BAY-598

Kinase experiment:

For SMYD2 inhibition, 10 µL of BAY-598 or DMSO is first incubated with recombinant SMYD2 in methylation buffer reaction for 1 h at 30°C, and then 2 µCi of 3H-AdoMet is added to the mix and incubated overnight at 30°C. The reaction mixture is resolved by SDS-PAGE followed by autoradiography, Coomassie stain, or MS analysis[1].

Cell experiment:

Cells are seeded in 96-well plates at 2000 cells per well (optimum density for growth) in a total volume of 100 μL of medium containing 2% fetal bovine serum. Serially diluted BAY-598 in 100 μL of medium is added to the cells 12 h later. After 72 h of incubation, cell viability is assessed by an MTT assay according to the manufacturer's instructions[1].

References:

[1]. Reynoird N, et al. Coordination of stress signals by the lysine methyltransferase SMYD2 promotes pancreaticcancer. Genes Dev. 2016 Apr 1;30(7):772-85.
[2]. Eggert E, et al. Discovery and Characterization of a Highly Potent and Selective Aminopyrazoline-Based in Vivo Probe (BAY-598) for the Protein Lysine Methyltransferase SMYD2. J Med Chem. 2016 May 26;59(10):4578-600.