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

NF-κB:1.2 μM (IC50)

IMD-0354（<5 μM）抑制了HMC-1细胞中NF-κB的表达以及NF-κB向细胞核的转移。IMD-0354以时间和剂量依赖的方式抑制HMC-1细胞的增殖。IMD-0354（0.5 μM）几乎完全抑制了IC-2 g559细胞和IC-2V814细胞的增殖。IMD-0354（0.5 μM）导致HMC-1细胞周期在G0/G1阶段停滞。IMD-0354（1 μM）增加了HMC-1细胞中具有低二倍体DNA含量的细胞数。IMD-0354（<1 μM）降低了HMC-1细胞中处于S和G2/M阶段的细胞比例。IMD-0354（1 μM）降低了HMC-1细胞中Cyclin D3表达以及pRb磷酸化水平，且这一作用随时间依赖增加。在高浓度下，IMD-0354（<10 μM）对HMC-1细胞中STAT3和STAT6的信号无影响，而STAT1和STAT5的磷酸化略有抑制。在剂量依赖的方式下，IMD-0354抑制了CBhCMCs中NF-κB向细胞核的转移，持续24小时。[1]在HepG2细胞中，IMD-0354以10 μg/ml的浓度抑制了98.5%的NF-κB活性。[2]IMD-0354（1 μM）改善了在同时给予TNFα（6 nM）和胰岛素（100 nM）的情况下，TNFα引起的3T3-L1脂肪细胞星化12小时后培养基中的脂联素浓度下降，以及TNFα治疗下调的Akt磷酸化恢复。[3]IMD-0354（1 μM）抑制了由肿瘤坏死因子-α（TNF-α）引起的IκBα磷酸化和NF-κB核内转移，在培养的心肌细胞中显著减少TNF-α诱导的白介素-1β和单核细胞趋化蛋白-1的产生。[4]

IMD-0354以5 mg/kg的剂量显著降低了OVA致敏小鼠肺部的NF-κB水平，但其降低幅度低于20 mg/kg剂量。20 mg/kg的IMD-0354能够改善气道高反应性，减少OVA致敏小鼠气管内嗜酸性粒细胞和黏液产生细胞的数量。此剂量还能减少OVA致敏小鼠支气管肺泡灌洗液中的细胞总数和嗜酸性粒细胞数量，抑制如白细胞介素(IL)-5、IL-13及趋化素等Th2型细胞因子在气道和/或肺部的产生，但在相同实验条件下不影响如IL-12和干扰素-γ等Th1型细胞因子的恢复。20 mg/kg的IMD-0354使OVA致敏小鼠的血清IgE浓度部分降低。[2] IMD-0354在剂量依赖性方式下显著减少了接受高脂饲料喂养的KKAy小鼠的血浆葡萄糖水平，且不影响体重。[3] 10 mg/kg的IMD-0354显著以剂量依赖的方式减少梗塞区域/风险区域比率并保留了收缩分数比率。[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)