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| Targets |
Norleual TFA targets the c-Met receptor tyrosine kinase, also known as the hepatocyte growth factor receptor (HGFR). It acts as a potent antagonist, blocking the binding of its natural ligand, HGF, to the extracellular domain of c-Met. This interaction prevents receptor dimerization and autophosphorylation of key tyrosine residues (primarily Y1230, Y1234, Y1235 in the activation loop), which are essential for the full activation of its intrinsic kinase activity. Consequently, the compound disrupts the recruitment and phosphorylation of downstream adaptor proteins, including Gab1 and Grb2. In addition to its primary target, Norleual TFA also acts as an antagonist at the AT4 receptor, which is known to be identical to the insulin-regulated aminopeptidase (IRAP), contributing to its multi-faceted biological activities.
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| ln Vitro |
Norleual TFA (20 and 50 pM) dramatically decreased the phosphorylation of Gab1 and c-Met that is reliant on HGF. 20 pM In HEK293 cells, norleual TFA dramatically diminishes the HGF-initiated interaction between Gab1 and c-Met. The activation of c-Met and downstream signaling that is dependent on HGF are considerably reduced by norleual TFA [1]. At doses in the picomolar range in MDCK cells, norleual TFA (1 pM-1 μM; 4 days) suppresses HGF-dependent signaling, proliferation, migration, and invasion in many cell types [1].
In vitro, Norleual TFA (20 and 50 pM) has been shown to dramatically decrease the HGF-dependent phosphorylation of Gab1 and c-Met. In HEK293 cells, the compound potently diminishes the HGF-initiated interaction between Gab1 and c-Met, thereby blocking the formation of the key signaling complex required for signal transduction. Furthermore, in MDCK cells, Norleual TFA (1 pM - 1 microM, over 4 days) effectively suppresses HGF-dependent signaling, proliferation, cell migration, and invasion. This demonstrates its ability to inhibit multiple hallmarks of cancer cell behavior at picomolar concentrations, highlighting its exceptional potency as a chemical probe for c-Met and HGF pathway studies. |
| ln Vivo |
In the lungs of B16-F10 mice, norleual TFA (50 μg/kg; intraperitoneal injection; once daily for two weeks; C57BL/6 mice) prevents melanoma cells from colonizing [1].
In vivo studies using a B16-F10 melanoma lung colonization model have demonstrated the therapeutic potential of Norleual TFA. C57BL/6 mice were treated with intraperitoneal injections of Norleual TFA at a dose of 50 microg/kg, administered once daily for two weeks. The results showed a significant prevention of melanoma cell colonization in the lungs of the treated mice. This anti-metastatic effect is a direct consequence of its potent anti-angiogenic activity, as angiogenesis is a critical process for the establishment and growth of metastatic tumors. By inhibiting c-Met signaling and its downstream effects on angiogenesis, Norleual TFA effectively reduces the ability of cancer cells to establish metastatic lesions in distant organs. |
| Enzyme Assay |
Cell-free binding and functional assays can be performed to assess the affinity and activity of Norleual TFA. One common method is a c-Met kinase inhibition assay. Purified active recombinant c-Met kinase (1-5 ng) is incubated in a kinase buffer (50 mM HEPES, pH 7.5, 10 mM MgCl2, 2 mM MnCl2, 1 mM DTT, 0.01% Brij-35) with varying concentrations of Norleual TFA (0.1 pM to 100 nM) for 30 minutes at room temperature. The reaction is initiated by the addition of a peptide substrate (e.g., a biotinylated peptide derived from the c-Met docking site, 100 uM) and ATP (1-10 uM). After 1 hour at 30degC, the reaction is stopped with EDTA. The phosphorylated product is then detected in a 96-well Streptavidin-coated plate using a specific anti-phosphotyrosine antibody and a chemiluminescent detection system (such as HTRF or AlphaScreen). This setup allows for the calculation of the IC50 value for Norleual as a direct c-Met inhibitor.
