Analysis of binding site for the novel small-molecule TLR4 signal transduction inhibitor TAK-242 and its therapeutic effect on mouse sepsis model.
OBJECTIVE
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.


METHODS
Binding assay with [(3)H]-TAK-242 and nuclear factor-kappaB 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.


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
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.
Takashima
K
K
Pharmaceutical Research Division, Pharmacology Research Laboratories I, Takeda Pharmaceutical Company Limited, 2-17-85 Jusohonmachi, Yodogawa-ku, Osaka, Japan.
Matsunaga
N
N
Yoshimatsu
M
M
Hazeki
K
K
Kaisho
T
T
Uekata
M
M
Hazeki
O
O
Akira
S
S
Iizawa
Y
Y
Ii
M
M
D000818:Animals; D001665:Binding Sites; D001667:Binding, Competitive; D002460:Cell Line; D002522:Chlorocebus aethiops; D004926:Escherichia coli; D017930:Genes, Reporter; D006801:Humans; D008024:Ligands; D008070:Lipopolysaccharides / Q000494:pharmacology; D051379:Mice; D008810:Mice, Inbred C57BL; D009163:Mycobacterium bovis; D053594:Myeloid Differentiation Factor 88 / Q000502:physiology; D016328:NF-kappa B / Q000235:genetics; D011487:Protein Conformation; D055503:Protein Multimerization; D017434:Protein Structure, Tertiary; D011869:Radioligand Assay; D018805:Sepsis / Q000188:drug therapy* / Q000382:microbiology; D015398:Signal Transduction / Q000187:drug effects; D013449:Sulfonamides / Q000494:pharmacology* / Q000627:therapeutic use; D051197:Toll-Like Receptor 4 / Q000502:physiology*
D008024:Ligands; D008070:Lipopolysaccharides; D053594:Myeloid Differentiation Factor 88; D016328:NF-kappa B; D013449:Sulfonamides; D051197:Toll-Like Receptor 4; C507035:ethyl 6-(N-(2-chloro-4-fluorophenyl)sulfamoyl)cyclohex-1-ene-1-carboxylate