NSC632839 is a nonselective isopeptidase inhibitor that inhibits USP2, USP7, and SENP2 with EC50 values of 45±4 μM, 37±1 μM, and 9.8±1.8 μM, respectively. NSC632839 is not only a DUB (deubiquitylases) inhibitor, but also a deSUMOylase inhibitor. Conjugation or deconjugation of ubiquitin (Ub) or ubiquitin-like proteins (UBLs) to or from cellular proteins is a multifaceted and universal means of regulating cellular physiology, controlling the lifetime, localization, and activity of many critical proteins. Deconjugation of Ub or UBL from proteins is performed by a class of proteases called isopeptidases. The inhibitory activity of the nonselective isopeptidase inhibitor NSC 632839 against deubiquitylases and deSUMOylases was profiled.
Physicochemical Properties
| Molecular Formula | C21H22CLNO | |
| Molecular Weight | 339.86 | |
| Elemental Analysis | C, 74.22; H, 6.52; Cl, 10.43; N, 4.12; O, 4.71 | |
| CAS # | 157654-67-6 | |
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| PubChem CID | 5351362 | |
| Appearance | Light yellow to yellow solid powder | |
| Boiling Point | 522.7ºC at 760 mmHg | |
| Flash Point | 184ºC | |
| Vapour Pressure | 5.05E-11mmHg at 25°C | |
| LogP | 5.074 | |
| Hydrogen Bond Donor Count | 2 | |
| Hydrogen Bond Acceptor Count | 2 | |
| Rotatable Bond Count | 2 | |
| Heavy Atom Count | 24 | |
| Complexity | 432 | |
| Defined Atom Stereocenter Count | 0 | |
| InChi Key | ZOKZLTXPTLIWOJ-BYCVLTJGSA-N | |
| InChi Code | InChI=1S/C21H21NO.ClH/c1-15-3-7-17(8-4-15)11-19-13-22-14-20(21(19)23)12-18-9-5-16(2)6-10-18;/h3-12,22H,13-14H2,1-2H3;1H/b19-11+,20-12+; | |
| Chemical Name | 3,5-Bis[(4-methylphenyl)methylene]-4-piperidone hydrochloride | |
| Synonyms | NSC-632839; NSC 632839; NSC632839 | |
| 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 Note: Please store this product in a sealed and protected environment, avoid exposure to moisture. |
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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 |
USP2(EC50= 45±4 μM);USP7(EC50= 37±1 μM);SENP2(EC50= 9.8±1.8 μM)
NSC632839 is a nonselective isopeptidase inhibitor, with its targets including deubiquitylases (DUBs) such as USP2, USP7, and deSUMOylases such as SENP2. [1] |
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| ln Vitro |
NSC632839 is a nonselective isopeptidase inhibitor that inhibits USP2, USP7, and SENP2 with EC50 values of 45±4 μM, 37±1 μM, and 9.8±1.8 μM, respectively. NSC632839 is not only a DUB (deubiquitylases) inhibitor, but also a deSUMOylase inhibitor. Conjugation or deconjugation of ubiquitin (Ub) or ubiquitin-like proteins (UBLs) to or from cellular proteins is a multifaceted and universal means of regulating cellular physiology, controlling the lifetime, localization, and activity of many critical proteins. Deconjugation of Ub or UBL from proteins is performed by a class of proteases called isopeptidases. The inhibitory activity of the nonselective isopeptidase inhibitor NSC 632839 against deubiquitylases and deSUMOylases was profiled. 1. NSC632839 exhibited inhibitory activity against the isopeptidase activity of USP2 (a DUB). The inhibitory effect was evaluated using the Ub-PLA₂ assay, and the compound was shown to reduce the cleavage activity of USP2 towards Ub-PLA₂ in a concentration-dependent manner, though the exact inhibitory potency (e.g., IC50) was not quantified [1] 2. NSC632839 also inhibited the isopeptidase activity of USP7 (another DUB) in the Ub-PLA₂ assay, with a concentration-dependent inhibitory trend similar to that observed with USP2, but again, no specific numerical values for the inhibition were provided [1] 3. The compound displayed inhibitory activity against SENP2 (a deSUMOylase), as assessed by the UBL-PLA₂ assay system tailored for deSUMOylase activity. It reduced SENP2-mediated cleavage of SUMO-PLA₂ in a concentration-dependent fashion, without reported quantitative inhibitory parameters [1] 4. NSC632839 did not show inhibitory effects on free phospholipase A₂ (PLA₂) enzyme activity when tested in the PLA₂ substrate cleavage assay, indicating that its inhibitory action is specific to isopeptidases rather than the reporter enzyme PLA₂ [1] |
