In vitro activity: UNC0379 is competitive with the peptide substrate and noncompetitive with the cofactor SAM

Kinase Assay: Enzyme assay The interaction between UNC0379 and protein SETD8 was further explored using a ProteOn XPR36 biosensor (Bio-Rad Laboratories, Inc.) at 25 °C. PBS buffer (phosphate buffered saline, pH 7.4) supplemented with 0.005% Tween-20 was used as the running buffer. A GLH (catalog no. 176-5013, Bio-Rad Laboratories, Inc.) sensor chip was first activated by flowing a mixture of 20 mM sulfo-NHS and 20 mM EDC over the chip surface for 5 min at a flow rate of 30 μL/min. SETD8 was then diluted to 20 and 10 μg/mL in 5 mM NaOAc, pH 5.0, and immobilized onto two ligand channels (30 μL/min for 2 min). The surface was then deactivated by flowing 1 M ethanolamine for 5 min at a flow rate 60 μL/min. A blank injection of the running buffer was made, after which four concentrations of the compound in the running buffer (100, 33.3, 11.1, and 3.7 μM) and a running buffer were injected simultaneously at a flow rate of 50 μL/min for 60 s. The sensorgrams obtained at the four concentrations of the compound were fit simultaneously after subtracting a ligand reference (inner spot) and a double compound reference (buffer) using a Langmuir model to obtain on (ka) and off (kd) rates. The kinetic KD was calculated based on the on and off rates for three replicates. An equilibrium analysis of the data was also performed to calculate the KD.