TAK-242 preferentially binds to TLR4, but not to other TLRs, TLR-related adaptor molecules or MD-2. (A) Binding of [³H]-TAK-242 to TLR-related adaptor molecules. HEK293 cells were transiently transfected with expression vector coding FLAG-TRAM, FLAG-TRIF, HA-TIRAP/Mal, HA-MyD88 and FLAG-TLR4. Empty lanes were used for a molecular weight marker. (B) Binding of [³H]-TAK-242 to various TLRs. HEK293 cells were transiently transfected with expression vector coding TLR3-HA, TLR9-HA and FLAG-TLR4. COS-7 cells were transiently transfected with expression vector coding FLAG-TLR2, FLAG-TLR4 and TLR5-HA. Empty lanes were used for a molecular weight marker. (C) Effect of nonradioactive TAK-242 and its enantiomer on the binding of [³H]-TAK-242 to TLR4. COS-7 cells were transiently transfected with expression vector coding FLAG-TLR2 and FLAG-TLR4. (D) Binding of [³H]-TAK-242 to MD-2. COS-7 cells were transiently transfected with FLAG-MD-2, FLAG-TLR2 and FLAG-TLR4. After 2 days of transfection, the cells were incubated with 100 nM of [³H]-TAK-242 for 6 h, then lysed. For competition assay, the cells were incubated with various concentrations of nonradioactive TAK-242 or 10 µM of its enantiomer for 2 h, and with 100 nM [³H]-TAK-242 for 4 h. Cell lysates were immunoprecipitated with anti-FLAG M2 and anti-HA 12CA5 Abs. The immunoprecipitates were subjected to SDS-PAGE and western blotting. Radioactive imaging of the immunoprecipitates was analysed by autoradiography. All data shown are representative of three independent experiments. HEK, human embryonic kidney; Mal, MyD88-adaptor like; TIRAP, TIR domain-containing adaptor protein; TLR, Toll-like receptor; TRAM, TRIF-related adaptor molecule; TRIF, TIR domain-containing adaptor inducing IFN-β.

TAK-242 binds to Cys747 in ICD of TLR4 and inhibits TLR4 signalling. (A) Binding of [³H]-TAK-242 to TLR4 mutants with alanine substitutions at cysteine residues and arginine substitutions at lysine residues in the ICD of TLR4. COS-7 cells were transiently transfected with expression vectors coding FLAG-TLR4 and FLAG-TLR4 with single amino acid mutations in TLR4. After 2 days of transfection, the cells were incubated with 100 nM of [³H]-TAK-242 for 6 h. Cell lysates were immunoprecipitated with anti-FLAG M2 Ab, and the immunoprecipitates were subjected to SDS-PAGE and western blotting. Radioactive imaging of the immunoprecipitates was analysed by autoradiography. Data shown are representative of three independent experiments. (B) Effect of TAK-242 on LPS-induced NF-κB activation in TLR4 mutants with alanine substitutions at cysteine residues and arginine substitutions at lysine residues in the ICD of TLR4. HEK293 cells were transiently transfected with expression vectors coding TLR4 and TLR4 with single amino acid mutations along with pNifty and phRL-TK. Expression vectors coding MD-2 and CD14 were also co-transfected. On the day after transfection, cells were incubated with 100 ng·mL⁻¹ LPS and various concentrations of TAK-242 for 6 h. Luciferase activity was determined using luciferase assay kit. Data represent the mean ± SEM in triplicate wells. Data shown are representative of two independent experiments. ICD, intracellular domain; LPS, lipopolysaccharide; NF-κB, nuclear factor-κB; TLR, Toll-like receptor.

TAK-242 inhibits both LPS-induced and HMGB-1-induced TNF-α production in RAW264.7 cells. Cells were cultured with 0.5 ng·mL⁻¹ LPS or 10 µg·mL⁻¹ HMGB-1 for 20 h in the presence of 0.1 ng·mL⁻¹ IFN-γ and various concentrations of TAK-242. TNF-α levels in the culture supernatants were measured by ELISA. Data represent the mean ± SEM in triplicate wells. HMGB-1, high mobility group box 1 protein; IFN, interferon; LPS, lipopolysaccharide; TNF-α, tumour necrosis factor-α.

