PR-619 (2,6-Diamino-3,5-dithiocyanopyridine) is a DUB inhibitor that inhibits USP2/4/5/7/8 (EC50=7.2/3.93/8.61/6.86/4.9 μM). PR-619 induces endoplasmic reticulum stress and activates autophagy.

USP8:4.90 μM(EC50), SENP6 core:2.37 μM(EC50), UCHL3:2.95 μM(EC50), JOSD2:1.17 μM(EC50), USP4:3.93 μM(EC50)

方法: HEK293T 细胞用 PR-619 (5-50 µM) 处理 6 h，使用 Western Blot 检测靶点蛋白表达水平。
结果: PR-619 在 5 µM 及以上的浓度下干扰探针标记。PR-619 抑制了探针对 USP7 的标记，但同时靶向了相同浓度范围内的许多其他 DUB，这与其更广泛的抑制特征是一致的。[1]
方法: 白血病细胞系 K562 用 PR-619 (40 µM) 处理 2 h，使用 Immunofluorescence 检测靶标。
结果: PR-619 诱导了 TOP2A 和 TOP2B 荧光信号的不同分布，由大信号灶组成。FK2 泛素荧光信号与 TOP2A 和 TOP2B 部分重叠，SUMO2/3 也是如此。[2]

方法: 为检测体内抗肿瘤活性，将 PR-619 (10 mg/kg，每天一次) 和 cisplatin (10 mg/kg，每周三次) 腹腔注射给携带 T24 或 BFTC-905 异种移植物的 Nude 小鼠，持续三周。
结果: 与单药治疗相比，cisplatin 和 PR-619 的联合治疗对 T24 和 BFTC-905 的异种移植物肿瘤显示出最显著的抗肿瘤作用。[3]

Ub-PLA2 assay: Recombinant enzymes in 20 mM Tris-HCl, pH 8.0, 2 mM CaCl2 and 2 mM β-mercaptoethanol (DUB assay buffer) are preincubated with single doses or dose ranges of PR-619 or P22077 for 30 minutes in a 96 well plate before the addition of Ub-PLA2 and NBD C6-HPC. The liberation of a fluorescent product within the linear range of the assay is monitored at room temperature using a fluorescence plate reader. Vehicle (2%(v/v) DMSO) and 10 mM N-ethylmaleimide are included as controls. Where ≥60% inhibition is observed, EC50 values are determined using a sigmoidal dose response equation.

72 h hours later, 0.2 mg/mL resazurin prepared in phosphate-buffered saline is added to each well and the cells are incubated for an additional 3-6 h. The fluorescence of the resazurin reduction product is measured using Ex=535 nm and Em=590 nm filters on a fluorimeter. The EC50 values are calculated in Prism.(Only for Reference)