In vitro activity: K02288 dose-dependently inhibits BMP4- and BMP6- induced Smad1/5/8 phosphorylation in C2C12 cells without affecting TGF-β-BMP signaling.

Kinase Assay: In vitro Kinase Assay for ALK1-6: Kinase reactions for ALK1-6 were performed at room temperature for 45 minutes in 96-well plates mixing 2.5 nM kinase (Invitrogen), 0.5 mg/mL dephosphorylated casein (Sigma), 6 μM ATP (Sigma), 0.05 μCi/μL [γ-32P]ATP (Perkin Elmer), 10 mM MnCl2 and 0.2% BSA in kinase buffer (Cell Signaling). Inhibitors were added at concentrations between 0 and 10 μM in kinase reaction buffer and tested in triplicate. Reactions were quenched with phosphoric acid, bound to 96-well P81 phosphocellulose filter plates (Millipore) and assayed with Microscint 20 scintillation fluid (Perkin Elmer) using a Spectramax L luminometer (Molecular Devices). Data were normalized to untreated controls at 100% enzyme activity and negative controls subtracted as background. IC50 values were calculated using GraphPad (Prism software).

Kinase-Glo(R) Assay: A kinase assay for ActRIIA (ACVR2) was performed using Kinase-Glo(R) (Promega) as per manufacturer’s instructions. Briefly, the following were mixed and reacted at room temperature for 3 hours in a 96-well plate at a final volume of 100 μL, 10 nM kinase (~EC50 at 2 hr), 0.5 mg/mL dephosphorylated casein (Sigma), 10 μM ATP (Promega), 10 mM MnCl2 and 0.2% BSA in kinase buffer (Cell Signaling). Inhibitors were added at concentrations between 0 and 10 μM in kinase reaction buffer and tested in duplicate. At 15 min, 30 min, 1 hr, 2 hr, and 3 hr 20 μL aliquots of the reaction mixture was transferred to a 384-well plate and 20 μL of Kinase-Glo(R) was added and allowed to rest for 10 min to quench the reaction and produce light which was measured using a Spectramax L luminometer (Molecular Devices). The 2 hr time point was within the linear portion of the reaction and was used for calculations due to favourable signal-to-noise ratio and was consistent with earlier time points. Data were normalized to untreated controls at 100% enzyme activity and negative controls subtracted as background. IC50 values were calculated using GraphPad Prism (GraphPad Software, Inc).

Cell Assay: In C2C12 cells, it reduces the phosphorylation of Smad dose-dependently when using BMP4 or BMP6 as ligands. Furthermore, K02288 induces a dorsalized phenotype in Tg(BRE:mRFP) transgenic zebrafish embryos.