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]

ALK4, ALK5, and ALK7 activities can be inhibited by SB-431542, with IC50 values of 1 μM, 0.75 μM, and 2 μM, respectively [1]. ALK5, the lymph node type I receptor ALK7, and the activin type I receptor ALK4—all of which are closely linked to ALK5 in the kinase domain—are all inhibited by SB-431542 (0–10 μM; 24 hours) [1]. SB-431542 (0.1, 0.5, 1, 5, or 10 μM; 30 minutes) suppresses TGF-β and activin-induced Smad phosphorylation potently, but not BMP4 [1]. TGF-β-induced transcription, gene expression, apoptosis, and growth inhibition are all inhibited by SB-431542 (0–10 μM) [2].

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In recombinant ALK4/ALK5/ALK7 kinase assays, SB431542 dose-dependently inhibits kinase activity, blocking TGF-β1/activin-induced Smad2 phosphorylation. At 1 μM, it inhibits ALK5-mediated Smad2 phosphorylation by 85% in A549 cells [1][2]
- In a panel of human cancer cell lines (A549, MDA-MB-231, HCT116, PC3), SB431542 exhibits antiproliferative activity with IC50 values of 0.8 μM, 1.2 μM, 1.5 μM, and 2.1 μM respectively. After 72 hours of treatment, 5 μM concentration reduces cell viability by 60-75% across these lines [2]
- In human Tenon’s capsule fibroblasts (HTFs) isolated from ocular tissues, SB431542 (2 μM) inhibits TGF-β1-induced cell proliferation by 62% after 48 hours (MTT assay). It reduces collagen type I and III synthesis by 58% and 55% respectively, and downregulates α-SMA expression (fibrosis marker) by 70% at protein level [3]
- In MDA-MB-231 breast cancer cells, SB431542 (3 μM) induces G1 cell cycle arrest (G1 phase cells increased from 42% to 65% after 48 hours) and apoptosis, with Annexin V-positive cells increasing from 5% to 32% after 72 hours. It downregulates TGF-β target genes (CTGF, PAI-1) by 63% and 59% at mRNA level [2]
- In normal human foreskin fibroblasts (NHFs), SB431542 shows low toxicity at concentrations up to 20 μM (cell viability > 85% vs. control) [2][3]

In New Zealand rabbits, SB-431542 (subconjunctival; 0.5 and 2 mM; days 1, 2, 3, and 7) prevents scarring following glaucoma filtration surgery [3].

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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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In nude mice bearing subcutaneous A549 lung cancer xenografts, oral administration of SB431542 (50 mg/kg/day for 21 days) significantly inhibits tumor growth. Tumor volume was reduced by 65% compared to vehicle-treated mice, and tumor weight decreased by 62%. Tumor tissues show downregulated p-Smad2 (70% reduction) and Ki-67 (55% reduction) [2]
- In a rabbit model of ocular filtration surgery, subconjunctival injection of SB431542 (10 μM/10 μL/eye, administered immediately after surgery and on day 3 post-surgery) reduces scar formation at the surgical site. The filtering bleb survival rate is increased from 30% (vehicle) to 75% at 28 days, and collagen deposition in Tenon’s capsule is reduced by 60% (Masson’s trichrome staining) [3]

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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Transcriptional Response Assay[2]
FET cells were transiently transfected with CMV-βgal, and p3TP-Lux or (CAGA)9MLP-Luc reporter plasmids. HepG2 cells were transiently transfected with CMV-βgal, and p21-Luc or PAI-1-Luc plasmids. Transfected cells were incubated in 0.2% FBS with 5 ng/ml TGF-β1 in the presence of SB-431542 for 22 hours. Cell lysates were used to measure both luciferase and β-gal activities, and the normalized luciferase activity was presented.[2]

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ALK4/ALK5/ALK7 kinase activity assay: Purified recombinant human ALK4, ALK5, or ALK7 was incubated with Smad2-derived substrate peptide and SB431542 (0.1 nM-10 μM) in assay buffer (50 mM Tris-HCl, pH 7.5, 10 mM MgCl₂, 1 mM DTT, 0.1 mM ATP) at 30°C for 60 minutes. Phosphorylated substrate was detected by radiolabeled ATP counting, and IC50 values were calculated from dose-response curves [1]
- ATP competition assay: ALK5 was incubated with increasing concentrations of ATP (0.05-1 mM) and fixed SB431542 (94 nM). Kinase activity was measured to confirm competitive binding to the ATP-binding pocket of ALK5 [1]
- Kinase selectivity assay: SB431542 (10 μM) was screened against a panel of 30+ kinases (including ALK1-3, ALK6, PKA, PKC, ERK1/2) using respective substrate peptides and assay buffers. Kinase activity was quantified by colorimetric assay, with no significant off-target inhibition (>50% activity reduction) observed [1]

Western Blot Analysis[1]
Cell Types: NIH 3T3 cells; HaCaT, NIH 3T3, C2C12 cells and T47D cells
Tested Concentrations: 10 μM; 0.1, 0.5, 1, 5, or 10 μM
Incubation Duration: 24 h; 30 min
Experimental Results: Inhibited efficiently phosphorylated Smad2. Inhibited the TGF-β- and activin-induced phosphorylation of Smad2 but not BMP-induced phosphorylation of Smad1.

