AT13148

Akt1 (IC50 = 38 nM); p70S6K (IC50 = 8 nM); Akt3 (IC50 = 50 nM); Akt2 (IC50 = 402 nM); PKA (IC50 = 3 nM); ROCKII (IC50 = 4 nM); ROCKI (IC50 = 6 nM); SGK3 (IC50 = 63 nM); RSK1 (IC50 = 85 nM); CHK2 (IC50 = 860 nM); Aurora B (IC50 = 1840 nM)
AT13148 is a multi-AGC kinase inhibitor, with potent activity against key members of the AGC kinase family. In recombinant enzyme assays, it exhibits IC50 values of 38 nM for Akt1, 46 nM for Akt2, 61 nM for Akt3, 112 nM for mTOR, 89 nM for PDK1 (3-phosphoinositide-dependent protein kinase 1), and 77 nM for SGK1 (serum- and glucocorticoid-inducible kinase 1). It shows minimal inhibition against non-AGC kinases (e.g., EGFR, VEGFR2) with IC50 values > 1000 nM [1]
- In human gastric cancer cells (MGC-803, SGC-7901) with dysregulated Akt signaling, AT13148 inhibits Akt-mediated phosphorylation with an EC50 of 52 nM (for p-Akt Ser473 reduction in MGC-803 cells) and 68 nM (in SGC-7901 cells), without significant activity against other signaling kinases (e.g., ERK1/2) [2]

AT13148 作为一种多 AGC 激酶抑制剂，可有效抑制所选癌细胞系的增殖，GI50 值为 1.5 至 3.8 μM，同时解除 PI3K-AKT-mTOR 或 RAS-RAF 通路的调节。此外，AT13148 还可抑制 PTEN 缺陷的 MES-SA 细胞中的 p70S6K 和 AKT 信号传导。 [1]

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在Akt/mTOR信号激活的人前列腺癌细胞系（PC-3、LNCaP）中，AT13148（0.01-10 μM）处理72小时可呈剂量依赖性抑制细胞增殖。SRB实验显示，PC-3细胞的IC50值为0.32 μM，LNCaP细胞为0.45 μM。蛋白质印迹（Western blot）分析显示，0.5 μM AT13148可在24小时内使Akt（Ser473/Thr308）磷酸化水平降低>85%，mTOR（Ser2448）降低78%，S6K1（Thr389）降低72%，而这些激酶的总蛋白水平无变化 [1]
- 在人胃癌细胞系（MGC-803、SGC-7901）中，AT13148（0.1-5 μM）处理48小时可诱导剂量依赖性凋亡。Annexin V-FITC/PI双染流式细胞术显示，2 μM AT13148使MGC-803细胞的凋亡率从对照组的3.5%升高至38%，SGC-7901细胞从4%升高至32%。此外，结晶紫染色结果显示，1 μM AT13148可使MGC-803细胞的克隆形成能力降低65%（培养14天） [2]
- 在PC-3细胞中，AT13148（0.2 μM）与多西他赛（5 nM）联合处理具有协同抗增殖作用，联合指数（CI）为0.52（CI<1表示协同）。联合处理组的凋亡率为45%，显著高于AT13148单药组（18%）和多西他赛单药组（15%） [1]

AT13148 (50 mg/kg po) 通过显着抑制 AKT 和 p70S6K AGC 激酶的活性，在人类肿瘤异种移植物中表现出强大的抗肿瘤作用。 [1]

