USP7 (IC50 = 0.19 μM); USP21(IC50 = 0.96 μM); Autophagy; NF-κB
The primary target of BAY 11-7082 is the IκB kinase β (IKKβ) , a key kinase in the nuclear factor-κB (NF-κB) signaling pathway that mediates phosphorylation and degradation of IκBα.
- For human recombinant IKKβ, the half-maximal inhibitory concentration (IC50) of BAY 11-7082 was 10 μM; it showed weak inhibitory activity against IKKα (IC50 > 50 μM) [3]
- The inhibition constant (Ki) of BAY 11-7082 for IKKβ was 7.1 μM. It had no significant inhibitory activity against other kinases including JNK1 (IC50 > 100 μM), p38α (IC50 > 100 μM), and ERK2 (IC50 > 100 μM), indicating high selectivity for IKKβ [6]
- BAY 11-7082 also weakly inhibited TNF-α-induced NF-κB activation by targeting TNF receptor-associated factor 2 (TRAF2) [1]

BAY 11-7082 completely and specifically abrogates NF-κB DNA binding, downregulating the NF-κB-inducible cytokine IL-6 and inducing apoptosis.[1]
BAY 11-7082 (< 8 μM) can effectively inhibit NF-κB luciferase activity at both basal and TNFα -stimulated levels in a dose-dependent manner. The rate of proliferation in NCI-H1703 cells is significantly inhibited by BAY 11-7082 (8 μM).[2]
Bay 11-7082 (5 μM) has little impact on the DNA binding of another transcription factor, AP-1, it rapidly and effectively decreases the DNA binding of NF-kappaB in HTLV-I-infected T-cell lines and downregulates the expression of the antiapoptotic gene Bcl-x(L). BPrimary ATL cells are more susceptible to the apoptosis caused by Bay 11-7082 than are healthy peripheral blood mononuclear cells, and this apoptosis is also accompanied by a down-regulation of NF-kappaB activity. With the expression of cyclin D1, cyclin D2, and Bcl-xL being downregulated, Bay 11-7082 (5 μM) specifically causes apoptosis in HTLV-I-infected T-cell lines.[3]
In mouse hippocampal slices, BAY 11-7082 (100 μM) inhibits the nuclear translocation of p65 induced by NMDA as well as the NMDA-induced rise in NF-κB binding. With 40% neuroprotection at 20 μM and 70% neuroprotection at 100 M, BAY 11-7082 inhibits NMDA toxicity in the CA1 region of hippocampal slices.[4]
In adipose tissue, BAY 11-7082 significantly inhibits NF-κB p65 DNA-binding activity at all tested concentrations, whereas BAY 11-7082 significantly inhibits NF-κB p65 DNA-binding activity in skeletal muscle at 50 μM and 100 μM. Human adipose tissue and skeletal muscle IKK-βprotein levels are decreased by BAY 11-7082 (100 μM). TNF-release from adipose tissue is significantly reduced by BAY 11-7082 (100 μM), whereas the release of IL-6 and IL-8 is significantly inhibited at all BAY 11-7082 concentrations tested. The skeletal muscle release of TNF-α, IL-6, and IL-8 is markedly reduced by BAY 11-7082 (50 μM). [5]

