EW-7197 [N-((4-([1,2,4]triazolo[1,5-a]pyridin-6-yl)-5-(6-methylpyridin-2-yl)-1H-imidazol-2-yl)methyl)-2-fluoroaniline]
is a potent, orally available small-molecule inhibitor of TGF-β type I receptor
kinase (activin-like kinase 5; ALK5). EW-7197 inhibits the Smad pathway by
occupying the ATP binding site of the ALK5 kinase domain, which is essential
for the phosphorylation of its substrates, Smad2/Smad3 proteins. In biochemical
assays using HotSpot method it was revealed that EW-7197 is a highly potent,
selective, activin receptor-like kinase-4/-5 (ALK4/ALK5) inhibitor with IC50 of
13 nM and 11 nM, respectively [1, 2]. In a separate assay namely, 33PanQinase
activity assay, the enzyme IC50 for ALK5 was found to be 7 nM. Using
the same assay for MAPK14 protein kinase, which has one of the most homologous
kinase domains to that of ALK5, IC50 was found to be 1.72 uM.
EW-7197 did not inhibit other members of the family namely ALK1, ALK2, ALK3,
and ALK6 (IC50 greater than 10 µM).
Researchers expanded the selectivity profiling of EW-7197 using a panel of 320 protein kinases. The residual activity of each kinase was measured at four different concentrations of 0.01, 0.1, 1, and 10 µM. EW-7197 reduced the residual activity of 45 protein kinases to less than 50% and 11 protein kinases to less than10% of control at a concentration of 10 µM. The residual activity of these 11 protein kinases were less than 50% of control at 1 µM. Of these, at a lower concentration of 0.1 µM, ALK5, ALK4, RIPK2 (IC50 = 0.054 uM), and VEGF-R2 (IC50 = 0.097 uM) were significantly inhibited by EW-7197. At the lowest concentration of 0.01 µM, the residual activity of ALK5 and ALK4 only were less than 50% [1]. This suggests a strong selectivity for ALK5 and ALK4.
Using 33PanQinase activity assay, researchers verified EW-7197
inhibitory activity against ACVR1B/ALK4 with IC50 value as 17.3 nM.
EW-7197 inhibited ALK-2/ACV-R1 at concentrations comparable with ALK-5. As the
ATP-binding site of the ALK5 kinase domain is essential for phosphorylation of
the substrates Smad2/3, researchers evaluated the inhibitory effect of EW-7197
on Smad2/3 phosphorylation. EW-7197 blocked the TGFβ-induced phosphorylation of
Smad2 or Smad3 in a dose-dependent manner in 4T1 cells, NMuMG, and MDA-MB-231
cells. EW-7197 suppressed the TGFβ-induced nuclear translocation of Smad2/3 in
4T1 cells and MCF10A cells. The IC50 values of EW-7197, SB-505124,
LY-2157299, and IN-1130 on pSmad3 in 4T1 cells were 10 to 30, 300 to 500, 500
to 1,000, and 300 to 500 nM, respectively. EW-7197 showed a more potent
inhibitory effect on TGFβ-induced Smad2 or Smad3 phosphorylation than other
ALK5 inhibitors previously identified [2]. Moreover, the activity of
EW-7197 with those of other ALK5 kinase inhibitors was measured using a
reporter gene assay in 4T1-3TP-Lux cells where IC50 values 13.2,
45.8, more than 50, and more than 100 nM, were obtained for EW-7197, IN-1130,
SB-505124, and LY-2157299.
The activity of EW-7197 is as follows:
IC50 (ALK5 enzyme assay, HotSpot assay; 33PanQinase
activity assay) = 11 nM; 7 nM (also 12.9 nM [2])
IC50 (ALK4 enzyme assay, HotSpot assay; 33PanQinase
activity assay) = 13 nM; 17.3 nM
IC50 (MAPK14 enzyme assay, 33PanQinase
activity assay) = 1.77 uM; 1.72 uM
IC50 (VEGFR-1 enzyme assay) = 0.391 uM
IC50 (VEGFR-2 enzyme assay) = 0.097 uM
IC50 (VEGFR-3 enzyme assay) = 0.257 uM
Common Name: EW-7197
Common Name: EW-7197
Synonyms: EW-7197; EW 7197; EW7197;
IUPAC Name: N-(2-fluorophenyl)-5-(6-methyl-2-pyridinyl)-4-[1,2,4]triazolo[1,5-a]pyridin-6-yl-1H-imidazole-2-methanamine
CAS Number: 1352608-82-2); 1352610-25-3 (hydrochloride)
SMILES:CC1=CC=CC(=N1)C2=C(N=C(N2)CNC3=CC=CC=C3F)C4=CN5C(=NC=N5)C=C4
Mechanism of Action: Kinase Inhibitor; TGF-β type I Receptor Kinase
Inhibitor; ALK5 kinase Inhibitor; ALK4 Inhibitor
Indication: Various Cancers; Inhibition of Cancer Metastasis
Development Stage: Phase I
Company: Ewha Womans University (Korea)/
Transforming growth factor-β (TGF-β) belongs to the TGF-β superfamily,
which consists of TGF-β1, TGF-β2, TGF-β3, activins, inhibins, and bone
morphogenetic proteins. TGF-β is involved in many cellular processes, including
cell proliferation, cell migration, invasion, epithelial-mesenchymal transition
(EMT), extracellular matrix production, and immune suppression. TGF-β and its
receptors are often chronically overexpressed in various human diseases,
including cancer, tissue fibrosis, inflammation, and autoimmunity. Therefore,
blockade of TGF-β signaling pathway is an attractive target for drug
development. TGF-β signals via two related transmembrane type I and type II
serine/threonine kinase receptors. Following TGF-β binding to the
constitutively active type II receptor, the type I receptor (activin
receptor-like kinase 5 (ALK5)) is phosphorylated and creates a binding site for
Smad2/Smad3 proteins, which are further phosphorylated. Phosphorylated
Smad2/Smad3 proteins form a heteromeric complex with Smad4, which translocates
into the nucleus, assembles with specific DNA-binding cofactors and
comodulators, and binds to the promoters of TGF-β target genes involved in cell
differentiation, proliferation, apoptosis, migration, and extracellular matrix
production [1].
