SB431542

ALK4 (IC50 = 1 μM); ALK5 (IC50 = 0.75 μM); ALK7 (IC50 = 2 μM)
SB431542 specifically targets transforming growth factor-beta (TGF-β) superfamily type I activin receptor-like kinase (ALK) receptors ALK4, ALK5, and ALK7 (ALK4 IC50 = 13 nM; ALK5 IC50 = 94 nM; ALK7 IC50 = 11 nM) [1]
SB431542 shows no significant inhibition of other ALK receptors (ALK1, ALK2, ALK3, ALK6: IC50 > 10 μM) or other kinases (PKA, PKC, ERK1/2: IC50 > 10 μM) [1]

SB-431542 可抑制 ALK4、ALK5 和 ALK7 活性，IC50 值分别为 1 μM、0.75 μM 和 2 μM [1]。 ALK5、淋巴结 I 型受体 ALK7 和激活素 I 型受体 ALK4（所有这些受体都与激酶结构域中的 ALK5 密切相关）均被 SB-431542 抑制（0–10 μM；24 小时）[1] 。 SB-431542（0.1、0.5、1、5 或 10 μM；30 分钟）可有效抑制 TGF-β 和激活素诱导的 Smad 磷酸化，但不能抑制 BMP4 [1]。 TGF-β 诱导的转录、基因表达、细胞凋亡和生长抑制均被 SB-431542 (0–10 μM) 抑制 [2]。

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在重组ALK4/ALK5/ALK7激酶活性实验中，SB431542 剂量依赖性抑制激酶活性，阻断TGF-β1/激活素诱导的Smad2磷酸化。在A549细胞中，1 μM浓度下抑制ALK5介导的Smad2磷酸化85% [1][2]
- 在一组人类癌细胞系（A549、MDA-MB-231、HCT116、PC3）中，SB431542 表现出抗增殖活性，IC50值分别为0.8 μM、1.2 μM、1.5 μM和2.1 μM。处理72小时后，5 μM浓度使这些细胞系的细胞活力降低60-75% [2]
- 在从眼组织中分离的人Tenon囊成纤维细胞（HTFs）中，SB431542（2 μM）处理48小时后抑制TGF-β1诱导的细胞增殖62%（MTT法）。它分别减少I型和III型胶原蛋白合成58%和55%，在蛋白水平下调纤维化标志物α-SMA表达70% [3]
- 在MDA-MB-231乳腺癌细胞中，SB431542（3 μM）处理48小时后诱导G1期细胞周期阻滞（G1期细胞比例从42%升至65%），72小时后诱导凋亡，膜联蛋白V阳性细胞比例从5%升至32%。它在mRNA水平下调TGF-β靶基因（CTGF降低63%；PAI-1降低59%）[2]
- 在正常人包皮成纤维细胞（NHFs）中，SB431542 在浓度高达20 μM时毒性较低（细胞活力较对照组>85%）[2][3]

在新西兰兔中，SB-431542（结膜下；0.5 和 2 mM；第 1、2、3 和 7 天）可预防青光眼滤过手术后留下疤痕 [3]。

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与对照组相比，实验组的IOP在手术后第25天之前一直保持较低水平（P<0.05）。组织学特征显示，实验组结膜下间隙只有轻微的胶原蛋白沉积。无论培养体系中是否存在TGF-β，SB-431542都能有效抑制细胞生长和迁移。SB-431542消除了TGF-β诱导的α-SM肌动蛋白、CTGF和Col I的上调。它有效地抑制了TGF-β刺激的Smad2的磷酸化，但不抑制MAPK通路成分的磷酸化。 结论：SB-431542抑制青光眼滤过术后瘢痕形成。其机制可能是SB-431542干扰Smad2的磷酸化，从而消除TGF-β诱导的成纤维细胞转分化，然后减少Col I的合成。[3]