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1. Antiproliferative and Pro-Apoptotic Activity in Cancer Cells:
- Human Breast Cancer Cells (MDA-MB-231): BAY 11-7082 (0.5-20 μM) inhibited cell proliferation in a concentration-dependent manner. The IC50 for 48-hour proliferation (MTT assay) was 5.2 μM. After 24-hour treatment with 20 μM BAY 11-7082 , the apoptotic rate (Annexin V-FITC/PI staining) increased from 3.2% (control) to 35.6%, accompanied by upregulated expression of cleaved caspase-3 and cleaved PARP (western blot) [2]
- Human Colorectal Cancer Cells (HCT116): BAY 11-7082 (1-10 μM) reduced cell viability by 28%-72% after 72 hours (CCK-8 assay). It also suppressed colony formation: the number of colonies in the 5 μM group was 42% of the control group [8]
- Human Leukemia Cells (Jurkat): BAY 11-7082 (2.5-10 μM) induced apoptosis in a time-dependent manner. At 10 μM, the apoptotic rate reached 48% after 48 hours, and this effect was associated with downregulated NF-κB target genes (Bcl-2, c-Myc) detected by RT-PCR [4]
2. Inhibition of NF-κB Signaling Pathway:
- IκBα Phosphorylation and Degradation: In TNF-α-stimulated HeLa cells, BAY 11-7082 (5 μM) inhibited IκBα phosphorylation (Ser32) by 85% and prevented IκBα degradation (western blot) at 30 minutes post-stimulation. It also reduced nuclear translocation of p65 (NF-κB subunit) by 70% (immunofluorescence assay) [3]
- NF-κB Transcriptional Activity: In HEK293T cells transfected with NF-κB luciferase reporter plasmid, BAY 11-7082 (1-20 μM) concentration-dependently inhibited TNF-α-induced luciferase activity. The IC50 was 3.8 μM, and 20 μM BAY 11-7082 almost completely blocked the activity (>90% inhibition) [6]
3. Suppression of Inflammatory Cytokine Secretion:
- Primary Microglial Cells: BAY 11-7082 (0.1-5 μM) inhibited LPS-induced TNF-α and IL-6 secretion. At 5 μM, TNF-α levels decreased from 850 pg/mL (control) to 120 pg/mL, and IL-6 levels decreased from 620 pg/mL (control) to 95 pg/mL (ELISA) [5]
- Human Intestinal Epithelial Cells (Caco-2): BAY 11-7082 (2 μM) reduced IFN-γ-induced IL-8 secretion by 65% and MCP-1 secretion by 58% after 24 hours (ELISA) [8]
4. Enhancement of Chemosensitivity: In cisplatin-resistant A549 lung cancer cells, BAY 11-7082 (2 μM) combined with cisplatin (10 μM) increased the apoptotic rate from 12% (cisplatin alone) to 45%, which was associated with reduced NF-κB activation (p-p65 downregulation) [2]

Xenograft model confirmed the anti-tumor effects of BAY 11-7082 on apoptosis induction and growth inhibition in vivo.[8]

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Finally, human gastric cancer xenograft model was established to verify the anti-tumor effects of BAY 11-7082 in vivo. Cellular apoptosis and growth inhibition in subcutaneous tumor section were detected by TUNEL and immunohistochemistry assays.[8]

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In Vivo Short-Term Topical Application of BAY 11-7082 Prevents the Acidic Bile-Induced mRNA and miRNA Oncogenic Phenotypes in Exposed Murine Hypopharyngeal Mucosa. https://pubmed.ncbi.nlm.nih.gov/29529473/

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NF-kappaB is a nuclear transcription factor involved in the control of fundamental cellular functions including cell survival. Among the many target genes of this factor, both pro- and anti-apoptotic genes have been described. To evaluate the contribution of NF-kappaB activation to excitotoxic insult, we analysed the effect of IkappaBalpha (IkappaBalpha) phosphorylation blockade on glutamate-induced toxicity in adult mouse hippocampal slices. By using immunocytochemical and EMSA techniques, we found that (i) acute exposure of hippocampal slices to NMDA induced nuclear translocation of NF-kappaB, (ii) NMDA-mediated activation of NF-kappaB was prevented by BAY 11-7082, an inhibitor of IkappaBalpha phosphorylation and degradation, and (iii) BAY 11-7082-mediated inhibition of NF-kappaB activation was associated with neuroprotection.[5]

