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Purity: ≥98%
P22077 (P-22077; P 22077) is a potent, cell-permeable and selective inhibitor of ubiquitin-specific protease USP7 (ubiquitin-specific protease 7) with potential antitumor activity. It inhibits USP7 with an EC50 of 8.6 μM, and also inhibits the closely related USP47. P22077 potently induces apoptosis in NB cells with an intact USP7-HDM2-p53 axis but not in NB cells with mutant p53 or without human homolog of MDM2 (HDM2) expression. P22077 also significantly augmented the cytotoxic effects of doxorubicin (Dox) and etoposide (VP-16) in NB cells with an intact USP7-HDM2-p53 axis. Moreover, P22077 was found to be able to sensitize chemoresistant LA-N-6 NB cells to chemotherapy.
| Targets |
USP7(EC50=8.01 μM);USP47(EC50=8.74 μM)
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| ln Vitro |
P 22077 has EC50 values of 8.01 μM and 8.74 μM, respectively, making it an inhibitor of DUB USP47 and USP7. A much smaller subset of DUBs is inhibited by P 22077 (15-45 μM). P 22077 at 25 μM inhibits DUBs in HEK293T cells[1].Neuroblastoma (NB) cells, such as IMR-32, NGP, CHLA-255, and SH-SY5Y cells, have significantly reduced cell viability when exposed to P 22077 (0–20 μM), but not NB-19 and SK-N-AS cells. P 22077 (10 μM) causes NB cells that express HDM2 and p53 wild-type cells to undergo apoptosis and increase p53 activity. P 22077 (5 μM) increases the cytotoxic effect of VP-16 and Dox on NB cells as well as the p53-mediated apoptosis that is induced by Dox and VP-16[2].
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| ln Vivo |
In a xenograft mouse model with IMR-32-derived tumors, P 22077 (15 mg/kg, i.p. for 21 days) demonstrates strong antitumor activities. P 22077 also shows antitumor effects in mice with tumors derived from SH-SY5Y and NGP after treatment at 10 mg/kg for 14 days and 20 mg/kg for 12 days, respectively[2].
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| Enzyme Assay |
Recombinant full length catalytic cores for SENP2, JOSD2, USP5, USP2, DEN1, PLpro, and USP7 are produced. Escherichia coli expresses amino terminal His6 tagged USP4, USP8, USP28, UCH-L1, UCH-L3, UCH-L5, and MMP13. Sf9 cells express USP15, USP20, and USP47 that are N-terminally His6 tagged. By using chromatography, all of the recombinant proteins are purified. A variety of substrates are prepared, including free catalytically active PLA2, SUMO3-PLA2 (SUMO3-CHOP), ISG15-PLA2 (ISG15-CHOP), NEDD8-PLA2 (NEDD8-CHOP), Ub-EKL (Ub-CHOP2), and amino terminal tagged His6 Ub-PLA2 (Ub-CHOP)[1].
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| Cell Assay |
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]) is used to evaluate cell viability assays. In 96-well plates with a flat bottom, cells are seeded at a density of 1 × 10^4 per well. Increasing concentrations of P 22077, Dox, VP-16, or their combinations are added to the wells following a 24-hour incubation period at 37°C. After adding 10 μL of CCK-8 to each well, wait 24 hours. After an hour of incubation, use the microplate reader to measure the absorbance at 450 nm. Every experiment is run in six replicates. Only media background reading is utilized to normalize the outcomes.
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| Animal Protocol |
The assay makes use of the orthotopic Neuroblastoma (NB) mouse model. In brief, 5-week-old female NCR nude mice have their left renal capsule surgically injected with 1.5 × 10^6 human IMR-32, SH-SY5Y, or NGP cells expressing luciferase. After allowing the IMR-32, SH-SY5Y, and NGP-derived xenografts to grow for about two to three weeks, mice are randomized into two groups: one for control and the other for P 22077 treatment. There are three or six mice per group. DMSO or P 22077 is administered intraperitoneally (i.p.) to animals once a day for a duration of 12, 14, or 21 days. All of the mice are killed when the experiments are over. The right side control kidneys are removed, weighed, and photographed along with any tumors.
