Activin receptor-like kinase 5 (ALK-5), TGF-β type I receptor (IC₅₀ = 25 nM in cell-based transcriptional reporter assay) [1]
Activin receptor-like kinase 4 (ALK-4), activin type IB receptor [1]
Activin receptor-like kinase 7 (ALK-7), nodal type I receptor [1]

Treatment with A-77-01 (0.01-10 µM; 25 hours; Mv1Lu cells) potently suppresses the concentration-dependent transcriptional activation elicited by TGF-β [1]. The growth-inhibitory effects of TGF-β are successfully prevented when Mv1Lu cells are treated with A-77-01 (1 µM) for 24-72 hours [1]. TGF-β-induced Smad2 phosphorylation is inhibited when HaCaT cells are treated with A-77-01 (1 µM) for 30 minutes [1].

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In a cell-based luciferase reporter assay using Mv1Lu cells transfected with the 9xCAGA-luciferase construct, A-77-01 inhibited TGF-β-induced transcriptional activation in a concentration-dependent manner, with an IC₅₀ of 25 nM [1].
At a concentration of 1 μM, A-77-01 almost completely inhibited the transcriptional activation induced by constitutively active ALK-5 (ALK5-TD) in the 9xCAGA-luciferase reporter assay [1].
A-77-01 (1 μM) had no effect on BMP-induced transcriptional activity in C2C12 cells using the (BRE)₂-luciferase reporter construct [1].
In HaCaT cells, A-77-01 (1 μM) completely inhibited TGF-β-induced phosphorylation of Smad2, as detected by immunoblotting with an anti-phospho-Smad2 antibody [1].
In Mv1Lu cell proliferation assays, A-77-01 (1 μM) efficiently prevented the growth-inhibitory effects of TGF-β (1 ng/mL) over 72 hours [1].
A-77-01 (1 μM) completely inhibited TGF-β-induced epithelial-to-mesenchymal transition (EMT) in NMuMG cells, preventing the morphological change from cuboidal epithelial to fibroblastic morphology [1].
RT-PCR and immunoblotting analysis in NMuMG cells showed that A-77-01 (1 μM) restored TGF-β-induced downregulation of E-cadherin and repressed the upregulation of fibronectin and N-cadherin at both mRNA and protein levels [1].
A-77-01 had no significant effect on FBS-induced activation of ERK1/2 in HaCaT cells at concentrations up to 10 μM, as shown by immunoblotting for phospho-ERK1/2 [1].
A-77-01 had weak inhibitory effects on osmotic shock-induced p38 MAPK activation only at high concentrations (above 3 μM), with no effect at lower concentrations (0.3-1 μM) where TGF-β signaling is potently inhibited [1].

RT-PCR[1]
Cell Types: Mv1Lu Cell
Tested Concentrations: 0.01 µM, 0.03 µM, 0.1 µM, 0.3 µM, 1 µM, 3 µM, 10 µM
Incubation Duration: 25 hrs (hours)
Experimental Results: Inhibition of TGF-β-induced luciferase activity Concentration-dependent fashion.

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Cell viability assay [1]
Cell Types: Mv1Lu Cell
Tested Concentrations: 1 µM
Incubation Duration: 24 hrs (hours), 48 hrs (hours), 72 hrs (hours)
Experimental Results: Effectively prevents the growth inhibitory effect of TGF-β.

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Western Blot Analysis[1]
Cell Types: HaCaT cells
Tested Concentrations: 1 µM
Incubation Duration: 30 minutes
Experimental Results: Inhibition of TGF-β-induced Smad2 phosphorylation.