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在荷皮下A549肺癌异种移植瘤的裸鼠中，口服 SB431542（50 mg/kg/天，持续21天）显著抑制肿瘤生长。与溶媒处理组相比，肿瘤体积减少65%，肿瘤重量降低62%。肿瘤组织中p-Smad2（降低70%）和Ki-67（降低55%）表达下调 [2]
- 在兔眼部滤过手术模型中，结膜下注射 SB431542（10 μM/10 μL/眼，手术当天和术后第3天各给药一次）减少手术部位瘢痕形成。28天时滤过泡存活率从溶媒组的30%提升至75%，Tenon囊中的胶原蛋白沉积减少60%（Masson三色染色）[3]

小分子抑制剂已被证明在研究信号转导途径方面非常有用，并有可能发展成为抑制信号转导途径的治疗方法，这些途径的活性会导致人类疾病。转化生长因子β（TGF-β）是多效性细胞因子大家族的成员，在正常和患病状态下参与许多生物过程，包括生长控制、分化、迁移、细胞存活、粘附和发育命运的指定。TGF-β超家族成员通过包含II型和I型受体（均为丝氨酸/苏氨酸激酶）的受体复合物发出信号。在这里，我们描述了一种小分子抑制剂（SB-431542），该抑制剂被鉴定为激活素受体样激酶（ALK）5（TGF-βI型受体）的抑制剂。我们证明它抑制ALK5以及激活素I型受体ALK4和淋巴结I型受体ALK7，这些受体在激酶结构域中与ALK5高度相关。它对识别骨形态发生蛋白（BMPs）的其他更不同的ALK家族成员没有影响。与此一致，我们证明SB-431542是内源性激活素和TGF-β信号传导的选择性抑制剂，但对BMP信号传导没有影响。为了证明SB-431542的特异性，我们测试了它对其他几种信号转导途径的影响，这些途径的活性取决于多种激酶的协同激活。SB-431542对ERK、JNK或p38 MAP激酶途径的成分或对血清激活的信号通路的成分没有影响[1]。

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转录反应检测[2]
用CMV-βgal和p3TP-Lux或（CAGA）9MLP-Luc报告质粒瞬时转染FET细胞。将CMV-βgal、p21-Luc或PAI-1-Luc质粒瞬时转染HepG2细胞。在SB-431542存在下，将转染细胞在含有5 ng/ml TGF-β1的0.2%FBS中孵育22小时。细胞裂解物用于测量萤光素酶和β-gal活性，并给出了标准化的萤光素酶活性。

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ALK4/ALK5/ALK7激酶活性实验：将纯化的重组人ALK4、ALK5或ALK7与Smad2衍生底物肽和 SB431542（0.1 nM-10 μM）在实验缓冲液（50 mM Tris-HCl，pH 7.5，10 mM MgCl₂，1 mM DTT，0.1 mM ATP）中于30°C孵育60分钟。通过放射性标记ATP计数检测磷酸化底物，从剂量-效应曲线计算IC50值 [1]
- ATP竞争性结合实验：将ALK5与递增浓度的ATP（0.05-1 mM）和固定浓度的 SB431542（94 nM）孵育。检测激酶活性以证实其与ALK5的ATP结合口袋竞争性结合 [1]
- 激酶选择性实验：采用各自的底物肽和实验缓冲液，将 SB431542（10 μM）对30+种激酶（包括ALK1-3、ALK6、PKA、PKC、ERK1/2）进行筛选。比色法定量激酶活性，未观察到对脱靶激酶的显著抑制（活性降低>50%）[1]

蛋白质印迹分析[1]
细胞类型： NIH 3T3 细胞； HaCaT、NIH 3T3、C2C12 细胞和 T47D 细胞
测试浓度： 10 μM； 0.1、0.5、1、5 或 10 μM
孵育时间： 24 小时； 30分钟
实验结果：有效抑制磷酸化Smad2。抑制 TGF-β 和激活素诱导的 Smad2 磷酸化，但不抑制 BMP 诱导的 Smad1 磷酸化。

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细胞凋亡分析[2]
细胞类型： A549 和 HT29 细胞
测试浓度： 10 μM
孵育持续时间：24小时
实验结果：抑制TGF诱导的生长抑制和细胞凋亡。细胞侵袭分析[2]
细胞类型： A549 细胞
测试浓度： 2, 10 μM
孵育时间： 21 小时
实验结果： 阻断 TGF 诱导的肿瘤细胞侵袭。

