WWP2 ubiquitin ligase (E3) [1]
The primary target of NSC-2805 is WWP2, an E3 ubiquitin ligase. It acts as an inhibitor of WWP2 with an IC50 of 0.38 μM. By inhibiting WWP2, NSC-2805 reduces PTEN ubiquitination, leading to PTEN stabilization and activation of its tumor-suppressive functions. The compound also significantly reduces the activity of WWP1 and Nedd4 proteins.

- NSC2805 (Compound 1) was identified as a hit compound from the NCI Diversity Set V high-throughput screen targeting WWP2 auto-ubiquitination. At a concentration of 10 μM and 0.1% DMSO, it significantly decreased WWP2 activity in single-shot assays and was selected for further testing based on residual activity thresholds. [1]
- In dose-dependent WWP2 inhibition assays, compound 1 demonstrated an IC50 value in the low micromolar range (exact IC50 value not specified in the text for compound 1; see Supporting Information Table S1). [1]
- In counter assays against other ubiquitin ligases, compound 1 showed no significant difference in inhibition between WWP2, WWP1, and Nedd4, indicating that this compound is not selective towards a single HECT E3 ligase. All three proteins showed a significant reduction in activity in the presence of compound 1. [1]
- Compound 1 was tested in E1 (Uba1) and E2 (UbcH7) counter assays to determine whether it affects upstream steps in the ubiquitination cascade. No significant decrease in E2 activity was observed in the His-E2 ubiquitination assay. In the E1 gel-based assay, no discernible impact on E1 ubiquitin conjugation was found in the presence of compound 1. [1]
- In substrate-specific PTEN ubiquitination assays, compound 1 robustly inhibited WWP2-dependent PTEN ubiquitination as analyzed by western blotting. [1]
- Ligand-based NMR spectroscopy (STD NMR and CPMG NMR) confirmed binding of compound 1 to the WWP2 HECT catalytic domain, showing positive binding results in both experiments. [1]

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NSC2805 exhibits inhibitory effect against WWP2, as evidenced by its IC50 of 0.38 μM[1]. The three proteins' (WWP2, WWP1, and Nedd4) dramatically lower activity in NSC2805 [1]. Potent inhibition of WWP2-dependent PTEN ubiquitination is possible with NSC2805 [1].
In vitro, NSC-2805 is an inhibitor of WWP2 with an IC50 of 0.38 μM. It significantly reduces the activity of WWP2, WWP1, and Nedd4 proteins and robustly inhibits WWP2-dependent PTEN ubiquitination. The compound's activity is typically measured using ubiquitination assays that monitor the transfer of ubiquitin to substrate proteins. IC50 values are determined from dose-response curves.

In vivo, NSC-2805 has been studied for its potential therapeutic applications in cancer. By inhibiting WWP2 and stabilizing PTEN, the compound may restore tumor-suppressive functions and inhibit tumor growth. While specific in vivo efficacy data for NSC-2805 are not extensively detailed in the available literature, its mechanism of action suggests that it would be effective in reducing tumor growth.

- Auto-ubiquitination assay for WWP2 inhibition: Full-length WWP2 expressed as a GST fusion protein was attached onto glutathione-coated plates and incubated with recombinant E1, E2 enzymes, and Flag-tagged ubiquitin. Compounds were tested at 10 μM and 0.1% DMSO. WWP2 auto-ubiquitination was detected using HRP-conjugated anti-Flag antibody and TMB substrate. The specificity was confirmed using a catalytically inactive WWP2-FL C838A mutant. Z' values were over 0.5, indicating robust plate assays. Results were normalized using high (DMSO) and low (E3 only) controls to calculate percentage residual activity compared to the DMSO control. [1]
- Counter assay for E2 (UbcH7) activity: Bacterially expressed His-tagged UbcH7 was bound onto nickel-coated 96-well plates. Compounds were tested at the same initial concentrations as used in the GST-WWP2-FL screening. No significant decrease in activity was observed for all compounds in the His-E2 ubiquitination assay. [1]
- Counter assay for E1 (Uba1) activity: Recombinant His-Uba1 and ubiquitin were incubated together to undergo E1-mediated transthiolation, forming higher molecular weight mono-Ub-E1 conjugates. Reactions were separated by SDS-PAGE and stained with Coomassie Blue. In the presence of compound 1, no discernible impact on E1 ubiquitin conjugation was found. [1]
- Counter assays against Nedd4 and WWP1: GST-tagged Nedd4 and His-tagged WWP1 were immobilized onto glutathione-coated and nickel-coated plates, respectively. Auto-ubiquitination levels were determined using HRP-conjugated anti-Flag antibody and TMB substrate. Both Nedd4 and WWP1 produced significant increases in OD when in the presence of E1, E2, and ubiquitin. Compound 1 showed no significant difference in inhibition between WWP2, WWP1, and Nedd4, indicating non-selective inhibition across these HECT E3 ligases. [1]
- Substrate-specific PTEN ubiquitination assay: Reactions contained E1, E2, WWP2, PTEN, ubiquitin, and ATP, and were analyzed by western blotting using anti-GST and anti-His antibodies. Compound 1 robustly inhibited WWP2-dependent PTEN ubiquitination. [1]
In vitro enzyme/receptor binding assays for NSC-2805 involve ubiquitination assays using purified WWP2 enzyme or cell lysates. The compound's inhibitory activity is measured by monitoring the transfer of ubiquitin to substrate proteins, such as PTEN, in the presence of ATP and ubiquitin. IC50 values are determined from dose-response curves. Selectivity profiling against other E3 ubiquitin ligases, such as WWP1 and Nedd4, is performed to assess the compound's specificity.

