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| 1mg |
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| 5mg |
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| 10mg |
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| Targets |
TLRs
Pepinh-TRIF directly targets the adaptor protein TRIF (TICAM-1), which mediates downstream signaling of TLR3 and TLR4 (via the MyD88-independent pathway). By blocking TRIF recruitment and activation, the peptide selectively inhibits the TRIF-dependent branch of TLR signaling. Its specificity for TRIF makes it distinct from inhibitors that target other TLR pathway components such as MyD88. |
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| ln Vitro |
Pepinh-TRIF (40 μM, 6 hours) significantly suppresses the triggered IκB-α phosphorylation and degradation in HCECs and blocks the expression and generation of IL-33 driven by polyI:C or flagellin [1]. Pepinh-TRIF (40 μM, 6 hours) by nuclear translocation of p65 protein suppresses NF-κB activation [1].
In vitro experiments with chicken embryo kidney (CEK) cells demonstrate that Pepinh-TRIF treatment increases infectious bronchitis virus (IBV) replication after 36 hours by blocking TLR3-mediated antiviral immunity. The compound is used as a TLR3 pathway inhibitor alongside other agents such as celastrol, chloroquine, and BX795 to study viral immune evasion mechanisms. |
| ln Vivo |
In specific-pathogen-free (SPF) chickens infected with IBV strains, the TLR7-MYD88 pathway was inhibited while the TLR3-TIRF pathway was activated. Treatment with Pepinh-TRIF resulted in increased viral replication after 36 hours, confirming its effectiveness as an in vivo TLR3 pathway antagonist. The compound modulates host antiviral responses in animal models of viral infection.
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| Enzyme Assay |
Cell lysates are prepared from TLR3-stimulated cells (e.g., HEK293 cells expressing TLR3) and immunoprecipitation (IP) assays are performed using anti-TRIF antibodies. Pepinh-TRIF is added to the lysates at various concentrations (1-50 uM) to assess disruption of the TRIF-signaling complex. Western blotting is used to detect downstream signaling molecules such as IRF3 and NF-kappaB. Alternatively, ELISA-based binding assays can be developed to measure compound-TRIF interactions.
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| Cell Assay |
Western Blot Analysis[1]
Cell Types: HCECs Tested Concentrations: 40 μM Incubation Duration: 6 hrs (hours) Experimental Results: Blocked NF-κB activation with p65 nuclear translocation. HEK293 cells stably expressing TLR3 are plated in 96-well plates and treated with Pepinh-TRIF at concentrations ranging from 1-100 uM for 2-4 hours prior to stimulation with poly(I:C) (TLR3 agonist). After 6-24 hours of stimulation, cell supernatants are collected for cytokine measurement (e.g., IFN-beta, IL-6, TNF-alpha) by ELISA. Cell viability is assessed by MTT assay to ensure observed effects are not due to cytotoxicity. |
| Animal Protocol |
In SPF chickens, Pepinh-TRIF is administered prior to infection with IBV strains. Viral load in tissue samples is measured by qRT-PCR at various time points post-infection (e.g., 12, 24, 36, 48 hours). In mouse models of viral infection, the compound is delivered intraperitoneally at doses of 1-5 mg/kg. Inflammatory cytokine levels in serum are quantified by ELISA, and immune cell infiltration in tissues is assessed by histology.
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| ADME/Pharmacokinetics |
Detailed pharmacokinetic data for Pepinh-TRIF TFA are not available in the literature. As a cell-permeable peptide, its half-life in circulation is expected to be short due to proteolytic degradation. Most in vitro studies use the compound at concentrations ranging from 1-100 uM, while in vivo studies use doses around 1-5 mg/kg, though bioavailability and clearance remain to be fully characterized.
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| Toxicity/Toxicokinetics |
No systematic toxicity studies have been reported for Pepinh-TRIF TFA. Based on its mechanism as a TLR3 pathway inhibitor, potential safety concerns relate to increased susceptibility to viral infections due to suppressed antiviral immune responses. In experimental settings, no severe adverse effects have been documented, but long-term or high-dose toxicity data are currently unavailable.
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| References | |
| Additional Infomation |
Pepinh-TRIF is a research-grade peptide and has not been approved for clinical use. It serves as an important pharmacological tool to dissect the differential roles of TLR3 and TLR7 pathways in innate immunity and viral pathogenesis. The TFA salt formulation ensures enhanced solubility and stability for in vitro and in vivo applications. However, researchers should note that PEP-INH-TRIF also inhibits TLR4/TRIF signaling and is cell-permeable. Compound handling requires dissolution in sterile PBS or cell culture medium with brief sonication.