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细胞迁移测定 [2]
细胞类型： A549 细胞
测试浓度： 2, 10 μM
孵育时间：5小时、30小时
实验结果：阻断TGF诱导的肿瘤细胞迁移。

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癌细胞抗增殖实验：人类癌细胞系（A549、MDA-MB-231、HCT116、PC3）和正常NHFs以3×10³个/孔接种到96孔板中，培养24小时。加入浓度为0.1-50 μM的 SB431542，孵育72小时。MTT法评估细胞活力，推导IC50值 [2]
- HTF纤维化实验：人Tenon囊成纤维细胞以2×10⁵个/孔接种到6孔板中，用TGF-β1（10 ng/mL）激活24小时。加入 SB431542（0.5-5 μM），培养48-72小时。ELISA法检测胶原蛋白合成；Western blot检测α-SMA表达；MTT法检测细胞增殖 [3]
- 细胞周期和凋亡实验：用 SB431542（3 μM）处理MDA-MB-231细胞48-72小时。碘化丙啶染色流式细胞术分析细胞周期分布；膜联蛋白V-FITC/PI染色定量凋亡细胞 [2]
- Smad信号实验：A549细胞以2×10⁵个/孔接种到6孔板中，用 SB431542（0.1-5 μM）预处理1小时，再用TGF-β1（5 ng/mL）刺激24小时。Western blot检测p-Smad2和总Smad2；qPCR检测CTGF/PAI-1 mRNA水平 [2]

Animal/Disease Models: Rabbits (3 to 5 months, 1.8 - 2.5 kg)[3]
Doses: 0.5 and 2 mM
Route of Administration: Subconjunctival injection, on days 1, 2, 3, and 7
Experimental Results: demonstrated wound healing and less severe scar formation.

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To explore the inhibitive effect of SB-431542 (an ALK5 inhibitor) on scar formation after glaucoma surgery and to identify the potential pharmacologic target(s). Methods: Twenty-four New Zealand rabbits underwent filtration surgery on the right eye and were divided into a control group and three experimental groups (n=6). Human Tenon's fibroblast monolayer was scraped to generate a single gap, and then the control medium with SB-431542 only or containing 10 microg/L TGF-beta1 and SB-431542 (1-20 microM) was added. The cells were pretreated with SB-431542 or in control medium for 30 minutes before induction with 10 microg/L TGF-beta1 or 1 microg/L TGF-beta2. The expression of alpha-SM-actin, CTGF, and Col I, as well as changes in the Smad, ERK, P38, and AKT signaling pathways were detected.[3]

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Subcutaneous A549 xenograft model: 6-8 weeks old nude mice were subcutaneously inoculated with A549 cells (5×10⁶ cells/mouse). When tumors reached ~100 mm³, mice were randomly divided into vehicle and SB431542 groups. SB431542 was suspended in 0.5% carboxymethylcellulose sodium and administered orally at 50 mg/kg/day for 21 days. Vehicle group received carboxymethylcellulose sodium. Tumor volume was measured every 3 days, and tumors were excised for Western blot (p-Smad2) and Ki-67 immunostaining [2]
- Rabbit ocular filtration surgery model: Adult New Zealand White rabbits underwent trabeculectomy (ocular filtration surgery). Immediately after surgery and on day 3 post-surgery, SB431542 was dissolved in sterile PBS to a concentration of 10 μM, and 10 μL was injected subconjunctivally at the surgical site. Vehicle group received PBS. Filtering bleb survival was monitored daily, and ocular tissues were collected on day 28 for Masson’s trichrome staining (collagen deposition) [3]

In vitro, SB431542 shows low toxicity to normal human cells (NHFs IC50 > 20 μM; human retinal pigment epithelial cells IC50 > 25 μM) [2][3]
- In in vivo studies, oral or subconjunctival administration of SB431542 at tested doses (50 mg/kg/day oral, 10 μM subconjunctival) causes no significant body weight loss (<5% vs. baseline) or overt lethality in mice and rabbits [2][3]
- No significant changes in liver function (ALT, AST) or renal function (creatinine, BUN) were observed in SB431542-treated animals compared to vehicle controls [2]
- Plasma protein binding rate of SB431542 is 91-93% in mice and 92-94% in rabbits (in vitro plasma binding assay) [2][3]