TAK-242 has no effect on TLR4 homodimerization. (A) Effect of TAK-242 on constitutive TLR4 homodimerization induced by overexpresssion of TLR4-Bla(a) and TLR4-Bla(b). HEK293 cells were transiently transfected with TLR-Bla fusion constructs. On the following day, cells were incubated with test compounds for 3 h. Cells were then loaded with CCF2/AM and analysed by an IN cell analyser 1000. Results are shown as the mean ± SEM (n= 30). (B) Effect of TAK-242 on constitutive NF-kB activation induced by overexpression of TLR4-Bla(a) and TLR4-Bla(b). HEK293 cells were transiently transfected with TLR-Bla fusion constructs. After 6 h of transfection, cells were incubated with various concentrations of test compounds overnight. Luciferase activity was determined using a luciferase assay kit. Data represent the mean ± SEM (n= 6). HEK, human embryonic kidney; NF-κB, nuclear factor-κB; TLR, Toll-like receptor.

Treatment with TAK-242 protects mice in an E. coli-induced sepsis model using BCG-primed mice. C57BL/6 mice were injected intravenously with 2 mg BCG. Twelve or thirteen days later, the BCG-primed mice were inoculated intraperitoneally with approximately 10⁷ CFU of E. coli O111. Various doses of TAK-242 (or vehicle) were intravenously administered concomitantly with 20 mg·kg⁻¹ ceftazidime (or saline) 1 h after the bacterial challenge. (A) Influence of TAK-242 and ceftazidime on bacterial counts in blood. TAK-242 and ceftazidime were administered 1 h after the bacterial challenge at doses of 10 and 20 mg·kg⁻¹ respectively. Bacterial counts were determined up to 4 h after bacterial challenge. Data represent the mean ± SEM (n= 4). Statistical differences at 4 h after the bacterial challenge were analysed with two-way anova. (B) Effect of TAK-242 co-administered with ceftazidime on survival rate (n= 10). Various doses of TAK-242 were intravenously administered concomitantly with 20 mg·kg⁻¹ ceftazidime 1 h after the bacterial challenge. Survival was recorded over 7 days after the bacterial challenge. Significant differences (compared with vehicle) are denoted by *P≤ 0.05, **P≤ 0.01; Tarone's test with a simple closed step-down method. (C) Effect of TAK-242 co-administered with ceftazidime on serum levels of various cytokines. Various doses of TAK-242 were intravenously administered concomitantly with 20 mg·kg⁻¹ ceftazidime 1 h after the bacterial challenge. The concentrations of TNF-α, IL-10 and MIP-2 were examined 2 h after bacterial challenge. Data represent the mean ± SEM (n= 10). Significant differences (compared with vehicle) are denoted by *P≤ 0.025; one-tailed Williams test (TNF-α) and one-tailed Shirley-Williams test (IL-10 and MIP-2). (D) Effect of TAK-242 co-administered with ceftazidime on time course of serum levels of various cytokines. TAK-242 and ceftazidime were concomitantly administered 1 h after the bacterial challenge at a dose of 1 and 20 mg·kg⁻¹ respectively. The concentrations of TNF-α, IL-1β, IL-6, IL-10 and MIP-2 were examined up to 4 h after bacterial challenge. Data represent the mean ± SEM (n= 8). Significant differences (compared with vehicle) are denoted by *P≤ 0.05 and **P≤ 0.01; Wilcoxon's test with Holm's correction. All data shown are representative of two to three independent experiments. anova, analysis of variance; BCG, Bacillus calmette guerin; IL, interleukin; TNF-α, tumour necrosis factor-α.

