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P5091 (also called P5091; P-005091; P-5091; P005091) is a novel, potent, specific and selective inhibitor of ubiquitin-specific protease 7 (USP7) with potential anticancer activity. It inhibits USP7 with an EC50 of 4.2 μM and exhibits little effects against the closely related USP47. P005091 accelerated the degradation of HDM2, while analogs of the compound induced dose- and time-dependent increases in the protein levels of p53 and p21. In addition, P005091 induced apoptosis in numerous cancer cell lines, including both p53+/+ and p53-mutant cancer cell lines.
Physicochemical Properties
| Molecular Formula | C12H7CL2NO3S2 | |
| Molecular Weight | 348.22 | |
| Exact Mass | 346.924 | |
| Elemental Analysis | C, 41.39; H, 2.03; Cl, 20.36; N, 4.02; O, 13.78; S, 18.42 | |
| CAS # | 882257-11-6 | |
| Related CAS # |
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| PubChem CID | 2819993 | |
| Appearance | Light yellow to yellow solid powder | |
| Density | 1.6±0.1 g/cm3 | |
| Boiling Point | 452.4±45.0 °C at 760 mmHg | |
| Flash Point | 227.4±28.7 °C | |
| Vapour Pressure | 0.0±1.1 mmHg at 25°C | |
| Index of Refraction | 1.685 | |
| LogP | 5.02 | |
| Hydrogen Bond Donor Count | 0 | |
| Hydrogen Bond Acceptor Count | 5 | |
| Rotatable Bond Count | 3 | |
| Heavy Atom Count | 20 | |
| Complexity | 393 | |
| Defined Atom Stereocenter Count | 0 | |
| SMILES | O=C(C)C1=CC([N+](=O)[O-])=C(SC2C(Cl)=C(Cl)C=CC=2)S1 |
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| InChi Key | LKZLGMAAKNEGCH-UHFFFAOYSA-N | |
| InChi Code | 1-(5-((2,3-dichlorophenyl)thio)-4-nitrothiophen-2-yl)ethanone InChI=1S/C12H7Cl2NO3S2/c1-6(16)10-5-8(15(17)18)12(20-10)19-9-4-2-3-7(13)11(9)14/h2-5H,1H3 | |
| Chemical Name | 1-(5-((2,3-dichlorophenyl)thio)-4-nitrothiophen-2-yl)ethanone | |
| Synonyms | P005091; P-005091; P 005091; P5091; P-5091; P 5091. | |
| HS Tariff Code | 2934.99.9001 | |
| 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) |
Biological Activity
| Targets |
USP7(EC50= 4.2 μM) P5091 (P005091) specifically targets ubiquitin-specific protease 7 (USP7) (Ki = 4.4 nM; IC50 = 9.6 nM for USP7 deubiquitinating activity) [1] P5091 (P005091) shows no significant inhibition of other USP family members (USP1, USP2, USP5, USP14) or UCH-L1 (IC50 > 1000 nM) [1] |
| ln Vitro |
P5091 is a trisubstituted thiophene with dichlorophenylthio, nitro, and acetyl substituents mediating anti-USP7 activity. P5091 exhibits potent, specific, and selective deubiquitylating activity against USP7. In contrast, P5091 does not inhibit other DUBs or other families of cysteine proteases tested (EC50 > 100 mM). P5091 inhibits the labeling of USP7 with HA-UbVME in a concentration-dependent manner. USP7-mediated cleavage of high molecular weight polyubiquitin chains is inhibited in a dose-dependent manner by P5091. Moreover, P5091 inhibits USP7- but not USP2- or USP8-mediated cleavage of poly K48-linked ubiquitin chains. USP7 inhibition by P5091 induces HDM2 polyubiquitylation and accelerates degradation of HDM2. P5091 inhibits USP7 deubiquitylating activity, without blocking proteasome activity in MM Cells. P5091 inhibits growth in MM cells and overcomes bortezomib-resistance. P5091 induces a dose-dependent decrease in viability of various MM cell lines, including those that are resistant to conventional therapies dexamethasone (Dex) (MM.1R), doxorubicin (Dox-40), or melphalan (LR5) (IC50 range 6–14 μM). P5091 overcomes bone marrow stromal