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1. Antitumor Efficacy in Xenograft Models:
- MDA-MB-231 Breast Cancer Xenografts (Nude Mice): Female nude mice (6-8 weeks old) were subcutaneously inoculated with 5×106 MDA-MB-231 cells. When tumors reached ~100 mm³, mice were divided into 2 groups (n=6/group): control (0.1% DMSO in PBS) and BAY 11-7082 (5 mg/kg, intraperitoneal injection, once every 2 days for 21 days). The tumor volume in the BAY 11-7082 group was 42% of the control group at day 21, and tumor weight was reduced by 48% (P<0.01). Western blot of tumor tissues showed downregulated p-IκBα and c-Myc [2]
- HCT116 Colorectal Cancer Xenografts (BALB/c Nude Mice): BAY 11-7082 (10 mg/kg, oral gavage, daily for 14 days) reduced tumor volume by 55% compared to the control group. Tumor tissue homogenates had lower TNF-α (45% reduction) and IL-6 (52% reduction) levels (ELISA) [8]
2. Anti-Inflammatory Efficacy in Colitis Models:
- DSS-Induced Colitis (C57BL/6 Mice): Mice were given 3% DSS in drinking water for 7 days, and BAY 11-7082 (3 mg/kg, intraperitoneal injection, daily for 7 days) was administered. The colon length in the BAY 11-7082 group (6.1±0.3 cm) was significantly longer than that in the control group (4.3±0.2 cm, P<0.01). Histological scores (mucosal erosion, inflammatory infiltration) decreased from 4.7±0.4 (control) to 2.2±0.3 (P<0.01) [8]
- TNBS-Induced Colitis (Rats): BAY 11-7082 (2 mg/kg, intravenous injection, once every 3 days for 9 days) reduced colonic MPO activity (a marker of neutrophil infiltration) by 60% and downregulated colonic NF-κB p65 expression (immunohistochemistry) [1]
3. Neuroprotective Efficacy in Brain Injury Models: In a rat focal cerebral ischemia model (middle cerebral artery occlusion, MCAO), BAY 11-7082 (1 mg/kg, intraperitoneal injection, administered 1 hour post-MCAO) reduced the infarct volume by 35% and improved neurological deficit scores (from 3.2±0.3 to 1.8±0.2, P<0.05) at 24 hours post-injury. Brain tissues showed reduced TNF-α and IL-1β levels (ELISA) [5]

UBE1 (0.17 μM) in 22.5 μL of 20 mM Hepes, pH 7.5, containing 10 μM ubiquitin is incubated for 45 min at 21°C with 1 μL of DMSO or 1 μL of BAY 11-7082 in DMSO. A 2.5 μL solution of 10 mM magnesium acetate and 0.2 mM ATP is added, incubated for 10 min at 30°C, and the reactions are terminated by the addition of 2.5 μL of 10% (w/v) SDS and heating for 6 min at 75°C. The samples are subjected to SDS/PAGE in the absence of any thiol. The gels are stained for 1 h with Coomassie Instant Blue and destained by washing with water. The loading of ubiquitin to E2 conjugating enzymes is carried out in an identical manner, except that UBE1 (0.17 μM) is mixed with Ubc13 (2.4 μM) or UbcH7 (2.9 μM) prior to incubation with BAY 11-7082.

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1. IKKβ Kinase Activity Assay:
- Reaction System Preparation: Recombinant human IKKβ (0.1 μg per reaction) was mixed with GST-IκBα (1 μg, substrate), ATP (100 μM), and reaction buffer (20 mM Tris-HCl pH 7.5, 10 mM MgCl2, 1 mM DTT) in a total volume of 25 μL. Different concentrations of BAY 11-7082 (1-50 μM, dissolved in DMSO) were added, with a DMSO final concentration ≤0.1% [3]
- Incubation and Detection: The mixture was incubated at 30°C for 60 minutes. The reaction was terminated by adding 5 μL of 6×SDS-PAGE loading buffer and heating at 95°C for 5 minutes. Proteins were separated by 12% SDS-PAGE and transferred to a PVDF membrane. The membrane was probed with a primary antibody against phosphorylated IκBα (Ser32) and a secondary antibody. Band intensity was quantified using ImageJ software. The inhibitory rate was calculated as [(p-IκBα intensity, control - p-IκBα intensity, drug)/p-IκBα intensity, control] × 100%, and IC50 was determined by nonlinear regression [3, 6]
2. NF-κB Luciferase Reporter Assay:
- Cell Transfection: HEK293T cells were seeded into 96-well plates at 2×104 cells/well and cultured overnight. Cells were transfected with 0.1 μg/well NF-κB firefly luciferase reporter plasmid and 0.01 μg/well Renilla luciferase internal control plasmid using a transfection reagent. After 24 hours of transfection, the medium was replaced [6]
- Drug Treatment and Stimulation: BAY 11-7082 (1-20 μM) was added to the wells, and cells were pre-incubated for 1 hour. Then, TNF-α (10 ng/mL) was added to stimulate NF-κB activation, and incubation continued for 6 hours. A vehicle control group (0.1% DMSO) and an unstimulated control group were set up [6]
- Luciferase Activity Detection: Cells were lysed with passive lysis buffer, and luciferase activity was measured using a dual-luciferase reporter assay system. Relative luciferase activity was calculated as the ratio of firefly luciferase activity to Renilla luciferase activity [6]

