Entinostat (MS-275) is an HDAC class I selective inhibitor of HDAC1, HDAC2 and HDAC3 (IC50=243/453/248 nM) with oral activity. Entinostat has antitumor activity.

HDAC1:0.18 μM(cell free), HDAC3:0.74 μM(cell free)

方法: 多种肿瘤细胞用 Entinostat 处理 72 h，使用 Resazurin solution 检测细胞活力。
结果: Entinostat 抑制肿瘤细胞增殖，平均 IC50 为 2.57 µM。[1]
方法: 人胰腺癌细胞 PANC-1 和 SUIT2 Clone 1 用 Entinostat (1-50 µM) 处理 72 h，使用 Western Blot 方法检测靶点蛋白表达水平。
结果: Entinostat 引起细胞组蛋白 H3 乙酰化的剂量依赖性增加，对总组蛋白 H3 蛋白水平没有影响，证实了 Entinostat 抑制癌症细胞系中 HDAC 的去乙酰活性。[2]

方法: 为检测体内抗肿瘤活性，将 Entinostat (12.5-49 mg/kg，0.05 N HCl+0.1% Tween 80) 口服给药给携带人类肿瘤异种移植物的 nude 小鼠，每周五次，持续四周。
结果: 49 mg/kg的 Entinostat 对 KB-3-1、4-1St 和 St-4 肿瘤系显示出显著的抗肿瘤作用，对 Capan-1 肿瘤显示出中等的作用。24.5 mg/kg 和 12.3 mg/kg 的药物对这些肿瘤也显示出显著的效果。24.5 mg/kg 的剂量对 A2780 和 HT-29 也显著有效，对 Calu-3 也中等有效。[3]

The HDAC enzyme activity assay was done as described. Briefly, 40 μl HeLa cell nuclear extract, 29 μl enzyme buffer [15 mM Tris HCl pH 8.1, 0.25 mM EDTA, 250 mM NaCl, 10% (v/v) glycerol]; for recombinant HDAC isoenzymes, 0.1 mg/ml bovine serum albumin (BSA was added) and 1 μl compound were added per well of a microtiter plate. The reaction was started by addition of 30 μl substrate (Ac-NH-GGK(Ac)-AMC final 25 μM). After incubation for 90 min at 30°C, reaction was terminated by adding 25 μl stop solution (50 mM Tris HCl pH 8, 100 mM NaCl, 0.5 mg/ml trypsin, 2 μM TSA). After 40 min incubation at room temperature, fluorescence was measured using a Wallac Victor 1420 multilabel counter (Excitation 355 nm, Emission 460 nm). The HDAC1, 3, 6 and 8 assays were done with slight modifications. About 14 ng/well HDAC1, 2 ng/well HDAC3 or 10 ng/well HDAC6 were incubated with 6, 25 or 10 lM Ac-NH-GGK(Ac)-AMC, respectively, for 2 or 3 hr at 30°C. In contrast, 100 ng/well HDAC8 were incubated with 50 μM Ac-NH-RHK(Ac)K(Ac)-AMC for 3 hr at 30°C. Termination of the reaction and all further steps were done as described earlier for HeLa cell nuclear extracts. For the enzyme kinetic studies with HDAC1, selected HDAC inhibitor (around IC50 value), as well as Ac-NH-GGK(Ac)-AMC substrate (up to 100 μM) concentrations, were evaluated under standard conditions as described earlier [1].

Cancer cells (5 × 10^3) were seeded into each well of 96-well plates and were cultured with graded concentrations of the drugs for 3 days. The cells were stained with 0.1 mg/ml neutral red for 1 h in a CO2-incubator, and, after aspiration of the medium, OD540 of the neutral red solubilized with 50 μl of ethanol and 150 μl of 0.1 M Na2HPO4 was measured. The IC50 value was determined by plotting growth inhibition of the cells against the logarithm of the drug concentration [2].

A2780 cells (9 × 10^6) grown in vitro were suspended in PBS and were injected subcutaneously into the flank of nude mouse. For the other tumor lines, KB-3-1, HCT-15, 4-1St, Calu-3, St-4, Capan-1, and HT-29, tumors were passaged several times before starting in vivo antitumor testing, and a tumor lump (2–3 mm in diameter) was transplanted subcutaneously into the flank of a nude mouse by using a trocar needle. Treatment (four or five mice in each experimental group) with the drugs was started after the tumors were confirmed to have grown in the body (tumor size, 20–100 mm3). MS-27-275 and compound 2, both dissolved with 0.05 N HCl, 0.1% Tween 80, and 5-fluorouracil (5-FU) and diluted with physiological saline, were administered orally once daily 5 days per week for 4 weeks. Tumor length and width were monitored twice weekly, and tumor volume was calculated as described [2].