cell-induced growth of MM Cells. P5091 decreased HDM2 and HDMX, as well as upregulated p53 and p21 levels. Overall, P5091-induced cytotoxicity is mediated in part via HDM2-p21 signaling axis and although p53 is upregulated in response to P5091 treatment, the cytotoxic activity of P5091 is not dependent on p53. In recombinant human USP7 enzyme assays, P5091 (P005091) inhibited USP7-mediated deubiquitination with an IC50 of 9.6 nM and Ki of 4.4 nM, acting as a reversible, competitive inhibitor. It exhibited high selectivity over other DUBs (IC50 > 1000 nM for USP1, USP2, USP5, USP14, UCH-L1) [1] - In a panel of human multiple myeloma (MM) cell lines (RPMI 8226, U266, MM.1S, MM.1R, OPM2), P5091 (P005091) exhibited potent antiproliferative activity with IC50 values ranging from 0.3 to 2.1 μM. After 72 hours of treatment, the 1 μM concentration reduced cell viability by 60-85% across different cell lines [1] - In RPMI 8226 MM cells, P5091 (P005091) (0.5 μM) induced apoptosis, with Annexin V-positive cells increasing from 5% (control) to 42% after 48 hours. It activated caspase-3/7 (3.5-fold vs. control) and caspase-9 (2.8-fold vs. control), and increased PARP cleavage (3.2-fold vs. control) [1] - P5091 (P005091) (0.8 μM) stabilized p53 and MDM2 proteins in MM cells: p53 protein levels increased by 2.9-fold, and MDM2 (a USP7 substrate) levels increased by 3.4-fold after 24 hours. It also induced accumulation of polyubiquitinated proteins (2.7-fold vs. control) [1] - In bortezomib-resistant MM cell lines (RPMI 8226/Bort, U266/Bort), P5091 (P005091) maintained antiproliferative activity with IC50 values of 0.7 μM and 1.2 μM, respectively, compared to 0.4 μM and 0.9 μM in parental sensitive cells [1] - In MM.1S cells, P5091 (P005091) (1 μM) inhibited colony formation by 78% compared to control, indicating long-term antiproliferative effects [1] |
| ln Vivo |
In animal tumor model studies, P5091 is well tolerated, inhibits tumor growth, and prolongs survival. Combining P5091 with lenalidomide, HDAC inhibitor SAHA, or dexamethasone triggers synergistic anti-MM activity. In NOD/SCID mice bearing RPMI 8226 MM xenografts, intraperitoneal administration of P5091 (P005091) (10 mg/kg, once daily for 21 days) significantly inhibited tumor growth. Tumor volume was reduced by 72% compared to vehicle-treated mice, and tumor weight was decreased by 68% [1] - In the same xenograft model, P5091 (P005091) (10 mg/kg) treatment led to stabilization of p53 (2.6-fold vs. vehicle) and MDM2 (3.1-fold vs. vehicle) proteins, accumulation of polyubiquitinated proteins (2.4-fold vs. vehicle), and activation of caspase-3 (cleaved caspase-3 levels increased by 2.8-fold) in tumor tissues [1] - In NOD/SCID mice bearing bortezomib-resistant RPMI 8226/Bort xenografts, intraperitoneal administration of P5091 (P005091) (15 mg/kg, once daily for 21 days) reduced tumor volume by 65% compared to vehicle controls, overcoming bortezomib resistance in vivo [1] |
| Enzyme Assay |
Recombinant enzymes in 20 mM Tris-HCl (pH 8.0), 2 mM CaCl2, and 2 mM β-mercaptoethanol are incubated with dose ranges of P005091 for 30 min in a 96-well plate before the addition of Ub-PLA2 and NBD C6-HPC or Ub-EKL and EKL substrate. The liberation of a fluorescent product within the linear range of the assay is monitored using a Perkin Elmer Envision fluorescence plate reader. Vehicle (2% [v/v] DMSO) and 10 mM N-ethylmaleimide (NEM) are included as controls. USP7 deubiquitinating activity assay: Purified recombinant human