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TAK-242 Sale

(Synonyms: 瑞沙托维; TAK-242; CLI-095) 目录号 : GC16148

A TLR4 antagonist

TAK-242 Chemical Structure

Cas No.:243984-11-4

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10mM (in 1mL DMSO)
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5mg
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10mg
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50mg
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100mg
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实验参考方法

Cell experiment [1]:

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°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.

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°C.

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 experiment [2]:

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.

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]. 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.

产品描述

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]

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.

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]

Chemical Properties

Cas No. 243984-11-4 SDF
别名 瑞沙托维; TAK-242; CLI-095
化学名 ethyl (6R)-6-[(2-chloro-4-fluorophenyl)sulfamoyl]cyclohexene-1-carboxylate
Canonical SMILES CCOC(=O)C1=CCCCC1S(=O)(=O)NC2=C(C=C(C=C2)F)Cl
分子式 C15H17ClFNO4S 分子量 361.82
溶解度 ≥ 18.091 mg/mL in DMSO, ≥ 100.6 mg/mL in EtOH 储存条件 Store at -20°C
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1 mM 2.7638 mL 13.819 mL 27.6381 mL
5 mM 0.5528 mL 2.7638 mL 5.5276 mL
10 mM 0.2764 mL 1.3819 mL 2.7638 mL
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Research Update

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

TAK-242 (resatorvid), a small-molecule-specific inhibitor of Toll-like receptor (TLR) 4 signaling, inhibits the production of lipopolysaccharide-induced inflammatory mediators by binding to the intracellular domain of TLR4. Cys747 in TLR4 has been identified previously as the binding site of TAK-242. However, the mechanism by which TAK-242 inhibits TLR4 signaling after binding to TLR4 remains unknown. The present study demonstrated, using coimmunoprecipitation, that TAK-242 interferes with protein-protein interactions between TLR4 and its adaptor molecules. Among 10 different human TLRs, TAK-242 selectively bound to TLR4. The time course of the inhibitory effect of TAK-242 on inflammatory mediator production corresponded to that of the binding of TAK-242 to TLR4. TAK-242 inhibited the association of TLR4 with Toll/interleukin-1 receptor domain-containing adaptor protein (TIRAP) or Toll/interleukin-1 receptor domain-containing adaptor protein inducing interferon-β-related adaptor molecule (TRAM) in human embryonic kidney (HEK) 293 cells overexpressing TLR4, MD-2, and TIRAP or TRAM, respectively. TAK-242 inhibited the TIRAP-mediated activation of nuclear factor κB (NF-κB) and the TRAM-mediated activation of NF-κB and interferon-sensitive response element in HEK293 cells stably expressing TLR4, MD-2, and CD14. The activation of endogenous interleukin-1 receptor-associated kinase in RAW264.7 cells was also inhibited by TAK-242 treatment. These findings suggest that TAK-242 binds selectively to TLR4 and subsequently disrupts the interaction of TLR4 with adaptor molecules, thereby inhibiting TLR4 signal transduction and its downstream signaling events. This work proposes a novel paradigm of a small molecule capable of disrupting protein-protein interactions.

TAK-242, a specific inhibitor of Toll-like receptor 4 signalling, prevents endotoxemia-induced skeletal muscle wasting in mice

Circulating lipopolysaccharide (LPS) concentrations are often elevated in patients with sepsis or various endogenous diseases related to bacterial translocation from the gut. Systemic inflammatory responses induced by endotoxemia induce severe involuntary loss of skeletal muscle, termed muscle wasting, which adversely affects the survival and functional outcomes of these patients. Currently, no drugs are available for the treatment of endotoxemia-induced skeletal muscle wasting. Here, we tested the effects of TAK-242, a Toll-like receptor 4 (TLR4)-specific signalling inhibitor, on myotube atrophy in vitro and muscle wasting in vivo induced by endotoxin. LPS treatment of murine C2C12 myotubes induced an inflammatory response (increased nuclear factor-κB activity and interleukin-6 and tumour necrosis factor-α expression) and activated the ubiquitin-proteasome and autophagy proteolytic pathways (increased atrogin-1/MAFbx, MuRF1, and LC-II expression), resulting in myotube atrophy. In mice, LPS injection increased the same inflammatory and proteolytic pathways in skeletal muscle and induced atrophy, resulting in reduced grip strength. Notably, pretreatment of cells or mice with TAK-242 reduced or reversed all the detrimental effects of LPS in vitro and in vivo. Collectively, our results indicate that pharmacological inhibition of TLR4 signalling may be a novel therapeutic intervention for endotoxemia-induced muscle wasting.

