TLR2/Toll-like receptor 2

In HEK-TLR2 stable transfectants, C29 (10 or 50 μM; 1 hour) suppresses IL-8 mRNA produced by P3C and P2C in a dose-inducible manner. Significant inhibition of P3C- and P2C-induced IL-1β gene expression occurs at 1 and 4 hours following THP-1 cell stimulation with C29 (50 - 200 μM; 1 hour) [1]. Oxygen agonist Induced pro-inflammatory gene expression[1] by TNF-α mRNA[1] and IL-12 p40 protein[1]; C29 (50 μM; 1 hour) suppresses TLR2 in HEK-TLR2 cells and mouse macrophages; and C29 (25 or 50 μM; 1 hour) greatly lowers P3C sensing diode P2C sensing in primary mouse macrophages.

C29L (a C29 Derivative) Inhibits TLR2/1-Induced Inflammation in Vivo. [1]
One of the advantages of using C29L in vivo is that C29L is more soluble in water than C29. We next examined if C29L could inhibit TLR2/1-induced proinflammatory cytokines in vivo. Mice treated twice with C29L before administration of P3C significantly blocked IL-12 p40 and TNF-α liver cytokine mRNA and serum protein (Fig. 4). Importantly, C29L had a significant inhibitory effect at the later time point for IL-12 p40. Collectively, C29L blocks TLR2/1 signaling both in vitro and in vivo.

Transient Transfection and NF-κB Reporter Assay. [1]
HEK293T cells were cultured and plated overnight in 12-well tissue culture plates (2 × 105 cells per well). Transfection mixtures consisted of pcDNA3-YFP-hTLR2 or pcDNA3.1 control vector (1 μg per well each), pELAM (NF-κB)-luciferase (0.2 μg per well), and pRL-TK-Renilla luciferase (0.05 μg per well). Transfection was carried out using Superfect transfection reagent, and cells were allowed to recover for 48 h and treated for 5 h with medium or stimuli in the presence/absence of C29. Cells were lysed in a passive lysis buffer, and firefly luciferase and Renilla luciferase activities were measured using the Dual-Luciferase Reporter Assay System. Renilla luciferase was used for normalization, and all values were further standardized to medium-treated pcDNA3-YFP-hTLR2 transfectants to determine relative luciferase units.

Western Blot Analysis [1]
Cell Types: THP-1 Cell
Tested Concentrations: 150 μM
Incubation Duration: 1 hour
Experimental Results: Interaction between endogenous TLR2 and myeloid differentiation primary response gene 88 (MyD88) 15 and 30 minutes after stimulation with P3C The effect is weakened.

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Western Blot Analysis [1]
Cell Types: Murine peritoneal macrophages
Tested Concentrations: 50 μM
Incubation Duration: 1 hour
Experimental Results: Strong blockade of MAPK activation at 30 minutes and diminished NF-κB activation from 5 to 30 minutes. Prevents P3C-induced IκBα degradation at 15 and 30 min.

In Vivo Studies of TLR2 Inhibitor.[1]
Female C57BL/6J mice (6–8 wk old) were purchased from The Jackson Laboratory and (n = 3 mice per group) received PBS, H2O, or C29L (in H2O; C29L is a C29 Derivative) administered i.p. (1.314 mM/g). After 1 h, mice received a second injection of PBS, H2O, or C29L administered i.p. (1.314 mM/g) and were subsequently challenged i.p. with PBS or P3C (100 μg) for 1 or 3 h. Mice were bled, and sera were prepared. Livers were also extracted for qRT-PCR analysis.

[1]. Inhibition of TLR2 signaling by small molecule inhibitors targeting a pocket within the TLR2 TIR domain. Proc Natl Acad Sci U S A. 2015 Apr 28;112(17):5455-60.

[2]. Synthesis, structure-activity relationships and preliminary mechanism study of N-benzylideneaniline derivatives as potential TLR2 inhibitors. Bioorg Med Chem. 2018;26(8):2041-2050.