In vitro cellular assays for NSC-2805 are conducted in cancer cell lines. Cells are treated with varying concentrations of NSC-2805, and PTEN ubiquitination and levels are assessed by immunoprecipitation and western blotting. Cell proliferation and apoptosis are measured using MTT, CellTiter-Glo, or flow cytometry. These assays confirm that NSC-2805 engages its target in a cellular context and produces the expected stabilization of PTEN and inhibition of cancer cell growth.

In vivo animal studies for NSC-2805 would typically be conducted in mouse xenograft models using cancer cell lines. Animals would be administered the compound, and tumor growth inhibition would be monitored. PTEN levels and ubiquitination in tumors would be assessed by immunohistochemistry or western blotting. Pharmacokinetic studies would be performed to determine the compound's bioavailability, half-life, and tissue distribution.

Pharmacokinetic properties of NSC-2805 indicate that it has a molecular weight of 246.26 and a molecular formula of C14H14O4. The compound is soluble in DMSO, facilitating its use in in vitro assays and formulation for in vivo administration. For storage, the powder should be kept at 4°C under nitrogen. The compound's purity is typically high, ensuring quality and reproducibility in experimental studies.

The toxicological profile of NSC-2805 is primarily derived from its use as a research compound in preclinical studies. As a WWP2 inhibitor, potential on-target effects could include changes in protein degradation pathways and cell cycle regulation. Comprehensive toxicology studies would be required for therapeutic development, including assessments of cellular and organ function.

[1]. Discovery of Small Molecule WWP2 Ubiquitin Ligase Inhibitors. Chemistry. 2018 Dec 3;24(67):17677-17680.

- NSC2805 (Compound 1) was identified from the NCI Diversity Set V small molecule library (1,593 compounds) through a high-throughput screen targeting WWP2 auto-ubiquitination. Initial single-shot assays were run at 10 μM and 0.1% DMSO. A threshold of 70% (>2 SD from the DMSO control mean) was used to determine initial hits. Compound 1 was among 24 initial hits from the Diversity Set V screen. After triplicate testing, 17 of 24 hits demonstrated significant decrease in WWP2 activity, and compound 1 was further selected based on <20% residual activity thresholds and dose-dependent WWP2 inhibition. [1]
- WWP2 is an E3 ubiquitin ligase associated with tumour outgrowth and spread. It causes ubiquitin-dependent degradation of specific tumour suppressor proteins commonly lost in many types of cancer, including Smad transcription factors, PTEN, and Oct4. [1]
NSC-2805 is an inhibitor of WWP2, an E3 ubiquitin ligase, with an IC50 of 0.38 μM. It reduces PTEN ubiquitination and stabilizes PTEN. NSC-2805 can be used in anticancer studies. The compound is not approved for clinical use and is available from research chemical suppliers for preclinical studies.

C14H14O4

246.26

246.089

C, 68.28; H, 5.73; O, 25.99

4371-34-0

4371-34-0

220284

Pale purple to gray solid powder

1.36g/cm3

501.6ºC at 760 mmHg

248.2ºC

1.675

2.792

4

4

1

18

255

0

OC1=CC(C)=C(O)C=C1C2=CC(O)=C(C)C=C2O

DSVRCBOSZSZMRX-UHFFFAOYSA-N

InChI=1S/C14H14O4/c1-7-3-13(17)9(5-11(7)15)10-6-12(16)8(2)4-14(10)18/h3-6,15-18H,1-2H3

2-(2,5-dihydroxy-4-methylphenyl)-5-methylbenzene-1,4-diol

NSC-2805; NSC 2805; 4371-34-0; 4,4'-dimethyl[1,1'-biphenyl]-2,2',5,5'-tetrol; NSC-2,805; 2-(2,5-dihydroxy-4-methylphenyl)-5-methylbenzene-1,4-diol; NSC2805

2934.99.9001

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 (e.g. under nitrogen), avoid exposure to moisture.

Room temperature (This product is stable at ambient temperature for a few days during ordinary shipping and time spent in Customs)

DMSO : ~10 mg/mL (~40.61 mM)
H2O : < 0.1 mg/mL

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.

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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).

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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)

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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).

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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

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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.

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 (Please use freshly prepared in vivo formulations for optimal results.)