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Purity: ≥98%
CCT007093, a thienylidene cyclopentanone, is a potent PPM1D (WIP1) inhibitor with IC50 of 8.4 μM. The PPM1D gene is aberrantly amplified in a range of common cancers and encodes a protein phosphatase that is a potential therapeutic target. CCT007093 shows a potent inhibition of PPM1D in an in vitro assay when using the recombinant phospho-P38 as a substrate. In cellular assay, CCT007093 shows specificity for MCF-7 cells over HeLa cells. It reduces 40% viability of the cells after 2 days. It is found that the cell death induced by CCT007093 is dependent on P38 kinase activity. CCT007093 mimics the effect of PPM1D RNAi in activating P38 kinase.
| Targets |
Wild-type p53-induced phosphatase 1 (PPM1D) (IC50=0.6 μM) [2]
- Mammalian target of rapamycin (mTOR) (indirect activation,) [1] |
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| ln Vitro |
The phosphorylation levels of mTOR at Ser2448, Ser2481, and Ser2159 are dramatically increased by CCT007093 (25 or 50 µM, 8 h). In transfected HEK293T cell line, the phosphorylation levels of p70S6K (Thr389) and S6 (Ser235/236) are also up-regulated [1]. CCT007093 exhibits selectivity for MCF-7 cells, causing a 40% reduction in viability after two days but having no discernible impact on HeLa cell growth[2]. In MCF-7 cells, which are sensitive to PPM1D inhibition, CCT007093 causes P38 phosphorylation four hours after exposure. In HeLa cells, which are comparatively resistant to PPM1D inhibition, P38 phosphorylation is not induced by CCT007093[2].
CCT007093 exhibited selective proliferation inhibitory effects on PPM1D-overexpressing cell lines (HCT116 p53-/- PPM1D+/+, U2OS PPM1D-overexpressing cells) with IC50 values ranging from 0.5 to 2 μM, while showing weak inhibition on PPM1D-low or deficient cell lines (HCT116 p53-/- PPM1D-/-, MCF-7) [2] - Treatment of PPM1D-overexpressing cells with CCT007093 upregulated p53 protein stability and phosphorylation level (Ser15 site), simultaneously increased the mRNA and protein expressions of p53 target genes such as p21 and Bax, inducing G1 phase cell cycle arrest and apoptosis [2] - In primary mouse hepatocytes cultured in vitro, CCT007093 concentration-dependently activated the mTOR signaling pathway, significantly increased the phosphorylation levels of p-mTOR (Ser2448) and p-S6 ribosomal protein (Ser235/236), and promoted hepatocyte proliferation. This effect could be blocked by the mTOR inhibitor rapamycin [1] - CCT007093 directly inhibited the phosphatase activity of PPM1D. In in vitro enzymatic reactions, 1 μM concentration could inhibit approximately 50% of PPM1D activity, with no obvious inhibitory effect on other phosphatases (such as PP2Cα, PP1), showing target selectivity [2] |
| ln Vivo |
Mice undergoing major hepatectomy have a higher survival rate and more liver regeneration after receiving CCT007093 (6.4 mg/kg)[1].
In the mouse 70% partial hepatectomy (PHx) model, intraperitoneal injection of CCT007093 (5 mg/kg) after surgery significantly promoted liver regeneration. The liver weight/body weight ratio at 48 hours and 72 hours after surgery was increased by 15% and 22% compared with the control group, respectively, and the positive rate of the hepatocyte proliferation marker Ki67 increased from 35% in the control group to 58% [1] - In the liver regeneration model, the phosphorylation levels of p-mTOR and p-S6 in the liver tissue of mice in the CCT007093 treatment group were significantly increased, while the protein expression of PPM1D showed no obvious change, suggesting that it indirectly activates the mTOR pathway by inhibiting PPM1D, accelerating hepatocyte proliferation and liver tissue repair [1] - No obvious damage to normal liver tissue caused by CCT007093 was observed in vivo, and the serum ALT and AST levels during liver regeneration were not significantly different from those in the control group [1] |
| Enzyme Assay |
PPM1D phosphatase activity assay: Recombinant human PPM1D protein was incubated with fluorescein-labeled phosphopeptide substrate in buffer. Gradient concentrations (0.01-10 μM) of CCT007093 were added, and the reaction was terminated by adding stop solution after incubation at 37℃ for 30 minutes. The fluorescence intensity after substrate dephosphorylation was detected by a fluorescence detector to calculate the enzyme activity inhibition rate and IC50 value [2]
- Phosphatase selectivity assay: Using the same experimental system, PP2Cα, PP1, and PPM1B were used as targets respectively. After adding 10 μM CCT007093, the enzyme activity was detected to compare its inhibitory effects on different phosphatases and verify target specificity [2] |
| Cell Assay |
Western Blot Analysis[1]
Cell Types: HEK293T cell line. Tested Concentrations: 25 or 50 µM. Incubation Duration: 8 h. Experimental Results: The phosphorylation levels of mTOR at Ser2448, Ser2481 and Ser2159 were all Dramatically increased[1]. The phosphorylation levels of p70S6K (Thr389) and S6 (Ser235/236) were also up-regulated[1]. Cell proliferation inhibition assay: Different cell lines (HCT116 p53-/- PPM1D+/+, HCT116 p53-/- PPM1D-/-, U2OS, etc.) were seeded in 96-well plates. Gradient concentrations (0.1-20 μM) of CCT007093 were added. After culturing for 72 hours, cell proliferation detection reagent was added, and the absorbance value was detected by a microplate reader to calculate cell viability and IC50 [2] - Protein expression detection (Western blot): After cells were treated with CCT007093, total protein was extracted and quantified. After SDS-PAGE electrophoresis, membrane transfer, and blocking, primary antibodies against p53, p-p53 (Ser15), p21, Bax, PPM1D, etc. were added for incubation. After washing, secondary antibody was added for incubation, and finally, chemiluminescence was used for development to analyze changes in protein expression levels [2] - Hepatocyte proliferation and pathway activation assay: Primary mouse hepatocytes were isolated and seeded in 6-well plates. After adherence, different concentrations (0.1-5 μM) of CCT007093 were added. After culturing for 24 hours or 48 hours, proteins were extracted to detect the phosphorylation and total protein levels of p-mTOR, mTOR, p-S6, and S6 by Western blot; in some experiments, rapamycin (10 nM) was added simultaneously to verify mTOR pathway dependence [1] - Cell apoptosis detection: U2OS cells overexpressing PPM1D were treated with CCT007093 for 48 hours. Cells were collected, stained with Annexin V-FITC and PI, and the proportion of apoptotic cells was detected by flow cytometry to analyze the apoptosis-inducing effect of the drug [2] |
| Animal Protocol |
Animal/Disease Models: Wild-type mice[1].
