BMS-345541 (IKK Inhibitor III) is a highly selective inhibitor of the catalytic subunits of IKK-2 and IKK-1 with IC50 of 0.3 μM and 4 μM, respectively.

IKK2:0.3 μM, IKK1:4 μM

75 mg/kg BMS-345541处理过的肿瘤小鼠表现对SK-MEL-5,A375和Hs 294T生长的有效抑制和对照组相比抑制率分别为86％,69％和67％.BMS-345541（100 mg/kg）将累积性关节炎损伤评分从4.4降低至0,伴随着较低的胫骨关节退化和炎症严重程度,滑膜增生,骨吸收和软骨侵蚀.BMS-345541剂量依赖性地抑制了IL-1β信号,100 mg/kg剂量组动物显示与无疾病对照动物相当的水平.口服100 mg/kg BMS-345541时,降低了葡聚糖硫酸钠诱导的结肠炎的小鼠的重量比,临床肠道评分,平均损伤评分和平均炎症评分,分别是0.86（vs对照组的0.77）,1.0（vs对照组的2.5）,5.66（vs对照组的8.52）,6.82（vs对照组的12.33）.

5 μM BMS-345541在BE-13和DND-41细胞中,诱导细胞停滞在细胞周期中的G2/M期,并阻碍RPMI-8402细胞停留在亚-G1期。5 μM BMS-345541处理16小时导致凋亡细胞的增多,伴随着procaspase-8,procaspase-3和聚（ADP-核糖）时间依赖性的解离。5 μM BMS-34554诱导IκBα和p65的时间依赖性的去磷酸化。10 μM BMS-345541抑制正常人类表皮黑素细胞和转移性黑色素瘤细胞（SK-MEL-5,A375和HS294T）的生长,72小时抑制率分别是由96％和99％。10 μM BMS-345541导致IKK活性降低76％,NF-kB活性降低95％,以及CXCL1产生。 BMS-345541剂量依赖性地抑制THP-1单核细胞中TNF-α刺激的IκBα的磷酸化,IC50为4 μM。BMS-345541在人脐静脉内皮细胞中,抑制TNFα-诱导的ICAM-1和VCAM-1的表达,IC50为5 μM。BMS-345541结合到IKK-1和IKK-2的变构位点,进而影响亚基的不同的活性位点。BMS-345541影响几种有丝分裂细胞周期转换,包括进入有丝分裂,早中期至后期的进展和胞质分裂。在细胞中加入BMS-345541导致细胞停留在G期,并抑制Aurora A,B和C的激活,Cdk1的激活和组蛋白H3的磷酸化。BMS-34554处理导致T-ALL细胞中FOXO3a的核易位,包括控制p21 CIP1基因的表达水平。BMS-345541还抑制儿童患者的T-ALL原代细胞的生长,IC50为2-6 μM。

Enzyme Assays: Assays measuring the enzyme-catalyzed phosphorylation of GST-IκBα are performed by adding enzyme (a final concentration of 0.5 μg/mL) at 30 ℃ to solutions of 100 μg/mL GST- IκBα and 5 μM [33P]ATP in 40 mM Tris HCl, pH 7.5, containing 4 mM MgCl2, 34 mM sodium phosphate, 3 mM NaCl, 0.6 mM potassium phosphate, 1 mM KCl, 1 mM dithiothreitol, 3% (w/v) glycerol, and 250 μg/mL bovine serum albumin. The specific activity of [33P]ATP used in the assay is 100 Ci/mmol. After 5 min, the kinase reactions are stopped by the addition of 2× Laemmli sample buffer and heat-treated at 90 ℃ for 1 min. The samples are then loaded on to NuPAGE 10% BisTris gels. After completion of SDS-PAGE, gels are dried on a slab gel dryer. The bands are then detected using a 445Si PhosphorImager, and the radioactivity is quantified using ImageQuant software. Under these conditions, the degree of phosphorylation of GST-IκBα is linear with time and concentration of enzyme.

1×105 cells per well are plated in six-well plates with 10% fetal bovine serum medium overnight to allow cell adhesion. Cells ae cultured in medium containing BMS-345541 for 72 hours of treatment. Cells are counted with a hemocytometer.(Only for Reference)