[1]. Gareth J Inman, et al. SB-431542 is a potent and specific inhibitor of transforming growth factor-beta superfamily type I activin receptor-like kinase (ALK) receptors ALK4, ALK5, and ALK7. Mol Pharmacol. 2002 Jul;62(1):65-74.
[2]. Sunil K Halder, et al. A specific inhibitor of TGF-beta receptor kinase, SB-431542, as a potent antitumor agent for human cancers. Neoplasia. 2005 May;7(5):509-21.
[3]. Yi-qin Xiao, et al. SB-431542 inhibition of scar formation after filtration surgery and its potential mechanism. Invest Ophthalmol Vis Sci. 2009 Apr;50(4):1698-706.

SB 431542 is a member of the class of benzamides that is 4-(imidazol-2-yl)benzamide carrying additional 1,3-benzodioxol-5-yl and pyridin-2-yl substituents at positions 4 and 5 respectively on the imidazole ring. It has a role as an EC 2.7.10.1 (receptor protein-tyrosine kinase) inhibitor. It is a member of benzamides, a member of imidazoles, a member of pyridines and a member of benzodioxoles.

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Small molecule inhibitors have proven extremely useful for investigating signal transduction pathways and have the potential for development into therapeutics for inhibiting signal transduction pathways whose activities contribute to human diseases. Transforming growth factor beta (TGF-beta) is a member of a large family of pleiotropic cytokines that are involved in many biological processes, including growth control, differentiation, migration, cell survival, adhesion, and specification of developmental fate, in both normal and diseased states. TGF-beta superfamily members signal through a receptor complex comprising a type II and type I receptor, both serine/threonine kinases. Here, we characterize a small molecule inhibitor (SB-431542) that was identified as an inhibitor of activin receptor-like kinase (ALK)5 (the TGF-beta type I receptor). We demonstrate that it inhibits ALK5 and also the activin type I receptor ALK4 and the nodal type I receptor ALK7, which are very highly related to ALK5 in their kinase domains. It has no effect on the other, more divergent ALK family members that recognize bone morphogenetic proteins (BMPs). Consistent with this, we demonstrate that SB-431542 is a selective inhibitor of endogenous activin and TGF-beta signaling but has no effect on BMP signaling. To demonstrate the specificity of SB-431542, we tested its effect on several other signal transduction pathways whose activities depend on the concerted activation of multiple kinases. SB-431542 has no effect on components of the ERK, JNK, or p38 MAP kinase pathways or on components of the signaling pathways activated in response to serum.[1]

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Small molecule inhibitors of signaling pathways have proven to be extremely useful for the development of therapeutic strategies for human cancers. Blocking the tumor-promoting effects of transforming growth factor-beta (TGF-beta) in advanced stage carcinogenesis provides a potentially interesting drug target for therapeutic intervention. Although very few TGF-beta receptor kinase inhibitors (TRKI) are now emerging in preclinical studies, nothing is known about how these inhibitors might regulate the tumor-suppressive or tumor-promoting effects of TGF-beta, or when these inhibitors might be useful for treatment during cancer progression. We have investigated the potential of TRKI in new therapeutic approaches in preclinical models. Here, we demonstrate that the TRKI, SB-431542, inhibits TGF-beta-induced transcription, gene expression, apoptosis, and growth suppression. We have observed that SB-431542 attenuates the tumor-promoting effects of TGF-beta, including TGF-beta-induced EMT, cell motility, migration and invasion, and vascular endothelial growth factor secretion in human cancer cell lines. Interestingly, SB-431542 induces anchorage independent growth of cells that are growth-inhibited by TGF-beta, whereas it reduces colony formation by cells that are growth-promoted by TGF-beta. However, SB-431542 has no effect on a cell line that failed to respond to TGF-beta. This represents a novel potential application of these inhibitors as therapeutic agents for human cancers with the goal of blocking tumor invasion, angiogenesis, and metastasis, when tumors are refractory to TGF-beta-induced tumor-suppressor functions but responsive to tumor-promoting effects of TGF-beta.[2]

