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Purity: ≥98%
SIS3 HCl is a potent and selective inhibitor of Smad3 which is a receptor-regulated intracellular protein that functions downstream of TGF-β and activin receptors and mediates their signaling, playing a role in cell proliferation, differentiation, apoptosis and formation of extracellular matrix. SIS3 can block excessive ECM production from the TGF-β1-treated normal fibroblasts and scleroderma fibroblasts, the model of cells with autocrine TGF-β signaling in vitro. Addition of SIS3 attenuates the effects of TGF-β1 by reducing the transcriptional activity. SIS3 also inhibits the myofibroblast differentiation of fibroblasts by TGF-β1.
| Targets |
SIS3 HCl targets Smad3 protein (R-Smad family, a key mediator of TGF-β signaling); [1]
SIS3 HCl inhibits the function of Hco-DAF-8 (an R-Smad homologue of Smad3 in Haemonchus contortus) involved in TGF-β signaling pathway[4] |
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| ln Vitro |
(E)-SIS3 (0.3–10 μM; for 1 hour) reduces the phosphorylation of Smad3 and its interaction with Smad4 caused by TGF-beta1[1]. The expression of FN and α-SMA is significantly suppressed by (E)-SIS3 (0.1, 10, 50 μM; 30 min), but not that of Sphk2, which is stimulated by TGF-β1[2]. In primary human dermal fibroblasts, (E)-SIS3 (10 μM; 24 hours) significantly reduces the expression of palladin, which is enhanced by TWEAK, as well as α-SMA[3]. In a dose-dependent manner, (E)-SIS3 significantly inhibits L4 development at five concentrations ranging from as low as 2 µM to 50 µM (5 µM, 10 µM, 20 µM, and 50 µM)[4].
1. SIS3 HCl abrogated the increased luciferase activity of p3TP-lux reporter plasmid induced by overexpression of constitutively active ALK-5 (TGF-β type I receptor) in a dose-dependent manner; it attenuated TGF-β1-induced phosphorylation of Smad3 and the interaction between Smad3 and Smad4 in human dermal fibroblasts, but did not affect Smad2 phosphorylation [1] 2. SIS3 HCl attenuated TGF-β1-induced up-regulation of type I procollagen in human dermal fibroblasts by reducing its transcriptional activity; it also inhibited TGF-β1-induced myofibroblast differentiation of fibroblasts [1] 3. SIS3 HCl completely diminished constitutive phosphorylation of Smad3 and up-regulated type I collagen expression in scleroderma fibroblasts [1] 4. SIS3 HCl (pretreated before TWEAK stimulation) abrogated TWEAK-induced upregulation of α-smooth muscle actin (α-SMA) and palladin (myofibroblastic differentiation markers) in cultured human dermal fibroblasts (Western blotting analysis, P<0.05 vs PBS group) [3] 5. SIS3 HCl affected the development of Haemonchus contortus from exsheathed third-stage larvae (xL3s) to fourth-stage larvae (L4s) in vitro: seven groups were set (blank control, DMSO control, 2/5/10/25/50 μM SIS3 HCl); the percentage of L4s developed from xL3s was significantly reduced in 5/10/25/50 μM groups (P<0.001 vs blank/DMSO groups), with no significant difference in 2 μM group [4] |
| ln Vivo |
SIS3 inhibits Smad3 activation in streptozotocin(STZ)-induced diabetic nephropathy in Tie2-Cre;Loxp-EGFP mice. It also reduces AGE-induced EndoMT and decreases EndoMT in STZ-induced diabetic nephropathy in Tie2-Cre;Loxp-EGFP mice. SIS3 significantly reduces collagen IV and fibronectin expression in the glomeruli and tubulointerstitium of STZ-injected Tie2-Cre;Loxp-EGFP mice, suggesting that SIS3 retards the early development of STZ-induced diabetic glomerulosclerosis and tubulointerstitial fibrosis. However, SIS3 administration does not reduce proteinuria.