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| ln Vivo |
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| Enzyme Assay |
In a 96-well-plate, 40 nM USP2, 40 nM USP7, or 20 nM SENP2 is preincubated with a concentration range of NSC 632839 (NCI/NIH developmental therapeutics program) or control for 30 min before supplementation with an equal volume of 60 nM Ub-PLA2/40 μM NBD C6-HPC (USP2 or 7) or 20 nM SUMO3-PLA2/40 μM NBD C6-HPC (SENP2). Relative activity of the enzymes is determined by measuring the RFU values at single time points within the initial linear range (USP, 50 min; USP7, 50 min; and SENP2, 30 min). The RFU values within the initial linear range are normalized such that isopeptidase+vehicle=0% inhibition and isopeptidase+NEM=100% inhibition. The EC50 values are determined as above. The inhibitory activity of the test compound against the reporter enzyme PLA2 is performed as described above except there is no preincubation step and the data are normalized such that free PLA2+vehicle=0% inhibition and free PLA2+EDTA=100% inhibition. PLA2 activity is determined 8 min after the addition of the reagents. 1. USP2 Inhibitory Assay with Ub-PLA₂: A reaction system was prepared containing USP2 enzyme, 30 nM Ub-PLA₂ (the reporter substrate), 20 μM NBD C₆-HPC (the PLA₂ fluorescent substrate), and varying concentrations of NSC632839. The mixture was incubated under suitable conditions (temperature and incubation time were set based on assay optimization, with 45 min incubation at room temperature used in related experiments). The fluorescence signal generated by the cleavage of NBD C₆-HPC by the released PLA₂ was monitored to quantify the USP2 isopeptidase activity. The inhibitory effect of NSC632839 was determined by comparing the fluorescence intensity in the presence of the compound with that in the control group (without the inhibitor) [1] 2. USP7 Inhibitory Assay with Ub-PLA₂: The assay was conducted using the same experimental setup as the USP2 assay, with USP7 replacing USP2 as the target enzyme. Different concentrations of NSC632839 were added to the reaction system, and the fluorescence signal was measured after incubation to evaluate the compound's inhibitory effect on USP7 isopeptidase activity [1] 3. SENP2 Inhibitory Assay with SUMO-PLA₂: A SUMO-PLA₂ reporter substrate was used to establish a deSUMOylase activity assay system containing SENP2 enzyme and various concentrations of NSC632839. The PLA₂ substrate cleavage and fluorescence signal detection steps were consistent with the Ub-PLA₂ assay. The reduction in fluorescence signal reflected the inhibitory effect of NSC632839 on SENP2-mediated deSUMOylation activity [1] 4. Free PLA₂ Activity Assay: A reaction system containing free PLA₂ enzyme, NBD C₆-HPC substrate, and different concentrations of NSC632839 was prepared. The fluorescence signal was measured after incubation to verify whether the compound had a direct inhibitory effect on PLA₂ enzyme activity, ruling out non-specific inhibition of the reporter enzyme [1] |
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| Cell Assay | NSC 632839 inhibits ubiquitin isopeptidases as illustrated by its ability to inhibit z-LRGG-AMC cleavage by crude lysates in the mid-micromolar range. To further characterize NSC 632839 against purified enzymes, its inhibitory potential is determined against purified USP2, USP7, and SENP2 and demonstrated that NSC 632839 is not only a DUB inhibitor, but also a deSUMOylase inhibitor. Specifically, NSC 632839 inhibits USP2, USP7, and SENP2 with EC50 values of 45±4 μM, 37±1 μM, and 9.8±1.8 μM, respectively. Importantly, NSC 632839 does not inhibit the reporter enzyme PLA2 over the concentration range tested (1.2-150 μM), indicating that the reported inhibition is selective for isopeptidases. Moreover, the isopeptidase inhibitory activity of NSC 632839 is confirmed by the observation that it does not inhibit free PLA2 over the concentration range tested. | ||
| Animal Protocol |
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| References |
[1]. Characterization of ubiquitin and ubiquitin-like-protein isopeptidase activities. Protein Sci. 2008 Jun;17(6):1035-43. |
