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Proinflammatory mediators such as cytokines and NO play pivotal roles in various inflammatory diseases. To combat inflammatory diseases successfully, regulation of proinflammatory mediator production would be a critical process. In the present study, we investigated the in vitro effects of ethyl (6R)-6-[N-(2-chloro-4-fluorophenyl)sulfamoyl]cyclohex-1-ene-1-carboxylate (TAK-242), a novel small molecule cytokine production inhibitor, and its mechanism of action. In RAW264.7 cells and mouse peritoneal macrophages, TAK-242 suppressed lipopolysaccharide (LPS)-induced production of NO, tumor necrosis factor-␣ (TNF-␣), and interleukin (IL)-6, with 50% inhibitory concentration (IC 50 ) of 1.1 to 11 nM. TAK-242 also suppressed the production of these cytokines from LPSstimulated human peripheral blood mononuclear cells (PBMCs) at IC 50 values from 11 to 33 nM. In addition, the inhibitory effects on the LPS-induced IL-6 and IL-12 production were similar in human PBMCs, monocytes, and macrophages. TAK-242 inhibited mRNA expression of IL-6 and TNF-␣ induced by LPS and interferon-␥ in RAW264.7 cells. The phosphorylation of mitogen-activated protein kinases induced by LPS was also inhibited in a concentration-dependent manner. However, TAK-242 did not antagonize the binding of LPS to the cells. It is noteworthy that TAK-242 suppressed the cytokine production induced by Toll-like receptor (TLR) 4 ligands, but not by ligands for TLR2, -3, and -9. In addition, IL-1␤-induced IL-8 production from human PBMCs was not markedly affected by TAK-242. These data suggest that TAK-242 suppresses the production of multiple cytokines by selectively inhibiting TLR4 intracellular signaling. Finally, TAK-242 is a novel small molecule TLR4 signaling inhibitor and could be a promising therapeutic agent for inflammatory diseases, whose pathogenesis involves TLR4.Cytokines and NO are involved in a variety of inflammatory diseases, including sepsis, rheumatoid arthritis (RA), atherosclerosis, inflammatory bowel disease (IBD), asthma, and chronic obstructive pulmonary disease. In RA, for example, interleukin (IL)-1 is considered to be a mediator. Plasma concentrations of IL-1 in patients with RA are elevated, and they correlate with disease activity (Eastgate et al., 1988). The expression of IL-1 in the bronchial epithelium of patients Article, publication date, and citation information can be found at

Phosphoinositide 3-kinase (PI 3-kinase) is a key signaling enzyme implicated in variety of receptor-stimulated cell responses. Receptors with intrinsic or associated tyrosine kinase activity recruit heterodimeric PI 3-kinases consisting of a 110-kDa catalytic subunit (p110) and an 85-kDa regulatory subunit (p85). We separated a PI 3-kinase that could be stimulated by the ␤␥ subunits of G protein (G␤␥) from rat liver. The G␤␥-sensitive PI 3-kinase appeared to be a heterodimer consisting of p110␤ and p85 (or their related subunits). The stimulation by G␤␥ was inhibited by the GDP-bound ␣ subunit of the inhibitory GTP-binding protein. Moreover, the stimulatory action of G␤␥ was markedly enhanced by the simultaneous addition of a phosphotyrosyl peptide synthesized according to the amino acid sequence of the insulin receptor substrate-1. Such enzymic properties could be observed with a recombinant p110␤/p85␣ expressed in COS-7 cells with their cDNAs. In contrast, another heterodimeric PI 3-kinase consisting of p110␣ and p85 in the same rat liver, together with a recombinant p110␣/p85␣, was not activated by G␤␥, although their activities were stimulated by the phosphotyrosyl peptide. These results indicate that p110␤/ p85 PI 3-kinase may be regulated in a cooperative manner by two different types of membrane receptors, one possessing tyrosine kinase activity and the other activating GTP-binding proteins.Phosphoinositide 3-kinase (PI 3-kinase) 1 is a key signaling enzyme implicated in the regulation of a broad array of biological responses including receptor-stimulated mitogenesis, oxidative burst, membrane ruffling, and glucose uptake (1, 2). The activation of PI 3-kinase results in an increase in cellular levels of D-3 phosphorylated phosphoinositides, such as PtdIns(3)P, PtdIns(3,4)P 2 , and PtdIns(3,4,5)P 3 . These products, however, do not serve as the substrates of phospholipase C (3) and thus have been proposed to act as second messengers. In this regard, recent studies have indicated that PtdIns(3,4)P 2 can directly activate certain protein kinase C and Akt (4, 5) and PtdIns(3,4,5)P 3 is capable of binding to the Pleckstrin homology domain of guanine nucleotide exchange factor of the small GTP-binding protein ARF1 (6 -8).At least two types of PI 3-kinase, in terms of mode of the activation, have been described in mammalian cells (2). One is stimulated by membrane-bound receptors activating tyrosine kinase, whereas the other is under the direct control of the heterotrimeric GTP-binding proteins. The well known former type has been structurally characterized as a heterodimer consisting of a 110-kDa catalytic subunit (p110) and an 85-kDa regulatory subunit (p85); the regulatory subunit contains one SH3 and two SH2 domains. Stimulation of tyrosine kinase receptors by extracellular signals phosphorylates specific tyrosine residues located in the YMXM motifs of their own receptors or adaptor molecules, such as insulin receptor substrate-1. These phosphorylated proteins bind to the SH2 domains of p85 and stimulate the...

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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-kB 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.