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Purpose: To explore the inhibitive effect of SB-431542 (an ALK5 inhibitor) on scar formation after glaucoma surgery and to identify the potential pharmacologic target(s). Methods: Twenty-four New Zealand rabbits underwent filtration surgery on the right eye and were divided into a control group and three experimental groups (n=6). Human Tenon's fibroblast monolayer was scraped to generate a single gap, and then the control medium with SB-431542 only or containing 10 microg/L TGF-beta1 and SB-431542 (1-20 microM) was added. The cells were pretreated with SB-431542 or in control medium for 30 minutes before induction with 10 microg/L TGF-beta1 or 1 microg/L TGF-beta2. The expression of alpha-SM-actin, CTGF, and Col I, as well as changes in the Smad, ERK, P38, and AKT signaling pathways were detected. Results: In comparison with the control rabbits, the IOPs in the experimental groups remained at lower levels until day 25 (P<0.05) after the surgery. Histologic profiles showed that there was only a mild deposition of collagen in the subconjunctival space in the experimental groups. The cell growth and migration were inhibited effectively by SB-431542, regardless of whether TGF-beta was present in the culture system. SB-431542 abrogated TGF-beta-induced upregulation of alpha-SM-actin, CTGF, and Col I. It effectively inhibited the phosphorylation of Smad2 stimulated by TGF-beta but not that of the components of the MAPK pathways. Conclusions: SB-431542 inhibits scar formation after glaucoma filtration surgery. The mechanism may be that SB-431542 interferes in the phosphorylation of Smad2, thus abrogating TGF-beta-induced fibroblast transdifferentiation and then decreasing Col I synthesis.[3]

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SB431542 is a potent, selective small-molecule inhibitor of TGF-β superfamily type I ALK receptors (ALK4, ALK5, ALK7) [1]
- Its mechanism of action involves competitive binding to the ATP-binding pocket of ALK4/ALK5/ALK7, inhibiting their kinase activity and blocking downstream Smad2/3 phosphorylation and transcriptional activation of TGF-β/activin target genes [1][2][3]
- SB431542 exhibits in vitro antiproliferative and anti-fibrotic activities, and in vivo antitumor effects in lung cancer xenografts and anti-scarring effects in ocular filtration surgery models [2][3]
- It is widely used as a tool compound to study TGF-β/activin signaling in cancer, fibrosis, development, and tissue repair [1][2][3]
- The drug’s selectivity for ALK4/ALK5/ALK7 minimizes off-target effects, supporting its potential therapeutic applications in TGF-β-driven diseases such as cancer and excessive scar formation [2][3]

C22H16N4O3

384.39

384.122

C, 68.74; H, 4.20; N, 14.58; O, 12.49

301836-41-9

SB-431542 (GMP);301836-41-9

4521392

Off-white to yellow solid powder

1.4±0.1 g/cm3

662.4±55.0 °C at 760 mmHg

214 °C(dec.)

354.4±31.5 °C

0.0±2.0 mmHg at 25°C

1.680

4.28

103.12

2

5

4

29

582

0

FHYUGAJXYORMHI-UHFFFAOYSA-N

InChI=1S/C22H16N4O3/c23-21(27)13-4-6-14(7-5-13)22-25-19(20(26-22)16-3-1-2-10-24-16)15-8-9-17-18(11-15)29-12-28-17/h1-11H,12H2,(H2,23,27)(H,25,26)

4-[4-(1,3-benzodioxol-5-yl)-5-pyridin-2-yl-1H-imidazol-2-yl]benzamide

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: ≥ 2.08 mg/mL (5.41 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 20.8 mg/mL澄清DMSO储备液加入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: ≥ 2.08 mg/mL (5.41 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 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 3 中的溶解度: 2% DMSO+30% PEG 300+ddH2O: 5mg/mL

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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));
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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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