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| References |
| Molecular Formula |
C12H7F2NO3S2
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| Molecular Weight |
315.32
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| Exact Mass |
314.98
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| Elemental Analysis |
C, 45.71; H, 2.24; F, 12.05; N, 4.44; O, 15.22; S, 20.34
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| CAS # |
1247819-59-5
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| Related CAS # |
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| Appearance |
Solid powder
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| SMILES |
CC(C1=CC([N+]([O-])=O)=C(SC2=CC=C(F)C=C2F)S1)=O
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| InChi Key |
RMAMGGNACJHXHO-UHFFFAOYSA-N
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| InChi Code |
InChI=1S/C12H7F2NO3S2/c1-6(16)11-5-9(15(17)18)12(20-11)19-10-3-2-7(13)4-8(10)14/h2-5H,1H3
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| Chemical Name |
1-(5-((2,4-difluorophenyl)thio)-4-nitrothiophen-2-yl)ethanone
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| Synonyms |
P22077; P-22077; P 22077.
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| HS Tariff Code |
2934.99.9001
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| Storage |
Powder -20°C 3 years 4°C 2 years In solvent -80°C 6 months -20°C 1 month |
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| Shipping Condition |
Room temperature (This product is stable at ambient temperature for a few days during ordinary shipping and time spent in Customs)
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| Solubility (In Vitro) |
DMSO : 50~63 mg/mL ( 158.57~199.79 mM)
Ethanol : ~1 mg/mL |
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| Solubility (In Vivo) |
Solubility in Formulation 1: ≥ 2.5 mg/mL (7.93 mM) (saturation unknown) in 10% DMSO + 90% Corn Oil (add these co-solvents sequentially from left to right, and one by one), clear solution.
For example, if 1 mL of working solution is to be prepared, you can add 100 μL of 25.0 mg/mL clear DMSO stock solution to 900 μL of corn oil and mix evenly. Solubility in Formulation 2: 2% DMSO+30% PEG300+2% Tween80+66% ddH2O: 3mg/ml View More
Solubility in Formulation 3: 5 mg/mL (15.86 mM) in 50% PEG300 50% Saline (add these co-solvents sequentially from left to right, and one by one), suspension solution; with ultrasonication (<60°C). |
| Preparing Stock Solutions | 1 mg | 5 mg | 10 mg | |
| 1 mM | 3.1714 mL | 15.8569 mL | 31.7138 mL | |
| 5 mM | 0.6343 mL | 3.1714 mL | 6.3428 mL | |
| 10 mM | 0.3171 mL | 1.5857 mL | 3.1714 mL |
*Note: Please select an appropriate solvent for the preparation of stock solution based on your experiment needs. For most products, DMSO can be used for preparing stock solutions (e.g. 5 mM, 10 mM, or 20 mM concentration); some products with high aqueous solubility may be dissolved in water directly. Solubility information is available at the above Solubility Data section. Once the stock solution is prepared, aliquot it to routine usage volumes and store at -20°C or -80°C. Avoid repeated freeze and thaw cycles.
Calculation results
Working concentration： mg/mL；
Method for preparing DMSO stock solution： mg drug pre-dissolved in μL DMSO (stock solution concentration mg/mL). Please contact us first if the concentration exceeds the DMSO solubility of the batch of drug.
Method for preparing in vivo formulation:：Take μL DMSO stock solution, next add μL PEG300, mix and clarify, next addμL Tween 80, mix and clarify, next add μL ddH2O，mix and clarify.
(1) Please be sure that the solution is clear before the addition of next solvent. Dissolution methods like vortex, ultrasound or warming and heat may be used to aid dissolving.
(2) Be sure to add the solvent(s) in order.
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