P 22077 是一种可渗透细胞的泛素特异性蛋白酶 7 (USP7) 抑制剂，EC50 为 8.01 μM。 P 22077 还抑制 USP47，EC50 为 8.74 μM。
Description:IC50: 8.6 nM (singleplexed); 7.8 nM (multiplexed)
USP7, also known as HAUSP, has been found to be critical in cancer progression due to its influence on the stability of the tumor suppressor p53. P022077 Inhibits ubiquitin-specific protease (USP) 7 and also inhibits the closely related deubiquitinase (DUB) USP4.
In vitro: P022077 had negligible activity versus DEN1 and SENP2core over the concentration range tested, but inhibited USP7 with an IC50 of 8 μM [1]. In another study Inhibiting, USP7 with the small-molecule inhibitor P22077 attenuates the p53-dependent apoptotic pathway by destabilizing Tip60. P22077, however, is still cytotoxic, and this is partly due to destabilization of Tip60 [2].
In vivo: P022077 is currently in in-vitro investigation and no animal in vivo study is ongoing.
Clinical trial: P022077 is currently in the preclinical development and no clinical trial is ongoing.
参考文献:
[1] Tian X, Isamiddinova NS, Peroutka RJ, Goldenberg SJ, Mattern MR, Nicholson B, Leach C. Characterization of selective ubiquitin and ubiquitin-like protease inhibitors using a fluorescence-based multiplex assay format. Assay Drug Dev Technol. 2011;9(2):165-73.
[2] Dar A, Shibata E, Dutta A. Deubiquitination of Tip60 by USP7 determines the activity of the p53-dependent apoptotic pathway. Mol Cell Biol. 2013;33(16):3309-20.
Kinase experiment: | Recombinant full length USP7, USP2 core, USP5, JOSD2, DEN1, PLpro core, and SENP2 catalytic core are generated. Amino terminal His6 tagged USP4, USP8, USP28, UCH-L1, UCH-L3, UCH-L5, and MMP13 are expressed in Escherichia coli. N-terminal His6 tagged USP15, USP20, and USP47 are expressed in Sf9 cells. All the recombinant proteins are purified by chromatography. Amino terminal tagged His6 Ub-PLA2 (Ub-CHOP), SUMO3-PLA2 (SUMO3-CHOP), ISG15-PLA2 (ISG15-CHOP), NEDD8-PLA2 (NEDD8-CHOP), Ub-EKL (Ub-CHOP2), and free catalytically active PLA2 are prepared[1]. |
Cell experiment: | Cell viability assays are assessed using the Cell Counting Kit-8 (CCK-8, WST-8[2-(2-methoxy-4-nitrophenyl)-3-(4-nitrophenyl)-5-(2,4-disulfophenyl)-2?H-tetrazolium, monosodium salt]). Cells are seeded in 96-well flat-bottomed plates at the density of 1 × 104 per well. After 24?h of incubation at 37°C, increasing concentrations of P 22077, Dox, VP-16, or their combinations are added to the wells. Twenty-four hours later, 10?μL of CCK-8 is added into each well and after 1?h of incubation, the absorbance is measure at 450?nm using the microplate reader. Each experiment is performed in replicates of six. Background reading of media only is used to normalize the results[2]. |
Animal experiment: | The orthotopic Neuroblastoma (NB) mouse model is used in the assay. Briefly, 1.5 × 106 human IMR-32, SH-SY5Y, or NGP cells with luciferase expression are surgically injected into the left renal capsule of 5-week-old female NCR nude mice. IMR-32, SH-SY5Y, and NGP-derived xenografts are allowed to grow for ~2-3 weeks before randomizing the mice into a control group and a P 22077 treatment group. Each group consists of three or six mice. Animals are treated with DMSO or P 22077 by intraperitoneal (i.p.) injection every day for 12, 14, or 21 days. At the end of the experiments, all mice are killed. Tumors and the right side control kidneys are resected, weighed, and photographed[2]. |
参考文献: [1]. Altun M, et al. Activity-based chemical proteomics accelerates inhibitor development for deubiquitylating enzymes. Chem Biol. 2011 Nov 23;18(11):1401-12. | |
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