Background and purpose:  TAK‐242, a novel synthetic small‐molecule, suppresses production of multiple cytokines by inhibiting Toll‐like receptor (TLR) 4 signalling. In this study, we investigated the target molecule of TAK‐242 and examined its therapeutic effect in a mouse sepsis model.

Experimental approach:  Binding assay with [³H]‐TAK‐242 and nuclear factor‐κB reporter assay were used to identify the target molecule and binding site of TAK‐242. Bacillus calmette guerin (BCG)‐primed mouse sepsis model using live Escherichia coli was used to estimate the efficacy of TAK‐242 in sepsis.

Key results:  TAK‐242 strongly bound to TLR4, but binding to TLR2, 3, 5, 9, TLR‐related adaptor molecules and MD‐2 was either not observed or marginal. Mutational analysis using TLR4 mutants indicated that TAK‐242 inhibits TLR4 signalling by binding to Cys747 in the intracellular domain of TLR4. TAK‐242 inhibited MyD88‐independent pathway as well as MyD88‐dependent pathway and its inhibitory effect was largely unaffected by lipopolysaccharide (LPS) concentration and types of TLR4 ligands. TAK‐242 had no effect on the LPS‐induced conformational change of TLR4‐MD‐2 and TLR4 homodimerization. In mouse sepsis model, although TAK‐242 alone did not affect bacterial counts in blood, if co‐administered with ceftazidime it inhibited the increases in serum cytokine levels and improved survival of mice.

Conclusions and implications:  TAK‐242 suppressed TLR4 signalling by binding directly to a specific amino acid Cys747 in the intracellular domain of TLR4. When co‐administered with antibiotics, TAK‐242 showed potent therapeutic effects in an E. coli‐induced sepsis model using BCG‐primed mice. Thus, TAK‐242 may be a promising therapeutic agent for sepsis.