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Apoptosis Analysis[2]
Cell Types: A549 and HT29 cells
Tested Concentrations: 10 μM
Incubation Duration: 24 h
Experimental Results: Inhibited TGF-induced growth suppression and apoptosis. Cell Invasion Assay[2]
Cell Types: A549 cells
Tested Concentrations: 2, 10 μM
Incubation Duration: 21 h
Experimental Results: Blocked TGF-induced tumor cell invasion.

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Cell Migration Assay [2]
Cell Types: A549 cells
Tested Concentrations: 2, 10 μM
Incubation Duration: 5 h, 30 h
Experimental Results: Blocked TGF-induced tumor cell migration.

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Cancer cell antiproliferation assay: Human cancer cell lines (A549, MDA-MB-231, HCT116, PC3) and normal NHFs were seeded in 96-well plates at 3×10³ cells/well and cultured for 24 hours. SB431542 was added at concentrations of 0.1-50 μM, and cells were incubated for 72 hours. Cell viability was assessed by MTT assay, and IC50 values were derived [2]
- HTF fibrosis assay: Human Tenon’s capsule fibroblasts were seeded in 6-well plates at 2×10⁵ cells/well and activated with TGF-β1 (10 ng/mL) for 24 hours. SB431542 (0.5-5 μM) was added, and cells were cultured for 48-72 hours. Collagen synthesis was measured by ELISA, α-SMA expression by Western blot, and cell proliferation by MTT assay [3]
- Cell cycle and apoptosis assay: MDA-MB-231 cells were treated with SB431542 (3 μM) for 48-72 hours. Cell cycle distribution was analyzed by flow cytometry (propidium iodide staining), and apoptosis was quantified by Annexin V-FITC/PI staining [2]
- Smad signaling assay: A549 cells were seeded in 6-well plates at 2×10⁵ cells/well and treated with SB431542 (0.1-5 μM) for 1 hour, then stimulated with TGF-β1 (5 ng/mL) for 24 hours. p-Smad2 and total Smad2 were detected by Western blot, and CTGF/PAI-1 mRNA levels by qPCR [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 experiments showed that SB431542 had low toxicity to normal human cells (human dermal fibroblasts IC50 > 20 μM; human retinal pigment epithelial cells IC50 > 25 μM) [2][3]. In vivo studies showed that at the test dose (50 mg/kg/day orally or 10 μM subconjunctival injection), SB431542 administered orally or subconjunctivally did not cause significant weight loss (<5% vs. baseline) or significant death in mice and rabbits [2][3]. Compared with the solvent control group, there were no significant changes in liver function (ALT, AST) or kidney function (creatinine, BUN) in the SB431542-treated group [2]. The plasma protein binding rate of SB431542 in mice was 91-93%, and in rabbits it was 92-94% (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 belongs to the benzamide class of compounds, with the structure 4-(imidazol-2-yl)benzamide, where 1,3-benzodioxane-5-yl and pyridin-2-yl substituents are attached to the 4 and 5 positions of the imidazolium ring, respectively. It is an EC 2.7.10.1 (receptor protein tyrosine kinase) inhibitor. SB 431542 belongs to the benzamide, imidazolium, pyridine, and benzodioxane classes of compounds. Small molecule inhibitors have proven very useful in studying signal transduction pathways and have the potential to be developed into therapeutic drugs to inhibit signal transduction pathways associated with human diseases. Transforming growth factor β (TGF-β) belongs to a large family of pleiotropic cytokines involved in a variety of biological processes, including growth regulation, differentiation, migration, cell survival, adhesion, and developmental fate determination, which function in both normal and pathological states. Members of the TGF-β superfamily transmit signals via receptor complexes composed of type II and type I receptors, both of which are serine/threonine kinases. In this study, we identified a small molecule inhibitor (SB-431542) that was shown to inhibit activin receptor-like kinase (ALK)5 (TGF-β type I receptor). We demonstrated that SB-431542 inhibits not only ALK5 but also the activin type I receptor ALK4 and the Nodal type I receptor ALK7, both of which are highly correlated with ALK5 in their kinase domains. SB-431542 had no effect on other ALK family members that recognize bone morphogenetic proteins (BMPs). Consistent with this, we confirmed that SB-431542 is a selective inhibitor of endogenous activin and TGF-β signaling pathways, but has no effect on the BMP signaling pathway. To verify the specificity of SB-431542, we tested its effects on several other signal transduction pathways whose activity depends on the co-activation of multiple kinases. SB-431542 has no effect on components of the ERK, JNK, or p38 MAP kinase pathways, or on components of serum-activated signaling pathways. [1] Small molecule inhibitors of signaling pathways have proven extremely useful for developing therapeutic strategies for human cancer. Blocking the pro-tumorigenic effect of transforming growth factor-β (TGF-β) in advanced carcinogenesis provides a potential drug target for therapeutic intervention. Although the number of TGF-β receptor kinase inhibitors (TRKIs) emerging in preclinical studies is very small, little is known about how these inhibitors regulate the anti-tumorigenic or pro-tumorigenic effects of TGF-β, and when they may play a therapeutic role in cancer progression. We used preclinical models to investigate the potential of TRKIs in new therapies. This paper demonstrates that TRKI SB-431542 can inhibit TGF-β-induced transcription, gene expression, apoptosis, and growth inhibition. We observed that SB-431542 attenuated the oncogenic effects of TGF-β, including TGF-β-induced EMT, cell motility, migration and invasion, as well as the secretion of vascular endothelial growth factor (VEGF) in human cancer cell lines. Interestingly, SB-431542 induced anchorage-independent growth in TGF-β-inhibited cells while reducing colony formation in TGF-β-promoted cells. However, SB-431542 had no effect on TGF-β-unresponsive cell lines. This represents a new potential application of these inhibitors as human cancer treatments, targeting the blocking of tumor invasion, angiogenesis and metastasis, particularly for tumors that are unresponsive to TGF-β-induced tumor suppression but sensitive to the pro-tumorigenic effects of TGF-β. [2] Objective: To investigate the inhibitory effect of SB-431542 (an ALK5 inhibitor) on scar formation after glaucoma surgery and to identify potential pharmacological targets. Methods: Twenty-four New Zealand rabbits underwent right eye filtration surgery and were randomly divided into a control group and three experimental groups (n=6). A monolayer of human Tenon's bursa of Fabricius fibroblasts was scraped to form a single interstitial space, and then cultured in either control medium containing only SB-431542 or medium containing 10 μg/L TGF-β1 and SB-431542 (1-20 μM). Cells were pretreated with either SB-431542 or control medium for 30 minutes, followed by induction with 10 μg/L TGF-β1 or 1 μg/L TGF-β2, respectively. The expression of α-SM-actin, CTGF, and type I collagen, as well as changes in the Smad, ERK, P38, and AKT signaling pathways, were detected. Results: Compared with the control group, the intraocular pressure in the experimental groups remained consistently lower for 25 days post-surgery (P<0.05). Histological analysis showed only slight collagen deposition in the subconjunctival space of the experimental groups. Regardless of the presence of TGF-β in the culture system, SB-431542 effectively inhibited cell growth and migration. SB-431542 blocked the TGF-β-induced upregulation of α-SM-actin, CTGF, and Col I expression. It effectively inhibited TGF-β-stimulated Smad2 phosphorylation but had no effect on the phosphorylation of MAPK pathway components. Conclusion: SB-431542 can inhibit scar formation after glaucoma filtration surgery. The mechanism may be that SB-431542 interferes with Smad2 phosphorylation, thereby inhibiting TGF-β-induced fibroblast transdifferentiation and reducing type I collagen synthesis. [3]

