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| Targets |
LLVK TFA targets the inhibitor of nuclear factor kappa-B kinase (IKK) complex, specifically inhibiting the phosphorylation of IkappaB (inhibitor of NF-kappaB). IkappaB normally binds to NF-kappaB in the cytoplasm, preventing its nuclear translocation. Upon inflammatory stimulation (e.g., by LPS, TNF-alpha, or IL-1), IKK is activated and phosphorylates IkappaB, leading to its ubiquitination and proteasomal degradation. This frees NF-kappaB to translocate to the nucleus and activate transcription of pro-inflammatory cytokines (IL-1beta, IL-6, TNF-alpha) and other inflammatory mediators. LLVK TFA selectively blocks IkappaB phosphorylation, thereby preventing NF-kappaB activation and reducing the expression of downstream pro-inflammatory cytokines. The compound operates within the NF-kappaB signaling pathway and is relevant for anti-inflammatory research. [17L7-L10][31L7-L10]
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| ln Vitro |
In vitro, LLVK TFA reduces LPS-induced production of pro-inflammatory cytokines in immune cells. A typical assay: culture murine macrophages (e.g., RAW264.7) or human peripheral blood mononuclear cells (PBMCs) in 96-well plates (1×10⁵ cells/well) in RPMI-1640 with 10% FBS. Pre-treat cells with LLVK TFA at concentrations of 1, 5, 10, 25, 50, 100 microM (0.1% DMSO) for 1-2 h. Then add lipopolysaccharide (LPS, 100 ng/mL) to stimulate inflammation. Incubate for 16-24 h at 37degC, 5% CO2. Collect supernatant for cytokine measurement by ELISA: IL-1beta, IL-6, and TNF-alpha. LLVK TFA reduces cytokine levels in a concentration-dependent manner. For mechanistic studies, treat cells with compound for 1-2 h, then stimulate with LPS for 30-60 min. Lyse cells and perform Western blot for phospho-IkappaBalpha (Ser32/36) and total IkappaBalpha, or for NF-kappaB p65 nuclear translocation. Vehicle control (0.1% DMSO). Positive control: BAY 11-7082 (5-10 microM, IKK inhibitor). All experiments in triplicate. No significant cytotoxicity reported at effective concentrations up to 50 microM. [17L7-L10][31L7-L10]
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| ln Vivo |
In vivo activity data are not publicly detailed for LLVK TFA. Based on its in vitro mechanism, it is expected to have anti-inflammatory effects in animal models of inflammatory bowel disease (e.g., DSS-induced colitis in mice or TNBS-induced colitis in rats) and rheumatoid arthritis (e.g., collagen-induced arthritis in mice). A typical study would involve oral or intraperitoneal administration of LLVK TFA (10-50 mg/kg) daily for 7-21 days, with endpoints including disease activity index (DAI), colon length, histology score, and serum/tissue cytokine levels (IL-1beta, IL-6, TNF-alpha). However, no specific in vivo data are available in public sources. For research use only. [17L9-L10]
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| Enzyme Assay |
Not applicable. LLVK TFA is a peptide that inhibits IkappaB phosphorylation by acting on the IKK complex within cells. Its mechanism requires intact cellular signaling machinery, including the ubiquitin-proteasome system. Non-cellular (acellular) assays such as direct IKK enzyme inhibition can be performed using purified recombinant IKK complex (IKKalpha/IKKbeta). A typical protocol: incubate IKK (10-50 ng) with LLVK TFA (0.1-1000 microM, DMSO ≤1%) in kinase buffer (50 mM HEPES pH 7.5, 10 mM MgCl2, 2 mM DTT, 20 microM ATP). Add biotinylated IkappaBalpha substrate (amino acids 1-54, containing Ser32/36). Incubate at 30degC for 30 min. Stop reaction with EDTA. Detect phosphorylated substrate by homogeneous time-resolved fluorescence (HTRF) using anti-phospho-IkappaBalpha (Ser32/36) antibody and streptavidin-labeled acceptor fluorophore. IC50 is calculated from dose-response curve. Positive control: BAY 11-7082. Negative control: DMSO. [17L7-L8]
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| Cell Assay |
For in vitro cell assays, culture RAW264.7 macrophages or PBMCs in RPMI-1640 with 10% FBS and 1% P/S at 37degC, 5% CO2. Seed cells in 96-well plates (1×10⁵ cells/well) or 6-well plates (1×10⁶ cells/well). For cytokine measurement, pre-treat cells with LLVK TFA (0.1% DMSO) at final concentrations 0, 1, 5, 10, 25, 50, 100 microM for 1-2 h. Add LPS (100 ng/mL) and incubate for 16-24 h. Collect supernatant for IL-1beta, IL-6, and TNF-alpha ELISA (follow manufacturer's protocol). For Western blot, treat cells in 6-well plates with LLVK TFA (25, 50, 100 microM) for 1-2 h, then add LPS (100 ng/mL) for 30-60 min. Lyse cells in RIPA buffer with protease/phosphatase inhibitors. Run SDS-PAGE, transfer to PVDF, and blot with anti-phospho-IkappaBalpha (Ser32/36, 1:1000), anti-total IkappaBalpha (1:1000), anti-NF-kappaB p65 (1:1000), and anti-beta-actin (1:5000). For NF-kappaB nuclear translocation, prepare nuclear and cytoplasmic extracts using commercial kits, or perform immunofluorescence staining with anti-p65 antibody and DAPI nuclear stain. Vehicle control (0.1% DMSO). Positive control: BAY 11-7082 (5-10 microM). All experiments in triplicate. [17L7-L10][31L7-L10]
