GSK126 (GSK2816126A) is an EZH2 methyltransferase inhibitor (IC50=9.9 nM) that is potent and selective. GSK126 has antitumor activity, inhibiting tumor cell proliferation and suppressing angiogenesis.

EZH2:9.9 nM

方法：ALL 细胞系用 GSK126 (70 pmol/L-36 µmol/L) 处理 6 天，使用 CellTiter-Glo 检测细胞生长。
结果：8 种 ALL 细胞系中，A687V EZH2 突变体 SUP-B8 细胞系对 GSK126 表现出最大的敏感性，IC50 为 441 nmol/L。EZH2 WT 细胞系对 GSK126 表现出一定范围的敏感性，IC50 值在 2.5-7.8 µmol/L 之间。[1]
方法：人类 B 细胞淋巴瘤细胞 KARPAS-422 用 Gilteritinib (25-2000 nM) 处理 72 h，使用 Western Blot 检测靶点蛋白表达水平。
结果：GSK126 对 H3K27me3 的抑制作用最强，H3K27me1 在最高抑制剂浓度下仅被微弱还原。[2]

方法：为测试体内抗肿瘤活性，将 GSK126 (50-150 mg/kg 每天一次；或 300 mg/kg 每周两次) 腹腔注射给携带人类 B 细胞淋巴瘤 KARPAS-422 的小鼠，持续五周。
结果：GSK126 显著抑制小鼠中 EZH2 突变的 DLBCL 异种移植物的生长。[2]

EZH2 assay: The five-member PRC2 complex (Flag–EZH2, EED, SUZ12, AEBP2, RbAp48) containing either wild-type or mutant EZH2 is prepared. GSK126 is dissolved in DMSO and tested at concentrations of 0.6?nM to 300?nM with a final DMSO concentration of 2.5%. In contrast to wild-type EZH2 which prefers H3K27me0 as a substrate in vitro, EZH2 Y641 mutants prefer H3K27me2 and have little activity with H3K27me0 or H3K27me1. The A677 g mutant is distinct from both the wild-type and Y641 mutant forms of EZH2 in that it efficiently methylates H3K27me0, H3K27me1, and H3K27me2; therefore, histone H3 peptides (residues 21–44; 10?μM final) with either K27me0 (wild type, A677 g EZH2), K27me1 (A677 g EZH2), or K27me2 (A677 g, Y641N, Y641C, Y641H, Y641S and Y641F EZH2) are used as methyltransferase substrates. GSK126 is added to plates followed by addition of 6?nM EZH2 complex and peptide. As the potency of GSK126 is at or near the tight binding limit of an assay run at [SAM] = Km, IC50 values are measured at a high concentration of the competitive substrate SAM relative to its Km (7.5 μM SAM where the SAM Km is 0.3?μM). Under these conditions, the contribution from the enzyme concentration becomes relatively small and accurate estimates of Ki can be calculated. Reactions are initiated with [3H]-SAM, incubated for 30 min, quenched with the addition of 500-fold excess unlabelled SAM, and the methylated product peptide is captured on phosphocellulose filters according to the vendor supplied protocol for MSPH Multiscreen plates. Plates are read on a TopCount after adding 20?μL of Microscint-20 cocktail. Apparent Ki values are calculated using the Cheng–Prusoff relationship for a competitive inhibitor. IC50=Ki (1+[S]/Km)+[E]/2, where E is the enzyme and S is the substrate.

The optimal cell seeding is determined empirically for all cell lines by examining the growth of a wide range of seeding densities in a 384-well format to identify conditions that permitted proliferation for 6?days. Cells are then plated at the optimal seeding density 24 h before treatment (in duplicate) with a 20-point two fold dilution series of GSK126 or 0.15% DMSO. Plates are incubated for 6?days at 37°C in 5% CO2. Cells are then lysed with CellTiter-Glo (CTG) and chemiluminescent signal is detected with a TECAN Safire2 microplate reader. In addition, an untreated plate of cells is harvested at the time of compound addition (T0) to quantify the starting number of cells. CTG values obtained after the 6?day treatment are expressed as a percent of the T0 value and plotted against compound concentration. Data are fit with a four-parameter equation to generate a concentration response curve and the concentration of GSK126 required to inhibit 50% of growth (growth IC50) is determined.(Only for Reference)