Journal: The FEBS journal

Article Title: Toll-like receptor 2 antagonists identified through virtual screening and experimental validation.

doi: 10.1111/febs.14124

Figure Lengend Snippet: Fig. 1. Workflow of the current study. Steps followed to identify non-peptide Toll-like receptor 2 (TLR2) antagonists.

Article Snippet: Human recombinant proteins TLR2, TLR1, TLR4/MD2 complex, and TLR3 (R&D systems, Minneapolis, MN, USA) were immobilized by amine coupling onto surfaces of a GLH or/and GLM sensor chips.

Techniques:

Journal: The FEBS journal

Article Title: Toll-like receptor 2 antagonists identified through virtual screening and experimental validation.

doi: 10.1111/febs.14124

Figure Lengend Snippet: Fig. 2. TLR2-TLR1 residues subjected to computational mutation binding energy calculations. Sequence alignment of the active site in human TLR2 and TLR1, based on structure. Details of the residues that bind Pam3CSK4 in the 2Z7X crystal structure, and the results obtained from computational alanine scanning are given. Residues with mutation energies > 0.5 kcal/mol were considered destabilizing.

Article Snippet: Human recombinant proteins TLR2, TLR1, TLR4/MD2 complex, and TLR3 (R&D systems, Minneapolis, MN, USA) were immobilized by amine coupling onto surfaces of a GLH or/and GLM sensor chips.

Techniques: Mutagenesis, Binding Assay, Sequencing

Journal: The FEBS journal

Article Title: Toll-like receptor 2 antagonists identified through virtual screening and experimental validation.

doi: 10.1111/febs.14124

Figure Lengend Snippet: Fig. 3. Receptor-ligand-based pharmacophore model. (A) Crystal structure of TLR2-TLR1-Pam3CSK4 (PDB ID: 2Z7X). (B) All pharmacophore features generated from Pam3CSK4 bound to TLR2-TLR1. (C) The five selected pharmacophore features for the receptor-ligand-based model, and the residues around the features are labeled. Asterisks indicate TLR1 residues. The green, magenta, and cyan spheres indicate hydrogen bond acceptors (HBA), hydrogen bond donors (HBD), and hydrophobic features (HYD), respectively. The gray exclusion spheres indicate the spaces used by the proteins. (D) Receptor-ligand-based pharmacophore model, showing only features and geometric constraints. The distances between two features are given in angstroms (Å).

Article Snippet: Human recombinant proteins TLR2, TLR1, TLR4/MD2 complex, and TLR3 (R&D systems, Minneapolis, MN, USA) were immobilized by amine coupling onto surfaces of a GLH or/and GLM sensor chips.

Techniques: Generated, Labeling

Journal: The FEBS journal

Article Title: Toll-like receptor 2 antagonists identified through virtual screening and experimental validation.

doi: 10.1111/febs.14124

Figure Lengend Snippet: Fig. 6. Molecular docking results of identified TLR2 antagonists. The TLR2 and TLR1 residues are shown in blue and green, respectively. The dark green dotted lines represent stronger hydrogen bonds, and the light green dotted lines represent weaker hydrogen bonds. The hydrogen bond distances in Angstroms (Å) are written next to the dotted lines. Asterisks indicate TLR1 residues. The green dashed arrow shows the hydrogen bond interactions of the ligand with the main chains of amino acids. The orange line indicates the Pi interactions. (A) C13. (B) C11.

Article Snippet: Human recombinant proteins TLR2, TLR1, TLR4/MD2 complex, and TLR3 (R&D systems, Minneapolis, MN, USA) were immobilized by amine coupling onto surfaces of a GLH or/and GLM sensor chips.

Techniques:

Journal: The FEBS journal

Article Title: Toll-like receptor 2 antagonists identified through virtual screening and experimental validation.

doi: 10.1111/febs.14124

Figure Lengend Snippet: Fig. 7. The identified TLR2-TLR1 antagonists, and the results of surface plasmon resonance (SPR) analysis performed on the GLH sensor chip. (A) Two-dimensional structures of the two non-peptide TLR2-TLR1 antagonists. (B) The binding kinetics of the compounds with TLR2, measured through SPR. The binding signals were captured between TLR2 and different concentrations of the compounds. (C) Sensograms show the association and dissociation of varying concentrations of the compounds with TLR2.

Article Snippet: Human recombinant proteins TLR2, TLR1, TLR4/MD2 complex, and TLR3 (R&D systems, Minneapolis, MN, USA) were immobilized by amine coupling onto surfaces of a GLH or/and GLM sensor chips.

Techniques: SPR Assay, Binding Assay

Journal: The FEBS journal

Article Title: Toll-like receptor 2 antagonists identified through virtual screening and experimental validation.

doi: 10.1111/febs.14124

Figure Lengend Snippet: Fig. 8. The identified TLR2-TLR1 antagonists, and the results of surface plasmon resonance (SPR) analysis performed on the GLM sensor chip. (A) Sensograms show the association and dissociation of varying concentrations of the compounds with TLR2, TLR4-MD2 and TLR3. (B) Two-dimensional structures of CU-CPT22, C11 and C13.

