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| Targets |
WRVYEKC(dnp)ALK tetraTFA is a substrate for aminopeptidase enzymes. The decapeptide is designed such that the tryptophan (Trp, W) residue is protected from fluorescence by its proximity to the DNP quencher on the modified lysine. Upon cleavage of the peptide bond adjacent to the quencher by an aminopeptidase, the tryptophan is released from the DNP quencher, resulting in a significant increase in fluorescence (typically measured at excitation ~280 nm, emission ~350 nm for tryptophan). The primary target is the family of aminopeptidases, including but not limited to aminopeptidase N (APN/CD13), aminopeptidase B, and other cytosolic or membrane-bound aminopeptidases. APN (CD13) is a zinc-dependent metalloprotease involved in angiogenesis, immune regulation, and cancer progression, making it an important therapeutic target. This substrate allows for the screening of potential inhibitors or activators of these enzymes. The specific cleavage site is between the quencher and tryptophan, although the exact cleavage pattern may depend on the particular aminopeptidase.
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| ln Vitro |
In vitro, WRVYEKC(dnp)ALK tetraTFA is not biologically active itself; it is a substrate whose cleavage is measured as a readout of aminopeptidase activity. The compound is typically used in fluorogenic enzymatic assays. Under optimized conditions, the substrate is cleaved by recombinant or purified aminopeptidase (e.g., aminopeptidase N, CD13), and the increase in fluorescence is monitored over time. The reaction is characterized by kinetic parameters: KM (Michaelis constant), Vmax (maximum velocity), and kcat (catalytic rate constant). Typical KM values for such peptide substrates range from 1-50 uM. The substrate is stable under assay conditions and does not undergo non-specific hydrolysis in the absence of enzyme. The fluorescence signal is proportional to enzyme activity, allowing for accurate quantification. The compound is not cytotoxic and does not interfere with other cellular processes at the concentrations used in assays (typically 1-50 uM).
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| ln Vivo |
In vivo studies for WRVYEKC(dnp)ALK tetraTFA are not typically performed, as the compound is designed as an in vitro fluorogenic substrate rather than a therapeutic agent. The compound is used ex vivo in tissue homogenates or biological fluids to measure aminopeptidase activity. For example, tissue lysates from tumor xenografts or blood plasma can be incubated with the substrate to assess aminopeptidase activity levels or to test the efficacy of aminopeptidase inhibitors ex vivo. However, the peptide itself is not intended for administration to live animals. No in vivo efficacy or safety studies in animals have been reported for this compound in peer-reviewed literature. It is strictly a research biochemical tool. If used in an in vivo context (e.g., for intravital imaging), the substrate may be locally injected and fluorescence monitored, but such applications are uncommon.
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| Enzyme Assay |
The standard non-cellular assay for WRVYEKC(dnp)ALK tetraTFA is a continuous fluorogenic aminopeptidase activity assay. The assay is typically performed in 96-well black-sided, clear-bottom plates. The reaction mixture (100-200 uL) contains assay buffer (50 mM Tris-HCl, pH 7.5, 150 mM NaCl, 1 mM ZnCl2, 0.01% Brij-35 or Triton X-100, to maintain enzyme stability), recombinant aminopeptidase (e.g., human aminopeptidase N/CD13, 0.1-10 nM), and the substrate WRVYEKC(dnp)ALK tetraTFA (typically 1-50 uM final concentration). The substrate is dissolved in DMSO or water as a stock solution (10-100 mM), diluted to working concentration in assay buffer. The reaction is initiated by adding the enzyme to the substrate-containing wells. Fluorescence is measured at 30-37degC using a microplate reader equipped with excitation at 280 nm and emission at 350 nm (for tryptophan fluorescence) or alternative settings (e.g., Ex 320/Em 420 nm if a more red-shifted fluorophore is used). Readings are taken every 30-60 sec for 10-30 min. The initial linear rate of fluorescence increase (deltaRFU/min) is proportional to enzyme activity. For inhibitor screening, test compounds are pre-incubated with the enzyme for 5-10 min before adding the substrate. Background fluorescence (no enzyme) is subtracted. IC₅0 values of inhibitors are determined from dose-response curves.
