SD-208 (ALK5 Inhibitor V), a selective TGF-βRI (ALK5) inhibitor (IC50: 48 nM), is >100-fold selectivity over TGF-βRII.

TGFβRI:48 nM

SD-208 (1 mg/mL, p.o.) significantly prolonged the median survival rate of mice loaded with SMA-560 glioma. In syngeneic 129S1 mice, SD-208 (60 mg/kg, p.o.) inhibited the growth of primary R3T tumors and reduced the number and size of lung metastases. Additionally, in a murine aortic allograft model, SD-208 effectively reduced the formation of intimal hyperplasia associated with transplant arteriosclerosis.

In vitro, SD-208 inhibits TGF-β-induced receptor-associated Smads, Smad2/3 phosphorylation, and stimulates differentiation, migration, and invasion from epithelial to mesenchymal cells into the basal membrane. Additionally, SD-208 blocks the protective effects of TGF-β on the migration and proliferation of neointimal smooth muscle-like cells. In both mouse SMA-560 and human SMA-560 glioma cells, SD-208 suppresses cell growth, constitutive and TGF-β-induced migration and infiltration, and enhances immunogenicity.

Kinase assay: Various kinase activities are assayed by measuring the incorporation of radiolabeled ATP into a peptide or protein substrate. The reactions are performed in 96-well plates and included the relevant kinase, substrate, ATP, and appropriate cofactors. The reactions are incubated and then stopped by the addition of phosphoric acid. Substrate is captured onto a phosphocellulose filter, which is washed free of unreacted ATP. The counts incorporated are determined by counting on a microplate scintillation counter. The ability of SD-208 to inhibit the respective kinase is determined by comparing counts incorporated in the presence of compound with those incorporated in the absence of compound.

Glioma cells are cultured in the absence or presence of SD-208 (1 μM) for 48 hours. The cells are pulsed for the last 24 hours with [methyl-3H]thymidine (0.5 μCi) and harvested, and incorporated radioactivity is determined in a liquid scintillation counter.(Only for Reference)