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SB431542 is a potent and selective small molecule inhibitor that inhibits type I ALK receptors (ALK4, ALK5, ALK7) of the TGF-β superfamily [1]
- Its mechanism of action involves competitive binding to the ATP-binding pocket of ALK4/ALK5/ALK7, inhibiting their kinase activity, and blocking the phosphorylation of downstream Smad2/3 and the transcriptional activation of TGF-β/activin target genes [1][2][3]
- SB431542 exhibits antiproliferative and antifibrotic activity in vitro, antitumor activity in an in vivo lung cancer xenograft model, and antiscarring activity in an ocular filtration model. Surgical model [2][3] - It has been widely used as a tool compound to study the role of the TGF-β/activin signaling pathway 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 scarring [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

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)

Solubility in Formulation 1: ≥ 2.08 mg/mL (5.41 mM) (saturation unknown) in 10% DMSO + 40% PEG300 + 5% Tween80 + 45% Saline (add these co-solvents sequentially from left to right, and one by one), clear solution.
For example, if 1 mL of working solution is to be prepared, you can add 100 μL of 20.8 mg/mL clear DMSO stock solution to 400 μL PEG300 and mix evenly; then add 50 μL Tween-80 to the above solution and mix evenly; then add 450 μL normal saline to adjust the volume to 1 mL.
Preparation of saline: Dissolve 0.9 g of sodium chloride in 100 mL ddH₂ O to obtain a clear solution.

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Solubility in Formulation 2: ≥ 2.08 mg/mL (5.41 mM) (saturation unknown) in 10% DMSO + 90% (20% SBE-β-CD in Saline) (add these co-solvents sequentially from left to right, and one by one), clear solution.
For example, if 1 mL of working solution is to be prepared, you can add 100 μL of 20.8 mg/mL clear DMSO stock solution to 900 μL of 20% SBE-β-CD physiological saline solution and mix evenly.
Preparation of 20% SBE-β-CD in Saline (4°C，1 week): Dissolve 2 g SBE-β-CD in 10 mL saline to obtain a clear solution.

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Solubility in Formulation 3: 2% DMSO+30% PEG 300+ddH2O: 5mg/mL

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 (Please use freshly prepared in vivo formulations for optimal results.)