Nutlin-3 is an MDM2 antagonist that inhibits MDM2-p53 interaction (Ki=90 nM) and activates p53. Nutlin-3 binds preferentially to the p53-binding pocket of MDM2, leading to stabilization of p53 and activation of the p53 pathway. Nutlin-3 has antitumor activity.

MDM2-p53:0.09 μM

方法: 22RV1 (p53 WT)、DU145 (p53 Mut) 和 PC-3 (p53 Null) 细胞用 Nutlin-3 (2-10 µM) 处理 48 h，通过 Hoechst 33342 染色检测细胞凋亡。
结果: Nutlin-3 诱导 22RV1 细胞凋亡显著增加，而 p53 缺陷型 DU145 或 PC-3 细胞凋亡没有显著增加。Nutlin-3 在表达 WT p53 的细胞中优先增加凋亡。[1]
方法: H460 和 HCT116 p53+/+ 细胞用 Nutlin-3 (0-100 µM) 和 Inauhzin (0-100 µM) 处理 72 h，通过 WST assay 检测细胞活力。
结果: Inauhzin 和 Nutlin-3 在抑制这两种肿瘤细胞系的生长方面显示出明显的协同作用，因为在有效剂量范围内的 CI 值明显小于 1，特别是在使用较低剂量的 Nutlin-3 时。[2]

方法: 为检测体内抗肿瘤活性，将 Nutlin-3 (150 mg/kg，EtOH: Tween: 5% Glucose = 5:5:90；口服给药，每天两次，连续四天停止两天) 和 Inauhzin (15 mg/kg，腹腔注射，每天一次) 给药给携带 HCT116 p53+/+ 异种移植物的 SCID 小鼠，持续 21 天。
结果: 每种化合物在治疗 21 天后可以单独将平均肿瘤体积减少不到 20%。然而，当以相同剂量组合两种治疗方法时，肿瘤的最终平均体积减少了 60%。Inauhzin 和 Nutlin-3 可以激活 p53 并抑制动物异种移植物肿瘤的生长。[2]

Biacore study: Competition assay is performed on a Biacore S51. A Series S Sensor chip CM5 is utilized for the immobilization of a PentaHis antibody for capture of the His-tagged p53. The level of capture is ~200 response units (1 response unit corresponds to 1 pg of protein per mm 2). The concentration of MDM2 protein is kept constant at 300 nM. Nutlin-3 is dissolved in DMSO at 10 mM and further diluted to make a concentration series of Nutlin-3 in each MDM2 test sample. The assay is run at 25°C in running buffer (10 mM Hepes, 0.15 M NaCl, 2% DMSO). MDM2-p53 binding in the presence of Nutlin-3 is calculated as a percentage of binding in the absence of Nutlin-3 and IC50 is calculated

Nutlin-3 (NUT) is dissolved with DMSO (100 mM) and diluted with appropriate media[2]. Human non-small-cell lung carcinoma wild type p53-containing H460 and A549, human non-small-cell lung carcinoma p53-null H1299, and human colon cancer HCT116 (p53+/+ and p53-/-) cells are used. Cells (1.5×105) are plated into 6-well plates, and incubated at 37°C overnight. After treatment of Inauhzin and Nutlin-3 at the indicated concentrations for 48 h, cells are harvested, fixed in 70% ice-cold ethanol overnight at -20°C, resuspended in propidium iodide-solution (50 μg/mL PI, 0.1 mg/mL RNase A, 0.05% Tritin X-100 in PBS) for 40 min at 37°C, then analyzed for DNA content using a flow cytometer and proprietary software[2].