hek blue htlr8 reporter cells Search Results


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Compound 10 inhibits TLR8-mediated inflammation and signaling. (A) hTLR-HEK293 reporter cells were preincubated with or without compound 10 (10 μM) for 1 h and then stimulated with ligands for TLR2/1 (Pam 3 CSK 4 , 10 ng/mL), TLR2/6 (Pam 2 CSK 4 , 1 ng/mL), TLR3 (poly­(I:C) HMW, 10 μg/mL), TLR4 (LPS E. coli , 10 ng/mL), TLR5 (flagellin B. subtilits , 100 ng/mL), TLR7 (CL307, 0.8 μM), TLR8 (TL8-506, 1.5 μM), or TLR9 (ODN2006, 5 μM). Supernatants were analyzed for TLR-mediated NF-κB activation by the SEAP reporter assay using QuantiBlue (OD 620 ) and normalized to the respective TLR agonist alone. Mean + SEM ( n = 3). (B) THP-1 macrophages were preincubated with 10 μM of compound 10 for 1 h and then stimulated with TL8-506 (6 μM) for 1 h. IL1B and TNF gene expression was normalized to the housekeeping gene GAPDH , and values were compared to control (designated with a value of 1). Bar graphs show mean + SEM ( n = 3). (C,D) THP-1 macrophages were primed with TL8-506 (0.6 μM) for 3 h and then stimulated for 3 h with ATP (5 mM). Compound 10 (10 μM) was added after priming for 1 h before stimulation. (C) IL-1β release in the cell culture supernatants was determined by ELISA. (D) LDH release was determined in cell culture supernatants. Results are shown as percentage of the maximum LDH release. Mean + SEM ( n = 4). (E) THP-1 macrophages were incubated with compound 10 (10 μM) for 1 h and stimulated with TL8-506 (0.6 μM) for 15 min. Whole-cell lysates were used for WB. Bar graphs were obtained by densitometric analysis of Western blot data. Uncropped Western blots are shown in Figure S10A . Mean + SEM ( n = 4). One-way ANOVA followed by Tukey’s post-test, ns ≥0.05, * P ≤ 0.05, ** P ≤ 0.01 *** P ≤ 0.001, **** P ≤ 0.0001. (F) THP1-Dual TLR4/MD-2/CD14 cells (THP-1 Dual cells) were preincubated with 10 μM of compound 10 for 1 h and then stimulated with TL8-506 (6 μM) or LPS from E. coli (10 ng/mL) for 24 h. Supernatants were analyzed for NF-κB activation by the SEAP reporter assay using QuantiBlue (OD 620 ) or for IRF activation by lucia luciferase using QuantiLuc. (G) THP-1 macrophages were incubated with compound 10 (10 μM) for 1 h, followed by TL8-506 (0.6 μM) stimulation for 30 min. Co-IP was performed using <t>anti-hTLR8</t> antibodies and is depicted as Western blots (WB) for hTLR8 and MyD88. Uncropped Western blots are shown in Figure S10B . Mean + SEM ( n = 4). (H) Inhibitory concentration–response curves resulting from DMR traces. THP1-Dual TLR4/MD-2/CD14 cells (THP-1 Dual cells) were preincubated with increasing concentration of compound 10 and afterward stimulated with 6 μM TL8-506. The concentration–response curve was derived from DMR signals recorded at 250 min shown in Figure S9B . Mean ± SEM ( n = 3).