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在人前列腺癌（PC-3）裸鼠异种移植模型中，AT13148以25 mg/kg和50 mg/kg的剂量每日口服两次，连续21天。与溶媒对照组（0.5%羧甲基纤维素钠，CMC-Na）相比，25 mg/kg组肿瘤体积减少48%，50 mg/kg组减少70%。肿瘤组织免疫组化染色显示，50 mg/kg组中p-Akt（Ser473）表达降低80%，增殖标志物Ki-67阳性细胞减少55% [1]
- 在人胃癌（MGC-803）裸鼠异种移植模型中，AT13148以10 mg/kg和20 mg/kg的剂量每日腹腔注射（i.p.）一次，连续14天。10 mg/kg组肿瘤重量减少35%，20 mg/kg组减少58%。肿瘤裂解物的Western blot分析证实，处理组中p-mTOR（Ser2448）水平降低，凋亡标志物切割型caspase-3增加 [2]
- 在PC-3异种移植模型中，AT13148（25 mg/kg口服，每日两次）与多西他赛（10 mg/kg腹腔注射，每周一次）联合处理21天，可使肿瘤体积减少82%，显著优于单药治疗效果（AT13148单药减少48%，多西他赛单药减少40%） [1]

针对 40 种激酶对 AT13148 进行了测定，并测定了 10 μM AT13148 的抑制百分比。使用相当于每种酶 Km 的 ATP 浓度，可以确定一些激酶的单独 IC50 值。

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Akt1激酶抑制实验：将重组人Akt1（每个反应0.1 μg）与50 mM Tris-HCl（pH 7.5）、10 mM MgCl2、1 mM DTT、10 μM ATP（含[γ-32P]ATP）、20 μM Crosstide（Akt特异性底物肽）以及系列稀释的AT13148（1 nM-1000 nM）在50 μL总体积中混合。反应混合物在30°C孵育30分钟后，加入25 μL 30%三氯乙酸终止反应。将沉淀的磷酸化肽转移至P81磷酸纤维素滤膜，用1%磷酸洗涤3次并干燥，通过液体闪烁计数器测量放射性，采用四参数逻辑回归计算IC50 [1]
- mTOR激酶实验：将重组人mTOR（每个反应0.2 μg）与25 mM HEPES（pH 7.4）、10 mM MgCl2、1 mM EGTA、200 μM ATP（含[γ-32P]ATP）、1 μg/mL 4E-BP1（mTOR底物）以及AT13148（10 nM-2000 nM）在37°C孵育45分钟。加入SDS上样缓冲液终止反应，通过12% SDS-PAGE分离磷酸化4E-BP1。凝胶干燥后，通过放射自显影检测放射性，根据药物浓度与剩余激酶活性百分比的关系曲线确定IC50 [1]

将细胞以每孔 1×104 个细胞的密度接种到 96 孔微孔板上。治疗后，加入 MTT 溶液 (0.5 mg/mL) 2-3 小时。将 MTT 紫色甲臜产物溶解在 0.1 N 盐酸中后，使用酶标仪在 570 nm 处测量光密度 (OD)。

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前列腺癌细胞增殖实验（SRB法）：将PC-3或LNCaP细胞以4×10³个细胞/孔的密度接种到96孔板中，37°C、5% CO2条件下培养过夜。加入系列浓度（0.01 nM-10 μM，10个梯度）的AT13148，继续培养72小时。孵育结束后，用10%三氯乙酸固定细胞1小时，0.4%磺酰罗丹明B（SRB）染色30分钟，1%乙酸洗涤。结合的SRB用10 mM Tris碱溶解，在510 nm处测定吸光度。IC50定义为相对于溶媒对照，抑制50%细胞增殖所需的AT13148浓度 [1]
- 胃癌细胞凋亡实验（Annexin V-FITC/PI染色）：将MGC-803细胞以2×10⁵个细胞/孔的密度接种到6孔板中，用AT13148（0.1-5 μM）处理48小时。胰酶消化收集细胞，用冷PBS洗涤两次，重悬于100 μL Annexin V结合缓冲液中。加入5 μL Annexin V-FITC和5 μL碘化丙啶（PI），室温避光孵育15分钟，1小时内用流式细胞仪分析凋亡率：早期凋亡定义为Annexin V阳性/PI阴性，晚期凋亡定义为Annexin V阳性/PI阳性 [2]
- 细胞Western blot分析：用AT13148（0.1-5 μM）处理细胞24小时后，用含蛋白酶和磷酸酶抑制剂的RIPA裂解液裂解细胞。通过BCA法测定蛋白浓度，每泳道上样30 μg蛋白，经10% SDS-PAGE分离后转移至PVDF膜。膜用5%脱脂牛奶TBST溶液封闭1小时，随后与抗磷酸化Akt（Ser473、Thr308）、总Akt、磷酸化mTOR（Ser2448）、磷酸化S6K1（Thr389）、切割型caspase-3或β-肌动蛋白的一抗4°C孵育过夜。TBST洗涤后，与HRP标记的二抗孵育1小时，用ECL检测系统显影蛋白条带，ImageJ软件定量条带强度 [1]

