TLR2 signaling inhibitor

Inhibitor of TLR2 signaling

Inhibition of TLR2 signaling by TL2-C29

TL2-C29 is a small-molecule inhibitor of Toll-like receptor 2 (TLR2) [1]. TLR2 plays an essential role in detecting a diverse range of microbial pathogen-associated molecular patterns (PAMPs) from bacteria, fungi, parasites, and viruses [2]. TLR1 and TLR6 co-receptors are crucial for TLR2 signaling and ligand specificity. Cell surface TLR2/TLR1 and TLR2/TLR6 heterodimers bind tri- and diacylated lipoproteins, respectively [3,4]. TLR2 signaling is initiated by ligand-induced dimerization of the essential cytoplasmic TIR (Toll/interleukin-1 receptor) domains of the TLR2 heterodimers and adapter proteins. Subsequent signaling cascades lead to a pro-inflammatory response.

Mode of action:

TL2-C29 binds a pocket in the BB loop within the TLR2 TIR domain, thereby inhibiting TLR2 interaction with the MyD88 adapter molecule and downstream MAPK and NF-κB activation [1]. In human TLR2 signaling, TL2-C29 blocks both TLR2/1 and TLR2/6 pathways. In murine TLR2 signaling, TL2-C29 preferentially inhibits the TLR2/1 pathway [1, and in-house data].

 More details

Key features:

• Potent inhibitor of human TLR2/1 and TLR2/6 signaling
• Potent inhibitor of murine TLR2/1 signaling
• No substantial cellular toxicity
• Inhibition of NF-κB/AP-1 signaling validated in cellular assays
• InvitroFit™ grade: each lot is highly pure (≥ 95%) and functionally tested

Learn more about TLR2 heterodimers.

References:

1. Mistry, P. et al., 2015. Inhibition of TLR2 signaling by small molecule inhibitors targeting a pocket within the TLR2 TIR domain. PNAS. 112(17):5455-5460.
2. Oliveira-Nascimento L. et al., 2012. The role of TLR2 in infection and Immunity. Front Immunol. 3(79): doi:10.3389/fimmu.2012.00079.
3. Takeuchi O. et al., 2001. Discrimination of bacterial lipoproteins by Toll-like receptor 6. Int Immunol. 13: 933-940.
4. Takeuchi O. et al., 2002. Cutting edge: role of Toll-like receptor 1 in mediating immune response to microbial lipoproteins. J Immunol. 169: 10-14.

Figures

TL2-C29 is a potent inhibitor of human TLR2 signaling pathways
HEK-Blue™ hTLR2 cells were cultured in the presence of increasing concentrations of TL2-C29 for 3 hours at 37°C. Cells were then incubated overnight with 10 ng/ml Pam3CSK4 (TLR2/TLR1 agonist) or 0.1 ng/ml FSL-1 (TLR2/TLR6 agonist). The neutralizing ability of TL2-C29 was determined by measuring the reduction of SEAP production in the supernatant using the QUANTI‑Blue™ Solution detection reagent. Data are shown as a percentage (%) of maximal TLR2 activation with each agonist.

TL2-C29 specifically inhibits human TLR2 signaling pathways.
HEK-Blue™ hTLR2, HEK-Blue™ hTLR3, HEK-Blue™ hTLR4, or HEK-Blue™ hTLR7 cells were cultured with specific agonists either alone (blue bars) or in the presence of 50 µM TL2-C29 (red bars): 1 ng/ml Pam2CSK4 (TLR2/TLR6 agonist), 10 ng/ml Pam3CSK4 (TLR2/TLR1 agonist), 1 µg/ml Poly(I:C)(TLR3 agonist), 1 ng/ml LPS-EK Ultrapure (TLR4 agonist), or CL264 (TLR7 agonist). After overnight incubation, the neutralizing activity of TL2-C29 was determined by measuring the reduction of SEAP production in the supernatant using the QUANTI‑Blue™ Solution detection reagent. The optical density (OD) at 630 nm is shown as mean ± SEM.

TL2-C29 specifically inhibits human TLR2/1 and TLR2/6 signaling pathways.
HEK-Blue™ hTLR2-TLR1 (A) or HEK-Blue™ hTLR2-TLR6 cells (B) were cultured in the presence of increasing concentrations of TL2-C29 for 3 hours at 37°C. Cells were then incubated overnight with 10 ng/ml Pam3CSK4, 10 µM CU-T12-9, 107 CFU/ml heat-killed pseudomonas aeruginosa (HKPA), 1 ng/ml Pam2CSK4, 10 ng/ml FSL-1, or 107 CFU/ml heat-killed listeria monocytogenes (HKLM)). The neutralizing activity of TL2-C29 was determined by measuring the reduction of SEAP production in the supernatant using the QUANTI‑Blue™ Solution detection reagent. Data are shown as a percentage (%) of maximal response with each ligand.

Specifications

Synonyms: C29; TLR2-IN-C29; 3-[[(2-hydroxy-3-methoxyphenyl)methylene]amino]-2-methyl-benzoic acid

CAS number: 363600-92-4

Formula: C₁₆H₁₅NO₄

Molecular weight: 285.3 g/mol

Solubility: 50 mM (14.3 mg/ml) in DMSO

Working concentration: 25 - 200 µM for cell culture assays.

Quality control:

• Purity ≥ 95% (NMR)
• Inhibition of human TLR2/1 and TLR2/6 signaling has been confirmed using cellular assays
• The absence of bacterial contamination (e.g. lipoproteins and endotoxins) has been confirmed using HEK-Blue™ TLR2 and HEK‑Blue™ TLR4 cells.

Contents

• 5 mg TL2-C29 (provided as a dried powder)

 TL2-C29 is shipped at room temperature.

 Store at -20°C.

 The resuspended product is stable for 2 months when properly stored.

 Avoid repeated freeze-thaw cycles.

Details

TL2-C29 chemical structure (CAS 363600-92-4)

TL2-C29 is a potent inhibitor of human TLR2/1 and TLR2/6 signaling, and preferentially inhibits murine TLR2/1 signaling

Inhibition of TLR2 signaling by TL2-C29

TL2-C29 has been described to mediate a TLR2 signaling inhibition in a species-dependent manner between humans and mice [1].

We assessed the species-specific inhibition potency of TL2-C29 using HEK-Blue™ hTLR2 and HEK-Blue™ mTLR2 cells, stably expressing human TLR2 and mouse TLR2, respectively (see Figure). We observed that TL2-C29-mediated inhibition of TLR2/TLR1 signaling is highly similar in cells expressing hTLR2 or mTLR2, using two doses of Pam3CSK4 (panel A). However, TL2-C29-mediated inhibition of TLR2/TLR6 signaling is stronger in cells expressing hTLR2 than in cells expressing mTLR2, especially at a low dose of FSL-1 (panel B).

1. Mistry, P. et al., 2015. Inhibition of TLR2 signaling by small molecule inhibitors targeting a pocket within the TLR2 TIR domain. PNAS. 112(17):5455-5460.