Ubc13-Uev1A complex (also known as UBE2N-UBE2V1) [1]

NSC697923 selectively inhibits PMA-induced NF-κB activation, and also inhibits IκBα phosphorylation by both RANKL and LPS. NSC697923 impedes the formation of the Ubc13 and ubiquitin thioester conjugate and suppresses constitutive NF-κB activity in ABC-DLBCL cells. In addition, NSC697923 inhibits the proliferation and survival of ABC-DLBCL cells and GCB-DLBCL cells, and also induces apoptosis of primary DLBCL cells. In neuroblastoma (NB) cell lines, NSC697923 shows cytotoxic effect, and inhibits both anchorage-dependent and -independent colony formation

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NSC697923 inhibits PMA-induced NF-κB activation in 293T-NF-luc cells at 2 µM but does not inhibit TNFα-induced activation (Figure 1). [1]
It inhibits IκBα phosphorylation induced by RANKL (15 ng/mL) and LPS (1.5 µg/mL) in RAW264.7 and MEF cells (Figure 3). [1]
It inhibits the formation of Ubc13~Ub thioester conjugate in OCI-Ly10 cells at 0.5-2 µM for 3 hours (Figure 5A). [1]
It inhibits constitutive NF-κB signaling in ABC-DLBCL cells: at 0.5-2 µM for 3.5 hours, it decreases phosphorylation of IKK and IκBα, and reduces expression of NF-κB targets p100 and Mcl-1 in OCI-Ly10 cells (Figure 5B). [1]
It inhibits proliferation and survival of ABC-DLBCL cells (OCI-Ly3, OCI-Ly10, HBL-1): treatment with 0.5-2 µM for 24 hours causes a dramatic decrease of live cells and increase of dead cells (Figure 5C). [1]
It induces apoptosis in OCI-Ly10 cells: at 1-2 µM for 5 hours, it activates caspase-3 and cleaves PARP (Figure 5D); at 0.5-2 µM for 24 hours, it increases Annexin V-positive cells (Figure 5E). [1]
It inhibits cell cycle progression: treatment of OCI-Ly10 cells with 1-2 µM for 5 hours results in a marked decrease of cells in S phase (Figure 5F). [1]
It also inhibits proliferation and survival of GCB-DLBCL cells (OCI-Ly7, SUDHL-6): at 0.5-2 µM for 24 hours, it reduces live cells and induces apoptosis (Figure 6A-B). [1]
It induces apoptosis in primary DLBCL cells from two patients: treatment with 0.5-2 µM for 24 hours increases Annexin V-positive cells (Figure 7). [1]

NSC697923 (5 mg/kg/day, i.p.) demonstrates potent antitumor efficacy in both NB SH-SY5Y and NGP xenografts.

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The reactions are carried out at 37°C for 40 minutes in a buffer containing 50mM Tris-HCl, pH 7.5, 5mM MgCl2, 200μM ATP, 120μM Ub (U-100H), and 0.1μM E1 (E-304). For Ubc13-mediated Ub chain synthesis, the reaction mixture includes 0.2 μM Ubc13 (E2-600) and 0.2 mM Uev1A (E2-662) with or without GST-TRAF6. For UbcH5c-catalyzed ubiquitination, UbcH5c (E2-627) instead of Ubc13 and Uev1A is used in the reaction. Compound NSC697923 is added into the reaction mixtures at the indicated concentrations. The reactions are terminated with an equal volume of SDS-PAGE sample buffer and the products are analyzed by Western blotting with a Ub-specific antibody. For detecting the E2-Ub (Ubc13∼Ub or UbcH5c∼Ub) thioester conjugates, the reactions are carried out as described in “In vitro ubiquitination assay” without GST-TRAF6. The reactions are terminated by the addition of the SDS-PAGE sample buffer without a reducing agent unless specified. The products are analyzed by Western blotting with an anti-Ubc13 or anti-UbcH5c antibody.

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In vitro ubiquitination assays: Reactions were carried out at 37°C for 40 minutes in a buffer containing 50 mM Tris-HCl (pH 7.5), 5 mM MgCl2, 200 µM ATP, 120 µM ubiquitin, and 0.1 µM E1. For Ubc13-mediated ubiquitin chain synthesis, the reaction mixture included 0.2 µM Ubc13 and 0.2 mM Uev1A with or without GST-TRAF6. NSC697923 was added at indicated concentrations (0.5, 1, 2 µM). Products were analyzed by western blotting with a ubiquitin-specific antibody. The compound inhibited polyubiquitin chain formation in a dose-dependent manner (Figure 4A-B). [1]
For detecting E2-ubiquitin thioester conjugates, reactions were carried out as above without GST-TRAF6, terminated with SDS-PAGE sample buffer without reducing agent, and analyzed by western blotting with anti-Ubc13 or anti-UbcH5c antibody. NSC697923 inhibited formation of the Ubc13~Ub conjugate but not UbcH5c~Ub (Figure 4E-F). [1]
In vitro interaction assay between Ubc13 and Uev1A: GST-Ubc13 bound on glutathione-agarose beads was incubated with OCI-Ly10 cell lysates with or without NSC697923 (1, 2 µM) at 4°C for 1 hour. Beads were washed and bound Uev1A analyzed by western blotting. The compound showed no inhibitory effect on complex formation (Figure 4D). [1]

Cell viability is measured by trypan blue exclusion assay.

