ALK5

GW-6604 (0-10 μM) efficiently blocks the Smad shock response by inhibiting TGF-β PAI-1 sleep and sleep in HepG2 cells with an IC50 of 500 nM [1].
In HepG2 cells stably transfected with the TGF-β-responsive wild-type PAI-1 promoter driving a luciferase reporter gene, GW6604 (see structure in Figure 1) inhibited TGF-β-induced PAI-1 transcription and secretion at submicromolar concentrations (IC50=500 nm; Figure 2a). GW6604 selectivity versus activin and BMP, two members of the TGF-β superfamily, was addressed using activin- and BMP-specific cellular reporter assays. GW6604 showed a five-fold selectivity versus activin (IC50 of 2.5 μm) and did not inhibit BMP signaling (IC20 >10 μm) (Figure 2a)[1].
Transduction of TGF-β signaling results in Smad protein translocation to the nucleus and transcription of TGF-β-responsive genes. The Smad binding sequence in the promoter region of TGF-β-responsive genes is critical for TGF-β/Smad-induced transcription (Dennler et al., 1998; Shi & Massague, 2003). Using HepG2 cells transfected with an artificial reporter comprising nine adjacent copies of the Smad3/Smad4 binding sites derived from the PAI-1 promoter, we show that GW6604 inhibited the TGF-β-induced response with a potency comparable to that measured on the wild-type PAI-1 promoter, suggesting that GW6604 efficiently blocked a Smad-dependent response (Figure 2b). To further investigate the mechanism by which GW6604 inhibits TGF-β signaling, an ALK5 autophosphorylation assay was performed using a purified recombinant kinase domain. As shown in Figure 2c, GW6604 inhibited ALK5 activity with an IC50 of 140 nm, a potency comparable to that measured in an ALK5 binding assay (IC50=107 nm (Table 1). The selectivity of GW6604 for ALK5 versus other kinases was tested using a panel of other kinases including P38MAPK, VEGFR2, P56lck, ITK, Src, and TGF-β type II receptor; GW6604 demonstrated no significant activity on these kinases at concentrations up to 10 μm (Table 1)[1].

In the temporary DMN model of SD, GW-6604 (po, 25-80 mg/kg, twice daily, 3 weeks) lowers transcriptional expression by 80% and overexpresses the transcription factor COL IA1. enhanced basal penis, successfully decreased liver fibrosis, and maintained normal liver weight [1].

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GW6604 increases liver regeneration in TGF-β1-overexpressing mice[1]
To evaluate whether GW6604 effectively blocked TGF-β effects in an in vivo physiological context, mice overexpressing TGF-β1 under the control of the liver-specific albumin promoter (Sanderson et al., 1995) were treated with GW6604 before and after partial hepatectomy and animals were killed 40 h after hepatectomy. Liver regeneration was quantified via BrdU labeling on five representative microscopic fields per animal. Treatment with GW6604 induced a 4.7-fold increase in hepatocyte proliferation in partially hepatectomized TGF-β transgenic animals (1.8±0.4 and 8.5±2.2 stained nuclei per field in animals treated with vehicle and GW6604, respectively, P=0.02 (t-test)) (Figure 3). In hepatectomized nontransgenic animals and in nonhepatectomized animals treatment with GW6604 did not increase BrdU staining (data not shown). These results show that the anti-TGF-β activity of GW6604 observed in vitro in HepG2 cells translates into an effect on mouse hepatocytes in vivo.[1]

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Effect of GW6604 in an acute DMN model[1]
During fibrogenesis, increase in COL IA1 synthesis is mainly triggered by TGF-β acting on hepatic stellate cells. In rats given DMN for 3 consecutive days, liver COL IA1 mRNA expression measured by quantitative RT–PCR was increased by about 10-fold at day 8. GW6604 (25–80 mg kg−1 p.o., b.i.d.) given on days 6, 7, and 8 to DMN-pretreated rats dose dependently inhibited COL IA1 overexpression (Figure 4).[1]

