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19563534
Analysis of binding site for the novel small-molecule TLR4 signal transduction inhibitor TAK-242 and its therapeutic effect on mouse sepsis model.

Br J Pharmacol, 2009/8;157(7):1250-62.

Takashima K[1], Matsunaga N, Yoshimatsu M, Hazeki K, Kaisho T, Uekata M, Hazeki O, Akira S, Iizawa Y, Ii M

Affiliations

PMID: 19563534DOI: 10.1111/j.1476-5381.2009.00297.x

Impact factor: 9.473

Abstract
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 [(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.
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.
MeSH terms
Animals; Binding Sites; Binding, Competitive; Cell Line; Chlorocebus aethiops; Escherichia coli; Genes, Reporter; Humans; Ligands; Lipopolysaccharides; Mice; Mice, Inbred C57BL; Mycobacterium bovis; Myeloid Differentiation Factor 88; NF-kappa B; Protein Conformation; Protein Multimerization; Protein Structure, Tertiary; Radioligand Assay; Sepsis; Signal Transduction; Sulfonamides; Toll-Like Receptor 4
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