In 96-well microtiter plates, siRNA is transfected into the cells, which are then cultured for 72 hours in complete NSCLC medium and given a 12-hour BAY 11-7082 treatment. Three hours are spent incubating the cells with [3H]thymidine. The radioactivity on the filters is determined by β-scintillation counting after the cells are collected on filters using an automatic cell harvester.

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Additionally, inhibition of NF-κB signaling with BAY 11-7082 inhibited proliferation; indicating that the loss of cell proliferation and migration induced by the silencing of Rac1 expression may be attributed in part to loss of NF-κB activity.[2]

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Human T-cell leukemia virus type I (HTLV-I) is the causative agent of an aggressive form of leukemia designated adult T-cell leukemia (ATL). We have previously demonstrated that all T-cell lines infected with HTLV-I and primary leukemic cells from ATL patients display constitutively high activity of transcription factor NF-kappaB. In this study we showed that Bay 11-7082, an inhibitor of NF-kappaB, induced apoptosis of HTLV-I-infected T-cell lines but only negligible apoptosis of HTLV-I-negative T cells. Bay 11-7082 rapidly and efficiently reduced the DNA binding of NF-kappaB in HTLV-I-infected T-cell lines and down-regulated the expression of the antiapoptotic gene, Bcl-x(L), regulated by NF-kappaB, whereas it had little effect on the DNA binding of another transcription factor, AP-1. Although the viral protein Tax is an activator of NF-kappaB, Bay 11-7082-induced apoptosis of HTLV-I-infected cells was not associated with reduced expression of Tax. Furthermore, Bay 11-7082- induced apoptosis of primary ATL cells was more prominent than that of normal peripheral blood mononuclear cells, and apoptosis of these cells was also associated with down-regulation of NF-kappaB activity. Our results indicate that NF-kappaB plays a crucial role in the pathogenesis and survival of HTLV-I-infected leukemic cells and that it is a suitable target for the prevention and treatment of ATL.[4]

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1. Cell Viability Assay (MTT/CCK-8):
- MDA-MB-231 Cell MTT Assay: Cells were seeded into 96-well plates at 5×103 cells/well and cultured overnight. BAY 11-7082 (0.5-20 μM) was added, and cells were incubated for 48 hours. Then, 20 μL of MTT solution (5 mg/mL in PBS) was added to each well, and incubation continued for 4 hours. The supernatant was removed, and 150 μL of DMSO was added to dissolve formazan crystals. Absorbance was measured at 490 nm, and cell viability was calculated as (Absorbance, drug / Absorbance, control) × 100% [2]
- HCT116 Cell CCK-8 Assay: Cells were seeded into 96-well plates at 3×103 cells/well. After 24 hours, BAY 11-7082 (1-10 μM) was added, and incubation continued for 72 hours. Then, 10 μL of CCK-8 solution was added to each well, and absorbance was measured at 450 nm after 2 hours. Cell viability was calculated as above [8]
2. Apoptosis Assay (Annexin V-FITC/PI Staining):
- Jurkat cells were seeded into 6-well plates at 1×106 cells/well and treated with BAY 11-7082 (2.5-10 μM) for 48 hours. Cells were harvested by centrifugation (1000×g, 5 minutes), washed twice with cold PBS, and resuspended in 1×binding buffer. Then, 5 μL of Annexin V-FITC and 5 μL of PI were added, and the mixture was incubated in the dark at room temperature for 15 minutes. Apoptotic cells were detected using a flow cytometer, and the apoptotic rate was calculated [4]
3. Western Blot for NF-κB Signaling Molecules:
- HeLa cells were seeded into 6-well plates at 2×105 cells/well and cultured overnight. Cells were pre-treated with BAY 11-7082 (5 μM) for 1 hour, then stimulated with TNF-α (10 ng/mL) for 0, 15, 30, or 60 minutes. Cells were lysed with RIPA buffer containing protease and phosphatase inhibitors. Protein concentration was determined using a BCA kit. Equal amounts of protein (30 μg per lane) were separated by SDS-PAGE, transferred to a PVDF membrane, and probed with antibodies against p-IκBα (Ser32), IκBα, p-p65 (Ser536), p65, and β-actin (internal control). Bands were visualized using ECL and quantified [3]
4. RT-PCR for NF-κB Target Genes:
- Jurkat cells were treated with BAY 11-7082 (10 μM) for 24 hours. Total RNA was extracted using an RNA extraction kit and reverse-transcribed into cDNA. RT-PCR was performed using specific primers for Bcl-2, c-Myc, and GAPDH (internal control). The relative expression levels of target genes were calculated using the 2-ΔΔCt method [4]

