Furthermore, this treatment preserved survival signals and attenuated death and inflammatory signals. These data suggest that GSK-3 plays an important role in the pathogenic mechanisms of ALS and that inhibition of GSK-3 could be a potential therapeutic candidate for ALS.[2]

C12H12N4O4S

308.3131

308.057

C, 46.75; H, 3.92; N, 18.17; O, 20.76; S, 10.40

487021-52-3

AR-A014418-d3;1216908-63-2

448014

Light yellow to yellow solid powder

1.5±0.1 g/cm3

208-210?C (dec.)

1.666

1.43

137.31

2

6

4

21

371

0

S1C(=C([H])N=C1N([H])C(N([H])C([H])([H])C1C([H])=C([H])C(=C([H])C=1[H])OC([H])([H])[H])=O)[N+](=O)[O-]

YAEMHJKFIIIULI-UHFFFAOYSA-N

InChI=1S/C12H12N4O4S/c1-20-9-4-2-8(3-5-9)6-13-11(17)15-12-14-7-10(21-12)16(18)19/h2-5,7H,6H2,1H3,(H2,13,14,15,17)

1-[(4-methoxyphenyl)methyl]-3-(5-nitro-1,3-thiazol-2-yl)ure

GSK-3β Inhibitor VIII; GSK-3beta Inhibitor VIII; SN 4521; SN-4521; SN4521; AR-A014418; AR-A 014418; AR-A-014418; AR-AO-14418; 487021-52-3; 1-(4-methoxybenzyl)-3-(5-nitrothiazol-2-yl)urea; N-(4-METHOXYBENZYL)-N'-(5-NITRO-1,3-THIAZOL-2-YL)UREA; GSK-3beta Inhibitor VIII; 1-[(4-methoxyphenyl)methyl]-3-(5-nitro-1,3-thiazol-2-yl)urea;

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: ~62 mg/mL (~201.1 mM)
Water: <1 mg/mL
Ethanol: <1 mg/mL

配方 1 中的溶解度: ≥ 2.5 mg/mL (8.11 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；
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