CPI-169 racemate (CPI 169) is a potent, and selective EZH2 inhibitor with IC50 of 0.24 nM, 0.51 nM, and 6.1 nM for EZH2 WT, EZH2 Y641N, and EZH1, respectively.

EZH2 (Y641N):0.51 nM, EZH2 (WT):0.24 nM, EZH1:6.1 nM

在KARPAS-422细胞中，CPI-169呈剂量依赖性地抑制细胞活力，并在与ABT-199联合使用时产生协同抗增殖活性。在25种NHL细胞系中的16种中，CPI-169还以GI50 <5 μM的效果抑制细胞生长。[1]

在携带KARPAS-422异种移植瘤的小鼠中，CPI-169（200 mg/kg, s.c.）有效降低了H3K27me3的水平，并导致淋巴瘤肿瘤的退缩，同时未影响体重或引起任何明显的不良反应。[1]

Biochemical Assays: Compound potency is also assessed through incorporation of 3H-SAM into a biotinylated H3 peptide. Specifically, PRC2 containing either EZH1 (160 pM), wt EZH2 (40 pM), or Y641N mutant EZH2 (80 pM, both EZH2 prepared in-house) is pre-incubated with 3H-SAM (0.9 μM), 2 μM H3K27me3 activating peptide (H2N-RKQLATKAAR(Kme3)SAPATGGVKKP-amide) and compounds (as 10 point duplicate dose response titrations) for 120 min in a buffer consisting of 50 mM Tris (pH 8.5), 1 mM DTT, 0.07 mM Brij-35, 0.1% BSA, and 0.8% DMSO in a total volume of 12.5 μl in a black 384 well plate. Reaction is initiated with biotinylated H3 substrate peptides (H3K27me1 for wt EZH2, H3K27me2 for Y641N mutant EZH2; H2N-RKQLATKAAR(Kmen)SAPATGGVKKP-NTPEGBiot) as a 2 μM stock in 12.5 μL and allowed to react at room temperature for 5 h. Quenching is accomplished by addition of 20 μl of STOP solution (50 mM Tris (pH 8.5), 200 mM EDTA, 2 mM SAH). 35 μL of the quenched solution is transferred to Streptavidin Flashplates, incubated overnight, washed, and read in a TopCount Reader. For titrations all compound dilutions are in DMSO, final DMSO concentrations are 0.8% (v/v), and turnover is kept to less than < 5%. IC50s are calculated using non-linear least square four parameter fits (GraphPad 6.0).

Relative cell numbers are assessed by Cell Titer-Glo (CTG) luminescent cell viability assay using an Envision instrument. GraphPad Prism 6.0 is used for curve fitting, IC50/GI50 and Hill coefficient (H) calculations. The GI90 is calculated using the formula: EC90 = (90 /100-90)1/H * EC50.(Only for Reference)

Animal Models: Mice bearing KARPAS-422 subcutaneous xenograftsFormulation: 10% DMSO + 60% polytheylene glycol 400 + 30% ddH2ODosages: 200 mg/kg, BIDAdministration: s.c.