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Compound 10 inhibits TLR8-mediated inflammation and signaling. (A) hTLR-HEK293 reporter cells were preincubated with or without compound 10 (10 μM) for 1 h and then stimulated with ligands for TLR2/1 (Pam 3 CSK 4 , 10 ng/mL), TLR2/6 (Pam 2 CSK 4 , 1 ng/mL), TLR3 (poly­(I:C) HMW, 10 μg/mL), TLR4 (LPS E. coli , 10 ng/mL), TLR5 (flagellin B. subtilits , 100 ng/mL), TLR7 (CL307, 0.8 μM), TLR8 (TL8-506, 1.5 μM), or TLR9 (ODN2006, 5 μM). Supernatants were analyzed for TLR-mediated NF-κB activation by the SEAP reporter assay using QuantiBlue (OD 620 ) and normalized to the respective TLR agonist alone. Mean + SEM ( n = 3). (B) THP-1 macrophages were preincubated with 10 μM of compound 10 for 1 h and then stimulated with TL8-506 (6 μM) for 1 h. IL1B and TNF gene expression was normalized to the housekeeping gene GAPDH , and values were compared to control (designated with a value of 1). Bar graphs show mean + SEM ( n = 3). (C,D) THP-1 macrophages were primed with TL8-506 (0.6 μM) for 3 h and then stimulated for 3 h with ATP (5 mM). Compound 10 (10 μM) was added after priming for 1 h before stimulation. (C) IL-1β release in the cell culture supernatants was determined by ELISA. (D) LDH release was determined in cell culture supernatants. Results are shown as percentage of the maximum LDH release. Mean + SEM ( n = 4). (E) THP-1 macrophages were incubated with compound 10 (10 μM) for 1 h and stimulated with TL8-506 (0.6 μM) for 15 min. Whole-cell lysates were used for WB. Bar graphs were obtained by densitometric analysis of Western blot data. Uncropped Western blots are shown in Figure S10A . Mean + SEM ( n = 4). One-way ANOVA followed by Tukey’s post-test, ns ≥0.05, * P ≤ 0.05, ** P ≤ 0.01 *** P ≤ 0.001, **** P ≤ 0.0001. (F) THP1-Dual TLR4/MD-2/CD14 cells (THP-1 Dual cells) were preincubated with 10 μM of compound 10 for 1 h and then stimulated with TL8-506 (6 μM) or LPS from E. coli (10 ng/mL) for 24 h. Supernatants were analyzed for NF-κB activation by the SEAP reporter assay using QuantiBlue (OD 620 ) or for IRF activation by lucia luciferase using QuantiLuc. (G) THP-1 macrophages were incubated with compound 10 (10 μM) for 1 h, followed by TL8-506 (0.6 μM) stimulation for 30 min. Co-IP was performed using anti-hTLR8 antibodies and is depicted as Western blots (WB) for hTLR8 and MyD88. Uncropped Western blots are shown in Figure S10B . Mean + SEM ( n = 4). (H) Inhibitory concentration–response curves resulting from DMR traces. THP1-Dual TLR4/MD-2/CD14 cells (THP-1 Dual cells) were preincubated with increasing concentration of compound 10 and afterward stimulated with 6 μM TL8-506. The concentration–response curve was derived from DMR signals recorded at 250 min shown in Figure S9B . Mean ± SEM ( n = 3).