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TGF-β-Induced Transcriptional Activation (Luciferase Reporter) Assay: Mv1Lu cells were seeded in 12-well plates and transiently transfected with the 9xCAGA-luciferreporter construct using a transfection reagent. Eight hours after transfection, cells were pretreated for 1 hour with various concentrations of A-77-01 or DMSO control. Cells were then stimulated with TGF-β (1 ng/mL) for 24 hours. Luciferase activity was measured using a dual-luciferase reporter system and normalized to TK-Renilla activity. The IC₅₀ value was calculated from the dose-response curve [1].
BMP-Induced Transcriptional Activation Assay: C2C12 cells were transfected with the (BRE)₂-luciferase reporter construct. Eight hours after transfection, cells were pretreated for 1 hour with various concentrations of A-77-01 or 1 μM SB-431542, then stimulated with BMP4 (20 ng/mL) for 24 hours. Luciferase activity was measured as described above [1].
Cell Proliferation Assay: Mv1Lu cells were seeded in 24-well plates at a density of 2.5 × 10⁴ cells/well. The next day, cells were pretreated for 1 hour with 1 μM A-77-01 and then cultured with TGF-β (1 ng/mL) for 24, 48, or 72 hours. Cells were trypsinized and counted using a Coulter counter [1].
Western Blot Analysis (Smad2 Phosphorylation): HaCaT cells were pretreated for 30 minutes with 1 μM A-77-01 and then treated with TGF-β (1 ng/mL) for 1 hour. Cell lysates were prepared, separated by SDS-PAGE, and transferred to membranes. Membranes were probed with anti-phospho-Smad2 antibody and anti-Smad2/3 antibody. Protein bands were visualized and analyzed [1].
Western Blot Analysis (MAPK Pathways): For ERK1/2 analysis, serum-starved HaCaT cells were pretreated for 30 minutes with various concentrations of A-77-01 or 10 μM U0126, then stimulated with 20% FBS for 30 minutes. For p38 analysis, HaCaT cells were pretreated for 30 minutes with various concentrations of A-77-01 or 10 μM SB-203580, then subjected to osmotic shock with 0.5 M NaCl for 5 minutes. Cell lysates were immunoblotted with anti-phospho-ERK1/2, anti-ERK1/2, anti-phospho-p38, and anti-p38 antibodies [1].
Epithelial-to-Mesenchymal Transition (EMT) Assay - Morphology: NMuMG cells were seeded in 6-well plates at 6 × 10⁴ cells/well. The next day, cells were stimulated with TGF-β (3 ng/mL) in the presence or absence of 1 μM A-77-01. After 48 hours, cell morphology was observed and photographed under a microscope [1].
EMT Assay - RT-PCR Analysis: NMuMG cells were treated with TGF-β (3 ng/mL) for 24 hours with or without 1 μM A-77-01. Total RNA was extracted and reverse transcribed to cDNA. PCR was performed using specific primers for E-cadherin, fibronectin, N-cadherin, and GAPDH. PCR products were separated by agarose gel electrophoresis and visualized with ethidium bromide [1].
EMT Assay - Western Blot Analysis: NMuMG cells were treated with TGF-β (3 ng/mL) for 48 hours with or without 1 μM A-77-01. Cell lysates were prepared and immunoblotted with antibodies against E-cadherin, fibronectin, N-cadherin, and tubulin (loading control) [1].

[1]. 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.

A-77-01 (compound 11) is a small molecule inhibitor structurally similar to previously reported ALK-5 inhibitors developed by Sawyer et al. It was synthesized and characterized in this study as part of a screen for inhibitors of TGF-β signaling [1].
A-77-01 inhibits signaling of type I serine/threonine kinase receptors ALK-5 (TGF-β type I receptor), ALK-4 (activin type IB receptor), and ALK-7 (nodal type I receptor), the kinase domains of which are structurally highly related [1].
The compound prevents Smad2/3 phosphorylation and the growth inhibition induced by TGF-β, while having little or no effect on BMP type I receptors, p38 MAPK, or ERK at concentrations that effectively block TGF-β signaling [1].
A-77-01 inhibits TGF-β-induced epithelial-to-mesenchymal transition (EMT) in NMuMG cells, restoring E-cadherin expression and suppressing fibronectin and N-cadherin expression. This suggests potential utility in preventing cancer progression and metastasis [1].
A-77-01 was found to be five to ten times more potent than the previously described ALK-5 inhibitor SB-431542 in inhibiting TGF-β-induced transcriptional activation [1].

C18H14N4

286.33

286.122

607737-87-1

10469233

Off-white to light yellow solid powder

1.254g/cm3

481.63ºC at 760 mmHg

256.391ºC

1.685

3.995

1

3

2

22

374

0

KJTYZDORHCDZPS-UHFFFAOYSA-N

InChI=1S/C18H14N4/c1-12-5-4-8-17(21-12)18-15(11-20-22-18)13-9-10-19-16-7-3-2-6-14(13)16/h2-11H,1H3,(H,20,22)

4-[5-(6-methylpyridin-2-yl)-1H-pyrazol-4-yl]quinoline

A77-01 A-77-01 A 77-01

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)

DMSO : ~25 mg/mL (~87.31 mM)

Solubility in Formulation 1: ≥ 2.5 mg/mL (8.73 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 25.0 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: ≥ 2.5 mg/mL (8.73 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 25.0 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: ≥ 2.5 mg/mL (8.73 mM) (saturation unknown) in 10% DMSO + 90% Corn Oil (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 25.0 mg/mL clear DMSO stock solution to 900 μL of corn oil and mix evenly.

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