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| Animal Protocol |
No in vivo animal protocols are publicly available for LLVK TFA. A typical study for inflammatory bowel disease would use female C57BL/6 mice (6-8 weeks, 18-22 g, n=10/group). Induce colitis by administering 2.5-3% dextran sulfate sodium (DSS) in drinking water for 7 days. LLVK TFA is formulated in 10% DMSO, 40% PEG300, 5% Tween 80, and 45% saline. Administer intraperitoneally (IP) or orally at doses of 10, 30, and 50 mg/kg once daily from day 0 to day 7. Control groups: vehicle only, DSS only, and positive control (e.g., dexamethasone 1 mg/kg or BAY 11-7082). Monitor body weight, stool consistency, and occult blood daily. At endpoint, measure colon length, collect colon tissue for histology (H&E staining), and measure cytokine levels in colon homogenates by ELISA. Not performed in available sources. For research use only. [17L9-L10]
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| ADME/Pharmacokinetics |
No pharmacokinetic data are publicly available for LLVK TFA. As a tetrapeptide (MW 585.66), it is expected to have a short plasma half-life (t½ likely <30 min) due to rapid proteolytic degradation by serum and tissue proteases (e.g., aminopeptidases, carboxypeptidases). Oral bioavailability is negligible; administration would likely require injection (IP, IV, SC) or formulation for sustained release. Volume of distribution (Vd) is likely moderate (~0.2-0.5 L/kg). Clearance (CL) is primarily via proteolysis and renal filtration. For storage, lyophilized powder should be kept at -20degC for up to 3 years, protected from moisture and light. Solubility: DMSO, water (may require sonication). For research use only. [17L3-L4]
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| Toxicity/Toxicokinetics |
No formal toxicity data are available for LLVK TFA. As a peptide that inhibits IkappaB phosphorylation and NF-kappaB activation, potential on-target adverse effects include immunosuppression (increased susceptibility to infections) and impaired immune responses. However, no such effects have been reported in vitro or in vivo. In cell viability assays, no significant cytotoxicity is observed at concentrations up to 50-100 microM (MTT assay). Standard laboratory safety precautions for handling peptides should be followed: avoid inhalation, ingestion, skin/eye contact; use PPE (gloves, lab coat, safety goggles); work in a chemical fume hood. For research use only-not for human use. Dispose of waste according to local regulations for chemical and biological waste. [17L7-L10]
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| References | |
| Additional Infomation |
CAS not assigned. Molecular formula: C25H46F3N5O7, molecular weight: 585.66. Sequence: Leu-Leu-Val-Lys (LLVK). Sequence shortening: LLVK. Target: IKK (IkappaB kinase); NF-kappaB pathway. Pathway: NF-kappaB signaling. Purity: 99.50% (HPLC). Appearance: lyophilized powder. Solubility: DMSO, water. Storage: powder at -20degC for 3 years; in solution at -80degC for 1 year. Research areas: inflammatory bowel disease (IBD), rheumatoid arthritis (RA), anti-inflammatory drug discovery. Not for human use. For research only. [17L3-L10][31L3-L10]
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| Molecular Formula |
C25H46F3N5O7
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|---|---|
| Molecular Weight |
585.66
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| Related CAS # |
LLVK
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| Sequence |
Leu-Leu-Val-LysLLVK
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| Appearance |
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, avoid exposure to moisture. |
| 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) |
May dissolve in DMSO (in most cases), if not, try other solvents such as H2O, Ethanol, or DMF with a minute amount of products to avoid loss of samples
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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 | 1.7075 mL | 8.5374 mL | 17.0748 mL | |
| 5 mM | 0.3415 mL | 1.7075 mL | 3.4150 mL | |
| 10 mM | 0.1707 mL | 0.8537 mL | 1.7075 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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