Article Snippet: Human recombinant proteins TLR2, TLR1, TLR4/MD2 complex, and TLR3 (R&D systems, Minneapolis, MN, USA) were immobilized by amine coupling onto surfaces of a GLH or/and GLM sensor chips.

Techniques: SPR Assay

Journal: The FEBS journal

Article Title: Toll-like receptor 2 antagonists identified through virtual screening and experimental validation.

doi: 10.1111/febs.14124

Figure Lengend Snippet: Fig. 9. Initial screening of compounds for TLR2-induced cytokine response. HEK293-hTLR2 cells were co-treated with Pam3CSK4 (50 nM) and the compounds (at 1 and 5 μM) for 24 hrs. IL-8 secretion was detected by ELISA. All data shown represent the mean ± SEM of three independent experiments (*P < 0.05, **P < 0.01).

Article Snippet: Human recombinant proteins TLR2, TLR1, TLR4/MD2 complex, and TLR3 (R&D systems, Minneapolis, MN, USA) were immobilized by amine coupling onto surfaces of a GLH or/and GLM sensor chips.

Techniques: Enzyme-linked Immunosorbent Assay

Journal: The FEBS journal

Article Title: Toll-like receptor 2 antagonists identified through virtual screening and experimental validation.

doi: 10.1111/febs.14124

Figure Lengend Snippet: Fig. 10. Dose-dependent TLR2 inhibitory effects and cytotoxic properties of the identified TLR2-TLR1 antagonists. HEK293-TLR2 cells were first treated with the compounds at three different concentrations (1 , 5, and 10 μM) for 1 hr, and then with 50 nM of Pam3CSK4. (A) After 24 hrs, culture supernatants were collected and IL-8 secretion was measured with an ELISA kit. The experiments were conducted independently (n = 3), and are shown as mean ± S.D. (n = 1, *P < 0.05 or **P < 0.01). (B) After 24 hrs, cell viability was determined by measuring the optical density at 490 nm with CellTiter 96 AQueous One Solution Cell Proliferation Assay (MTS). The percentage of cell viability compared to control is presented in the histogram; experiments were conducted independently (n = 3), and are shown as the mean ± S.E.M. of independent experiments.

Article Snippet: Human recombinant proteins TLR2, TLR1, TLR4/MD2 complex, and TLR3 (R&D systems, Minneapolis, MN, USA) were immobilized by amine coupling onto surfaces of a GLH or/and GLM sensor chips.

Techniques: Enzyme-linked Immunosorbent Assay, Proliferation Assay, Control

Journal: The FEBS journal

Article Title: Toll-like receptor 2 antagonists identified through virtual screening and experimental validation.

doi: 10.1111/febs.14124

Figure Lengend Snippet: Fig. 11. Cytokine inhibitory effects of C11 and C13 in human and mouse cells. (A) HEK293-hTLR2 and HEK293-Null cells were pre-treated with 10 μM of CU-CPT22, C11 and C13 for 1 hr, and then with 50 nM of Pam3CSK4. After 24 hrs, culture supernatants were collected and IL-8 secretion was measured with an ELISA kit. (B) The expression level of TNF-α secretion was measured by ELISA assay when the compounds CU-CPT22, C11 and C13 were co-treated with TLR2/TLR1 (Pam3CSK4), or TLR2/6 (FSL-1) or TLR3 [Poly (I:C)] or TLR4 (LPS) agonists in RAW 264.7 cells. CU-CPT22 was used as a positive control. The data shown represent at least three independent experiments (n ≥ 3), and bars represent means ± S.E.M. (*P < 0.05, **P < 0.01).

Article Snippet: Human recombinant proteins TLR2, TLR1, TLR4/MD2 complex, and TLR3 (R&D systems, Minneapolis, MN, USA) were immobilized by amine coupling onto surfaces of a GLH or/and GLM sensor chips.

Techniques: Enzyme-linked Immunosorbent Assay, Expressing, Positive Control

Journal: The FEBS journal

Article Title: Toll-like receptor 2 antagonists identified through virtual screening and experimental validation.

doi: 10.1111/febs.14124

Figure Lengend Snippet: Fig. 12. Two-dimensional structures of the already reported and identified TLR2-TLR1 antagonists.

Article Snippet: Human recombinant proteins TLR2, TLR1, TLR4/MD2 complex, and TLR3 (R&D systems, Minneapolis, MN, USA) were immobilized by amine coupling onto surfaces of a GLH or/and GLM sensor chips.

Techniques:

Journal: International Journal of Environmental Research and Public Health

Article Title: DNA Methylation of Candidate Genes (ACE II, IFN-γ, AGTR 1, CKG, ADD1, SCNN1B and TLR2) in Essential Hypertension: A Systematic Review and Quantitative Evidence Synthesis

doi: 10.3390/ijerph16234829

Figure Lengend Snippet: DNA methylation (mDNA) process and selective candidate genes in essential HTN.

Article Snippet: Mao, T., et al. (2017) TLR2 Hypo-mDNA in e-HTN , Blood sample–antecubital vein , Bisulfite pyrosequencing of TLR2 CpGs. , 96 controls and 96 incident essential HTN cases , Hypo-methylation and increased transcription.

Techniques: DNA Methylation Assay, Methylation