Glycochemistry & Glycobiology Lab, Shanghai Institute of Materia Medica, Chinese Academy of Sciences, Shanghai 201203, China.
Acta Pharmacol Sin. 2012 Sep;33(9):1204-16. doi: 10.1038/aps.2012.56. Epub 2012 Jun 11.
To characterize a small molecule compound HK-156 as a novel inhibitor of the nuclear factor κB (NF-κB) signaling pathway.
THP-1 monocytes and HEK293/hTLR4A-MD2-CD14 cells were tested. HK-156 and compound 809, an HK-156 analogue, were synthesized. A luciferase assay was used to evaluate the transcriptional activity of NF-κB. The levels of cytokines were measured with cytokine arrays, ELISA and quantitative PCR. An electrophoretic mobility shift assay (EMSA), immunofluorescence, Western blot and mass spectrometry were used to investigate the molecular mechanisms underlying the actions of the agent. BALB/c mice challenged with lipopolysaccharide (LPS, 15 mg/kg, ip) were used as a mouse experimental endotoxemia model.
In HEK293hTLR4/NF-κB-luc cells treated with LPS (1000 ng/mL), HK-156 inhibited the transcriptional activity of NF-κB in a concentration-dependent manner (IC₅₀=6.54 ± 0.37 μmol/L). Pretreatment of THP-1 monocytes with HK-156 (5, 10 and 20 μmol/L) significantly inhibited LPS-induced release and production of TNF-α and IL-1β, attenuated LPS-induced translocation of NF-κB into the nucleus and its binding to DNA, and suppressed LPS-induced phosphorylation and degradation of IκBα, and phosphorylation of IKKβ and TGFβ-activated kinase (TAK1). Meanwhile, HK-156 (5, 10 and 20 μmol/L) slightly suppressed LPS-induced activation of p38. The effect of HK-156 on LPS-induced activation of NF-κB signaling was dependent on thiol groups of cysteines in upstream proteins. In mouse models of sepsis, pre-injection of HK-156 (50 mg/kg, iv) significantly inhibited TNFα production and reduced the mortality caused by the lethal dose of LPS.
HK-156 inhibits LPS-induced activation of NF-κB signaling by suppressing the phosphorylation of TAK1 in vitro, and exerts beneficial effects in a mouse sepsis model. HK-156 may therefore be a useful therapeutic agent for treating sepsis.
将小分子化合物 HK-156 鉴定为核因子 κB（NF-κB）信号通路的新型抑制剂。
检测 THP-1 单核细胞和 HEK293/hTLR4A-MD2-CD14 细胞。合成 HK-156 和其类似物化合物 809。采用荧光素酶检测试剂盒评估 NF-κB 的转录活性。细胞因子阵列、ELISA 和定量 PCR 检测细胞因子水平。电泳迁移率变动分析（EMSA）、免疫荧光、Western blot 和质谱分析用于研究该药物作用的分子机制。脂多糖（LPS，15mg/kg，腹腔注射）处理的 BALB/c 小鼠作为小鼠实验性内毒素血症模型。
在 LPS（1000ng/mL）处理的 HEK293hTLR4/NF-κB-luc 细胞中，HK-156 呈浓度依赖性抑制 NF-κB 的转录活性（IC₅₀=6.54±0.37μmol/L）。HK-156（5、10 和 20μmol/L）预处理 THP-1 单核细胞可显著抑制 LPS 诱导的 TNF-α和 IL-1β释放和产生，减弱 LPS 诱导的 NF-κB 向核内易位及其与 DNA 的结合，抑制 LPS 诱导的 IκBα磷酸化和降解，以及 IKKβ和 TGFβ激活激酶（TAK1）磷酸化。同时，HK-156（5、10 和 20μmol/L）轻度抑制 LPS 诱导的 p38 激活。HK-156 对 LPS 诱导的 NF-κB 信号转导的激活作用依赖于上游蛋白半胱氨酸巯基。在脓毒症小鼠模型中，HK-156（50mg/kg，静脉注射）预处理可显著抑制 TNFα产生，并降低致死剂量 LPS 引起的死亡率。
HK-156 通过抑制 TAK1 的磷酸化在体外抑制 LPS 诱导的 NF-κB 信号转导，在小鼠脓毒症模型中发挥有益作用。因此，HK-156 可能是治疗脓毒症的一种有用的治疗剂。