Journal: Journal of Medicinal Chemistry

Article Title: Discovery of Novel Isoxazole-Based Small-Molecule Toll-Like Receptor 8 Antagonists

doi: 10.1021/acs.jmedchem.4c03148

Figure Lengend Snippet: Compound 10 inhibits TLR8-mediated inflammation and signaling. (A) hTLR-HEK293 reporter cells were preincubated with or without compound 10 (10 μM) for 1 h and then stimulated with ligands for TLR2/1 (Pam 3 CSK 4 , 10 ng/mL), TLR2/6 (Pam 2 CSK 4 , 1 ng/mL), TLR3 (poly­(I:C) HMW, 10 μg/mL), TLR4 (LPS E. coli , 10 ng/mL), TLR5 (flagellin B. subtilits , 100 ng/mL), TLR7 (CL307, 0.8 μM), TLR8 (TL8-506, 1.5 μM), or TLR9 (ODN2006, 5 μM). Supernatants were analyzed for TLR-mediated NF-κB activation by the SEAP reporter assay using QuantiBlue (OD 620 ) and normalized to the respective TLR agonist alone. Mean + SEM ( n = 3). (B) THP-1 macrophages were preincubated with 10 μM of compound 10 for 1 h and then stimulated with TL8-506 (6 μM) for 1 h. IL1B and TNF gene expression was normalized to the housekeeping gene GAPDH , and values were compared to control (designated with a value of 1). Bar graphs show mean + SEM ( n = 3). (C,D) THP-1 macrophages were primed with TL8-506 (0.6 μM) for 3 h and then stimulated for 3 h with ATP (5 mM). Compound 10 (10 μM) was added after priming for 1 h before stimulation. (C) IL-1β release in the cell culture supernatants was determined by ELISA. (D) LDH release was determined in cell culture supernatants. Results are shown as percentage of the maximum LDH release. Mean + SEM ( n = 4). (E) THP-1 macrophages were incubated with compound 10 (10 μM) for 1 h and stimulated with TL8-506 (0.6 μM) for 15 min. Whole-cell lysates were used for WB. Bar graphs were obtained by densitometric analysis of Western blot data. Uncropped Western blots are shown in Figure S10A . Mean + SEM ( n = 4). One-way ANOVA followed by Tukey’s post-test, ns ≥0.05, * P ≤ 0.05, ** P ≤ 0.01 *** P ≤ 0.001, **** P ≤ 0.0001. (F) THP1-Dual TLR4/MD-2/CD14 cells (THP-1 Dual cells) were preincubated with 10 μM of compound 10 for 1 h and then stimulated with TL8-506 (6 μM) or LPS from E. coli (10 ng/mL) for 24 h. Supernatants were analyzed for NF-κB activation by the SEAP reporter assay using QuantiBlue (OD 620 ) or for IRF activation by lucia luciferase using QuantiLuc. (G) THP-1 macrophages were incubated with compound 10 (10 μM) for 1 h, followed by TL8-506 (0.6 μM) stimulation for 30 min. Co-IP was performed using anti-hTLR8 antibodies and is depicted as Western blots (WB) for hTLR8 and MyD88. Uncropped Western blots are shown in Figure S10B . Mean + SEM ( n = 4). (H) Inhibitory concentration–response curves resulting from DMR traces. THP1-Dual TLR4/MD-2/CD14 cells (THP-1 Dual cells) were preincubated with increasing concentration of compound 10 and afterward stimulated with 6 μM TL8-506. The concentration–response curve was derived from DMR signals recorded at 250 min shown in Figure S9B . Mean ± SEM ( n = 3).

Article Snippet: Human embryonic Kidney (HEK)-Blue Null1, HEK-Blue hTLR2-TLR1, HEK-Blue hTLR2-TLR6, HEK-Blue hTLR4, HEK-Blue hTLR5, HEK-Blue hTLR7, HEK-Blue hTLR8, and HEK-Blue hTLR9 (InvivoGen, Toulouse, France) were cultured in Dulbecco’s modified Eagle’s medium (PAN-Biotech, Aidenbach, Germany) containing 10% (v/v) heat inactivated fetal bovine serum (FBS; S0615), 100 U/mL penicillin, 100 mg/mL streptomycin (P4333), 2 mM 1-glutamine (G7513) (all from Sigma-Aldrich, Taufkirchen, Germany), 100 μg/mL Normocin and selective antibiotics HEK-Blue Selection (hTLR2-TLR1, hTLR2-TLR6, hTLR4), 100 μg/mL zeocin (Null1), 100 μg/mL zeocin with 10 μg/mL (hTLR7, hTLR9), and 30 μg/mL (hTLR5, hTLR8) blasticidin (all from InvivoGen, Toulouse, France).

Techniques: Activation Assay, Reporter Assay, Gene Expression, Control, Cell Culture, Enzyme-linked Immunosorbent Assay, Incubation, Analysis, Western Blot, Luciferase, Co-Immunoprecipitation Assay, Concentration Assay, Derivative Assay, Real-time Polymerase Chain Reaction

