Background
TAK 242 is a toll-like receptor 4 (TLR4) signaling inhibitor. Binds to intracellular domain of TLR4. Inhibits LPS-induced cytokine production in vitro (IC50 values are 1.3, 1.3 and 3.2 nM for IL-6, TNFα and NO production).?TAK-242 binds to the TIR domain of TLR4 via Cys747,[1] and inhibits MyD88 and TRIF-dependent pathway. Previous studies showed that TAK-242 prevented acute kidney injury and lung injury in LPS-injected sheep and mice. What’s more, TAK-242 has also been tested in a clinical trial of patients with sepsis and was found to be well tolerated. It is also researched to have the usage as a therapeutic strategy for endotoxemia-associated muscle weakness.[2]
In vitro study demonstrated that TAK-242 disrupts the interactions of TLR4 with its adaptor molecules, TIRAP and TRAM. TAK-242 inhibited the association of TIRAP with TLR4 and the association of TRAM with TLR4. The inhibitory effect of TAK-242 may result from the direct action of TAK-242 on TLR4 without requiring the induction of intermediate gene expression or de novo protein synthesis. Moreover, strong evidence was provided that TAK-242 modulates the formation of the signaling complex containing the proximal elements in TLR4 signaling. TAK-242 inhibited TIRAP mediated NF-γB activation and TRAM-mediated NF-γB and ISRE activation in the presence of TLR4/MD-2. In addition, TAK-242 also inhibited LPS-induced NF-γB and ISRE activations in HEK293-hTLR4/MD2-CD14 cells. These results indicated that TAK-242 impairs the ability of TLR4 to associate with adaptor molecules and blocks subsequent signal transduction.[1]
In vivo experiments indicated that TAK-242 effectively reduced the severity of acute liver failure and increased the survival rate of FH mice. Mechanistically, the hepatoprotective effect of TAK-242 was related to inhibiting inflammation, reducing oxidative stress, and increasing the proportion of MDSCs. Results suggested that TAK-242 is a potent TLR4 inhibitor and would be a potential drug to protect against acute liver failure.[3]
参考文献:
[1]. Matsunaga N, et al. TAK-242 (resatorvid), a small-molecule inhibitor of Toll-like receptor (TLR) 4 signaling, binds selectively to TLR4 and interferes with interactions between TLR4 and its adaptor molecules. Mol Pharmacol. 2011 Jan;79(1):34-41.
[2]. Ono Y, et al. TAK-242, a specific inhibitor of Toll-like receptor 4 signalling, prevents endotoxemia-induced skeletal muscle wasting in mice. Sci Rep. 2020 Jan 20;10(1):694.
[3]. Wang H, et al. Toll-like Receptor 4 Inhibitor TAK-242 Improves Fulminant Hepatitis by Regulating Accumulation of Myeloid-Derived Suppressor Cell. Inflammation. 2021 Apr;44(2):671-681.