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Cell culture: HEK293T cells grown in DMEM +10% FBS. DLBCL cells (SUDHL-6, OCI-Ly3, OCI-Ly7, OCI-Ly10, HBL-1) cultured as previously described. Primary DLBCL cells prepared from fresh patient tumors by mechanical disruption and filtration. [1]
NF-κB luciferase reporter assay: 293T-NF-luc cells (carrying stably integrated NF-κB-luciferase reporter and constitutively expressing GFP) seeded in 96-well plates at 30,000 cells/well. Compounds added at 2 µM final concentration 1.5 hours before stimulation with PMA (100 ng/mL) or TNFα (10 ng/mL). After 6 hours, cells lysed and luciferase activity measured and normalized to GFP fluorescence. NSC697923 inhibited PMA-induced but not TNFα-induced NF-κB activation (Figure 1B-C). [1]
Cell viability assay: Cells seeded at 3×10^5 cells/mL in 6-well plates, treated with DMSO or NSC697923 (0.5, 1, 2 µM) for 24 hours, then live/dead cells counted by trypan blue exclusion. [1]
Apoptosis assay: Cells treated as above, stained with 7AAD/Annexin V, and analyzed by flow cytometry. [1]
Cell cycle analysis: Cells treated with NSC697923 for 4.5 hours, then BrdU added for 30 minutes before harvest, stained with propidium iodide and BrdU, and analyzed by flow cytometry. [1]
Western blotting: Cells treated with NSC697923 for indicated times, lysed, and proteins detected with specific antibodies against phospho-IκBα, phospho-IKK, IκBα, IKK, Mcl-1, p100, cleaved caspase-3, PARP, etc. [1]

Dissolved in DMSO; 5 mg/kg; i.p. injection SH-SY5Y and NGP xenografts

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NSC697923 shows much less toxic effects on the proliferation and survival of other cell types such as breast cancer and osteosarcoma cells compared to its effect on DLBCL cells (Supplemental Figure S6). [1]

Blood.2012 Aug 23;120(8):1668-77;Cell Death Dis.2014 Feb 20;5:e1079.

NSC697923 was identified from the NCI Mechanistic Set (879 compounds) via a cell-based screen for inhibitors of PKCβ signaling and NF-κB activation. Structure-activity relationship analysis indicates that the nitrofuran moiety is important but not sufficient for its inhibitory activity (Figure 2). [1]
The compound acts by specifically inhibiting the formation of the Ubc13~Ub thioester conjugate, thereby blocking Ubc13-Uev1A-mediated K63-linked polyubiquitination. [1]
In ABC-DLBCL cells, inhibition of Ubc13 by NSC697923 leads to NF-κB suppression, p53 activation (increased nuclear p53 and p21 expression, Supplemental Figure S3A), and apoptosis. In GCB-DLBCL cells (which are NF-κB-independent and lack functional p53), the compound still induces toxicity, possibly via MAP kinase pathways. [1]
NSC697923 may also sensitize cancer cells to radiation therapy or chemotherapy by inhibiting DNA double-strand break repair (Supplemental Figure S4). It exhibits additive cytotoxic effect on DLBCL cells with CHOP agents (Supplemental Figure S5). [1]
Potential therapeutic target for DLBCL, especially ABC-DLBCL which harbors oncogenic mutations in CBM complex and may be resistant to PKCβ inhibition. [1]

C11H9NO5S

267.255

267.02

C, 49.44; H, 3.39; N, 5.24; O, 29.93; S, 12.00

343351-67-7

394449

White to light yellow solid powder

3.933

0

5

2

18

402

0

O=S(C1=CC=C([N+]([O-])=O)O1)(C2=CC=C(C)C=C2)=O

GAUHIPWCDXOLCZ-UHFFFAOYSA-N

InChI=1S/C11H9NO5S/c1-8-2-4-9(5-3-8)18(15,16)11-7-6-10(17-11)12(13)14/h2-7H,1H3

2-[(4-Methylphenyl)sulfonyl]-5-nitrofuran

NSC697923; NSC 697923; NSC-697923

2934.99.9001

Powder      -20°C    3 years

                     4°C     2 years

In solvent   -80°C    6 months

                  -20°C    1 month

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

DMSO : 200~53 mg/mL (748.33 ~198.3 mM）

Solubility in Formulation 1: ≥ 5 mg/mL (18.71 mM) (saturation unknown) in 10% DMSO + 40% PEG300 + 5% Tween80 + 45% Saline (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 50.0 mg/mL clear DMSO stock solution to 400 μL PEG300 and mix evenly; then add 50 μL Tween-80 to the above solution and mix evenly; then add 450 μL normal saline to adjust the volume to 1 mL.
Preparation of saline: Dissolve 0.9 g of sodium chloride in 100 mL ddH₂ O to obtain a clear solution.

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Solubility in Formulation 2: ≥ 5 mg/mL (18.71 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 50.0 mg/mL clear DMSO stock solution to 900 μL of corn oil and mix evenly.

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