TGFβR-I (IC50 = 59 nM)

LY-364947 is an ATP competitive tight-binding inhibitor that, at a Ki of 28 nM, prevents P-Smad3 from being phosphorylated by TGFβR-I kinase. In NMuMg cells, LY-364947 inhibits Smad2 phosphorylation in vivo at an IC50 of 135 nM. In NMuMg cells, LY-364947 reverses TGF-β-mediated growth inhibition with an IC50 of 0.218 μM. In NMuMg cells, LY-364947 increases the xVent2-lux BMP4 response by 30% at as low as 0.25 μM. TGF-β-induced epithelial-to-mesenchymal transition in NMuMg cells is inhibited by LY-364947 (2 μM) [1]. Prox1 and LYVE-1 expression was induced in nearly all HDLECs by LY-364947 (3 μM) after a 24-hour period [2]. When leukemia-initiating cells have high Akt phosphorylation levels and low Smad2/3, LY-364947 stimulates Foxo3a's nuclear export. Following co-cultivation with OP-9 stromal cells, leukemia-initiating cells' capacity to form colonies is inhibited by LY-364947 (< 20 μM) [3].

In a mouse model of chronic peritonitis, LY-364947 (1 mg/kg, ip) significantly increased the LYVE-1-positive region, indicating that it promotes lymphangiogenesis. Using BxPC3 pancreatic cancer cells as a tumor xenograft model, LY-364947 (1 mg/kg, i.p.) dramatically enhanced the amount of LYVE-1-positive tissue in the tumor [2]. In CML-affected mice, LY-364947 (25 mg/kg) raises p-Akt and lowers nuclear Foxo3a in leukemia-initiating cells [3].

TβRI Kinase Domain Expression and Purification.[1]
The intracellular kinase domain of TβRI (ALK5) and its constitutively active mutation, T204D, were cloned and expressed in Sf9 insect cells by standard procedures as described. The proteins were purified from Sf9 cells on a single Ni-NTA-affinity column. Briefly, Sf9 cells expressing the TβRI kinase domain or its constitutively active mutation, T204D, were harvested and lysed in buffer consisting of 50 mM Tris-HCl at pH 7.5, 150 mM NaCl, 50 mM NaF, 0.5% NP-40, 20 mM β-mercaptoethenol, 10 mM imidazole, 1 mM PMSF, and 1× EDTA-free protease inhibitor cocktail. After centrifugation, the lysate was loaded onto a Ni-NTA column and the protein was eluted with a linear 0−200 mM imidazole gradient prepared in kinase buffer containing 50 mM Tris-HCl, 150 mM NaCl, 4 mM MgCl2, 1 mM NaF, and 2 mM β-mercaptoethenol. The purity of the purified kinase was analyzed by SDS−PAGE, and selected fractions were pooled and utilized for kinase assays.

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Gel-Based Peptide-Substrate Phosphorylation Assay. [1]
Reaction mixtures (40 μL), containing 50 mM HEPES at pH 7.5, 1 mM NaF, 10 mM MgCl2, 8 μM ATP, 1 μCi [γ-32P]ATP (2200 Ci/mmol), 100 μM peptide substrate, and 100 nM TβRI kinase or the T204D mutant were incubated at 30 °C for 30 min. The reactions were terminated by the addition of an equal volume of 2× SDS−PAGE sample buffer. A total of 20 μL of the mixture was then loaded on a 10−20% SDS−polyacrylamide-gradient gel. After electrophoresis, the gel was fixed with 50% methanol and 15% acetic acid, dried by a GelAir Dryer from Bio-Rad, and exposed to X-ray film overnight or a phosphoimaging screen for 1−2 h. Quantitation was performed with a phosphoimager and Quantity One software from Bio-Rad.

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HPLC−Mass Spectrum Analysis of Peptide Substrate pSmad3(−3) and Its Phosphorylated Product. [1]
Typically, reactions were performed in a 40 μL reaction mixture containing 50 mM HEPES at pH 7.5, 1 mM NaF, 5 mM MgCl2, 8 μM ATP, 100 μM peptide substrate pSmad3(−3), and 100 nM TβRI T204D. The reactions were incubated for 60 min at 30 °C and terminated by the addition of 100 mM EDTA. The reaction mixtures were analyzed by HPLC using a Hewlett−Packard Series 1100 system equipped with an autosampler. The reaction mixtures (20 μL) were injected into a reversed-phase column (Vydac C18), and the substrate and its phosphorylated product were separated using a 15−35% linear acetonitrile gradient in 0.1% TFA with a flow rate 1 mL/min. Peak detection was accomplished by monitoring the absorbance at 214 nm, and the molecular mass of each peak was detected by electrospray mass spectrometry.