TAK 242 是一种 Toll 样受体 4 (TLR4) 信号转导抑制剂。结合 TLR4 的细胞内结构域。在体外抑制 LPS 诱导的细胞因子产生（IL-6、TNFα 和 NO 产生的 IC50 值为 1.3、1.3 和 3.2 nM）。 TAK-242 通过 Cys747[1] 与 TLR4 的 TIR 结构域结合，并抑制 MyD88 和 TRIF 依赖性通路。先前的研究表明，TAK-242 可预防注射 LPS 的绵羊和小鼠的急性肾损伤和肺损伤。此外，TAK-242 还在脓毒症患者的临床试验中进行了测试，发现耐受性良好。还研究了将其用作内毒素血症相关肌肉无力的治疗策略。[2]
体外研究表明，TAK-242 破坏了 TLR4 与其衔接分子 TIRAP 和 TRAM 的相互作用。 TAK-242 抑制 TIRAP 与 TLR4 的结合以及 TRAM 与 TLR4 的结合。 TAK-242 的抑制作用可能源于 TAK-242 对 TLR4 的直接作用，而不需要诱导中间基因表达或蛋白质从头合成。此外，提供了强有力的证据表明 TAK-242 调节包含 TLR4 信号转导中近端元件的信号转导复合物的形成。 TAK-242 在 TLR4/MD-2 存在的情况下抑制 TIRAP 介导的 NF-γB 激活和 TRAM 介导的 NF-γB 和 ISRE 激活。此外，TAK-242 还抑制 HEK293-hTLR4/MD2-CD14 细胞中 LPS 诱导的 NF-γB 和 ISRE 激活。这些结果表明，TAK-242 削弱了 TLR4 与衔接分子结合的能力，并阻断了后续的信号转导。[1]
体内实验表明，TAK-242 可有效降低急性肝衰竭的严重程度并提高 FH 小鼠的存活率。从机制上讲，TAK-242 的保肝作用与抑制炎症、减少氧化应激和增加 MDSCs 的比例有关。结果表明，TAK-242 是一种有效的 TLR4 抑制剂，有望成为预防急性肝衰竭的潜在药物。[3]
Protocol
| Cell experiment [1]: |
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Cell lines
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HEK293, HEK293-hTLR4/MD2-CD14
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Preparation Method
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HEK293 cells were cultured in DMEM supplemented with 10% heat-inactivated FCS, and 50 μg/ml gentamicin. HEK293-hTLR4 /MD2-CD14 cells were cultured in DMEM supplemented with 10% FCS, 10 μg/ml blasticidin, 50 μg/ml HygroGold, and 100 μg/ml Normocin. Cells were plated in a 96-well plate at a density of ~5 x 105 cells/well in RPMI 1640 supplemented with 10% FCS and incubated for 2 h at 37°C in a humidified incubator containing 5% CO2. Then the cells were washed with RPMI 1640 containing 10% FCS to eliminate nonadherent cells. TAK-242 was dissolved in N,N-dimethylformamide.
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Reaction Conditions
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HEK293 cells were transfected with expression vectors encoding FLAG-TLR4, MD-2, and FLAG-TIRAP or expression vectors encoding FLAG-TLR4, MD-2, and FLAG-TRAM. Six hours after transfection, the cells were incubated with various concentrations of TAK-242 (10, 100, 1000 nM) at 37°C.
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Applications
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TAK-242 inhibited the association of TIRAP with TLR4 and the association of TRAM with TLR4. TAK-242 also modulated the formation of the signaling complex containing the proximal elements in TLR4 signaling. TAK-242 also interferes with the interaction of TRAM and TLR4, inhibiting both NF-γB and ISRE activation and cytokine/interferon gene expression.
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| Animal experiment [2]: |
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Animal models
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Male C57BL/6 mice, 6–8 weeks old, weighing 22–25 g
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Preparation Method
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C57BL/6 mice were randomly assigned to the LPS/D-GalN administration group and the TAK-242 + LPS/DGalN treatment group. Mice in the pretreatment group were injected with TAK-242 intraperitoneally 3h before mice were injected intraperitoneally with LPS (10 μg/kg)/D-GalN.
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Dosage form
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5 mg/kg
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Applications
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TAK-242 pretreatment improved LPS/D-GalN-induced FH in mice. Moreover, TAK-242 has anti-inflammatory effects in mice with concanavalin A-induced acute hepatitis. TAK-242 protected the liver by regulating MDSCs and the inhibition of TLR4 provided by TAK-242 significantly reduced the inflammatory response in FH.
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参考文献:
[1]. Matsunaga N, et al. TAK-242 (resatorvid), a small-molecule inhibitor of Toll-like receptor (TLR) 4 signaling, binds selectively to TLR4 and interferes with interactions between TLR4 and its adaptor molecules. Mol Pharmacol. 2011 Jan;79(1):34-41.
[2]. Wang H, et al. Toll-like Receptor 4 Inhibitor TAK-242 Improves Fulminant Hepatitis by Regulating Accumulation of Myeloid-Derived Suppressor Cell. Inflammation. 2021 Apr;44(2):671-681.
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