Compound 10 is a competitive antagonist of TL8-506. (A) HEK293 reporter control cells were transfected with mutant (G = Glycin351, V = Valin378, F = Phenylalanine495; GV, GF, VF, and GVF = dual or triple mutations of amino acids) or wildtype TLR8 plasmid (TLR8 WT ). Cells were stimulated with indicated concentrations of TL8-506 for 24 h. Supernatants were analyzed for TLR8-mediated NF-κB activation by SEAP reporter assay using QuantiBlue (OD 620 ) and normalized to TL8-506. Mean + SEM ( n = 3). (B) HEK293 reporter control cells were transfected with mutant or wildtype TLR8 plasmid. Cells were preincubated with either compound 10 (2 μM) or Enpatoran (1 μM) for 1 h and then stimulated with TL8-506 (3 μM) or CL075 (4 μM) for 24 h. Supernatants were analyzed for TLR8-mediated NF-κB activation by the SEAP reporter assay using QuantiBlue (OD 620 ) and normalized to TL8-506 or CL075, respectively. Mean + SEM ( n = 4). (C) hTLR8-HEK293 reporter cells were preincubated with indicated concentrations of compound 10 for 1 h and stimulated with increasing concentrations of TL8-506 for 24 h. Supernatants were analyzed for TLR8-mediated NF-κB activation by the SEAP reporter assay using QuantiBlue (OD 620 ). Calculated pharmacological parameters of the concentration–effect curves are depicted in Table S6 . (D) Schild plot of (C). The lines show linear regression, with Schild slopes unconstrained (solid) or constrained to unity (dashed) ( n = 3).

Journal: Journal of Medicinal Chemistry

Article Title: Discovery of Novel Isoxazole-Based Small-Molecule Toll-Like Receptor 8 Antagonists

doi: 10.1021/acs.jmedchem.4c03148

Figure Lengend Snippet: Compound 10 is a competitive antagonist of TL8-506. (A) HEK293 reporter control cells were transfected with mutant (G = Glycin351, V = Valin378, F = Phenylalanine495; GV, GF, VF, and GVF = dual or triple mutations of amino acids) or wildtype TLR8 plasmid (TLR8 WT ). Cells were stimulated with indicated concentrations of TL8-506 for 24 h. Supernatants were analyzed for TLR8-mediated NF-κB activation by SEAP reporter assay using QuantiBlue (OD 620 ) and normalized to TL8-506. Mean + SEM ( n = 3). (B) HEK293 reporter control cells were transfected with mutant or wildtype TLR8 plasmid. Cells were preincubated with either compound 10 (2 μM) or Enpatoran (1 μM) for 1 h and then stimulated with TL8-506 (3 μM) or CL075 (4 μM) for 24 h. Supernatants were analyzed for TLR8-mediated NF-κB activation by the SEAP reporter assay using QuantiBlue (OD 620 ) and normalized to TL8-506 or CL075, respectively. Mean + SEM ( n = 4). (C) hTLR8-HEK293 reporter cells were preincubated with indicated concentrations of compound 10 for 1 h and stimulated with increasing concentrations of TL8-506 for 24 h. Supernatants were analyzed for TLR8-mediated NF-κB activation by the SEAP reporter assay using QuantiBlue (OD 620 ). Calculated pharmacological parameters of the concentration–effect curves are depicted in Table S6 . (D) Schild plot of (C). The lines show linear regression, with Schild slopes unconstrained (solid) or constrained to unity (dashed) ( n = 3).

Article Snippet: Human embryonic Kidney (HEK)-Blue Null1, HEK-Blue hTLR2-TLR1, HEK-Blue hTLR2-TLR6, HEK-Blue hTLR4, HEK-Blue hTLR5, HEK-Blue hTLR7, HEK-Blue hTLR8, and HEK-Blue hTLR9 (InvivoGen, Toulouse, France) were cultured in Dulbecco’s modified Eagle’s medium (PAN-Biotech, Aidenbach, Germany) containing 10% (v/v) heat inactivated fetal bovine serum (FBS; S0615), 100 U/mL penicillin, 100 mg/mL streptomycin (P4333), 2 mM 1-glutamine (G7513) (all from Sigma-Aldrich, Taufkirchen, Germany), 100 μg/mL Normocin and selective antibiotics HEK-Blue Selection (hTLR2-TLR1, hTLR2-TLR6, hTLR4), 100 μg/mL zeocin (Null1), 100 μg/mL zeocin with 10 μg/mL (hTLR7, hTLR9), and 30 μg/mL (hTLR5, hTLR8) blasticidin (all from InvivoGen, Toulouse, France).

Techniques: Control, Transfection, Mutagenesis, Plasmid Preparation, Activation Assay, Reporter Assay, Concentration Assay