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Therapeutic effect of GW6604 in a chronic DMN model of liver fibrosis[1]
To demonstrate a potential therapeutic benefit of GW6604 treatment on matrix deposition and liver function, rats were given DMN for 6 consecutive weeks and received GW6604 (80 mg kg−1 p.o., b.i.d.) for the last 3 weeks. During the first 3 weeks of DMN administration, liver disease developed as described (Wu & Norton, 1996; George et al., 2001); weight progression was only slightly decreased by DMN. All rats treated with GW6604 in the DMN group (DMN-GW6604) survived the 6 weeks, whereas mortality approached 50% in the DMN-vehicle-treated group (DMN-vehicle) (Table 2). A significant decrease in liver weight was observed in the DMN-vehicle group compared to the saline-vehicle group. GW6604 prevented this decrease and maintained a normal liver weight (9±1.4 g for the DMN-vehicle versus 17.3±0.5 g for DMN-GW6604-treated animals) (Table 2). However, body weight gain was totally inhibited in both DMN-GW6604- and DMN-vehicle-treated groups (Figure 5). Immunostaining of liver section with PCNA antibodies showed very few regenerating hepatocytes in DMN-vehicle-treated rats, whereas a 10-fold increase in proliferating hepatocytes was observed in DMN-GW6604 group (Figure 6). Long-term DMN administration is known to cause chronic liver disease characterized by extensive fibrosis leading to cirrhosis (Wu & Norton, 1996; George et al., 2001). Analysis of liver gene expression showed that DMN treatment greatly increased mRNA encoding for matrix components (COL IA1, COL IA2, and COL III) as well as for TIMP-1 without interfering with 18S, which was used as a reference gene. In DMN-GW6604-treated animals, overexpression of mRNA encoding for TGF-β1, collagens, and TIMP-1 was reduced by 50–75% compared to DMN-vehicle group, indicating a reduction in the fibrotic process as well as an increased matrix degradation (Table 2).

Fluorescence polarization kinase binding assays[1]
Compound binding to ALK5 was tested on purified recombinant GST-ALK5 (residues 198–503). Displacement of a rhodamine green fluorescently labeled ATP-competitive inhibitor (described in patent application WO02/24680, 2000) by different concentrations of test compounds was used to calculate a binding pIC50. GST-ALK5 was added to a buffer containing 62.5 mm Hepes (pH 7.5), 1 mm DTT, 12.5 mm MgCl2, 1.25 mm CHAPS (all reagents obtained from XXX), and 1 nm rhodamine green-labeled ligand so that the final ALK5 concentration is 10 nm based on active site titration of the enzyme. A measure of 40 μl of the enzyme/ligand reagent was added to 384-well assay plates containing 1 μl of different concentrations of test compound. The plates are read immediately on an LJL Acquest fluorescence reader with excitation, emission, and dichroic filters of 485, 530, and 505 nm, respectively. The fluorescence polarization for each well is calculated by the Acquest and is then imported into curve fitting software for construction of concentration–response curves. The same assay conditions were used to measure binding of GW6604 to other kinases.[1]

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Alk5 autophosphorylation assay[1]
The kinase domain of ALK5 (Franzen et al., 1993) (amino acids 162–503) was cloned by PCR and expressed in a baculovirus/Sf9 cells system. The protein was 6-His tagged in C-terminus and purified by affinity chromatography using an Ni2+ column. The material thus obtained was used to assess the ability of test compounds to inhibit ALK5 autophosphorylation.[1]
Purified enzyme (10 nm) in 50 μl of Tris buffer (Tris 50 mm, pH 7.4; NaCl 100 mm; MgCl2 5 mm; MnCl2 5 mm; DTT 10 mm) was preincubated with different concentrations of compounds (0.1% dimethylsulfoxide (DMSO) final concentration in the test) for 10 min at 37°C. The reaction was initiated by the addition of 3 μm ATP (0.5 μCi gamma-33P-ATP). After 15 min at 37°C, phosphorylation was stopped by the addition of SDS–PAGE sample buffer (50 mm Tris-HCl (pH 6.9), 2.5% glycerol, 1% SDS, 5% beta-mercaptoethanol). Samples were boiled for 5 min at 95°C and run on a 12% SDS–PAGE. Dried gels were exposed to a phosphor screen overnight. ALK5 autophosphorylation was quantified using a Storm imaging system.[1]

