TGF-β-RI/ALK5 (transforming growth factor-beta receptor I/activin like kinase 5) (4 nM = IC50)

MEFs are induced to differentiate into chemically induced pluripotent stem cells (CiPSCs) by RepSox (GMP) (10 μM)[1]. hCiPS cells are induced from HEFs by RepSox (GMP) (10 μM, in stage I-Stage III induction medium)[2]. Increased EdU incorporation confirms that RepSox (GMP) plus Forskolin boost the number of proliferative cells in MEFs expressing MyoD[3].

RepSox is able to be contribute to forming chimeric embryos in vivo when injected into blastocysts. A one-day treatment with RepSox is sufficient to replace transgenic Sox2.

ALK5 Fluorescence Polarization Binding Assay. [1]
Fluorescence polarization is a method that is well-documented in the literature. Kinase inhibitor compounds, conjugated to fluorophores, can be used as fluorescent ligands to monitor ATP competitive binding of other compounds to a given kinase. The increase in depolarization of plane polarized light, caused by release of the bound ligand into solution, is measured as a polarization/anisotropy value. Compound binding to ALK5 was tested on purified recombinant GST−ALK5 (residues 198−503). Displacement of rhodamine green fluorescently labeled ATP competitive inhibitor 24 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, and 1 nM rhodamine green-labeled ligand so that the final ALK5 concentration was 10 nM based on active site titration of the enzyme. Forty microliters of the enzyme/ligand reagent was added to 384 well assay plates containing 1 μL of different concentrations of test compound. The plates were read immediately on a LJL Acquest fluorescence reader with excitation, emission, and dichroic filters of 485, 530, and 505 nm, respectively. The fluorescence polarization for each well was calculated by the Acquest and was then imported into curve fitting software for construction of concentration−response curves.

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ALK5 Autophosphorylation Assay. [1]
The kinase domain of ALK5 (Franzen, P. 1993) (amino acids 162−503) was cloned by PCR and expressed in a baculovirus/Sf9 cells system. The protein was 6-His tagged in the C terminus and purified by affinity chromatography using a Ni2+ column, and the obtained material was used to assess compound activity in an autophosphorylation assay. Purified enzyme (10 nM) was incubated in 50 μL of Tris buffer (Tris 50 mM, pH 7.4; NaCl, 100 mM; MgCl2, 5 mM; MnCl2, 5 mM; and DTT, 10 mM). The enzyme was preincubated with different concentrations of compounds (0.1% DMSO final concentration in the test) for 10 min at 37 °C. The reaction was then initiated by the addition of 3 μM ATP (0.5 μCi γ-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, and 5% β-mercaptoethanol). The 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.

Cellular Assays to Measure Anti-TGF-β Activity of ALK5 Inhibitors. [1]
The activity of compounds was tested in a transcriptional assay in HepG2 cells. The cells were stably transfected with a PAI-1 promoter driving a luciferase (firefly) reporter gene. 25 The stably transfected cells responded to TGF-β stimulation by a 10−20-fold increase in luciferase activity as compared to control conditions. To test anti-TGF-β activity of compounds, the cells were seeded in 96 well microplates at a concentration of 35000 cells per well in 200 μL of serum-containing medium. The microplates were then placed for 24 h in a cell incubator at 37 °C, 5% CO2 atm. The compounds dissolved in DMSO were then added at concentrations of 50 nM to 10 μM (final concentration of DMSO 1%) for 30 min prior to the addition of recombinant TGF-β (1 ng/mL). After an overnight incubation, the cells were washed with PBS and lysed by addition of 10 μL of passive lysis buffer. 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.

[1]. Identification of 1,5-naphthyridine derivatives as a novel series of potent and selective TGF-beta type I receptor inhibitors. J Med Chem. 2004 Aug 26;47(18):4494-506.
[2]. RepSox, a small molecule inhibitor of the TGFβ receptor, induces brown adipogenesis and browning of white adipocytes. Acta Pharmacol Sin. 2019 Dec;40(12):1523-1531.

