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| 5mg |
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Purity: ≥98%
NSC232003 is a novel, highly potent and cell-permeable UHRF1 inhibitor, which inhibits DNA methylation in vitro and disrupts DNMT1/UHRF1 interactions at a cellular level. Compound NSC232003, a uracil derivative freely available by the NCI/DTP Repository, provides a versatile lead for developing highly potent and cell-permeable UHRF1 inhibitors that will enable dissection of DNA methylation inheritance.
| Targets |
SRA domain of UHRF1 (Ubiquitin-like with PHD and RING finger domains 1). NSC232003 is proposed to bind to the 5-methylcytosine (5mC) binding cavity of this domain, potentially inhibiting its interaction with hemi-methylated DNA and disrupting the UHRF1-DNMT1 functional complex. [1]
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| ln Vitro |
Freely downloadable from the NCI/DTP repository, UHRF1 gene NSC232003 offers a flexible starting point for creating a cell-permeable UHRF1 that facilitates the analysis of DNA methylation inheritance. In fact, NSC232003 is an effective scaffold for DNA methylation, suggesting that specialized scaffolds can offer a flexible foundation for the development of effective UHRF1. Given that NSC232003's scaffold bicyclic ring contains more acidic imine nitrogen with a pKa value of 7.6, it is anticipated that NSC232003 will partially deprotonate at pH 7. The most potent chemical, NSC232003, signaled through U251 neuroastromas cells, which after 4 hours considerably reduced DNMT1/UHRF1 responses, showing 50% response inhibition at 15 μM and total DNA tyrosine demethylation evaluated by ELISA induction [1].
Treatment of U251 glioma cells with NSC232003 at 15 µM for 4 hours significantly reduced the in situ interaction between DNMT1 and UHRF1 proteins, as measured by the Proximity Ligation In Situ Assay (P-LISA). The compound achieved approximately 50% inhibition of DNMT1/UHRF1 interactions at this concentration. [1] In U251 glioma cells treated with 15 µM NSC232003 for 72 hours, a global DNA cytosine demethylation effect was observed. ELISA measurements showed a notable 50% decrease in genomic 5mC content compared to controls. [1] In MCF7 breast cancer cells, treatment with 15 µM NSC232003 for 72 hours led to a reduction in global genomic 5-methylcytosine (5mC) levels, as quantified by HPLC-MS/MS analysis. This demethylating activity was comparable to or better than the positive control 5-azacytidine in the initial screening phase that identified its precursor compound. [1] A virtual screening (structure-based docking and ligand-based 3D similarity) and subsequent experimental validation pipeline identified NSC232003 as a hit compound capable of modulating DNA methylation in cells. [1] |
| Enzyme Assay |
The study attempted to use a Fluorescent Thermal Shift Assay (FTSA/Differential Scanning Fluorimetry) to evaluate direct binding of compounds to the purified SRA domain of UHRF1. The SRA domain protein was overexpressed in bacterial cells, purified using nickel-affinity chromatography followed by histidine tag cleavage and size-exclusion chromatography. Binding was assessed using a fluorescent dye and a methylated oligonucleotide as a ligand. However, the assay could not be successfully validated for high-throughput screening due to challenges including the moderate affinity of the protein for the methylated oligonucleotide (Kd ~19 µM) and the lack of a suitable positive control inhibitor. [1]
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| Cell Assay |
DNA Demethylation Screening (PCR-MS & HPLC-MS/MS): MCF7 cells were treated with test compounds (dissolved in DMSO, final concentration 15 µM) for 72 hours, with fresh medium changes every other day. Genomic DNA was then extracted. For locus-specific analysis, DNA was bisulfite-converted and subjected to methylation-specific PCR (MS-PCR) for the RARβ gene exon 2. For global analysis, another portion of DNA was digested to individual nucleosides using nuclease P1 and alkaline phosphatase. The resulting nucleoside mixture was analyzed by HPLC-MS/MS to quantify the ratio of 5-methyl-2'-deoxycytidine (5mdCyd) to total deoxycytidine (dCyd), providing a measure of global DNA methylation levels. [1]