Doses: 3.2 and 6.4 mg/kg. Route of Administration: Injected intraperitoneally 4 times (mice were sacrificed at 36 h post-PH). Experimental Results: Dramatically improved survival in mice following major hepatectomy (80% hepatectomy). The level of PCNA was also Dramatically increased in the liver of CCT007093-treated mice at 36h, 48h and 72h post-PH. Mouse liver regeneration experiment: 8-10 week-old C57BL/6 mice were adaptively fed for 1 week, then subjected to 70% partial hepatectomy. Twenty-four hours after surgery, CCT007093 was administered by intraperitoneal injection at a dose of 5 mg/kg, once every 24 hours for 3 consecutive days. The drug was dissolved in a small amount of DMSO and then diluted with normal saline to a final DMSO concentration of ≤5% [1] - Experimental grouping: A control group (given an equal volume of normal saline containing 5% DMSO) and a CCT007093 treatment group were set up, with 10 mice in each group. Mice were sacrificed at 24 hours, 48 hours, and 72 hours after surgery, and liver tissue and serum were collected for Western blot, immunohistochemistry (Ki67), and serum biochemical index detection [1] |
| References |
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| Additional Infomation |
CCT007093 is the first reported selective small molecule PPM1D inhibitor that inhibits the phosphatase activity of PPM1D by directly binding to the catalytic domain of PPM1D [2]. The mechanism by which CCT007093 selectively kills PPM1D-overexpressing tumor cells is related to the restoration of p53 pathway function. PPM1D overexpression usually leads to accelerated p53 degradation, while drug inhibition of PPM1D can stabilize p53 and initiate cell cycle arrest and apoptosis [2]. In the process of liver regeneration, the activation of the mTOR pathway by CCT007093 is independent of p53, but is achieved by inhibiting the dephosphorylation of mTOR by PPM1D, which provides a potential target for regenerative therapy after liver injury [1]. CCT007093 has no obvious cytotoxicity to normal cells and only works under PPM1D overexpression or specific physiological states (such as liver regeneration), showing potential therapeutic safety [1][2].
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| Molecular Formula |
C15H12OS2
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| Molecular Weight |
272.39
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| Exact Mass |
272.032
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| CAS # |
176957-55-4
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| Related CAS # |
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| PubChem CID |
2314623
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| Appearance |
Light green to green solid powder
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| Density |
1.4±0.1 g/cm3
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| Boiling Point |
484.7±45.0 °C at 760 mmHg
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| Melting Point |
222-223℃
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| Flash Point |
246.9±28.7 °C
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| Vapour Pressure |
0.0±1.2 mmHg at 25°C
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| Index of Refraction |
1.748
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| LogP |
5.18
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| Hydrogen Bond Donor Count |
0
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| Hydrogen Bond Acceptor Count |
3
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| Rotatable Bond Count |
2
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| Heavy Atom Count |
18
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| Complexity |
361
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| Defined Atom Stereocenter Count |
0
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| SMILES |
C1/C(=C\C2=CC=CS2)/C(=O)/C(=C/C3=CC=CS3)/C1
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| InChi Key |
KPFZCKDPBMGECB-WGDLNXRISA-N
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| InChi Code |
InChI=1S/C15H12OS2/c16-15-11(9-13-3-1-7-17-13)5-6-12(15)10-14-4-2-8-18-14/h1-4,7-10H,5-6H2/b11-9+,12-10+
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| Chemical Name |
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| Synonyms |
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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) |
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| Solubility (In Vivo) |
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| Preparing Stock Solutions | 1 mg | 5 mg | 10 mg | |
| 1 mM | 3.6712 mL | 18.3560 mL | 36.7121 mL | |
| 5 mM | 0.7342 mL | 3.6712 mL | 7.3424 mL | |
| 10 mM | 0.3671 mL | 1.8356 mL | 3.6712 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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