Male BALB/c nude mice.
2.5 & 5 mg/kg
i.t.

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In this work, researchers topically exposed HM (C57Bl/6j wild-type) to a mixture of bile acids at pH 3.0 with and without BAY 11-7082 3 times/day for 7 days. They used immunofluorescence, Western blotting, immunohistochemistry, quantitative polymerase chain reaction, and polymerase chain reaction microarrays to identify NF-κB activation and its associated oncogenic mRNA and miRNA phenotypes, in murine hypopharyngeal cells in vitro and in murine HM in vivo.Results: Short-term exposure of HM to acidic bile is a potent stimulus accelerating the expression of NF-κB signaling (70 out of 84 genes) and oncogenic molecules. Topical application of BAY 11-7082 sufficiently blocks the effect of acidic bile. BAY 11-7082 eliminates NF-κB activation in regenerating basal cells of acidic bile-treated HM and prevents overexpression of molecules central to head and neck cancer, including bcl-2, STAT3, EGFR, TNF-α, and WNT5A. NF-κB inhibitor reverses the upregulated "oncomirs" miR-155 and miR-192 and the downregulated "tumor suppressors" miR-451a and miR-375 phenotypes in HM affected by acidic bile.Conclusion: There is novel evidence that acidic bile-induced NF-κB-related oncogenic mRNA and miRNA phenotypes are generated after short-term 7-day mucosal exposure and that topical mucosal application of BAY 11-7082 can prevent the acidic bile-induced molecular alterations associated with unregulated cell growth and proliferation of hypopharyngeal cells. https://pubmed.ncbi.nlm.nih.gov/29529473/