Cellular assays to measure anti-TGF-β activity of ALK5 inhibitors[1]
Compound activity was tested in a transcriptional assay in HepG2 cells (ATCC). Cells were stably transfected with a reporter construct comprising the human PAI-1 promoter (−806 to +72 region) driving a luciferase (firefly) reporter gene or with a construct containing nine adjacent copies of a previously described Smad binding site (Dennler et al., 1998). The stably transfected cell lines were obtained by a limiting dilution method and clonal expansion of a selected clone. The cell line containing the human PAI-1 promoter (−806 to +72) responded to TGF-β stimulation with a 10- to 20-fold increase in luciferase activity compared to control conditions, whereas the cell line containing the nine adjacent Smad binding sites was highly responsive to TGF-β stimulation with a >500-fold increase in luciferase activity following the addition of TGF-β.
To test anti-TGF-β activity of a compound, cells were seeded in 96-well microplates at a concentration of 35,000 cells per well in 200 μl of serum-containing medium. Microplates were then placed for 24 h in a cell incubator at 37°C, 5% CO2 atmosphere. Cells were then cultured in serum-free BME medium and GW6604 was added at concentrations of 10 nm to 10 μm (final concentration of DMSO 1%) 30 min prior to the addition of recombinant TGF-β1 (1 ng ml−1). After an overnight incubation, cells were washed with PBS and lysed by the addition of 10 μl of passive lysis buffer (Promega, Carbonières, France). Inhibition of luciferase activity relative to control groups was used as a measure of compound activity. A concentration–response curve was constructed from which an IC50 value was determined graphically.

Acute DMN model[1]
Acute DMN model was set up after careful study of the DMN-induced fibrosis development. The aim was to be able to detect as early as possible gene changes related to fibrogenesis and ultimately test compound activity in vivo in a short time frame.[1]
Male Sprague–Dawley rats weighing 200–225 g were treated for 3 consecutive days (days 1–3) with 12.5 mg kg−1 i.p. DMN, or saline. Animals were then treated twice a day with GW6604 p.o. or its vehicle (20% HCl 1 n, 80% hydroxypropyl-methylcellulose (0.5%), Tween 80 (5%), adjusted to pH 4) in a volume of 4 ml kg−1 on days 6, 7, and 8. Animals were killed by CO2 inhalation on day 8, 2 h after the fifth administration of GW6604 or vehicle. Livers were collected for collagen IA1 (COL IA1) mRNA quantification by RT–PCR. Results are reported as the percent inhibition of DMN-induced increase in COL IA1 compared to control. COL IA1 mRNA was quantified relative to ribosomal 18S. Four to six rats were used in each group.[1]

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Chronic DMN model[1]
Male Sprague–Dawley rats weighing 200–225 g were treated for 6 weeks, 3 consecutive days each week with 10 mg kg−1 i.p. of DMN, or saline. After 3 weeks of DMN administration, treatment with GW6604 (80 mg kg−1 p.o., b.i.d.) or its vehicle in a volume of 4 ml kg−1 was initiated and continued for 3 weeks. DMN administration was continued during the 3-week treatment period. At the end of the 6-week period, animals were killed by CO2 inhalation. Blood was collected for laboratory analysis of ALAT, ASAT, total bilirubin, alkaline phosphatase, and hyaluronic acid. Livers were collected for RT–PCR quantification of genes encoding for COL IA1, collagen IA2 (COL IA2), collagen III alpha1 chain (COL III), TIMP-1, TGF-β1, and for histologic assessment. Changes in liver messenger RNA levels relative to 18S are reported as fold induction compared to saline-vehicule, group. Mortality is reported at the end of the 6-week treatment period. All animals survived the initial 3 weeks of DMN treatment.

[1]. Inhibition of TGF-beta signaling by an ALK5 inhibitor protects rats from dimethylnitrosamine-induced liver fibrosis. Br J Pharmacol. 2005 May;145(2):166-77.

In conclusion, although liver fibrosis is a multicomponent disease, TGF-β appears to play a major role as underlined by the results obtained with GW6604, a novel ALK5 inhibitor. GW6604 inhibits TGF-β signaling in vitro and blocks TGF-β effects in vivo as evidenced by its ability to increase hepatocyte proliferation following liver injury or partial hepatectomy, and also to reduce matrix gene expression in acute and chronic models of liver fibrosis. In a chronic model of DMN-induced fibrosis, GW6604 was a valuable therapeutic tool as it reduced mortality and decreased fibrosis, leading to an overall amelioration in liver function. These data suggest that blocking TGF-β effect through ALK5 inhibition represents a promising approach for the treatment of chronic liver diseases.[1]

C19H14N4

298.34126

298.121

C, 76.49; H, 4.73; N, 18.78

452342-37-9

9861063

Off-white to light yellow solid powder

1.2±0.1 g/cm3

505.2±50.0 °C at 760 mmHg

272.5±16.5 °C

0.0±1.2 mmHg at 25°C

1.650

3.18

1

3

3

23

368

0

BDCBRQYHYNUWAM-UHFFFAOYSA-N

InChI=1S/C19H14N4/c1-2-6-14(7-3-1)18-12-15(9-11-21-18)16-13-22-23-19(16)17-8-4-5-10-20-17/h1-13H,(H,22,23)