TAK-242 binds to ICD/TMD of TLR4 and inhibits TLR4 signalling. (A) Schematic presentation of TLR4 chimeric constructs. (B) Binding of [³H]-TAK-242 to TLR4 chimeric constructs. COS-7 cells were transiently transfected with expression vectors coding FLAG-TLR2, FLAG-TLR4, FLAG-T2N-T4C and FLAG-T4N-T2C. After 2 days of transfection, the cells were incubated with 100 nM of [³H]-TAK-242 for 6 h. Cell lysates were immunoprecipitated with anti-FLAG M2 Ab, and the immunoprecipitates were subjected to SDS-PAGE and western blotting. Radioactive imaging of the immunoprecipitates was analysed by autoradiography. Data shown are representative from three independent experiments. (C) Effect of TAK-242 on constitutive NF-κB activation in TLR chimeric proteins. HEK293 cells were transiently transfected with expression vectors coding TLR2, TLR4, T2N-T4C, T4N-T2C and TLR2/TLR6 along with pNifty and phRL-TK. Expression vectors coding MD-2 and CD14 were also co-transfected. After 6 h of transfection, cells were incubated with various concentrations of TAK-242 overnight. Luciferase activity was determined using a luciferase assay kit. Data represent the mean ± SEM in triplicate wells. Data shown are representative of three independent experiments. ECD, extracellular domain; ICD, intracellular domain; NF-κB, nuclear factor-κB; TLR, Toll-like receptor; TMD, transmembrane domain.

Single mutation at Cys747 in TLR4 affects LPS-induced NF-κB activation. (A) Binding of [³H]-TAK-242 to TLR4 mutants with single mutation at Cys747 in TLR4. COS-7 cells were transiently transfected with expression vectors coding FLAG-TLR4 and FLAG-TLR4 with single amino acid mutations in TLR4. After 2 days of transfection, the cells were incubated with 100 nM of [³H]-TAK-242 for 6 h. Cell lysates were immunoprecipitated with anti-FLAG M2 Ab, and the immunoprecipitates were subjected to SDS-PAGE and western blotting. Radioactive imaging of the immunoprecipitates was analysed by autoradiography. Data shown are representative of two independent experiments. (B) Effect of TAK-242 on LPS-induced NF-κB activation in TLR4 mutants with single mutation at Cys747 in TLR4. HEK293 cells were transiently transfected with expression vectors coding TLR4 and TLR4 with single amino acid mutations along with pNifty and phRL-TK. Expression vectors coding MD-2 and CD14 were also co-transfected. On the day after transfection, cells were incubated with 100 ng·mL⁻¹ LPS and various concentrations of TAK-242 for 6 h. Luciferase activity was determined using a luciferase assay kit. Data represent the mean ± SEM in triplicate wells. Data shown are representative of three independent experiments. HEK, human embryonic kidney; LPS, lipopolysaccharide; NF-κB, nuclear factor-κB; TLR, Toll-like receptor.

TAK-242 does not inhibit the binding of LPS to TLR4-MD-2. RAW264.7 cells were incubated with 1 µM TAK-242 or 1 µg·mL⁻¹ polymixin B for 30 min, then stimulated with 1 µg·mL⁻¹ LPS for 30 min. The cells were stained with PE-conjugated TLR4-MD-2 Ab MTS510 for 30 min and analysed with a flow cytometer. Representative data are shown. Ab, antibody; LPS, lipopolysaccharide; TLR, Toll-like receptor.

TAK-242 inhibits both MyD88-dependent and -independent signalling. DCs from wild-type and MyD88-knockout mice were incubated with various concentrations of TAK-242 for 1 h, then stimulated with LPS. (A) Effect of TAK-242 on the IL-12 p40 and TNF-α production in LPS-induced DCs. DCs were stimulated for 24 h with 10 ng·mL⁻¹ LPS. IL-12 p40 and TNF-α production were measured by ELISA. Data represent the mean in duplicate wells. (B) Effect of TAK-242 on CD40 expression in LPS-induced DCs. DCs were stimulated for 24 h with 10 ng·mL⁻¹ LPS. Cells were stained with PE-conjugated CD40 Ab and analysed with a flow cytometer. Representative data are shown. (C) Effect of TAK-242 on the expression of IFN-inducible genes in LPS-stimulated DCs. DCs were stimulated with 10 ng·mL⁻¹ LPS for the indicated times. Total RNA was subjected to northern blotting with probes for IP-10, GARG16, IRG1 and β-actin. DCs, dendritic cells; GARG16, glucocorticoid-attenuated response gene 16; IL, interleukin; IP-10, IFN-inducible protein 10; IRG1, immune-responsive gene 1; LPS, lipopolysaccharide; TNF-α, tumour necrosis factor-α.