Pifithrin-α hydrobromide (Pifithrin-α hydrobromide) is a p53 inhibitor, inhibiting p53-dependent transactivation of p53-responsive genes.

小鼠腹腔注射3.6 μg/kg Pifithrin-α,抑制Dex诱导的胸腺退化.Pifithrinα（2 mg/kg）与对照组相比,在大脑中动脉暂时闭塞的大鼠中导致运动障碍程度显著降低.在小鼠大脑中动脉闭塞治疗前30分钟,Pifithrin-α（2 mg/kg i.p.）减少缺血性脑损伤并保护海马神经元免受兴奋毒性损伤.C57BL和Balb/c小鼠腹腔注射2.2 mg/kg Pifithrin-α,使小鼠完全免受60％致死率的γ射线照射造成的损伤.通过Tunel和caspase 3染色,观察到 Pifithrin-α明显降低大鼠的细胞凋亡.当在中风发作后一小时内施用药物时,Pifithrinα处理的动物具有较少的运动障碍和较小的梗塞.与空白对照组相比,Pifithrin-α处理7天后,明显降低大鼠的运动障碍评分.

10 μM Pifithrin-α抑制Dox,Etoposide,Taxol和Cytosine arabinoside诱导的C8细胞凋亡。10 μM Pifithrin-α降低热休克转录因子的激活,且增强细胞对热的敏感性。10 μM Pifithrin-α降低HeLa细胞中糖皮质激素受体的激活,且保护小鼠胸腺免受Dexamethasone诱导的凋亡。100–200 nM Pifithrin-α完全抑制海马细胞中Camptothecin诱导的p53 DNA结合水平和p53应答基因Bax的增加。200 nM Pifithrin-α保护培养的海马神经元免受DNA损伤剂诱导的死亡。200 μM Pifithrin-α稳定线粒体功能,抑制caspase活化,保护培养的海马神经元免受谷氨酸和β-淀粉样肽诱导的死亡。 Pifithrin-α抑制人类二倍体成纤维细胞对DNA损伤的p53依赖性生长抑制,但对p53缺陷型成纤维细胞没有影响。Pifithrin-α可调节p53的核输入或输出（或两者同时）,也可降低核p53的稳定性。Pifithrin-α可以抑制热休克和糖皮质激素受体信号,但不影响NF-κB信号。

The ligand binding competition assays are performed. Cytosolic cell extracts from Hepa-1 cells are generated by the resuspension of the cell pellets in HEDG buffer [25 mM Hepes, 1 mM EDTA, 1 mM dithiothreitol, and 10% (v/v) glycerol, pH 7.5] containing 0.4 mM leupeptin, 4 mg/mL aprotinin, and 0.3 mM phenylmethylsulfonyl fluoride, homogenization, and centrifugation at 100,000 g for 45 min. Aliquots of the supernatant (120 μg) are incubated at room temperature for 2 h with the indicated concentrations of Pifithrin-α in the presence of 3 nM [3H]TCDD in HEDG buffer. After incubation on ice with hydroxyapatite for 30 min, HEDG buffer with 0.5% Tween 80 is added. The samples are centrifuged, washed twice, resuspended in 0.2 mL of scintillation fluid, and subjected to scintillation counting. Nonspecific binding is determined using a 150-fold molar excess of TCDF and subtracted from the total binding to obtain the specific binding. The specific binding is reported relative to [3H]TCDD alone[2].

At the end of cell treatments, the number of attached cells is estimated by staining with 0.25% crystal violet in 50% methanol, followed by elution of the dye with 1% SDS. Optical density (530 nm) reflecting the number of stained cells is determined with a Bio-Tek EL311 microplate reader. Cell viability in suspension of short term culture of primary thymocytes is determined by their staining with 0.1% of methyl blue and microscopic counting of blue (dead) cells.(Only for Reference)