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Filter-Binding Assay. [1]
Reactions were performed in a polypropylene 96-well U-bottom microtiter plate. Reactions (40 μL) containing 50 mM HEPES at pH 7.5, 1 mM NaF, 10 mM MgCl2, and different concentrations of ATP, pKSmad3(−3), TβRI kinase, and inhibitors were incubated for 30 min at 30 °C. The reactions were terminated by the addition of 100 μL of 0.5% phosphoric acid. The mixtures were then transferred to a 96-well MultiScreen-MAPH filter plate, which was pre-equilibrated with 0.5% phosphoric acid for 30 min and incubated for 30 min at room temperature. The plate was then washed 3 times by filtration with 300 μL of 0.5% phosphoric acid with a vacuum filtration apparatus from Millipore. After washing, the filter plate was transferred to a MultiScreen Adaptor plate, 100 μL of Microscint 20 was added to each well, and the radioactivity was determined on a Microplate Scintillation Counter from Packard.

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Km Determination. [1]
The Km values for ATP and peptide substrates were determined by a gel-based assay as described above. To measure the Km of ATP, reactions containing 50 mM HEPES at pH 7.5, 1 mM NaF, 10 mM MgCl2, 400 μM pSmad3(−3), 100 nM TβRI kinase or the T204D mutant, and a dilution series of ATP from 0 to 500 μM were incubated for 30 min at 30 °C and terminated by the addition of an equal volume of SDS−sample buffer. To measure the Km values of peptide substrates, reactions containing 50 mM HEPES at pH 7.5, 1 mM NaF, 10 mM MgCl2, 100 μM ATP, 100 nM of the T204D mutant, and a dilution series of peptide substrate from 0 to 1000 μM were incubated for 30 min at 30 °C and terminated by the addition of SDS−sample buffer. The reaction mixtures (20 μL) were then loaded onto a 10−20% SDS−polyacrylamide-gradient gel. After electrophoresis, gels were fixed by 50% methanol and 15% acetic acid and dried with a GelAir Dryer from Bio-Rad. After exposure to a phosphoimager for 1−2 h, the radioactive bands were analyzed by a phosphoimager equipped with Quantity One software from Bio-Rad. The Km was calculated in SigmaPlot using the enzyme kinetics module.

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Determination of Substrate Specificity of pSmad3(−3). [1]
To determine the specificity of peptide substrate pSmad3(−3), we investigated if TβRII was able to catalyze the phosphorylation of this peptide substrate. The kinase domain of TβRII was expressed and purified from Sf9 cells with one step Ni-NTA-affinity column (Yingling et al., unpublished data). The gel-based assay as described above was utilized for this specificity determination. Briefly, 40 μL reaction mixtures, containing 50 mM HEPES at pH 7.5, 1 mM NaF, 10 mM MgCl2, 8 μM ATP 1 μCi [γ-32P]ATP (2200 Ci/mmol), 100 μM peptide substrate, and 100 nM TβRI kinase or different concentrations of TβRII kinase, were incubated at 30 °C for 30 min. The autophosphorylation and peptide-substrate phosphorylation were visualized by SDS−PAGE and autoradiography.

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Ki Determination and Mechanism of Action Studies. [1]
Ki values and mechanism of action of selected pyrazoles were determined by the filter-binding assay. In a 96-well microtiter plate, an appropriate titration of the inhibitor, ATP, and substrate pSmad3(−3) was utilized. A 6-point titration was generally utilized with the inhibitor, ATP, and the peptide substrate. The titration used for each inhibitor varied depending upon the potency of the compound. To analyze if the compound competed with ATP, an ATP titration with concentrations of 0, 6.25, 12.5, 25, 50, and 100 μM ATP was used and the substrate pKSmad3(−3) and T204D mutant enzyme were fixed at 200 μM and 100 nM, respectively. To analyze if an inhibitor competed with substrate pKSmad3(−3), a pKSmad3(−3) titration with concentrations of 0, 50, 100, 200, 400 and 800 μM was used and ATP and T204D mutant were fixed at 25 μM and 100 nM, respectively. The reactions were incubated for 30 min at 30 °C and terminated by the addition of 0.5% phosphoric acid. The Ki and competitive mechanism of each inhibitor were determined by kinetic analyses with Mathematica (version 4.1) software from Microsoft.