TAK-242 Ameliorates Hepatic Fibrosis by Regulating the Liver-Gut Axis

Objective: The aims of this study were to investigate the impact of TAK-242 on the Toll-like receptor 4 (TLR4)/myeloid differentiation factor 88 (MyD88)/nuclear transcription factor-κB (NF-κB) signal transduction pathway in rats with hepatic fibrosis (HF) using the liver gut axis and to investigate the molecular mechanism of its intervention on HF.
Methods: SPF grade SD male rats were randomly allocated to the control, model, and TAK-242 groups. For 8 weeks, the model and TAK-242 groups received 3 mL·kg-1 (the initial dose 5 mL·kg-1) intraperitoneal injections of 40% CCL4 olive oil solution. TAK-242 (5 mg·kg-1) was administered once a day for 5 days after modeling. The pathological alterations of liver and small intestine tissues in each group were observed using H&E and Masson staining. ELISA was used to measure serum levels of alanine aminotransferase (ALT), aspartate aminotransferase (AST), direct bilirubin (DBIL), total bilirubin (TBIL), interleukin-1β (IL-1β), interleukin-6 (IL-6), and tumor necrosis factor alpha (TNF-α). RT-qPCR was utilized to identify the mRNA expression level of IL-1β, IL-6, TNF-α, TLR4, MyD88, and NF-κB in rat liver and small intestine tissues. The protein level of IL-1β, IL-6, TNF-α, TLR4, MyD88, and NF-κB protein in rat liver and small intestine tissues was determined utilizing Western blot and IHC.
Results: TAK-242 significantly reduced AST, ALT, TBIL, and DBIL expression in HF rats' serum (P < 0.01) and alleviated liver tissue injury. Hematoxylin-eosin (H&E) and Masson staining revealed inflammatory cell infiltration and fibrous proliferation in the liver and small intestine tissue in the model group and partial cell swelling in the TAK-242 group, which indicated a considerable improvement compared to the model group. RT-qPCR, Western blot, and IHC data indicated that TAK-242 reduced the IL-1β, IL-6, TNF-α, TLR4, MyD88, and NF-κB expression in the liver and small intestine tissues of HF rats.
Conclusion: TAK-242 might downregulate the TLR4/MyD88/NF-κB signal pathway through the liver-gut axis, suppress the inflammatory response, and eventually alleviate HF in rats.

TAK-242 ameliorates epileptic symptoms in mice by inhibiting the TLR4/NF-κB signaling pathway

Background: There is evidence that immune inflammation plays an important role in the of epilepsy. The toll-like receptor 4 (TLR4)/nuclear factor kappa beta (NF-κB) signaling pathway is a target for the treatment of epilepsy. TAK-242 is a potent TLR4 inhibitor with neuroprotective effects. No study has examined whether TAK-242 has a protective effect on epilepsy.
Methods: Male C57BL/6 mice were randomly divided into the following 3 groups: (I) the control group; (II) the model [pentetrazol (PTZ)] group; and (III) the treatment group [PTZ + TAK-242 (3 mg/kg)], with 8 mice in each group. A mouse model of epilepsy was established via the intraperitoneal injection of PTZ (37 mg/kg). The behavioral changes of the mice were observed and scored using the Racine grading criteria. TAK-242 (3 mg/kg) was administered to establish the treatment model. The control group was intraperitoneally injected with normal saline at the same dose as the PTZ epileptogenic dose, and 3 mg/kg of normal saline was intragastrically administered. The messenger RNA (mRNA) and protein expressions of TLR4, NF-κB, and the NF-κB downstream gene tumor necrosis factor alpha (TNF-α) in the mouse brain tissues were detected by real-time quantitative polymerase chain reaction (RT-qPCR) and Western blot.
Results: Compared to the control group, the mice in the model group showed generalized tonic convulsion and involuntary falling after PTZ modeling. The results of the hematoxylin and eosin (H&E) staining showed that the treatment group had a higher number of normal neurons than the model group, and the neuronal cell morphology in the treatment group was closer to the control group. However, the occurrence of generalized tonic convulsion and involuntary falling in the mice in the treatment group was significantly improved. Comparing the Western Blot and RT-qPCR results, we detected higher TLR4, NF-KB, TNF-α protein and mRNA expression levels in the model group than the treatment group, and there was no significant difference between the control group and the treatment group.
Conclusions: TAK-242 treatment ameliorates epileptic symptoms in mice, and the mechanism by which this occurs may be related to the inhibition of the TLR4/NF-κB inflammatory pathway.

TAK-242 treatment and its effect on mechanical properties and gene expression associated with IVD degeneration in SPARC-null mice

Purpose: Intervertebral disc (IVD) degeneration is accompanied by mechanical and gene expression changes to IVDs. SPARC-null mice display accelerated IVD degeneration, and treatment with (toll-like receptor 4 (TLR4) inhibitor) TAK-242 decreases proinflammatory cytokines and pain. This study examined if chronic TAK-242 treatment impacts mechanical properties and gene expression associated with IVD degeneration in SPARC-null mice.
Methods: Male and female SPARC-null and WT mice aged 7-9 months were given intraperitoneal injections with TAK-242 or an equivalent saline vehicle for 8 weeks (3x/per week, M-W-F). L2-L5 spinal segments were tested in cyclic axial tension and compression. Gene expression analysis (RT-qPCR) was performed on male IVD tissues using Qiagen RT2 PCR arrays.
Results: SPARC-null mice had decreased NZ length (p = 0.001) and increased NZ stiffness (p < 0.001) compared to WT mice. NZ length was not impacted by TAK-242 treatment (p = 0.967) despite increased hysteresis energy (p = 0.024). Tensile stiffness was greater in SPARC-null mice (p = 0.018), and compressive (p < 0.001) stiffness was reduced from TAK-242 treatment in WT but not SPARC-null mice (p = 0.391). Gene expression analysis found upregulation of 13 ECM and 5 inflammatory genes in SPARC-null mice, and downregulation of 2 inflammatory genes after TAK-242 treatment.
Conclusions: TAK-242 had limited impacts on SPARC-null mechanical properties and did not attenuate NZ mechanical changes associated with IVD degeneration. Expression analysis revealed an increase in ECM and inflammatory gene expression in SPARCnull mice with a reduction in inflammatory expression due to TAK-242 treatment. This study provides insight into the role of TLR4 in SPARC-null mediated IVD degeneration.