Toll-like receptor (TLR) signaling is initiated by dimerization of intracellular Toll/IL-1 receptor resistance (TIR) domains. For all TLRs except TLR3, recruitment of the adapter, myeloid differentiation primary response gene 88 (MyD88), to TLR TIR domains results in downstream signaling culminating in proinflammatory cytokine production. Therefore, blocking TLR TIR dimerization may ameliorate TLR2-mediated hyperinflammatory states. The BB loop within the TLR TIR domain is critical for mediating certain protein-protein interactions. Examination of the human TLR2 TIR domain crystal structure revealed a pocket adjacent to the highly conserved P681 and G682 BB loop residues. Using computer-aided drug design (CADD), we sought to identify a small molecule inhibitor(s) that would fit within this pocket and potentially disrupt TLR2 signaling. In silico screening identified 149 compounds and 20 US Food and Drug Administration-approved drugs based on their predicted ability to bind in the BB loop pocket. These compounds were screened in HEK293T-TLR2 transfectants for the ability to inhibit TLR2-mediated IL-8 mRNA. C16H15NO4 (C29) was identified as a potential TLR2 inhibitor. C29, and its derivative, ortho-vanillin (o-vanillin), inhibited TLR2/1 and TLR2/6 signaling induced by synthetic and bacterial TLR2 agonists in human HEK-TLR2 and THP-1 cells, but only TLR2/1 signaling in murine macrophages. C29 failed to inhibit signaling induced by other TLR agonists and TNF-α. Mutagenesis of BB loop pocket residues revealed an indispensable role for TLR2/1, but not TLR2/6, signaling, suggesting divergent roles. Mice treated with o-vanillin exhibited reduced TLR2-induced inflammation. Our data provide proof of principle that targeting the BB loop pocket is an effective approach for identification of TLR2 signaling inhibitors.[1]

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Toll-like receptor 2 (TLR2) can recognize pathogen-associated molecular patterns to defense against invading organisms and has been represents an attractive therapeutic target. Until today, none TLR2 small molecule antagonist have been developed in clinical trial. Herein, we designed and synthesized 50 N-benzylideneaniline compounds with the help of CADD. And subsequent in vitro studies leading to the optimized compound SMU-A0B13 with most potent inhibitory activity to TLR2 (IC50=18.21 ± 0.87 μM). Preliminary mechanism studies indicated that this TLR2 inhibitor can work through the NF-κB signaling pathway with high specificity and low toxicity, and can also efficiently downregulate inflammatory cytokines, such as SEAP, TNF-α and NO in HEK-Blue hTLR2, human PBMC and Raw 264.7 cell lines. Additionally, the docking situation also indicate SMU-A0B13 can well bind to the TLR2-TIR (PDB: 1FYW) active domain, which probably explains the bioactivity.[2]

C16H15NO4

285.2946

285.1001

C, 67.36; H, 5.30; N, 4.91; O, 22.43

363600-92-4

363600-92-4;

3579893

Off-white to yellow solid powder

2.7

2

5

4

21

385

0

WTGMGRFVBFDHGQ-RQZCQDPDSA-N

InChI=1S/C16H15NO4/c1-10-12(16(19)20)6-4-7-13(10)17-9-11-5-3-8-14(21-2)15(11)18/h3-9,18H,1-2H3,(H,19,20)/b17-9+

3-[[(2-Hydroxy-3-methoxyphenyl)methylene]amino]-2-methyl-benzoic acid

TLR2-IN-C29; TLR2 IN C29; 3-[(2-hydroxy-3-methoxyphenyl)methylideneamino]-2-methylbenzoic acid; 3-((2-Hydroxy-3-methoxybenzylidene)amino)-2-methylbenzoic acid; 3-[(2-Hydroxy-3-methoxybenzylidene)amino]-2-methylbenzoic acid; 3-[(E)-[(2-HYDROXY-3-METHOXYPHENYL)METHYLIDENE]AMINO]-2-METHYLBENZOIC ACID; ChemDiv3_012645; TLR2INC29; TLR2-inhibitor-C29; TLR2 inhibitor-C29; TLR2 inhibitor C29

2934.99.9001

Powder      -20°C    3 years

                     4°C     2 years

In solvent   -80°C    6 months

                  -20°C    1 month

Room temperature (This product is stable at ambient temperature for a few days during ordinary shipping and time spent in Customs)

DMSO : ≥ 30 mg/mL (~105.16 mM)

Solubility in Formulation 1: ≥ 2.5 mg/mL (8.76 mM) (saturation unknown) in 10% DMSO + 40% PEG300 + 5% Tween80 + 45% Saline (add these co-solvents sequentially from left to right, and one by one), clear solution.
For example, if 1 mL of working solution is to be prepared, you can add 100 μL of 25.0 mg/mL clear DMSO stock solution to 400 μL PEG300 and mix evenly; then add 50 μL Tween-80 to the above solution and mix evenly; then add 450 μL normal saline to adjust the volume to 1 mL.
Preparation of saline: Dissolve 0.9 g of sodium chloride in 100 mL ddH₂ O to obtain a clear solution.

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Solubility in Formulation 2: ≥ 2.5 mg/mL (8.76 mM) (saturation unknown) in 10% DMSO + 90% (20% SBE-β-CD in Saline) (add these co-solvents sequentially from left to right, and one by one), clear solution.
For example, if 1 mL of working solution is to be prepared, you can add 100 μL of 25.0 mg/mL clear DMSO stock solution to 900 μL of 20% SBE-β-CD physiological saline solution and mix evenly.
Preparation of 20% SBE-β-CD in Saline (4°C，1 week): Dissolve 2 g SBE-β-CD in 10 mL saline to obtain a clear solution.

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 (Please use freshly prepared in vivo formulations for optimal results.)