Proximity Ligation In Situ Assay (P-LISA): U251 glioma cells were fixed and permeabilized on glass slides. Primary antibodies against DNMT1 and UHRF1 were applied, followed by species-specific secondary antibodies conjugated to unique DNA oligonucleotides. If the two target proteins are in close proximity (<40 nm), the attached DNA strands can hybridize to a connector oligonucleotide, forming a circular DNA template. This circle is then amplified via rolling circle amplification and detected with a fluorescently labeled probe, generating a discrete fluorescent signal (dot) at the site of protein-protein interaction. The number of fluorescent dots per cell was quantified to assess the level of DNMT1/UHRF1 interaction after compound treatment (e.g., 15 µM for 4 hours). [1] Global DNA Methylation ELISA: U251 cells were treated with compounds (e.g., 15 µM for 72 hours). Genomic DNA was then extracted and analyzed using a commercial global DNA methylation quantification ELISA kit. This method quantifies the relative amount of 5-methylcytosine in the total genomic DNA. [1] |
| References | |
| Additional Infomation |
NSC232003 is a uracil derivative (oxime-substituted) screened using a tandem virtual screening method (combining structure-based molecular docking and ligand-based three-dimensional similarity) of the National Cancer Institute/Targeted Therapy Program (NCI/DTP) compound library. [1] It is considered the first reported chemical tool compound to target the SRA domain of UHRF1 and regulate DNA methylation in the cellular environment. [1] Its mechanism of action may involve binding to the 5mC pocket of the UHRF1 SRA domain, thereby competitively inhibiting the domain's recognition of hemimethylated DNA. This inhibition is thought to interfere with the recruitment of DNMT1, thereby impairing the maintenance of DNA methylation patterns during cell division and ultimately leading to passive DNA demethylation. [1] Molecular modeling (QM polarization docking) suggests that the deprotonated NSC232003 (predicted pKa approximately 7.6) may bind to residues such as Y466 and Y478 within the 5mC cavity via a hydrogen bond network and π-π stacking interactions. Its oxime moiety may interact with the positively charged inlet within the cavity, which consists of residues such as R433, R484, and K540. [1] This compound is freely available from the NCI/DTP compound library and is considered a versatile lead compound that could be used to develop more effective and cell-penetrating UHRF1 inhibitors. [1]
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| Molecular Formula |
C6H7N3O3
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|---|---|
| Molecular Weight |
169.138080835342
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| Exact Mass |
169.048
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| CAS # |
1905453-18-0
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| PubChem CID |
135468142
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| Appearance |
White to off-white solid powder
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| LogP |
-0.8
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| Hydrogen Bond Donor Count |
3
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| Hydrogen Bond Acceptor Count |
4
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| Rotatable Bond Count |
1
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| Heavy Atom Count |
12
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| Complexity |
292
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| Defined Atom Stereocenter Count |
0
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| SMILES |
O=C1C(C(C)=NO)=CNC(N1)=O
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| InChi Key |
UDHCVAJUUVCYHW-YCRREMRBSA-N
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| InChi Code |
InChI=1S/C6H7N3O3/c1-3(9-12)4-2-7-6(11)8-5(4)10/h2,12H,1H3,(H2,7,8,10,11)/b9-3+
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| Chemical Name |
5-[(E)-N-hydroxy-C-methylcarbonimidoyl]-1H-pyrimidine-2,4-dione
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| Synonyms |
NSC232003; NSC-232003; NSC 232003
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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 |
| 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) |
H2O : ~2 mg/mL (~11.82 mM)
DMSO :< 1 mg/mL |
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| Solubility (In Vivo) |
Solubility in Formulation 1: 4.76 mg/mL (28.14 mM) in PBS (add these co-solvents sequentially from left to right, and one by one), clear solution; with sonication (<60°C).
(Please use freshly prepared in vivo formulations for optimal results.) |
| Preparing Stock Solutions | 1 mg | 5 mg | 10 mg | |
| 1 mM | 5.9123 mL | 29.5613 mL | 59.1226 mL | |
| 5 mM | 1.1825 mL | 5.9123 mL | 11.8245 mL | |
| 10 mM | 0.5912 mL | 2.9561 mL | 5.9123 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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