IMD-0354 (IKK2 Inhibitor V) is an IKKβ inhibitor and inhibits IκBα phosphorylation in NF-κB pathway.

IMD-0354 (< 5 μM) inhibits the expression of NF-κB as well as the translocation of NF-κB to the nucleus in HMC-1 cells. IMD-0354 suppresses cell proliferation in a time- and dose-dependent manner in HMC-1 cells. IMD-0354 (0.5 μM) almost inhibits the proliferation of IC-2 g559 cells and IC-2V814 cells. IMD-0354 (0.5 μM) results in arrest of the cell cycle at the G0/G1 phase in HMC-1 cells. IMD-0354 (1 μM) increases the number of cells with hypodiploid DNA content in HMC-1 cells. IMD-0354 (<1 μM) decreases the ratio of cells in S and G2/M phases in HMC-1 cells. IMD-0354 (1 μM) downregulates Cyclin D3 expression as well as pRb phosphorylation level in a time-dependent manner in HMC-1 cells. IMD-0354 (< 10 μM) has no influence on the signals of STAT3 and STAT6, whereas the phosphorylation of STAT1 and STAT5 is very slightly suppressed at high concentrations in HMC-1 cells. IMD-0354 suppresses the translocation of NF-κB to the nucleus in CBhCMCs after 24 hours in a dose-dependent manner. [1] IMD-0354 inhibits 98.5% of NF-κB activity at a concentration of 10 μg/ml in HepG2 cells. [2] IMD-0354 (1 μM) ameliorates the TNFα-induced decrease in the adiponectin concentration in the media, when the TNFα (6 nM) and insulin (100 nM) are administered simultaneously in 3T3-L1 adipocytes serum-starved for 12 h. IMD-0354 (1 μM) restores the phosphorylation of Akt down-regulated by the TNFα treatment, when the TNFα (6 nM) and insulin (100 nM) are administered simultaneously in 3T3-L1 adipocytes serum-starved for 12 h. [3] IMD-0354 (1 μM) inhibits phosphorylation of IκBα and nuclear translocation of nuclear factor-kappa B (NF-κB) induced by tumor necrosis factor-α (TNF-α) in cultured cardiomyocytes. IMD-0354 (1 μM) significantly reduces TNF-α-induced production of interleukin-1β and monocyte chemoattractant protein-1 from cultured cardiomyocytes. [4]

IMD-0354 at 5 mg/kg also significantly decreases NF-κB, but the magnitude of the decrease is lower than with 20 mg/kg IMD-0354 in lungs of OVA-sensitized mice. IMD-0354 (20 mg/kg) ameliorates airway hyperresponsiveness and reduces the numbers of bronchial eosinophils and mucus-producing cells in OVA-sensitized mice. IMD-0354 (20 mg/kg) also reduces the total numbers of cells and eosinophils in bronchoalveolar lavage fluid in OVA-sensitized mice. IMD-0354 (20 mg/kg) inhibits the production of Th2 cytokines such as interleukin (IL)-5 and IL-13 and eotaxin in the airways and/or lungs of OVA-sensitized mice, but it does not affect the restoration of Th1 cytokines such as IL-12 and interferon-gamma under the same experimental conditions. IMD-0354 (20 mg/kg) results in a partial decrease in serum IgE concentration in OVA-sensitized mice. [2] IMD-0354 significantly decreases the plasma glucose levels in KKAy mice treated with and fed an HF diet in an dose-dependent manner without influence of body weight. [3] IMD-0354 (10 mg/kg) results in a significant dose-dependent reduction of the infarction area/area at risk ratio and the preservation of fractional shortening ratio. [4]

In vitro mTOR kinase assays : The reaction mixture consisted of the following components in 10 μL assay buffer (50 mM Hepes pH 7.5, 10 mM MgCl 2, 3 mM MnCl 2, 1 mM EGTA, 2 mM DTT, 0.01%Tween-20): 0.10 μg/mL of in-house generated mTOR enzyme, 0.05 μM ULight-eIF4E-binding protein 1 (Thr37/46) peptide and 10 μM ATP. The mixture is incubated for 60 min at room temperature. 10 μL of Detection mixture consisted of 16 mM EDTA, 0.004 mM Eu-W1024-labeled Anti-Phospho-eIF4E-binding protein 1-(Thr37/46) antibody and 1X LANCE? Detection Buffer is then added and incubated for 60 min.

Cells (2×105 cells/mL) are incubated in phenol red free α-MEM containing 10% FCS (for HMC-1 and IC-2 cells) or 5% FCS (for CBhCMCs), and antibiotics with or without various concentrations of IMD-0354, STI571, or PDTC. IC-2WT cells and CBhCMCs are incubated in the presence of 100 ng/mL recombinant rat or recombinant human SCF. One hundred microliters of cell suspension is applied to each well of 96-well culture plates and are incubated for 24, 48, and 72 hours. Before 4 hours from the end of the culture, 10 μL of 5 mg/mL 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyl-tetrazolium bromide (MTT) dissolves in PBS is added to each well. The reaction is stopped with the addition of 100 μL of 10% SDS in 0.01 N HCl. Absorbance is measured at 577 nm with ImmunoMini NJ-2300.(Only for Reference)