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Kinetic Binding Type Determination of Selected Pyrazoles. [1]
To determine the kinetic binding type of pyrazole inhibitors, we first performed an enzyme titration experiment with constitutively active enzyme, TβRI T204D. Briefly, 40 μL reactions containing a dilution series of the T204D mutant at concentrations of 800, 600, 400, 300, 200, 150, 100, 75, 50, 37.5, 25, and 0 nM in 50 mM HEPES at pH 7.5, 1 mM NaF, 200 μM pKSmad3(−3), and 50 μM ATP were incubated at 30 °C for 30 min. The linear reaction range of enzyme was determined by a linear regression analysis. The IC50 of LY364947 and LY566578 at different enzyme concentrations were determined by the filter-binding assay. Typically, 40 μL reactions in 50 mM HEPES at pH 7.5, 1 mM NaF, 200 μM pKSmad3(−3), and 50 mM ATP containing a titration of each inhibitor with concentrations of 1600, 800, 400, 200, 100, 50, 25, and 0 nM were incubated at 30 °C for 30 min. The IC50 was calculated using a nonlinear regression method with GraphPad Prism software. The binding type was determined by plotting the correlation between enzyme concentrations and IC50 values.

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Autophosphorylation of TβRI Kinase and IC50 Determination. [1]
Autophosphorylation of TβRI kinase was performed as described ( 30). Briefly, 200 nM constitutively active RI T204D enzyme was incubated for 60 min at 30 °C in kinase buffer containing 50 mM Tris-HCl at pH 7.5, 150 mM NaCl, 4 mM MgCl2, 1 mM NaF, 2 mM β-mercaptoethanol, 4 μM ATP, and 1 μCi [γ-33P]ATP. Reactions were terminated by the addition of 25% TCA, and BSA was then added to a final concentration of 250 μg/mL. The autophosphorylated TβRI kinase was captured on MultiScreen-MAFB filter (pure borosilicate glass fiber) plates. The radioactivity was determined on a Microbeta JET Trilux counter from Wallac. For IC50 determination, a 10-point titration with a 1:2 dilution for each compound was selected. The IC50 is calculated using a nonlinear regression method with GraphPad Prism software.

Western Blot Analysis of in Vivo Phospho-Smad2 in NMuMg Cells.[1]
Western blot analysis of phospho-Smad2 protein in NMuMg mammary epithelial cells was performed by standard protocol. Briefly, 2 × 106 NMuMg cells were grown overnight on a 100 mm plate in DMEM with 10% FBS and then starved for 1 h by switching to DMEM containing 0.5% FBS and a dilution series of LY364947 (10 μM, 1:2 dilutions) or LY580276 (20 μM, 1:3 dilutions). Cells were then treated with 2.5 ng/mL (100 pM) TGF-β1 and incubated for 2 h at 37 °C. The cells were washed with 1× PBS and lysed in 150 μL lysis buffer containing 50 mM Tris-HCl at pH 7.5, 500 mM NaCl, 1% NP-40, 0.25% Na-deoxycholate, 20 mM NaF, protease inhibitor cocktail, and phosphatase inhibitor cocktail I and II (Sigma). A total of 50 μg of protein was loaded onto a 10% SDS−polyacrylamide gel. Western blot analysis was performed with the PS2 phospho-Smad2 antibody.

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Cellular Pathway Selectivity Analysis. [1]
The TGF-β growth inhibition assay in NMuMg cells was performed as described previously for mink lung epithelial cells. The xVent2-luciferase BMP4 reporter assay in NMuMg cells was performed as follows. A stable clone harboring the xVent2-lux plasmid was seeded at 1.2 × 104 cells per well in 96-well microtiter plates and allowed to adhere overnight. Cells were serum-starved in 0.5% FBS media containing a dilution series of inhibitor for 2 h prior to the addition of 500 pM BMP4. Luciferase activity was determined 24 h later as previously described. Western blot analysis of MAP kinases and their downstream effectors in HeLa cells was performed as follows. A total of 2.5 × 105 cells were seeded in 6-well plates in 1 mL of RPMI1640 containing 10% FBS and allowed to adhere overnight. Cells were treated with a dilution series of compound for 1 h prior to the addition of 10 ng/mL TNFα for 15 min. The cells were then washed with ice-cold PBS and lysed in 100 μL of lysis buffer. A total of 10 μg of cell lysate was loaded onto a 4−20% SDS−polyacrylamide gel, and Western blot analysis was performed according to standard protocols.