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| Enzyme Assay |
1. Smad3 phosphorylation assay (human dermal fibroblasts): Fibroblasts were treated with TGF-β1 in the presence/absence of SIS3 HCl; cell lysates were prepared for immunoprecipitation to detect Smad3 phosphorylation level and the interaction between Smad3 and Smad4; the phosphorylation level of Smad2 was also detected as a control to verify the selectivity of SIS3 HCl [1]
2. Reporter gene assay (p3TP-lux): Cells were transfected with p3TP-lux reporter plasmid and constitutively active ALK-5 expression vector, then treated with different concentrations of SIS3 HCl; luciferase activity was measured to evaluate the inhibitory effect of SIS3 HCl on Smad3-mediated TGF-β signaling [1] 3. Hco-DAF-8 functional assay (Haemonchus contortus larvae): Exsheathed third-stage larvae (xL3s) of H. contortus were incubated with different concentrations of SIS3 HCl (2/5/10/25/50 μM) in vitro; the developmental rate from xL3s to L4s was counted to assess the inhibitory effect of SIS3 HCl on Hco-DAF-8 (Smad3 homologue) function [4] |
| Cell Assay |
Western Blot Analysis[1]
Cell Types: Human dermal fibroblasts Tested Concentrations: 0.3, 1, 3, 10 μM Incubation Duration: For 1 hour Experimental Results: Attenuated the TGF-beta1-induced phosphorylation of Smad3 and interaction of Smad3 with Smad4. 1. Human dermal fibroblast culture and TGF-β1 stimulation assay: Human dermal fibroblasts were cultured in appropriate medium, then treated with TGF-β1 alone or combined with SIS3 HCl; total RNA and protein were extracted from cells to detect type I procollagen expression at transcriptional and translational levels (PCR/Western blotting) to evaluate the effect of SIS3 HCl on TGF-β1-induced extracellular matrix expression [1] 2. Scleroderma fibroblast assay: Scleroderma fibroblasts were cultured and treated with SIS3 HCl; cell lysates were prepared to detect Smad3 phosphorylation level (immunoprecipitation/Western blotting) and type I collagen expression (PCR/Western blotting) to verify the inhibitory effect of SIS3 HCl on constitutively active Smad3 [1] 3. Human dermal fibroblast myofibroblast differentiation assay: Fibroblasts were treated with TGF-β1 in the presence/absence of SIS3 HCl; the expression of myofibroblast markers (α-SMA) was detected by immunofluorescence/Western blotting to evaluate the effect of SIS3 HCl on myofibroblast differentiation [1] 4. TWEAK-induced fibroblast differentiation assay: Human dermal fibroblasts were pretreated with SIS3 HCl (Smad3 inhibitor) and other pathway inhibitors (NF-κB, Wnt/β-catenin, EGFR, p38 MAPK inhibitors), then stimulated with TWEAK (250 ng/ml) for 48 h; α-SMA and palladin protein levels were measured by Western blotting to assess the role of Smad3 in TWEAK-induced myofibroblastic differentiation [3] |
| Animal Protocol |
1, 2.5, or 5 μg/g;i.p.
Male C57BL/6J mice |
| References |
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| Additional Infomation |
SIS3 is the hydrochloride formed by the reaction of the free SIS3 base with 1 mol equivalent of hydrogen chloride. It is a Smad3 inhibitor. It contains the free SIS3 base (1+).
