In vitro activity: BAY 11-7082 completely and specifically abrogates NF-κB DNA binding, downregulating the NF-κB-inducible cytokine IL-6 and inducing apoptosis. BAY 11-7082 (< 8 μM) is able to effectively inhibit both basal and TNFα stimulated NFκB luciferase activity in a dose dependent manner. BAY 11-7082 (8 μM) strongly inhibits the rate of proliferation in NCI-H1703 cells. Bay 11-7082 (5 μM) rapidly and efficiently reduces the DNA binding of NF-kappaB in HTLV-I-infected T-cell lines and down-regulates the expression of the antiapoptotic gene, Bcl-x(L), whereas it has little effect on the DNA binding of another transcription factor, AP-1. Bay 11-7082-induced apoptosis of primary ATL cells is more prominent than that of normal peripheral blood mononuclear cells, and apoptosis of these cells is also associated with down-regulation of NF-kappaB activity. Bay 11-7082 (5 μM) selectively induces apoptosis of HTLV-I–infected T-cell lines associated with down-regulation of the expression of cyclin D1, cyclin D2, and Bcl-xL. BAY 11-7082 (100 μM) prevents the nuclear translocation of p65 elicited by NMDA and the NMDA-induced increase of NF-κB binding in mouse hippocampal slices. BAY 11-7082 prevents NMDA toxicity occurring in CA1 region of hippocampal slices with 40% neuroprotection at 20 μM and 70% neuroprotection at 100 μM. BAY 11-7082 at all concentrations tested significantly inhibits NF-κB p65 DNA-binding activity in adipose tissue, whereas in skeletal muscle, BAY 11-7082 at 50 μM and 100 μM significantly inhibits NF-κB p65 DNA-binding activity. BAY 11-7082 (100 μM) reduces IKK-β protein in human adipose tissue and skeletal muscle. BAY 11-7082 (100 μM) significantly decreases the release of TNF-α from adipose tissue, whereas the release of IL-6 and IL-8 is significantly inhibited at all concentrations of BAY 11-7082 tested. BAY 11-7082 (50 μM) significantly decreases the release of TNF-α, IL-6, and IL-8 in skeletal muscle. BAY 11-7082 is also found to inactivate the E2-conjugating enzymes Ubc (ubiquitin conjugating) 13 and UbcH7 and the E3 ligase LUBAC (linear ubiquitin assembly complex), and thus induces B-cell lymphoma and leukaemic T-cell death

Kinase Assay: 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.

Cell Assay: Experiments show that Bay 11-7821 is highly toxic at concentrations of effective IKK inhibition. It can induce cell necrosis in MM cells. Additionally, Bay 11-7821 is proved to have an anti-inflammatory ability via inducing death of B-cell lymphoma and leukaemic T-cell. It is also reported to inhibit the NALP3 inflammasome in macrophages