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TGF-β-Induced EMT and Immunofluorescence Microscopy. [1]
NMuMg cells were plated at a density of 1 × 104 cells in 10% FBS DMEM on chamber coverglass and allowed to adhere overnight. TGF-β1 (100 pM) was added for an additional 24 h prior to fixation in 1% formaldehyde prepared in 1× PBS for 10 min at room temperature. Fixed cells were washed twice for 10 min each with 1× PBS and then permeabilized in 1× PBS plus 1% BSA and 0.025% NP-40 for 15 min at room temperature. After permeabilization, cells were washed twice with 1× PBS and 1% BSA and then incubated for 30 min at room temperature with protein-blocker buffer. Phalloidin-Alexa 488 at 1:26 dilution and anti-E-cadherin at 1:75 dilution were added and incubated for 1 h in a dark humid chamber. After the primary incubation, the cells were again washed with 1× PBS and 1% BSA 3 times and the E-cadherin samples were then incubated for 1 h with anti-rabbit IgG conjugated with Alexa 488 at a 1:500 dilution. After washing with 1× PBS and 1% BSA, the cells were counter-stained with propidium iodide at a 1:20 dilution. After a final wash with 1× PBS, the cells were visualized by confocal microscopy.

Cancer xenograft models[2]
BALB/c nude mice 5 to 6 weeks of age were obtained from CLEA Japan and Sankyo Laboratory. Parental, or VEGF-C- or TGF-β1-expressing tumor cells (5 × 106) in 100 μL PBS were implanted subcutaneously into male nude mice and allowed to grow for 2 to 3 weeks to reach proliferative phase, before initiation of TβR-I inhibitor administration. TβR-I inhibitor LY364947, dissolved in 5 mg/mL in DMSO and diluted with 100 μL PBS, or the vehicle control, was injected intraperitoneally at 1 mg/kg, 3 times a week for 3 weeks. Excised samples were directly frozen in dry-iced acetone for immunohistochemistry. Frozen samples were further sectioned at 10-μm thickness in a cryostat and subsequently incubated with primary and secondary antibodies as described above. Samples were observed using a confocal microscope.

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Dissolved in 5 mg/mL in DMSO and diluted with 100 μL PBS; 1 mg/kg; i.p. administration

Tumor xenograft models with BxPC3 pancreatic adenocarcinoma cells.aminonucleoside-induced renal fibrosis

[1]. Kinetic characterization of novel pyrazole TGF-beta receptor I kinase inhibitors and their blockade of the epithelial-mesenchymal transition. Biochemistry, 2005, 44(7), 2293-2304.

[2]. Inhibition of endogenous TGF-beta signaling enhances lymphangiogenesis. Blood, 2008, 111(9), 4571-4579.

[3]. TGF-beta-FOXO signalling maintains leukaemia-initiating cells in chronic myeloid leukaemia. Nature, 2010, 463(7281), 676-680.

LY 364947 is a member of the class of pyrazoles carrying pyridin-2-yl and quinolin-4-yl substituents at positions 3 and 4 respectively. It has a role as a TGFbeta receptor antagonist. It is a member of pyrazoles, a member of pyridines and a member of quinolines.
4-(3-Pyridin-2-YL-1H-pyrazol-4-YL)quinoline has been reported in Exserohilum rostratum with data available.

C17H12N4

272.3

272.106

C, 74.98; H, 4.44; N, 20.58

396129-53-6

447966

Typically exists as off-white to yellow solids at room temperature

1.3±0.1 g/cm3

490.8±45.0 °C at 760 mmHg

268.7±15.1 °C

0.0±1.2 mmHg at 25°C

1.700

2.35

1

3

2

21

348

0

N1([H])C(C2=C([H])C([H])=C([H])C([H])=N2)=C(C([H])=N1)C1=C([H])C([H])=NC2=C([H])C([H])=C([H])C([H])=C12

IBCXZJCWDGCXQT-UHFFFAOYSA-N

InChI=1S/C17H12N4/c1-2-6-15-13(5-1)12(8-10-19-15)14-11-20-21-17(14)16-7-3-4-9-18-16/h1-11H,(H,20,21)

4-(3-(pyridin-2-yl)-1H-pyrazol-4-yl)quinoline

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: ≥ 1.25 mg/mL (4.59 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 12.5 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: ≥ 1.25 mg/mL (4.59 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 12.5 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.)