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Selective inhibition of Toll-like receptor 4 signaling by the small molecule TAK-242


Sepsis is a clinical syndrome whose pathophysiology reflects the activation of an innate host response to infection. Toll-like receptors (TLRs) recognize structural components of various microbes and activate inflammatory signaling. TLR4 detects Gram-negative bacteria through recognition of the lipid A moiety of lipopolysaccharide (LPS). The cyclohexene derivative TAK-242 inhibits multiple inflammatory mediators produced by LPS-stimulated murine RAW264.7 macrophages. We determined the selectivity of TAK-242 against the TLR family and its efficacy in vitro and in vivo.


The selectivity of TAK-242 against the TLR family was examined in a NF-κB reporter gene assay using 293 cells transfected with human TLR expression vectors. The amount of nitric oxide (NO) in the medium was determined by measuring the fluorescence of 2,3-diaminonaphthalene; cytokine levels in the medium and activation of the signal pathway of TLR4 were examined by BioPlex cytokine and phosphoprotein panel assay systems. To evaluate the efficacy and selectivity of TAK-242 in vivo, cytokine levels in sera of mice injected intraperitoneally with LPS, lipoteichoic acid (LTA), or peptidoglycan (PGN) (10 mg/kg each) were measured by ELISA. TAK-242 was administered intravenously 1 hour before the challenge.


TAK-242 almost completely inhibited production of NO and cytokines (e.g. TNF-α, IL-1α, and IL-6) in LPS-stimulated RAW264.7 cells; the IC50 for LPS-induced NO production was 7.0 nmol/l. The LPS-induced activation of IkB kinase (IKK), p38, extracellular signal-regulated kinase (ERK), and c-Jun N-terminal kinase (JNK) pathways were also markedly inhibited by TAK-242, but it showed little effect on NO production induced by Pam3CSK4 (TLR1/2), PGN (TLR6/2), double-strand RNA (TLR3), or CpG oligonucleotide (TLR9). The NF-κB reporter gene assay showed that LPS-induced TLR4 activation was inhibited by TAK-242 in 293 cells transiently expressing TLR4 and the TLR4 co-receptors MD2 and CD14. TAK-242 also inhibited LPS-independent NF-κB activation resulting from overexpression of TLR4, but showed little effect on NF-κB activation by other TLRs (TLR1/2, TLR6/2, TLR3, TLR5, TLR7 and TLR9). TLR4 selectivity was also observed in vivo. Intravenous administration of TAK-242 to mice at a dose of 1 mg/kg inhibited the increase in serum IL-6 levels induced by TLR4 ligands LPS or LTA, but not by the TLR6/2 ligand PGN even at a dose of 10 mg/kg.


TAK-242 is a potent and selective inhibitor of TLR4 signaling in vitro and in vivo, and therefore represents a novel therapeutic approach to the treatment of sepsis.

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Kawamoto, T., Sha, T., Ii, M. et al. Selective inhibition of Toll-like receptor 4 signaling by the small molecule TAK-242. Crit Care 10 (Suppl 1), P154 (2006).

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