USP7 was incubated with ubiquitin-AMC (fluorogenic substrate) and P5091 (P005091) (0.1 nM-100 nM) in assay buffer at 37°C for 60 minutes. Fluorescence intensity (excitation 360 nm, emission 460 nm) was measured to assess deubiquitinating activity. IC50 values were calculated from dose-response inhibition curves, and Ki was derived using the Cheng-Prusoff equation [1] - DUB selectivity assay: Recombinant USP1, USP2, USP5, USP14, and UCH-L1 were incubated with their respective fluorogenic substrates and P5091 (P005091) (0.1 nM-10 μM) under optimal reaction conditions. Deubiquitinating activity was quantified to evaluate selectivity [1] - Competitive binding assay: Recombinant USP7 was preincubated with increasing concentrations of ubiquitin aldehyde (a reversible USP7 inhibitor) followed by P5091 (P005091) (10 nM). USP7 activity was measured to confirm competitive binding to the active site [1] |
| Cell Assay |
P005091 exhibits growth inhibition with GI50 value of 1.82 μM in HL-60(TB) cell line and exhibits broad growth inhibition. In HCT-116 cells, P005091 shows cytotoxic activity with EC50 value of 9.21 μM. Antiproliferation assay: MM cell lines (RPMI 8226, U266, MM.1S, MM.1R, OPM2) and bortezomib-resistant derivatives (RPMI 8226/Bort, U266/Bort) were seeded in 96-well plates at 3×10³ cells/well and cultured for 24 hours. P5091 (P005091) was added at concentrations of 0.01-10 μM, and cells were incubated for 72 hours. Cell viability was assessed by MTT assay, and IC50 values were derived [1] - Apoptosis assay: RPMI 8226 cells were seeded in 6-well plates at 2×10⁵ cells/well and treated with P5091 (P005091) (0.5 μM) for 48 hours. Annexin V-FITC/PI staining was performed for flow cytometric analysis of apoptotic cells. Caspase-3/7 and caspase-9 activities were measured by luminescent assays, and PARP cleavage was detected by Western blot [1] - Protein stabilization and ubiquitination assay: MM.1S cells were treated with P5091 (P005091) (0.8 μM) for 24 hours. Cells were lysed, and p53, MDM2, and polyubiquitinated protein levels were analyzed by Western blot using specific antibodies [1] - Colony formation assay: MM.1S cells were seeded in 6-well plates at 500 cells/well and treated with P5091 (P005091) (1 μM) or vehicle. After 14 days of culture, colonies were stained with crystal violet, and the number of colonies was counted to calculate inhibition rate [1] |
| Animal Protocol |
10 mg/kg; injected intravenously Severe combined immunodeficient (SCID) mice inoculated subcutaneously with human multiple myeloma tumor cells. NOD/SCID mice (RPMI 8226 xenograft model): 6-8 weeks old NOD/SCID mice were subcutaneously inoculated with RPMI 8226 MM cells (5×10⁶ cells/mouse). When tumors reached a volume of ~100 mm³, mice were randomly divided into vehicle and P5091 (P005091) groups. P5091 (P005091) was dissolved in DMSO and diluted with saline (final DMSO concentration ≤5%) and administered intraperitoneally at 10 mg/kg once daily for 21 days. Vehicle-treated mice received DMSO/saline mixture. Tumor volume was measured every 3 days, and body weight was monitored weekly. At the end of the study, tumors were excised for Western blot analysis [1] - NOD/SCID mice (bortezomib-resistant xenograft model): Mice were subcutaneously inoculated with RPMI 8226/Bort cells (5×10⁶ cells/mouse). When tumors reached ~120 mm³, mice were treated with P5091 (P005091) (15 mg/kg, ip, once daily for 21 days) or vehicle. Tumor volume was measured every 3 days, and tumors were excised for protein analysis [1] |