EW-7197 inhibited TGF-β/Smad signaling, cell migration, invasion, and lung metastasis in MMTV/c-Neu mice and 4T1 orthotopic-grafted mice, and also inhibited the epithelial-to-mesenchymal transition (EMT) in TGF-β-treated breast cancer cells and 4T1 orthotopic-grafted mice. In addition, EW-7197 demonstrated its pronounced antifibrotic efficacy in animal models of liver fibrosis, lung fibrosis, and renal fibrosis. Thus, treatment with EW-7197 significantly reduced the expression levels of collagen, a-smooth muscle actin, fibronection, 4-hydroxy-2,3-nonenal, and integrins in the livers of CCl4-treated mice and bile duct-ligated rats, in the lungs of bleomycin-treated mice, and in the kidneys of unilateral ureteral obstruction (UUO) mice. It also significantly extended the lifespan of CCl4-treated mice, bile duct-ligated rats, and bleomycin-treated mice.
Pharmacokinetic study with EW-7197 in rats showed an oral bioavailability of 51% with high systemic exposure (AUC) of 1426 ng × h/mL and maximum plasma concentration (Cmax) of 1620 ng/mL. Oral administration of EW-7197 (2.5 mg/kg, daily) for 18 days not only blocked Smad2/Smad3 phosphorylation, but also induced ubiquitin-mediated degradation of Smad4 mainly in CD8+ T cells in melanoma-bearing mice. Treatment with EW-7197 significantly increased the proportions and numbers of CD8+ T cells in the draining lymph nodes and the protein expression of perforin and granzyme B in the draining lymph node CD8+ T cells, thus enhancing CTL activity. The T cell-specific deficiency of Smad4 suppressed the progression of primary melanomas and lympth node metastasis by derepressing eomesodermin, a T-box transcription factor regulating CTL function.
EW-7197 inhibited TGF-β/Smad signaling, cell migration, invasion, and lung metastasis in MMTV/c-Neu mice and 4T1 orthotopic-grafted mice, and also inhibited the epithelial-to-mesenchymal transition (EMT) in TGF-β-treated breast cancer cells and 4T1 orthotopic-grafted mice. In addition, EW-7197 demonstrated its pronounced antifibrotic efficacy in animal models of liver fibrosis, lung fibrosis, and renal fibrosis. Thus, treatment with EW-7197 significantly reduced the expression levels of collagen, a-smooth muscle actin, fibronection, 4-hydroxy-2,3-nonenal, and integrins in the livers of CCl4-treated mice and bile duct-ligated rats, in the lungs of bleomycin-treated mice, and in the kidneys of unilateral ureteral obstruction (UUO) mice. It also significantly extended the lifespan of CCl4-treated mice, bile duct-ligated rats, and bleomycin-treated mice.
Pharmacokinetic study with EW-7197 in rats showed an oral bioavailability of 51% with high systemic exposure (AUC) of 1426 ng × h/mL and maximum plasma concentration (Cmax) of 1620 ng/mL. Oral administration of EW-7197 (2.5 mg/kg, daily) for 18 days not only blocked Smad2/Smad3 phosphorylation, but also induced ubiquitin-mediated degradation of Smad4 mainly in CD8+ T cells in melanoma-bearing mice. Treatment with EW-7197 significantly increased the proportions and numbers of CD8+ T cells in the draining lymph nodes and the protein expression of perforin and granzyme B in the draining lymph node CD8+ T cells, thus enhancing CTL activity. The T cell-specific deficiency of Smad4 suppressed the progression of primary melanomas and lympth node metastasis by derepressing eomesodermin, a T-box transcription factor regulating CTL function.
References:
1. Cheng, H. J.; et. al. Discovery of N-((4-([1,2,4]triazolo[1,5-a]pyridin-6-yl)-5-(6-methylpyridin-2-yl)-1H-imidazol-2-yl)methyl)-2-fluoroaniline (EW-7197): a highly potent, selective, and orally bioavailable inhibitor of TGF-β type I receptor kinase as cancer immunotherapeutic/antifibrotic agent. J Med Chem 2014, 57(10), 4213-4238.
2. Son, J. Y.; et. al. EW-7197, a novel ALK-5 kinase inhibitor, potently inhibits breast to lung metastasis. Mol Cancer Ther 2014, 13(7), 1704-1716.
3. Kim, D. -K.; et. al. Methods of treating fibrosis, cancer and vascular injuries. US8513222B2
4. ClinicalTrials.gov First in Human Dose Escalation Study of TEW-7197 in Subjects With Advanced Stage Solid Tumors. NCT02160106 (retrieved 01-10-2015)