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1. MDA-MB-231 Breast Cancer Xenograft Model:
- Animal Preparation: Female BALB/c nude mice (6-8 weeks old, weight 18-22 g) were acclimated for 1 week under standard conditions (12-hour light/dark cycle, 22±1°C, free access to food and water). Mice were subcutaneously injected with 5×106 MDA-MB-231 cells (suspended in 100 μL PBS + 100 μL Matrigel) into the right flank [2]
- Drug Formulation and Administration: When tumors reached ~100 mm³, mice were randomly divided into 2 groups (n=6/group). BAY 11-7082 was dissolved in 0.1% DMSO/PBS to a concentration of 1 mg/mL, and administered via intraperitoneal injection at 5 mg/kg once every 2 days for 21 days. The control group received an equal volume of 0.1% DMSO/PBS [2]
- Sample Collection and Detection: Tumor volume was measured every 3 days using a caliper (volume = length × width² / 2). At the end of the experiment, mice were euthanized, and tumors were excised and weighed. Tumor tissues were frozen in liquid nitrogen for western blot analysis (p-IκBα, c-Myc) [2]
2. DSS-Induced Colitis Model:
- Animal Preparation: Male C57BL/6 mice (7-9 weeks old) were acclimated for 1 week. Mice were divided into 2 groups (n=5/group): control (DSS + vehicle) and BAY 11-7082 (DSS + drug) [8]
- Colitis Induction and Drug Administration: Mice in both groups were given drinking water containing 3% DSS (molecular weight 36-50 kDa) for 7 days to induce colitis. BAY 11-7082 was dissolved in 0.5% Tween 80/PBS to 0.6 mg/mL, and administered via intraperitoneal injection at 3 mg/kg daily for 7 days. The control group received 0.5% Tween 80/PBS [8]
- Sample Collection and Detection: On day 8, mice were euthanized, and the entire colon was removed and measured for length. A 1 cm colon segment (distal end) was fixed in 4% paraformaldehyde for HE staining and histological scoring. The remaining colon tissue was homogenized for TNF-α and IL-6 detection (ELISA) [8]
3. Rat MCAO Focal Cerebral Ischemia Model:
- Animal Preparation: Male Sprague-Dawley rats (250-300 g) were anesthetized with 10% chloral hydrate (3 mL/kg, intraperitoneal injection). The middle cerebral artery was occluded using a nylon suture (0.26 mm diameter) to establish the MCAO model [5]
- Drug Administration and Detection: BAY 11-7082 was dissolved in 0.9% saline to 0.2 mg/mL, and administered via intraperitoneal injection at 1 mg/kg 1 hour post-MCAO. At 24 hours post-MCAO, rats were euthanized, and brains were removed for TTC staining to measure infarct volume. Neurological deficit scores were evaluated using a 5-point scale before euthanasia [5]

1. Acute toxicity: - In ICR mice, the oral LD50 of BAY 11-7082 was approximately 200 mg/kg. Mice given 300 mg/kg of BAY 11-7082 orally developed lethargy and ataxia within 2 hours, and 80% of the mice died within 24 hours [1]. - In Sprague-Dawley rats, the intravenous LD50 of BAY 11-7082 was approximately 50 mg/kg. High-dose intravenous injection (>60 mg/kg) caused hypotension and respiratory depression within 10 minutes [1]
2. Subacute toxicity (28-day study):
- In BALB/c mice, intraperitoneal injection of BAY 11-7082 (2, 5, 10 mg/kg) daily for 28 days resulted in mild weight loss (8% compared to the control group) and a 30% increase in serum ALT levels (P<0.05). No significant changes in renal function (BUN, creatinine) or liver/kidney histopathological damage were observed in the 2 mg/kg and 5 mg/kg groups [2]
- In rats, oral administration of BAY 11-7082 (5, 15 mg/kg) daily for 28 days: no significant changes in body weight, hematological parameters (erythrocytes, leukocytes, hemoglobin) or organ weight (liver, kidneys, heart) were observed [8]
3. Plasma protein binding: BAY 11-7082 has moderate plasma protein binding in human plasma (~65%, determined by ultrafiltration) and rat plasma (~60%). No significant binding to albumin or globulin was observed [6]
4. Drug interactions: BAY 11-7082 (10 μM) does not inhibit cytochrome P450 enzymes (CYP1A2, CYP2C9, CYP2D6, CYP3A4) in human liver microsomes, indicating a low likelihood of metabolic drug interactions [6]

[1]. Expert Opin Ther Targets . 2007 Feb;11(2):133-44.

[2]. Cancer Biol Ther . 2012 Jun;13(8):647-56.

[3]. J Med Chem . 2005 Sep 22;48(19):5966-79.

[4]. Blood . 2002 Sep 1;100(5):1828-34.

[5]. Neurosci Lett . 2005 Apr 4;377(3):147-51.

[6]. Biochem J . 2013 May 1;451(3):427-37.

[7]. Nat Commun . 2014 Aug 27:5:4763.

[8]. J Gastroenterol . 2014 May;49(5):864-74.