2-phenyl-4-(5-pyridin-2-yl-1H-pyrazol-4-yl)pyridine

GW 6604; GW-6604; 2-Phenyl-4-(3-(pyridin-2-yl)-1H-pyrazol-4-yl)pyridine; 2-phenyl-4-(3-pyridin-2-yl-1H-pyrazol-4-yl)pyridine; CHEMBL205736; 273G6SN31H; Pyridine, 2-phenyl-4-(3-(2-pyridinyl)-1H-pyrazol-4-yl)-; GW6604

2934.99.9001

Powder      -20°C    3 years

                     4°C     2 years

In solvent   -80°C    6 months

                  -20°C    1 month

Note: This product requires protection from light (avoid light exposure) during transportation and storage.

Room temperature (This product is stable at ambient temperature for a few days during ordinary shipping and time spent in Customs)

DMSO : ~100 mg/mL (~335.19 mM)

Note: Listed below are some common formulations that may be used to formulate products with low water solubility (e.g. < 1 mg/mL), you may test these formulations using a minute amount of products to avoid loss of samples.

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Injection Formulation 1: DMSO : Tween 80： Saline = 10 : 5 : 85 (i.e. 100 μL DMSO stock solution → 50 μL Tween 80 → 850 μL Saline)
*Preparation of saline: Dissolve 0.9 g of sodium chloride in 100 mL ddH ₂ O to obtain a clear solution.
Injection Formulation 2: DMSO : PEG300 ：Tween 80 : Saline = 10 : 40 : 5 : 45 (i.e. 100 μL DMSO → 400 μLPEG300 → 50 μL Tween 80 → 450 μL Saline)
Injection Formulation 3: DMSO : Corn oil = 10 : 90 (i.e. 100 μL DMSO → 900 μL Corn oil)
Example: Take the Injection Formulation 3 (DMSO : Corn oil = 10 : 90) as an example, if 1 mL of 2.5 mg/mL working solution is to be prepared, you can take 100 μL 25 mg/mL DMSO stock solution and add to 900 μL corn oil, mix well to obtain a clear or suspension solution (2.5 mg/mL, ready for use in animals).

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Injection Formulation 4: DMSO : 20% SBE-β-CD in saline = 10 : 90 [i.e. 100 μL DMSO → 900 μL (20% SBE-β-CD in saline)]
*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.
Injection Formulation 5: 2-Hydroxypropyl-β-cyclodextrin : Saline = 50 : 50 (i.e. 500 μL 2-Hydroxypropyl-β-cyclodextrin → 500 μL Saline)
Injection Formulation 6: DMSO : PEG300 : castor oil : Saline = 5 : 10 : 20 : 65 (i.e. 50 μL DMSO → 100 μLPEG300 → 200 μL castor oil → 650 μL Saline)
Injection Formulation 7: Ethanol : Cremophor : Saline = 10： 10 : 80 (i.e. 100 μL Ethanol → 100 μL Cremophor → 800 μL Saline)
Injection Formulation 8: Dissolve in Cremophor/Ethanol (50 : 50), then diluted by Saline
Injection Formulation 9: EtOH : Corn oil = 10 : 90 (i.e. 100 μL EtOH → 900 μL Corn oil)
Injection Formulation 10: EtOH : PEG300：Tween 80 : Saline = 10 : 40 : 5 : 45 (i.e. 100 μL EtOH → 400 μLPEG300 → 50 μL Tween 80 → 450 μL Saline)

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Oral Formulation 1: Suspend in 0.5% CMC Na (carboxymethylcellulose sodium)
Oral Formulation 2: Suspend in 0.5% Carboxymethyl cellulose
Example: Take the Oral Formulation 1 (Suspend in 0.5% CMC Na) as an example, if 100 mL of 2.5 mg/mL working solution is to be prepared, you can first prepare 0.5% CMC Na solution by measuring 0.5 g CMC Na and dissolve it in 100 mL ddH2O to obtain a clear solution; then add 250 mg of the product to 100 mL 0.5% CMC Na solution, to make the suspension solution (2.5 mg/mL, ready for use in animals).

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Oral Formulation 3: Dissolved in PEG400
Oral Formulation 4: Suspend in 0.2% Carboxymethyl cellulose
Oral Formulation 5: Dissolve in 0.25% Tween 80 and 0.5% Carboxymethyl cellulose
Oral Formulation 6: Mixing with food powders

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Note: Please be aware that the above formulations are for reference only. InvivoChem strongly recommends customers to read literature methods/protocols carefully before determining which formulation you should use for in vivo studies, as different compounds have different solubility properties and have to be formulated differently.

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