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.

---

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 the dimerization of the intracellular Toll/IL-1 receptor resistance (TIR) domain. Except for TLR3, the TIR domains of all TLRs recruit the adaptor protein Myeloid Differentiation Primary Response Gene 88 (MyD88), triggering downstream signaling and ultimately leading to the production of pro-inflammatory cytokines. Therefore, blocking TLR TIR dimerization may improve TLR2-mediated hyperinflammatory states. The BB loop within the TLR TIR domain is crucial for mediating certain protein-protein interactions. Analysis of the crystal structure of the human TLR2 TIR domain revealed a pocket near the highly conserved BB loop residues P681 and G682. We used computer-aided drug design (CADD) to identify small molecule inhibitors that could embed in this pocket and potentially interfere with TLR2 signaling. Based on predicted BB loop pocket binding ability, computer screening identified 149 compounds and 20 FDA-approved drugs. These compounds were screened in HEK293T-TLR2 transfected cells to test their ability to inhibit TLR2-mediated IL-8 mRNA. C16H15NO4 (C29) was identified as a potential TLR2 inhibitor. C29 and its derivative o-vanillin inhibited the TLR2/1 and TLR2/6 signaling pathways induced by synthetic and bacterial TLR2 agonists in human HEK-TLR2 and THP-1 cells, but only the TLR2/1 signaling pathway in mouse macrophages. C29 failed to inhibit other TLR agonist- and TNF-α-induced signaling pathways. Mutation analysis of BB loop pocket residues indicated that C29 plays an indispensable role in the TLR2/1 signaling pathway, but not in the TLR2/6 signaling pathway, suggesting a possible functional difference between C29 and the TLR2/6 signaling pathways. Mice treated with o-vanillin showed a reduction in TLR2-induced inflammation. Our data preliminarily confirm that targeting the BB loop pocket is an effective method for identifying TLR2 signaling pathway inhibitors. [1]

---

Toll-like receptor 2 (TLR2) can recognize pathogen-associated molecular patterns, thereby defending against invading pathogens, and has become a highly attractive therapeutic target. To date, no small molecule TLR2 antagonists have entered clinical trials. In this paper, we designed and synthesized 50 N-benzylaniline compounds using computer-aided drug design (CADD). Subsequent in vitro studies screened out the most effective compound, SMU-A0B13, which had the strongest inhibitory activity against TLR2 (IC50 = 18.21 ± 0.87 μM). Preliminary mechanistic studies showed that this TLR2 inhibitor exerts highly specific and low-toxicity effects through the NF-κB signaling pathway and can effectively downregulate inflammatory cytokines such as SEAP, TNF-α, and NO in HEK-Blue hTLR2, human PBMC, and Raw 264.7 cell lines. In addition, molecular docking results also showed that SMU-A0B13 can bind well to the active domain of TLR2-TIR (PDB: 1FYW), which may explain its biological activity. [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.

---

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.

---

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