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| Molecular Formula |
C180H279F3N58O40S2
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| Molecular Weight |
4016.63
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| Appearance |
White to off-white solid powder
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| HS Tariff Code |
2934.99.9001
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| Storage |
Powder -20°C 3 years 4°C 2 years In solvent -80°C 6 months -20°C 1 month Note: Please store this product in a sealed and protected environment (e.g. under nitrogen), avoid exposure to moisture and light. |
| Shipping Condition |
Room temperature (This product is stable at ambient temperature for a few days during ordinary shipping and time spent in Customs)
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| Solubility (In Vitro) |
H2O :~50 mg/mL (~12.45 mM)
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| Solubility (In Vivo) |
Note: Listed below are some common formulations that may be used to formulate products with low water solubility (e.g. < 1 mg/mL), you may test these formulations using a minute amount of products to avoid loss of samples.
Injection Formulations
Injection Formulation 1: DMSO : Tween 80： Saline = 10 : 5 : 85 (i.e. 100 μL DMSO stock solution → 50 μL Tween 80 → 850 μL Saline)(e.g. IP/IV/IM/SC) *Preparation of saline: Dissolve 0.9 g of sodium chloride in 100 mL ddH ₂ O to obtain a clear solution. Injection Formulation 2: DMSO : PEG300 ：Tween 80 : Saline = 10 : 40 : 5 : 45 (i.e. 100 μL DMSO → 400 μLPEG300 → 50 μL Tween 80 → 450 μL Saline) Injection Formulation 3: DMSO : Corn oil = 10 : 90 (i.e. 100 μL DMSO → 900 μL Corn oil) Example: Take the Injection Formulation 3 (DMSO : Corn oil = 10 : 90) as an example, if 1 mL of 2.5 mg/mL working solution is to be prepared, you can take 100 μL 25 mg/mL DMSO stock solution and add to 900 μL corn oil, mix well to obtain a clear or suspension solution (2.5 mg/mL, ready for use in animals). View More
Injection Formulation 4: DMSO : 20% SBE-β-CD in saline = 10 : 90 [i.e. 100 μL DMSO → 900 μL (20% SBE-β-CD in saline)] Oral Formulations
Oral Formulation 1: Suspend in 0.5% CMC Na (carboxymethylcellulose sodium) Oral Formulation 2: Suspend in 0.5% Carboxymethyl cellulose Example: Take the Oral Formulation 1 (Suspend in 0.5% CMC Na) as an example, if 100 mL of 2.5 mg/mL working solution is to be prepared, you can first prepare 0.5% CMC Na solution by measuring 0.5 g CMC Na and dissolve it in 100 mL ddH2O to obtain a clear solution; then add 250 mg of the product to 100 mL 0.5% CMC Na solution, to make the suspension solution (2.5 mg/mL, ready for use in animals). View More
Oral Formulation 3: Dissolved in PEG400 (Please use freshly prepared in vivo formulations for optimal results.) |
| Preparing Stock Solutions | 1 mg | 5 mg | 10 mg | |
| 1 mM | 0.2490 mL | 1.2448 mL | 2.4896 mL | |
| 5 mM | 0.0498 mL | 0.2490 mL | 0.4979 mL | |
| 10 mM | 0.0249 mL | 0.1245 mL | 0.2490 mL |
*Note: Please select an appropriate solvent for the preparation of stock solution based on your experiment needs. For most products, DMSO can be used for preparing stock solutions (e.g. 5 mM, 10 mM, or 20 mM concentration); some products with high aqueous solubility may be dissolved in water directly. Solubility information is available at the above Solubility Data section. Once the stock solution is prepared, aliquot it to routine usage volumes and store at -20°C or -80°C. Avoid repeated freeze and thaw cycles.
Calculation results
Working concentration： mg/mL；
Method for preparing DMSO stock solution： mg drug pre-dissolved in μL DMSO (stock solution concentration mg/mL). Please contact us first if the concentration exceeds the DMSO solubility of the batch of drug.
Method for preparing in vivo formulation:：Take μL DMSO stock solution, next add μL PEG300, mix and clarify, next addμL Tween 80, mix and clarify, next add μL ddH2O，mix and clarify.
(1) Please be sure that the solution is clear before the addition of next solvent. Dissolution methods like vortex, ultrasound or warming and heat may be used to aid dissolving.
(2) Be sure to add the solvent(s) in order.
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