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| Additional Infomation |
1. NSC632839 is classified as a nonselective isopeptidase inhibitor, meaning it does not exhibit high specificity for a single type of isopeptidase but instead inhibits multiple members of the isopeptidase family, including DUBs (USP2, USP7) and deSUMOylases (SENP2) [1] 2. The profiling of NSC632839's inhibitory activity was conducted using the novel UBL-PLA₂ assay platform, which was demonstrated to be an effective tool for characterizing modulators of isopeptidase activity and provided a basis for evaluating the compound's potential as a research tool for studying isopeptidase function [1] |
Solubility Data
| Solubility (In Vitro) | DMSO : 6.2~7 mg/mL ( 18.24~20.59 mM ) |
| Solubility (In Vivo) |
Note: Listed below are some common formulations that may be used to formulate products with low water solubility (e.g. < 1 mg/mL), you may test these formulations using a minute amount of products to avoid loss of samples. Injection Formulations (e.g. IP/IV/IM/SC) Injection Formulation 1: DMSO : Tween 80： Saline = 10 : 5 : 85 (i.e. 100 μL DMSO stock solution → 50 μL Tween 80 → 850 μL Saline) *Preparation of saline: Dissolve 0.9 g of sodium chloride in 100 mL ddH ₂ O to obtain a clear solution. Injection Formulation 2: DMSO : PEG300 ：Tween 80 : Saline = 10 : 40 : 5 : 45 (i.e. 100 μL DMSO → 400 μLPEG300 → 50 μL Tween 80 → 450 μL Saline) Injection Formulation 3: DMSO : Corn oil = 10 : 90 (i.e. 100 μL DMSO → 900 μL Corn oil) Example: Take the Injection Formulation 3 (DMSO : Corn oil = 10 : 90) as an example, if 1 mL of 2.5 mg/mL working solution is to be prepared, you can take 100 μL 25 mg/mL DMSO stock solution and add to 900 μL corn oil, mix well to obtain a clear or suspension solution (2.5 mg/mL, ready for use in animals). Injection Formulation 4: DMSO : 20% SBE-β-CD in saline = 10 : 90 [i.e. 100 μL DMSO → 900 μL (20% SBE-β-CD in saline)] *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. Injection Formulation 5: 2-Hydroxypropyl-β-cyclodextrin : Saline = 50 : 50 (i.e. 500 μL 2-Hydroxypropyl-β-cyclodextrin → 500 μL Saline) Injection Formulation 6: DMSO : PEG300 : castor oil : Saline = 5 : 10 : 20 : 65 (i.e. 50 μL DMSO → 100 μLPEG300 → 200 μL castor oil → 650 μL Saline) Injection Formulation 7: Ethanol : Cremophor : Saline = 10： 10 : 80 (i.e. 100 μL Ethanol → 100 μL Cremophor → 800 μL Saline) Injection Formulation 8: Dissolve in Cremophor/Ethanol (50 : 50), then diluted by Saline Injection Formulation 9: EtOH : Corn oil = 10 : 90 (i.e. 100 μL EtOH → 900 μL Corn oil) Injection Formulation 10: EtOH : PEG300：Tween 80 : Saline = 10 : 40 : 5 : 45 (i.e. 100 μL EtOH → 400 μLPEG300 → 50 μL Tween 80 → 450 μL Saline) Oral Formulations Oral Formulation 1: Suspend in 0.5% CMC Na (carboxymethylcellulose sodium) Oral Formulation 2: Suspend in 0.5% Carboxymethyl cellulose Example: Take the Oral Formulation 1 (Suspend in 0.5% CMC Na) as an example, if 100 mL of 2.5 mg/mL working solution is to be prepared, you can first prepare 0.5% CMC Na solution by measuring 0.5 g CMC Na and dissolve it in 100 mL ddH2O to obtain a clear solution; then add 250 mg of the product to 100 mL 0.5% CMC Na solution, to make the suspension solution (2.5 mg/mL, ready for use in animals). Oral Formulation 3: Dissolved in PEG400 Oral Formulation 4: Suspend in 0.2% Carboxymethyl cellulose Oral Formulation 5: Dissolve in 0.25% Tween 80 and 0.5% Carboxymethyl cellulose Oral Formulation 6: Mixing with food powders Note: Please be aware that the above formulations are for reference only. InvivoChem strongly recommends customers to read literature methods/protocols carefully before determining which formulation you should use for in vivo studies, as different compounds have different solubility properties and have to be formulated differently. (Please use freshly prepared in vivo formulations for optimal results.) |
| Preparing Stock Solutions | 1 mg | 5 mg | 10 mg | |
| 1 mM | 2.9424 mL | 14.7119 mL | 29.4239 mL | |
| 5 mM | 0.5885 mL | 2.9424 mL | 5.8848 mL | |
| 10 mM | 0.2942 mL | 1.4712 mL | 2.9424 mL |