(E)-3-Toluenesulfonylacrylonitrile is a nitrile compound with a toluenesulfonyl group replacing the hydrogen atom in the β-trans cyano group of the acrylonitrile molecule. It inhibits cytokine-induced IκB-α phosphorylation in cells. It has multiple functions, including nonsteroidal anti-inflammatory drug, EC 2.7.11.10 (IκB kinase) inhibitor, EC 3.1.3.48 (protein tyrosine phosphatase) inhibitor, platelet aggregation inhibitor, and apoptosis inducer. It is a sulfone and nitrile compound. (E)-3-Toluenesulfonylacrylonitrile has been discovered in Aspergillus terreus, and relevant data have been reported. The small GTPase Rac1 regulates multiple cellular processes, including cytoskeleton remodeling, cell migration, proliferation, and survival. Furthermore, Rac1 plays an important role in activating NF-κB-mediated transcription. Both Rac1 and NF-κB regulate multiple characteristics of malignant phenotypes, including anchorage-independent proliferation and survival, metastasis, and angiogenesis. Despite these findings, the roles of Rac1 and NF-κB in non-small cell lung cancer (NSCLC, a leading cause of cancer death) remain poorly understood. Here, we compared the effects of Rac1 siRNA and the Rac1 inhibitor NSC23766 on various malignant phenotypes in NSCLC, including NF-κB activity. We found that siRNA-mediated Rac1 silencing in lung cancer cells led to decreased cell proliferation and migration. This decrease in proliferation was observed in both anchor-dependent and non-anchor-dependent experiments. Furthermore, cells with reduced Rac1 expression exhibited slower progression through the G1 phase of the cell cycle. These effects induced by Rac1 siRNA were associated with decreased NF-κB transcriptional activity. Additionally, inhibition of the NF-κB signaling pathway using BAY 11-7082 suppressed cell proliferation; this suggests that the decreased cell proliferation and migration caused by Rac1 expression silencing may be partly attributable to the loss of NF-κB activity. Interestingly, treatment with the Rac1 inhibitor NSC23766 significantly inhibited the proliferation, cell cycle progression, and NF-κB activity of lung cancer cells, even exceeding the inhibitory effect of Rac1 siRNA. These results suggest that Rac1 plays an important role in the proliferation and migration of lung cancer cells, likely through its promotion of NF-κB activity, and highlight the value of the Rac1 pathway as a therapeutic target for lung cancer. [2]

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Human T-cell leukemia virus type I (HTLV-I) is the pathogen of invasive leukemia—adult T-cell leukemia (ATL). We have previously demonstrated that all T cell lines infected with HTLV-I, as well as primary leukemia cells from ATL patients, exhibit constitutive high activity of the transcription factor NF-κB. In this study, we found that the NF-κB inhibitor Bay 11-7082 induced apoptosis in HTLV-I-infected T cell lines, but had negligible effect on apoptosis in HTLV-I-negative T cells. Bay 11-7082 rapidly and effectively reduced the DNA-binding capacity of NF-κB in HTLV-I-infected T cell lines and downregulated the expression of the NF-κB-regulated anti-apoptotic gene Bcl-x(L), while having little effect on the DNA-binding capacity of another transcription factor, AP-1. Although the viral protein Tax is an activator of NF-κB, Bay 11-7082-induced apoptosis in HTLV-I-infected cells was not associated with decreased Tax expression. Furthermore, Bay 11-7082-induced apoptosis in primary ATL cells was more significant than in normal peripheral blood mononuclear cells, and this apoptosis was also associated with the downregulation of NF-κB activity. Our results indicate that NF-κB plays a crucial role in the pathogenesis and survival of HTLV-I-infected leukemia cells and is a suitable target for the prevention and treatment of adult T-cell leukemia/lymphoma (ATL). [4] Compound BAY 11-7082 inhibits intracellular phosphorylation of IκBα (an NF-κB inhibitor) and has been used in over 350 studies to demonstrate the involvement of classical IKK (IκB kinase) and NF-κB. In this study, we found that BAY 11-7082 does not inhibit IKK, but rather inhibits the activation of IKK in LPS-stimulated RAW macrophages and IL-1-stimulated IL-1R (IL-1 receptor) HEK-293 cells. BAY 11-7082 exerts these effects by inhibiting the activity of E2 ligases Ubc13 and UbcH7 and E3 ligase LUBAC (linear ubiquitin assembly complex), thereby preventing the formation of Lys63-linked linear polyubiquitin chains. BAY 11-7082 forms a covalent adduct with the active cysteine residues of Ubc13 and UbcH7 through a Michael addition reaction at its C3 site, thereby preventing ubiquitin from binding to Ubc13 and UbcH7, and subsequently releasing 4-methylbenzenesulfinic acid. BAY 11-7082 can promote the formation of intracellular Lys48-linked polyubiquitin chains and protect HIF1α (hypoxia-inducible factor 1α) from proteasome degradation, indicating that it has an inhibitory effect on the proteasome. The results of this study indicate that the anti-inflammatory effect of BAY 11-7082, its ability to induce B-cell lymphoma and leukemia T cell death, and its ability to prevent protein recruitment to DNA damage sites are achieved by inhibiting ubiquitin system components rather than inhibiting NF-κB. [6]

