AZ505 is an effective and specific SMYD2 inhibitor (IC50: 0.12 μM).

SMYD2:0.12 μM

AZ505对SMYD2的IC50为0.12μM，这是对其他组蛋白甲基转移酶（如SMYD3（IC50>83.3μM）、DOT1L（IC50>83.3μM）和EZH2（IC50>83.3μM））IC50的>600倍[1]。AZ505未能抑制一系列蛋白质赖氨酸甲基转移酶的酶活性。AZ505被提名进行ITC结合研究，Kd为0.5μM。相比之下，p53底物肽的计算Kd为3.7μM[2]。

SMYD2 is expressed in insect cells and purified. Methylation (12 μL) reactions are carried out in TDT buffer (50 mM Tris-HCl [pH 9.0], 2 mM DTT, and 0.01% Tween 20) at room temperature using 1.25 nM SMYD2 protein, 200 nM SAM, and 100 nM biotinylated p53 peptide substrate (Biotin-aminohexanoyl-GSRAHSSHLKSKKGQSTSRH) in low volume 384-well plates. Following a 75 min incubation period, reactions are quenched by the addition of 5 μL of detection solution (20 mM HEPES [pH 7.4], 1.7 mg/mL BSA, 340 mM NaCl, 680 μM SAH, 0.04 mg/mL Streptavidin-coated AlphaScreen donor, and Protein A-coated acceptor beads), and 1 nM of a custom p53K370me1 polyclonal antibody. Reaction plates are incubated overnight in the dark at room temperature and read using an Envision 2101 Multi-label Reader. Compounds showing >50% inhibition of SMYD2 are nominated for concentration dose-response determination and are also subjected to an artifact assay. Seven compound concentrations are selected beginning at 30 μM with six half-log dilution steps. The artifact assay conditions are identical to those in the SMYD2 enzymatic activity assay, except for the absence of SMYD2 protein and the presence of 1 nM methylated p53 peptide. IC50 values are calculated from dose-response data using in-house software [2].