1. SIS3 HCl is the first reported potent and selective inhibitor of Smad3 function. It specifically blocks the Smad3-mediated TGF-β signaling pathway without affecting the phosphorylation of Smad2[1] 2. Smad3 is a key mediator of the TGF-β signaling pathway, which regulates the expression of extracellular matrix (ECM) and the differentiation of myofibroblasts; SIS3 HCl is a useful tool for assessing the cellular mechanisms of TGF-β regulation by selectively inhibiting Smad3[1] 3. SIS3 HCl inhibits the upregulation of α-SMA and palladin in TWEAK-induced human dermal fibroblasts, indicating that Smad3 signaling is involved in the TWEAK/Fn14-mediated myofibroblast differentiation of dermal fibroblasts[3] 4. Hco-DAF-8 is the Smad3 inhibitor in Haemaphysalis contortus. R-Smad homologs are crucial for the development of Haemaphysalis contortus; SIS3 HCl (a specific inhibitor of human Smad3) affects the development of Haemaphysalis contortus from the xL3 to L4 stage in vitro, indicating that Hco-DAF-8 is a functional homolog of Smad3 and a potential target for antiparasitic drugs [4] |
| Molecular Formula |
C28H27N3O3.HCL
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| Molecular Weight |
489.99
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| Exact Mass |
489.181
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| CAS # |
521984-48-5
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| Related CAS # |
SIS3 free base;521985-36-4
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| PubChem CID |
16079005
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| Appearance |
Off-white to yellow solid powder
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| Boiling Point |
737.2ºC at 760 mmHg
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| Flash Point |
399.7ºC
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| LogP |
5.595
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| Hydrogen Bond Donor Count |
1
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| Hydrogen Bond Acceptor Count |
4
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| Rotatable Bond Count |
5
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| Heavy Atom Count |
35
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| Complexity |
724
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| Defined Atom Stereocenter Count |
0
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| SMILES |
CN1C(=C(C2=C1N=CC=C2)/C=C/C(=O)N3CCC4=CC(=C(C=C4C3)OC)OC)C5=CC=CC=C5.Cl
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| InChi Key |
CDKIEBFIMCSCBB-CALJPSDSSA-N
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| InChi Code |
InChI=1S/C28H27N3O3.ClH/c1-30-27(19-8-5-4-6-9-19)22(23-10-7-14-29-28(23)30)11-12-26(32)31-15-13-20-16-24(33-2)25(34-3)17-21(20)18-31;/h4-12,14,16-17H,13,15,18H2,1-3H3;1H/b12-11+;
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| Chemical Name |
(E)-1-(6,7-dimethoxy-3,4-dihydro-1H-isoquinolin-2-yl)-3-(1-methyl-2-phenylpyrrolo[2,3-b]pyridin-3-yl)prop-2-en-1-one;hydrochloride
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| Synonyms |
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| HS Tariff Code |
2934.99.9001
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| Storage |
Powder -20°C 3 years 4°C 2 years In solvent -80°C 6 months -20°C 1 month Note: Please store this product in a sealed and protected environment, avoid exposure to moisture. |
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| Shipping Condition |
Room temperature (This product is stable at ambient temperature for a few days during ordinary shipping and time spent in Customs)
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| Solubility (In Vitro) |
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| Solubility (In Vivo) |
Solubility in Formulation 1: ≥ 2.08 mg/mL (4.24 mM) (saturation unknown) in 10% DMSO + 40% PEG300 +5% Tween-80 + 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. (Please use freshly prepared in vivo formulations for optimal results.) |
| Preparing Stock Solutions | 1 mg | 5 mg | 10 mg | |
| 1 mM | 2.0409 mL | 10.2043 mL | 20.4086 mL | |
| 5 mM | 0.4082 mL | 2.0409 mL | 4.0817 mL | |
| 10 mM | 0.2041 mL | 1.0204 mL | 2.0409 mL |
*Note: Please select an appropriate solvent for the preparation of stock solution based on your experiment needs. For most products, DMSO can be used for preparing stock solutions (e.g. 5 mM, 10 mM, or 20 mM concentration); some products with high aqueous solubility may be dissolved in water directly. Solubility information is available at the above Solubility Data section. Once the stock solution is prepared, aliquot it to routine usage volumes and store at -20°C or -80°C. Avoid repeated freeze and thaw cycles.
Calculation results
Working concentration： mg/mL；
Method for preparing DMSO stock solution： mg drug pre-dissolved in μL DMSO (stock solution concentration mg/mL). Please contact us first if the concentration exceeds the DMSO solubility of the batch of drug.
Method for preparing in vivo formulation:：Take μL DMSO stock solution, next add μL PEG300, mix and clarify, next addμL Tween 80, mix and clarify, next add μL ddH2O，mix and clarify.
(1) Please be sure that the solution is clear before the addition of next solvent. Dissolution methods like vortex, ultrasound or warming and heat may be used to aid dissolving.
(2) Be sure to add the solvent(s) in order.
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