RepSox 是 TGFβR-1/ALK5 的选择性抑制剂， 能够抑制ALK5自磷酸化(IC50:4 nM)。

RepSox is a potent and selective of the TGFβR-1/ALK5 inhibitor.

RepSox注射到囊胚能够促成体内形成嵌合胚胎.用RepSox处理一天,足以取代转基因的Sox2.

RepSox能够通过抑制转化生长因子-β（Tgf-β）信号传导,从而诱导Nanog表达,从而成功代替Sox2进行重编程。RepSox诱导重编程不需要染色质重塑。RepSox对产生iPSCs是有效的。RepSox分别抑制ATP与ALK5和ALK5自磷酸化的结合,IC50分别为23 nM和4 nM。RepSox抑制TGF-β诱导的细胞PAI-1荧光素酶活性,IC50为18 nM。

The kinase domain of ALK5 is cloned by PCR and expressed in a baculovirus/Sf9 cells system. The protein is 6-His tagged in the C terminus and purified by affinity chromatography using a Ni2+column, and the obtained material is used to assess compound activity in an autophosphorylation assay. Purified enzyme (10 nM) is 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 is preincubated with different concentrations of RepSox (0.1% DMSO final concentration in the test) for 10 min at 37°C. The reaction is then initiated by the addition of 3 μM ATP (0.5 μCi γ-33P-ATP). After 15 min at 37°C, phosphorylation is 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 are boiled for 5 min at 95°C and run on a 12% SDS?PAGE. Dried gels are exposed to a phosphor screen overnight. ALK5 autophosphorylation is quantified using a Storm imaging system[1].

RepSox is dissolved in DMSO and stored, and then diluted with appropriate media (DMSO 1%) before use[1]. To test anti-TGF-β activity of compounds, HepG2 cells are seeded in 96 well microplates at a concentration of 35000 cells per well in 200 μL of serum-containing medium. The microplates are then placed for 24 h in a cell incubator at 37°C, 5% CO2 atm. RepSox dissolved in DMSO are 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 are washed with PBS and lysed by addition of 10 μL of passive lysis buffer. Inhibition of luciferase activity relative to control groups is used as a measure of compound activity. A concentration?response curve is constructed from which an IC50 value is determined graphically[1].

446859-33-2

C17H13N5

287.326

E-616452;SJN 2511;ALK5 Inhibitor II

Ethanol:<1 mgml
H2O:<1 mgml
DMSO:53 mg/mL (184.5 mM)
Powder: -20°C for 3 years
In solvent: -80°C for 2 years