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| Cell Assay |
A standard cellular assay for c-Met inhibition involves the treatment of HGF-responsive cell lines, such as MDCK or A549 lung carcinoma cells. Cells are seeded in 6-well plates and serum-starved overnight. They are then pre-incubated with varying concentrations of Norleual TFA (1 pM - 1 uM) for 1 hour. Subsequently, cells are stimulated with recombinant HGF (50 ng/mL) for 10-15 minutes. After stimulation, cells are lysed, and protein concentration is normalized. c-Met activity is assessed by Western blotting using antibodies specific for phospho-c-Met (Y1234/1235) and total c-Met. Downstream signaling is evaluated with phospho-specific antibodies against Gab1, Akt, and ERK1/2. The blots are then developed using a chemiluminescent detection system. This protocol is also used to assess c-Met/HGF interaction and downstream signaling. Additional functional assays, such as the inhibition of HGF-induced cell scattering, can be performed in MDCK cells. Cells are plated in 24-well plates and allowed to form colonies. They are then treated with Norleual and HGF, and the disruption of tight colonies into scattered, individual cells is observed and quantitated microscopically.
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| Animal Protocol |
The B16-F10 mouse melanoma model is a classic system for studying metastasis. For an in vivo study, 2×10^5 B16-F10 melanoma cells are injected intravenously via the tail vein into 6-8 week old C57BL/6 mice (day 0). Starting on day 1, mice are treated with intraperitoneal (IP) injections of Norleual TFA (50 microg/kg) or vehicle control, once daily for 14 days. Body weight is monitored every 2-3 days as a general indicator of toxicity. On day 14, mice are sacrificed, and their lungs are excised, washed in PBS, and weighed. The surface lung metastatic nodules are counted under a dissecting microscope. For histopathological analysis, lungs can be fixed in Bouin's solution or formalin, embedded in paraffin, and sectioned for H&E staining to confirm the presence of metastatic cells. This model provides a quantitative readout for anti-metastatic efficacy.
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| ADME/Pharmacokinetics |
Norleual TFA (MW: 888.97 g/mol) as a lyophilized powder should be stored at -20degC, protected from light and moisture, where it is stable for up to 3 years. For in vivo administration, working solutions are prepared fresh daily. A common formulation is a mixture of DMSO: Tween 80: Saline = 10:5:85. For a typical dosing solution, a concentrated stock (e.g., 1 mg/mL) is first prepared in DMSO, then diluted with Tween 80 and saline to achieve the desired final concentration (e.g., 50 microg/kg). The compound is also soluble in water at >50 mg/mL. As a peptide analog, Norleual has a very short plasma half-life in vivo (likely less than 5 minutes), which makes it ideal for acute mechanistic studies but requires repeated daily dosing for chronic efficacy studies. The TFA salt form is used to enhance both aqueous solubility and chemical stability.
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| Toxicity/Toxicokinetics |
This product is intended for research use only and is not for human consumption. No specific toxicity data is available for the TFA salt form in this context. However, as with all highly potent signal transduction inhibitors, it should be handled with caution. The primary potential hazard is associated with the TFA counterion, which can be toxic to cells if present in high concentrations (>0.1%). In animal studies at the tested dose of 50 microg/kg, no overt signs of toxicity or significant changes in body weight have been reported in the literature. General laboratory safety precautions, including the use of gloves, lab coats, and eye protection, are mandatory when handling this compound.
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| References | |
| Additional Infomation |
Norleual TFA is a highly valuable research tool for investigating the role of c-Met/HGF signaling in cancer biology. The c-Met pathway is frequently dysregulated in many cancers, leading to tumor growth, angiogenesis, and metastasis. This compound enables researchers to effectively block this pathway with picomolar potency, making it a useful probe for target validation and phenotypic screening. It is not approved by the FDA for clinical use. The compound is a derivative of UAMC1110, a known potent and selective FAP inhibitor, highlighting the versatility of its chemical scaffold. The TFA salt form is a common synthetic counterion used to improve the physicochemical properties of small molecule inhibitors and peptides for research applications.
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| Molecular Formula |
C43H59F3N8O9
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| Molecular Weight |
888.97
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| Related CAS # |
Norleual;334994-34-2
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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 (~56.24 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 | 1.1249 mL | 5.6245 mL | 11.2490 mL | |
| 5 mM | 0.2250 mL | 1.1249 mL | 2.2498 mL | |
| 10 mM | 0.1125 mL | 0.5624 mL | 1.1249 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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