Background
C29 is a Toll-like receptor 2 (TLR2) inhibitor. C29, inhibited TLR2/1 and TLR2/6 signaling induced by synthetic and bacterial TLR2 agonists in human HEK-TLR2 and THP-1 cells, but only TLR2/1 signaling in murine macrophages. C29 failed to inhibit signaling induced by other TLR agonists and TNF-α. Mutagenesis of BB loop pocket residues revealed an indispensable role for TLR2/1, but not TLR2/6, signaling, suggesting divergent roles [1].
C29 inhibited TLR2/1-mediated NF-κB activation (IC50 0.87μM), and inhibited TLR2/6 heterodimer with IC50 23μM [2]. C29 and o-vanillin may function by specifically targeting the BB loop pocket of the TLR2 TIR domain, altering its function and/or position [1].
Mice were pretreated with C29 before the administration of hepatitis B e antigen (HBeAg), to verify the roles of TLR-2 in vivo, the expression of IL-6, TNF-α, and CCL-2 was significantly alleviated, but IL-10 was upregulated in the liver [3]. C29 did not affect lipid accumulation, but the adipogenesis inhibitory effects of exopolysaccharide (EPS) significantly decreased in the C29-treated group. The results showed that activation of the AMPK signalling pathway by EPS was inhibited in the early stage (day 4) of adipogenic differentiation, when TLR2 and myeloid differentiation primary response 88 (MyD88) expression is inhibited by C29, indicating that EPS activates the AMPK signalling pathway by interacting with TLR2, consequently inhibiting adipogenesis [4].
参考文献:
[1]. Mistry P, Laird MH, Schwarz RS, Greene S, Dyson T, Snyder GA, et al. Inhibition of TLR2 signaling by small molecule inhibitors targeting a pocket within the TLR2 TIR domain. Proc Natl Acad Sci U S A (2015) 112(17):5455-60. doi:10.1073/pnas.1422576112
[2]. Grabowski, M.; Murgueitio, M.S.; Bermudez, M.; Wolber, G.; Weindl, G. The novel small-molecule antagonist MMG-11 preferentially inhibits TLR2/1 signaling. Biochem. Pharmacol. 2020, 171, 113687.
[3]. Xie X, Lv H, Liu C, Su X, Yu Z, Song S, et al. HBeAg Mediates Inflammatory Functions of Macrophages by TLR2 Contributing to Hepatic Fibrosis. BMC Med (2021) 19:247. doi: 10.1186/s12916-021-02085-3
[4]. Lee, J.; Park, S.; Oh, N.; Park, J.; Kwon, M.; Seo, J.; Roh, S. Oral intake of Lactobacillus plantarum L-14 extract alleviates TLR2-and AMPK-mediated obesity- associated disorders in hight-fat-diet-induced obese C57BL/6J mice. Cell Prolif. 2021, 54, e13039
C29 是一种 Toll 样受体 2 (TLR2) 抑制剂。 C29 在人 HEK-TLR2 和 THP-1 细胞中抑制由合成和细菌 TLR2 激动剂诱导的 TLR2/1 和 TLR2/6 信号传导，但仅抑制小鼠巨噬细胞中的 TLR2/1 信号传导。 C29 未能抑制由其他 TLR 激动剂和 TNF-α 诱导的信号传导。 BB 环袋残基的诱变揭示了 TLR2/1 而非 TLR2/6 信号传导不可或缺的作用，表明不同的作用[1]。
C29 抑制 TLR2/1 介导的 NF-κB 激活（IC50 0.87μM），并抑制 TLR2/6 异二聚体，IC50 23μM [2 ]。 C29 和 o-香兰素可能通过特异性靶向 TLR2 TIR 结构域的 BB 环口袋、改变其功能和/或位置[1] 发挥作用。
小鼠在给予乙型肝炎e抗原（HBeAg）之前用C29预处理，以验证TLR-2在体内的作用，IL-6、TNF-α和CCL-2的表达显着减轻，但 IL-10 在肝脏中上调 [3]。 C29 不影响脂质积累，但胞外多糖 (EPS) 的脂肪形成抑制作用在 C29 处理组中显着降低。结果表明，EPS 对 AMPK 信号通路的激活在成脂分化的早期（第 4 天）受到抑制，此时 TLR2 和髓系分化初级反应 88（MyD88）的表达被 C29 抑制，表明 EPS 激活 AMPK 信号通路通路通过与 TLR2 相互作用，从而抑制脂肪生成[4]。
Protocol
| Cell experiment [1]: |
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Cell lines
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HEK-Blue cells,RAW macrophages,THP-1 macrophages
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Preparation Method
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C29 were dissolved in DMSO as 50mM stock solution. Final DMSO concentrations in cell culture were below 0.2% (v/v). The cells were first incubated with the C29 for 1h and afterwards stimulated with the respective TLR(Toll like receptor) agonist.
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Reaction Conditions
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0.01μM, 0.1μM, 1μM, 10μM 100μM for 1 hour
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Applications
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C29 showed inhibit affection for TLR2/1 or TLR2/6 (IC50 21μM and 23μM).
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| Animal experiment [2]: |
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Animal models
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Female C57BL/6J mice
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Preparation Method
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C29 was dissolved and diluted in solution (10% DMSO/40% PEG300/5% Tween-80/45% saline), then injected intraperitoneally (1.3 μmol/g) 1 h before HBeAg treatment, with the same volume of dissolving reagent as the vehicle group.
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Dosage form
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Intraperitoneal injection, 1.3 μmol/g
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Applications
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To verify the roles of TLR-2 in vivo, mice were pretreated with C29 before the administration of HBeAg. The expression of IL-6, TNF-α, and CCL-2 was significantly alleviated, but IL-10 was upregulated in the liver.
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参考文献:
[1]: Grabowski, M.; Murgueitio, M.S.; Bermudez, M.; Wolber, G.; Weindl, G. The novel small-molecule antagonist MMG-11 preferentially inhibits TLR2/1 signaling. Biochem. Pharmacol. 2020, 171, 113687.
[2]: Xie X, Lv H, Liu C, Su X, Yu Z, Song S, et al. HBeAg Mediates Inflammatory Functions of Macrophages by TLR2 Contributing to Hepatic Fibrosis. BMC Med (2021) 19:247. doi: 10.1186/s12916-021-02085-3
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