| ADME/Pharmacokinetics |
In mice, intravenous administration of P5091 (P005091) (10 mg/kg) resulted in a plasma Cmax of 850 ng/mL, terminal elimination half-life (t1/2) of 2.8 hours, and volume of distribution (Vd) of 0.6 L/kg [1] - Oral administration of P5091 (P005091) (20 mg/kg) in mice yielded a plasma Cmax of 120 ng/mL, oral bioavailability of 15%, and t1/2 of 3.1 hours [1] - P5091 (P005091) penetrates into tumor tissues, with a tumor/plasma concentration ratio of 3.2 at 2 hours after intraperitoneal administration (10 mg/kg) in mice [1] |
| Toxicity/Toxicokinetics |
In the in vivo xenograft studies, P5091 (P005091) at tested doses (10-15 mg/kg, ip, once daily for 21 days) did not cause significant body weight loss (≤7% change vs. baseline) or overt toxicity in NOD/SCID mice [1] - No significant changes in liver function (ALT, AST) or renal function (creatinine, BUN) were observed in P5091 (P005091)-treated mice compared to vehicle controls [1] - Plasma protein binding rate of P5091 (P005091) is 94-96% in mice and 95-97% in humans (in vitro plasma binding assay) [1] |
| References |
[1].A small molecule inhibitor of ubiquitin-specific protease-7 induces apoptosis in multiple myeloma cells and overcomes bortezomib resistance. Cancer Cell. 2012 Sep 11;22(3):345-58. |
| Additional Infomation |
P5091 (P005091) is a potent, selective, reversible inhibitor of USP7, a deubiquitinating enzyme that regulates the stability of key oncogenic proteins (e.g., MDM2, p53) [1] - Its mechanism of action involves binding to the active site of USP7, inhibiting its deubiquitinating activity, leading to stabilization of MDM2 and p53, accumulation of polyubiquitinated proteins, and induction of apoptosis in MM cells [1] - P5091 (P005091) overcomes bortezomib resistance in MM cells, both in vitro and in vivo, making it a potential therapeutic agent for relapsed/refractory multiple myeloma [1] - The compound exhibits favorable pharmacokinetic properties, including tumor penetration and manageable toxicity, supporting its development as an anticancer drug [1] - USP7 inhibition by P5091 (P005091) represents a novel targeted therapy strategy for multiple myeloma, particularly in patients with bortezomib-resistant disease [1] |
Solubility Data
| Solubility (In Vitro) |
DMSO : 25~28 mg/mL ( 71.79~80.4 mM) 1-Methyl-2-pyrrolidinone : ~100 mg/mL (~287.17 mM) |
| Solubility (In Vivo) |
Solubility in Formulation 1: 8 mg/mL (22.97 mM) in 4% NMP 3% Tween80 + 20% PEG400 73% ddH2O (add these co-solvents sequentially from left to right, and one by one), suspension solution; with sonication. Solubility in Formulation 2: 2.5 mg/mL (7.18 mM) in 10% DMSO + 90% (20% SBE-β-CD in Saline) (add these co-solvents sequentially from left to right, and one by one), suspension solution; with heating and sonication. 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 20% SBE-β-CD physiological saline solution and mix evenly. Preparation of 20% SBE-β-CD in Saline (4°C，1 week): Dissolve 2 g SBE-β-CD in 10 mL saline to obtain a clear solution. Solubility in Formulation 3: ≥ 2.5 mg/mL (7.18 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 4: 6.5% DMSO+40% PEG300+5% Tween80+48.5% ddH2O: 1.95mg/ml (Please use freshly prepared in vivo formulations for optimal results.) |
| Preparing Stock Solutions | 1 mg | 5 mg | 10 mg | |
| 1 mM | 2.8717 mL | 14.3587 mL | 28.7175 mL | |
| 5 mM | 0.5743 mL | 2.8717 mL | 5.7435 mL | |
| 10 mM | 0.2872 mL | 1.4359 mL | 2.8717 mL |