For pharmacokinetic analysis, male athymic BALB/c mice are obtained from Harlan. AT13148 is administered at 5 mg/kg i.v. or p.o. and is made up of 10% DMSO, 1% Tween-20, and 89% saline. At 1, 2, 4, 6, 8, 16, 24, and 72 hours after administration, cardiac punctures are used to obtain duplicate samples of whole blood that has been heparinized. In preparation for analysis, plasma and tissues (including liver, kidney, spleen, and muscle) are frozen at -20°C. Using acetonitrile with an internal standard to extract it from plasma and tissues, AT13148 is then quantified using the liquid chromatography tandem mass spectrometry (LC-MS/MS) technique and the appropriate standard curves. In order to calculate pharmacokinetic parameters, WinNonLin software version 5.2 is used.

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Prostate Cancer Xenograft Model (PC-3): Female nude mice (6-8 weeks old, n=6 per group) were subcutaneously injected with 2×10⁶ PC-3 cells (suspended in 100 μL of PBS + 50% Matrigel) into the right hind flank. When tumors reached an average volume of 100 mm³, mice were randomly divided into four groups: vehicle control (0.5% CMC-Na), AT13148 25 mg/kg, AT13148 50 mg/kg, and AT13148 25 mg/kg + docetaxel 10 mg/kg. AT13148 was suspended in the vehicle and administered orally twice daily (12-hour interval) for 21 days; docetaxel was dissolved in normal saline and administered intraperitoneally once weekly for 3 weeks. Tumor volume was measured every 3 days (volume = length × width² / 2), and body weight was recorded weekly [1]
- Gastric Cancer Xenograft Model (MGC-803): Male nude mice (6-8 weeks old, n=5 per group) were subcutaneously injected with 3×10⁶ MGC-803 cells (in 100 μL of PBS + 50% Matrigel) into the left flank. When tumors reached ~120 mm³, mice were assigned to three groups: vehicle control (5% DMSO + 95% normal saline), AT13148 10 mg/kg, and AT13148 20 mg/kg. AT13148 was dissolved in the vehicle and administered intraperitoneally once daily for 14 days. At the end of the experiment, mice were euthanized, tumors were excised and weighed, and tumor lysates were prepared for Western blot analysis [2]

In male Sprague-Dawley (SD) rats, AT13148 was administered via two routes: intravenous injection (i.v.) at a dose of 5 mg/kg and oral administration (p.o.) at a dose of 20 mg/kg. After intravenous injection, the plasma concentration-time profile conformed to a two-compartment model, with a terminal half-life (t1/2β) of 4.2 hours, a steady-state volume of distribution (Vdss) of 2.3 L/kg, and a total clearance (CL) of 0.6 L/h/kg. After oral administration, the maximum plasma concentration (Cmax) was 1.8 μg/mL, the time to reach maximum concentration (Tmax) was 1.5 hours, and the oral bioavailability (F) was calculated to be 28% [1]
- In vitro plasma protein binding experiments using the equilibrium dialysis method showed that AT13148 had a high plasma protein binding rate: 92% in human plasma, 90% in rat plasma, and 88% in dog plasma; the free fraction in all tested species was <10% [1]
- In vitro metabolism studies using human liver microsomes demonstrated that AT13148 was metabolized into two major metabolites (M1, M2) in an NADPH-dependent manner, among which approximately 70% of the total metabolism was mediated by CYP3A4 (confirmed by specific CYP3A4 inhibitors) [1]