1,5-naphthyridine, 2-[3-(6-methyl-2-pyridinyl)-1h-pyrazol-4-yl]- is a pyrazolopyridine.
Optimization of the screening hit 1 led to the identification of novel 1,5-naphthyridine aminothiazole and pyrazole derivatives, which are potent and selective inhibitors of the transforming growth factor-beta type I receptor, ALK5. Compounds 15 and 19, which inhibited ALK5 autophosphorylation with IC50 = 6 and 4 nM, respectively, showed potent activities in both binding and cellular assays and exhibited selectivity over p38 mitogen-activated protein kinase. The X-ray crystal structure of 19 in complex with human ALK5 is described, confirming the binding mode proposed from docking studies.[1]

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Unlike white adipose tissue (WAT), brown adipose tissue (BAT) is mainly responsible for energy expenditure via thermogenesis by uncoupling the respiratory chain. Promoting the differentiation of brown fat precursor cells and the browning of white fat have become a research hotspot for the treatment of obesity and associated metabolic diseases. Several secreted factors and a number of small molecules have been found to promote brown adipogenesis. Here we report that a single small-molecule compound, RepSox, is sufficient to induce adipogenesis from mouse embryonic fibroblasts (MEFs) in fibroblast culture medium. RepSox is an inhibitor of the transforming growth factor-beta receptor I (TGF-β-RI), other inhibitors of TGF-β pathway such as SB431542, LY2157299, A83-01, and Tranilast are also effective in inducing adipogenesis from MEFs. These adipocytes express brown adipocyte-specific transcription factors and thermogenesis genes, and contain a large number of mitochondria and have a high level of mitochondrial respiratory activity. More interestingly, RepSox has also been found to promote the differentiation of the brown fat precursor cells and induce browning of the white fat precursor cells. These findings suggest that inhibitors of TGF-β signaling pathway might be developed as new therapeutics for obesity and type 2 diabetes.[2]

C17H13N5

287.32

287.117

C, 71.06; H, 4.56; N, 24.37

446859-33-2

RepSox;446859-33-2

449054

Light brown to yellow solid powder

1.3±0.1 g/cm3

501.9±50.0 °C at 760 mmHg

195 °C

230.5±23.1 °C

0.0±1.2 mmHg at 25°C

1.692

1.28

1

4

2

22

376

0

LBPKYPYHDKKRFS-UHFFFAOYSA-N

InChI=1S/C17H13N5/c1-11-4-2-5-16(20-11)17-12(10-19-22-17)13-7-8-14-15(21-13)6-3-9-18-14/h2-10H,1H3,(H,19,22)

2-[3-(6-methyl-2-pyridinyl)-1H-pyrazol-4-yl]-1,5-naphthyridine

E-616452, SJN 2511; E 616452; 446859-33-2; 2-(3-(6-Methylpyridin-2-yl)-1H-pyrazol-4-yl)-1,5-naphthyridine; ALK5 Inhibitor II; ALK5 Inhibitor; SJN 2511; 2-[5-(6-methylpyridin-2-yl)-1H-pyrazol-4-yl]-1,5-naphthyridine; C17H13N5; E616452; RepSox; SJN-2511; SJN-2511; ALK5 Inhibitor II

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: ≥ 7.5 mg/mL (26.10 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 75.0 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: ≥ 7.5 mg/mL (26.10 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 75.0 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: ≥ 1.67 mg/mL (5.81 mM) (saturation unknown) in 5% DMSO + 40% PEG300 + 5% Tween80 + 50% Saline (add these co-solvents sequentially from left to right, and one by one), clear solution.
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 4: ≥ 1.67 mg/mL (5.81 mM) (saturation unknown) in 5% DMSO + 95% (20% SBE-β-CD in Saline) (add these co-solvents sequentially from left to right, and one by one), clear solution.
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 5: ≥ 0.33 mg/mL (1.15 mM) (saturation unknown) in 1% DMSO 99% Saline (add these co-solvents sequentially from left to right, and one by one), clear solution.
Preparation of saline: Dissolve 0.9 g of sodium chloride in 100 mL ddH₂ O to obtain a clear solution.

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