In vitro activity: LDN193189 potently inhibits BMP4-mediated Smad1, Smad5 and Smad8 activation with IC50 of 5 nM, and efficiently inhibits transcriptional activity of the BMP type I receptors ALK2 and ALK3 with IC50 of 5 nM and 30 nM, respectively. Furthermore, LDN193189 also shows the inhibitory effect on the transcriptional activity induced by either constitutively active ALK2R206H or ALK2Q207D mutant proteins. A recent study shows that LDN-193189 blocks the production of reactive oxygen species induced by oxidized LDL during atherogenesis in human aortic endothelial cells.

Kinase Assay: LDN-193189 inhibits BMP4-mediated Smad1, Smad5 and Smad8 activation with greater potency than did dorsomorphin (IC50=5 nM versus 470 nM) while retaining 200-fold selectivity for BMP signaling versus TGF-β signaling (IC50 for TGF-β ≥1,000 nM). LDN-193189 efficiently inhibits transcriptional activity of the BMP type I receptors ALK2 and ALK3 (IC50=5 nM and 30 nM, respectively), with substantially weaker effects on activin and the TGF-β type I receptors ALK4, ALK5 and ALK7 (IC50≥500 nM) and increases selectivity for BMP signaling versus AMP-activated protein kinase, PDGFR and MAPK signaling pathways as compared to the parent compound. LDN-193189 blocks the transcriptional activity induced by either constitutively active ALK2R206H or ALK2Q207D mutant proteins. These findings suggest that LDN-193189 might affect BMP-induced osteoblast differentiation. In fact, LDN-193189 inhibits the induction of alkaline phosphatase activity in C2C12 cells by BMP4 even when administered 12 h after BMP stimulation, indicating sustained BMP signaling activity is needed for osteogenic differentiation. C

Cell Assay: 2C12 cells are seeded into 96-well plates at 2,000 cells per well in DMEM supplemented with 2% FBS. The wells are treated in quadruplicate with BMP ligands and LDN-193189 or vehicle. The cells are collected after 6 d in culture in 50 μL Tris-buffered saline and 1% Triton X-100. The lysates are added to p-nitro-phenylphosphate reagent in 96-well plates for 1 h and then evaluated alkaline phosphatase activity (absorbance at 405 nm). Cell viability are measured and quantity by Cell Titer Aqueous One (absorbance at 490 nm), using replicate wells treated identically to those used for alkaline phosphatase measurements.