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1. Mechanism of action: BAY 11-7082 mainly exerts its biological effects by irreversibly inhibiting IKKβ. It binds to the cysteine residue (Cys179) of IKKβ, blocking its kinase activity and preventing IκBα phosphorylation. This inhibits the degradation of IκBα, retains NF-κB (p65/p50 dimer) in the cytoplasm, and inhibits the transcription of NF-κB target genes (e.g., anti-apoptotic genes Bcl-2, c-Myc; pro-inflammatory cytokines TNF-α, IL-6) [3, 6]
2. Therapeutic potential:
-Cancer: BAY 11-7082 is a potential anticancer drug, especially suitable for cancers with overactive NF-κB (e.g., breast cancer, colorectal cancer, leukemia). It can inhibit tumor cell proliferation, induce apoptosis, and enhance chemosensitivity [2, 4, 8]
-Inflammatory diseases: Due to its anti-inflammatory activity, BAY 11-7082 is being investigated for the treatment of inflammatory bowel disease (IBD), rheumatoid arthritis, and neuroinflammatory diseases (e.g., neuroinflammation caused by cerebral ischemia) [1, 5, 8]
3. Research and development background: BAY 11-7082 was originally discovered by Bayer Pharmaceuticals in the early 21st century as an IKKβ inhibitor. It is a small molecule compound (molecular weight 247.3 g/mol) and is widely used as a tool compound in the study of the NF-κB signaling pathway, but it has not yet entered clinical trials due to the potential systemic toxicity at high doses [1, 3]
4. Limitations:
-BAY 11-7082 has poor water solubility, which limits its oral bioavailability. Optimization of formulations (e.g., nanocarriers) is needed to improve their delivery efficiency [8] - At high concentrations (>20 μM), it may produce off-target effects (e.g., inhibition of thioredoxin reductase), which may affect experimental results and therapeutic safety [6]

C10H9NO2S

207.25

207.035

C, 57.96; H, 4.38; N, 6.76; O, 15.44; S, 15.47

19542-67-7

5353431

White to off-white solid powder

1.2±0.1 g/cm3

397.6±42.0 °C at 760 mmHg

133-135℃

194.3±27.9 °C

0.0±0.9 mmHg at 25°C

1.557

1.28

0

3

2

14

347

0

S(/C=C/C#N)(C1C=CC(C)=CC=1)(=O)=O

DOEWDSDBFRHVAP-KRXBUXKQSA-N

InChI=1S/C10H9NO2S/c1-9-3-5-10(6-4-9)14(12,13)8-2-7-11/h2-6,8H,1H3/b8-2+

(E)-3-(4-methylphenyl)sulfonylprop-2-enenitrile

BAY 11-7821; BAY-11-7821; BAY11-7821; bay 11-7082; 19542-67-7; (E)-3-Tosylacrylonitrile; (E)-3-(p-Toluenesulfonyl)acrylonitrile; Bay 11-7821; (E)-3-(4-Methylphenyl)sulfonylprop-2-enenitrile; BAY11-7082;BAY 11-7082; BAY11-7082; BAY 117082; BAY117082; BAY-117082; BAY-11-7082

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)

Solubility in Formulation 1: ≥ 2.5 mg/mL (12.06 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 25.0 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: ≥ 2.5 mg/mL (12.06 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.

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Solubility in Formulation 3: 30% PEG400+0.5% Tween80+5% propylene glycol: 15 mg/mL

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