Cell lines

HEK293, HEK293-hTLR4/MD2-CD14

Preparation Method

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℃ 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.

Reaction Conditions

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℃.

Applications

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.

Animal models

Male C57BL/6 mice, 6–8 weeks old, weighing 22–25 g

Preparation Method

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.

Dosage form

5 mg/kg

Applications

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.

• Theriogenology (2022).

TAK 242 is a toll-like receptor 4 (TLR4) signaling inhibitor. Binds to intracellular domain of TLR4. Inhibits LPS-induced cytokine production in vitro (IC₅₀values 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]

References:
[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.