ALK5: 12 nM (IC50); ALK4: 45 nM (IC50); ALK7: 7.5 nM (IC50)

83-01 是 TGF-β I 型受体 ALK5 激酶、ALK4 和 ALK7 的强抑制剂。它还微弱地抑制由组成型活性 ALK-6、ALK-2、ALK-3 和 ALK-1 诱导的转录。在 Mv1Lu 细胞中，它降低 ALK-5 诱导的转录水平，IC50 为 12 nM。它还阻断 ALK4-TD 和 ALK7-TD 诱导的转录，在 R4-2 细胞中的 IC50 分别为 45 nM 和 7.5 nM。在 0.03–10 μM 浓度下，A 83-01 可有效抵消 TGF-β 的生长抑制作用，在 3 μM 浓度下，可完全消除这些作用。 A 83-01 (1–10 μM) 可抑制由 TGF-β 引起的 HaCaT 细胞 Smad 激活[1]。 83-01 (1 μM) 不会改变细胞增殖，但会降低 HM-1 细胞中 TGF-β1 诱导的细胞运动、粘附和侵袭[2]。

当腹腔内给予 83-01 剂量（50、150 和 500 μg/小鼠）时，没有体重或神经行为表现的小鼠表现出四分之一率大幅增加 [2]。在小鼠中，以0.5 mg/kg的剂量腹腔注射83-01 M109细胞具有很强的抗肿瘤作用[3]。

先前描述了哺乳动物表达载体中ALK-1至-7的组成型活性形式的原始构建。9xCAGA萤光素酶质粒包含驱动萤光素酶表达的CAGA-Smad结合元件的九个重复序列。（BRE）2-荧光素酶质粒包含Id1启动子的BMP响应元件的两个重复序列，所述启动子克隆在驱动荧光素素酶表达的最小启动子的上游。3GC2萤光素酶质粒包含来源于Smad6启动子的近端BMP响应元件的富含GC的序列的三个重复序列。[1]

Mv1Lu细胞以2.5×104个细胞/孔的密度接种在24孔板中，一式两份。第二天，用1µM小分子抑制剂预处理细胞1小时，然后用TGF-β1 ng/mL）培养24小时、48小时或72小时。用胰蛋白酶消化细胞并用Coulter计数器计数。为了探索小分子抑制剂是否以浓度依赖的方式降低TGF-β的生长抑制作用，如上所述接种Mv1Lu细胞，并用不同浓度的小分子抑制剂预处理1小时。预处理后，用TGF-β1 ng/mL）培养细胞48小时并计数。[1]

Female B6C3F1 mice used for the in vivo studies are maintained under specific pathogen-free conditions. To evaluate the effect of A 83-01 on the survival of mice bearing peritoneal dissemination, HM-1 cells (1×106) are injected into the abdominal cavity via the left flank of the mouse. Starting the next day, A 83-01 (150 μg/body) or vehicles (PBS with 0.5% DMSO) are injected into the abdominal cavity three times per week. Mice are euthanized before reaching the moribund state.

[1]. Tojo M, et al. The ALK-5 inhibitor A-83-01 inhibits Smad signaling and epithelial-to-mesenchymal transition by transforming growth factor-beta. Cancer Sci. 2005 Nov;96(11):791-800.
[2]. Yamamura S, et al. The activated transforming growth factor-beta signaling pathway in peritoneal metastases is a potential therapeutic target in ovarian cancer. Int J Cancer. 2012 Jan 1;130(1):20-8.
[3]. Taniguchi Y, et al. Enhanced antitumor efficacy of folate-linked liposomal Adriamycin with TGF-β type I receptor inhibitor. Cancer Sci. 2010 Oct;101(10):2207-13

Transforming growth factor (TGF)-beta signaling facilitates tumor growth and metastasis in advanced cancer. Use of inhibitors of TGF-beta signaling may thus be a novel strategy for the treatment of patients with such cancer. In this study, we synthesized and characterized a small molecule inhibitor, A-83-01, which is structurally similar to previously reported ALK-5 inhibitors developed by Sawyer et al. (2003) and blocks signaling of type I serine/threonine kinase receptors for cytokines of the TGF-beta superfamily (known as activin receptor-like kinases; ALKs). Using a TGF-beta-responsive reporter construct in mammalian cells, we found that A-83-01 inhibited the transcriptional activity induced by TGF-beta type I receptor ALK-5 and that by activin type IB receptor ALK-4 and nodal type I receptor ALK-7, the kinase domains of which are structurally highly related to those of ALK-5. A-83-01 was found to be more potent in the inhibition of ALK5 than a previously described ALK-5 inhibitor, SB-431542, and also to prevent phosphorylation of Smad2/3 and the growth inhibition induced by TGF-beta. In contrast, A-83-01 had little or no effect on bone morphogenetic protein type I receptors, p38 mitogen-activated protein kinase, or extracellular regulated kinase. Consistent with these findings, A-83-01 inhibited the epithelial-to-mesenchymal transition induced by TGF-beta, suggesting that A-83-01 and related molecules may be useful for preventing the progression of advanced cancers.[1]

C25H19N5S

421.52

421.136

C, 71.23; H, 4.54; N, 16.61; S, 7.61

909910-43-6

A 83-01 sodium;2828431-89-4;A 83-01;909910-43-6

16218924

Off-white to light yellow solid powder

1.27g/cm3

590ºC at 760 mmHg

310.6ºC

1.706

5.786

87.72

1

4

3

31

609

0

S=C(N1C=C(C2C3C(=CC=CC=3)N=CC=2)C(C2C=CC=C(C)N=2)=N1)NC1C=CC=CC=1

HIJMSZGHKQPPJS-UHFFFAOYSA-N

InChI=1S/C25H19N5S/c1-17-8-7-13-23(27-17)24-21(19-14-15-26-22-12-6-5-11-20(19)22)16-30(29-24)25(31)28-18-9-3-2-4-10-18/h2-16H,1H3,(H,28,31)

3-(6-methylpyridin-2-yl)-N-phenyl-4-(quinolin-4-yl)-1H-pyrazole-1-carbothioamide

A8301; A 8301; 909910-43-6; A 83-01; 3-(6-methylpyridin-2-yl)-N-phenyl-4-(quinolin-4-yl)-1H-pyrazole-1-carbothioamide; A-83-01; A83-01; Stemolecule A83-01; 3-(6-methylpyridin-2-yl)-N-phenyl-4-quinolin-4-ylpyrazole-1-carbothioamide; X3ZNM7QJ2Q; A-8301

2934.99.9001

Powder      -20°C    3 years

                     4°C     2 years

In solvent   -80°C    6 months

                  -20°C    1 month

注意： 该产品在溶液状态不稳定，请现配现用。

Room temperature (This product is stable at ambient temperature for a few days during ordinary shipping and time spent in Customs)

DMSO : ~25 mg/mL (~59.31 mM)
H2O : < 0.1 mg/mL

配方 1 中的溶解度: 2.08 mg/mL (4.93 mM) 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), 悬浮液；超声助溶。
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 2 中的溶解度: 1.25 mg/mL (2.97 mM) 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; 超声助溶.
例如，若需制备1 mL的工作液，可将 100 μL 12.5 mg/mL澄清的DMSO储备液加入到400 μL PEG300中，混匀；再向上述溶液中加入50 μL Tween-80，混匀；然后加入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 3 中的溶解度: ≥ 1.25 mg/mL (2.97 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 12.5 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。