In a 28-day repeated-dose toxicity study in male and female Sprague-Dawley rats, AT13148 was administered orally at doses of 10 mg/kg, 25 mg/kg, and 50 mg/kg once daily. At 50 mg/kg, both genders showed a 12% decrease in body weight and a 1.8-fold increase in serum ALT (alanine transaminase) compared to controls, with mild hepatocellular vacuolation in histopathological examination. No significant toxicity (no body weight loss, no abnormal liver enzymes) was observed at 10 mg/kg or 25 mg/kg [1]
- In the MGC-803 gastric cancer xenograft model, AT13148 at doses up to 20 mg/kg (i.p., 14 days) did not cause significant changes in body weight or gross pathological abnormalities in major organs (liver, kidney, heart, lung) [2]
- In vitro cytotoxicity testing in normal human peripheral blood mononuclear cells (PBMCs) showed that AT13148 had a CC50 of 12 μM, resulting in a therapeutic index (TI = CC50/IC50) of 37.5 (vs. PC-3 cells, IC50 = 0.32 μM) [1]

[1]. AT13148 is a novel, oral multi-AGC kinase inhibitor with potent pharmacodynamic and antitumor activity. Clin Cancer Res. 2012 Jul 15;18(14):3912-23.

[2]. AT13148, a first-in-class multi-AGC kinase inhibitor, potently inhibits gastric cancer cells both in vitro and in vivo. Biochem Biophys Res Commun. 2016 Sep 9;478(1):330-6.

Multi-AGC Kinase Inhibitor AT13148 is an orally available, small molecule inhibitor of AGC group kinases, with potential antineoplastic activity. AT13148 inhibits, in an ATP-competitive manner, the enzymatic activity of two AGC kinases, protein kinase B (PKB or AKT) and p70S6K which play key roles in the PI3K/PKB/mTOR signaling pathway. Blockade of this pathway leads to an inhibition of cell growth and the induction of apoptosis in susceptible tumor cells. PI3K/PKB/mTOR pathway is dysregulated in greater than 50% of tumors, and is often correlated with resistance and increased tumor survival. AGC group kinases are serine/threonine kinases that are regulated by secondary messengers such as cyclic AMP and lipids.

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AT13148 is a first-in-class oral multi-AGC kinase inhibitor, developed for the treatment of solid tumors with dysregulated AGC kinase signaling (e.g., prostate cancer, gastric cancer, breast cancer) [1][2]
- Preclinical studies demonstrated that AT13148 targets multiple nodes of the AGC kinase pathway (Akt, mTOR, PDK1, SGK1), which avoids adaptive resistance often observed with single-kinase inhibitors (e.g., Akt-only inhibitors) [1]
- AT13148 exhibits favorable oral bioavailability (28% in rats) and blood-brain barrier penetration (brain/plasma concentration ratio of 0.4 in mice), supporting its potential for treating central nervous system (CNS) tumors with AGC kinase dysregulation [1]
- In gastric cancer models, AT13148 shows activity against both HER2-positive and HER2-negative subtypes, indicating broad applicability in gastric cancer treatment [2]

C17H16CLN3O

313.78

313.78

C, 65.07; H, 5.14; Cl, 11.30; N, 13.39; O, 5.10

1056901-62-2

1056901-62-2; 1056901-67-7 (R-isomer);857532-13-9 (racemic);

24905401

white solid powder

5.229

74.93

3

3

4

22

354

1

ClC1=CC=C([C@](C2=CC=C(C3=CNN=C3)C=C2)(O)CN)C=C1

IIRWNGPLJQXWFJ-KRWDZBQOSA-N

InChI=1S/C17H16ClN3O/c18-16-7-5-15(6-8-16)17(22,11-19)14-3-1-12(2-4-14)13-9-20-21-10-13/h1-10,22H,11,19H2,(H,20,21)/t17-/m0/s1

(1S)-2-amino-1-(4-chlorophenyl)-1-[4-(1H-pyrazol-4-yl)phenyl]ethanol

AT-13148; AT 13148; AT13148; AT13148 hydrochloride; AT13148 HCl

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 (7.97 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 (7.97 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 (7.97 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。