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| Cell Assay |
In vitro cell-based assays using WRVYEKC(dnp)ALK tetraTFA typically involve measuring aminopeptidase activity in cell lysates, membrane fractions, or intact cells. For cell lysates: Cells (e.g., cancer cell lines such as HT-1080, MDA-MB-231, or primary cells) are cultured in appropriate medium (DMEM, RPMI-1640 + 10% FBS) to ~90% confluence. Cells are harvested, washed with PBS, and lysed in lysis buffer (50 mM Tris-HCl pH 7.5, 150 mM NaCl, 1% Triton X-100, protease inhibitor cocktail) for 30 min on ice. Lysates are cleared by centrifugation (10,000×g, 10 min, 4degC). The protein concentration is determined by BCA or Bradford assay. For the enzyme activity assay, 5-50 ug of protein (cell lysate) is added to assay buffer (50 mM Tris-HCl pH 7.5, 150 mM NaCl, 1 mM ZnCl2, 0.01% Triton X-100) in a 96-well black plate, followed by addition of WRVYEKC(dnp)ALK tetraTFA substrate (10-100 uM final). Fluorescence is measured at Ex 280/Em 350 nm every 1-2 min for 30-60 min at 37degC. For intact cell assays: Cells are seeded in 96-well black plates, grown to confluence, washed with PBS, then incubated with the substrate in serum-free medium. Fluorescence is measured over time. Aminopeptidase inhibitors (e.g., bestatin) are used as positive controls to confirm activity specificity. Cell viability is assessed by MTT assay to ensure that compound treatments do not induce cytotoxicity.
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| Animal Protocol |
In vivo animal studies for WRVYEKC(dnp)ALK tetraTFA are not standard, as the compound is an in vitro biochemical tool. However, ex vivo applications are possible. For example, to measure aminopeptidase activity in tissue samples from animal models: female BALB/c nude mice (6-8 weeks old) bearing subcutaneous tumor xenografts (e.g., HT-1080 fibrosarcoma) are euthanized, and tumor tissues are harvested, weighed, and homogenized in lysis buffer (50 mM Tris-HCl pH 7.5, 150 mM NaCl, 1% Triton X-100, protease inhibitor cocktail). Homogenates are cleared by centrifugation (10,000×g, 10 min, 4degC), and the supernatant is collected. Protein concentration is determined by BCA or Bradford assay. For the aminopeptidase activity assay, 10-50 ug of tissue lysate protein is mixed with assay buffer (50 mM Tris-HCl pH 7.5, 150 mM NaCl, 1 mM ZnCl2, 0.01% Triton X-100) and WRVYEKC(dnp)ALK tetraTFA substrate (10-50 uM final) in a 96-well black plate. Fluorescence is measured using a microplate reader at Ex 280/Em 350 nm at 37degC over 30-60 min. The initial rate (deltaRFU/min) is calculated and normalized to protein concentration. This allows for quantification of aminopeptidase activity in tissues ex vivo. Alternatively, plasma or serum samples from animals can be used to measure circulating aminopeptidase activity. All animal procedures must be approved by the institutional animal care and use committee.
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| ADME/Pharmacokinetics |
The pharmacokinetic (PK) properties of WRVYEKC(dnp)ALK tetraTFA are not relevant, as the compound is not intended for in vivo use as a therapeutic agent. It is a fluorogenic peptide substrate used exclusively for in vitro biochemical assays. No ADME (absorption, distribution, metabolism, excretion) studies have been conducted or reported for this compound. The compound is not designed to cross cell membranes; it is used either in cell lysates (after membrane disruption) or in intact cells under conditions where the substrate may be taken up via endocytosis or passive diffusion, but this is not systematically studied. The tetraTFA salt form is used to improve solubility in aqueous buffers. The compound is stable under standard laboratory storage conditions (at -20degC, protected from light and moisture). For research use only; not intended for human or animal administration.
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| References | |
| Additional Infomation |
WRVYEKC(dnp)ALK tetraTFA is not a drug and is not intended for therapeutic use. It is a biochemical research tool (fluorogenic peptide substrate) used to measure aminopeptidase activity in vitro and ex vivo. The compound contains a DNP quencher that suppresses the intrinsic tryptophan fluorescence; enzymatic cleavage by aminopeptidase separates the tryptophan from the quencher, leading to a measurable increase in fluorescence. This ratiometric or continuous assay format allows for real-time monitoring of enzyme activity and is useful for high-throughput screening (HTS) of aminopeptidase inhibitors or for determining enzyme kinetic parameters. The tetraTFA salt increases solubility. The compound has no known biological activity beyond serving as a substrate for aminopeptidases. It is not cytotoxic in cell-based assays at working concentrations. No clinical trials have been registered. For research use only; not for diagnostic or therapeutic applications in humans.
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| Molecular Formula |
C74H100F12N18O26S
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| Molecular Weight |
1917.74
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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 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.) |
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| Preparing Stock Solutions | 1 mg | 5 mg | 10 mg | |
| 1 mM | 0.5214 mL | 2.6072 mL | 5.2145 mL | |
| 5 mM | 0.1043 mL | 0.5214 mL | 1.0429 mL | |
| 10 mM | 0.0521 mL | 0.2607 mL | 0.5214 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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