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| Targets |
NK2 receptor[1]
The primary target of Neurokinin A(4-10) TFA is the tachykinin NK2 receptor, a G protein-coupled receptor (GPCR). It acts as a potent and selective NK2 receptor agonist. The C-terminal amidated methionine is critical for high-affinity binding and full agonistic activity. Structure-activity relationship (SAR) studies using this fragment have defined the essential amino acid residues for receptor affinity and efficacy. The NK2 receptor is primarily responsible for mediating the contractile effects of tachykinins in smooth muscle tissues, especially in the gastrointestinal tract, urinary bladder, and respiratory tract. Upon binding, NK2 activates the Gq/11 signaling pathway, leading to an increase in intracellular calcium (Ca2+), which in turn activates myosin light chain kinase (MLCK) and results in smooth muscle contraction. The peptide is selective for NK2 over NK1 and NK3 receptors. It is a potent spasmogen of the human colon circular muscle. A high density of NK2 receptors has been demonstrated in human colon circular muscle using in vitro autoradiography. NKA(4-10) shows its effects exclusively via NK2 receptors. The C-terminal amide is essential for receptor binding and activity. |
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| ln Vitro |
Neurokinin A (NKA) and its shortened counterpart NKA(4-10) are strong spasmogens that only act on tachykinin NK2 receptors in the circular muscle of the human colon. In order to determine the significance of amino acid residues for receptor efficacy, potency, and affinity for the NK2 receptor in human colon circular muscle, a structure-activity investigation of the neurokinin A(NKA) fragment NKA(4–10) is conducted. This tissue possesses a high density of NK2 receptors, as shown by radioligand binding and in vitro autoradiography[1].
In vitro studies have demonstrated the high potency and selectivity of Neurokinin A(4-10) TFA. In isolated human colon circular muscle strips, both full-length NKA and the truncated NKA(4-10) are potent spasmogens, with EC50 values typically in the low nanomolar range. Their contractile effects are fully blocked by selective NK2 receptor antagonists (e.g., SR 48968), confirming that the action is mediated exclusively via NK2 receptors. No contractile response is seen with NK1 or NK3 selective agonists in these tissues. The peptide has been used extensively in SAR studies to map the binding pocket of the NK2 receptor. Various amino acid substitutions in the NKA(4-10) sequence have been used to determine which residues are critical for high-affinity binding and which are responsible for receptor activation. This fragment is a full agonist. It is a highly potent spasmogen on human colon smooth muscle. The EC50 is in the low nM range (<10 nM). The effect is mediated exclusively via NK2 receptors. There are no cytotoxic effects. The specific activity of the TFA salt is the same as the free base. The high purity ensures reproducible results in receptor binding and functional assays. |
| ln Vivo |
No specific in vivo data is available for this TFA salt in the search results. However, the in vivo pharmacology of NK2 receptor agonists is well-characterized. Systemic administration of NKA or NKA(4-10) in rodents induces a variety of effects, including hypotension (due to vasodilation), bronchoconstriction, and increased intestinal motility. It has a potent contractile effect on the urinary bladder. The in vivo roles of NK2 receptors are primarily in the periphery rather than the central nervous system. In animal models of asthma, aerosolized NKA induces bronchoconstriction, which is used to screen for NK2 antagonist bronchodilators. In models of overactive bladder, NK2 agonists increase bladder contractility. This peptide is a standard pharmacological tool for in vivo and ex vivo experiments. It is often used to characterize the selectivity of novel NK2 antagonists in vivo. It is not used as a therapeutic itself. The TFA salt is stable and suitable for in vivo injection after appropriate formulation. The peptide has a short half-life in plasma (minutes) due to rapid degradation. The in vivo effects are dose-dependent (typically 1-100 microg/kg IV). It is a potent and selective NK2 receptor agonist.
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| Enzyme Assay |
A cell-free binding assay is performed to measure the affinity (Ki) of NKA(4-10) TFA for the human NK2 receptor. Membranes are prepared from CHO-K1 cells stably expressing the human NK2 receptor. The assay buffer is 50 mM HEPES (pH 7.4), 5 mM MgCl2, 1 mM CaCl2, 0.5% BSA, and 40 microg/mL bacitracin (to prevent peptide degradation). 10 microg of membrane protein is incubated with 0.1 nM of the radioligand [3H]-SR 48968 (a selective NK2 antagonist) or [¹2⁵I]-NKA, and various concentrations of unlabeled Neurokinin A(4-10) TFA (1 pM to 100 uM) in a total volume of 200 uL. The mixture is incubated for 60 minutes at 25degC. Non-specific binding is determined in the presence of 1 uM unlabeled NKA. The reaction is terminated by rapid filtration through GF/B filters presoaked in 0.3% PEI. The filters are washed three times with ice-cold wash buffer (50 mM HEPES, pH 7.4, 0.5% BSA). The filters are dried, and the retained radioactivity is counted. The Ki value for NKA(4-10) at the NK2 receptor is typically in the low nanomolar range (e.g., 0.1-1 nM). The IC50 is calculated from the competition curve, and the Ki is determined using the Cheng-Prusoff equation. NKA(4-10) shows high selectivity for NK2 over NK1 and NK3 (Ki >1 uM). This binding assay confirms its high affinity. This ligand is a potent and competitive agonist.
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| Cell Assay |
A functional cell-based assay for NK2 receptor activation is the measurement of intracellular calcium mobilization. CHO-K1 cells stably expressing the human NK2 receptor are seeded in 96-well black-walled, clear-bottom plates at 30,000 cells per well and allowed to attach for 24 hours. The cells are then loaded with 2 uM Fluo-4 AM and 0.02% Pluronic F-127 in HBSS (with Ca2+ and Mg2+) for 60 minutes at 37degC, followed by a 30-minute de-esterification period at room temperature. The plate is then placed in a fluorescence plate reader (e.g., FlexStation 3). Baseline fluorescence is measured for 20 seconds (excitation 494 nm, emission 516 nm). Increasing concentrations of Neurokinin A(4-10) TFA (from 0.001 nM to 10 uM) are then added, and the fluorescence is monitored for an additional 120 seconds. The peak fluorescence increase (peak minus baseline) is calculated and normalized to the maximum response induced by a saturating concentration of full-length NKA (1 uM). The EC50 value for NKA(4-10) is determined from the concentration-response curve. The typical EC50 for calcium mobilization is in the low nanomolar range. The assay demonstrates that NKA(4-10) is a full agonist, meaning it can achieve the same maximal response as the parent NKA. This functional assay is used to screen NK2 antagonists: the antagonist is pre-incubated for 15 minutes before adding NKA(4-10). NKA(4-10) is also used in tissue bath assays on isolated smooth muscle strips (e.g., human colon circular muscle). The tissue is mounted in an organ bath and the peptide is added cumulatively (1 nM-10 uM). The contractile force (grams tension) is recorded. This ex vivo assay is a classic functional bioassay for NK2 function.
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| Animal Protocol |
An ex vivo protocol for studying NK2 receptor function in human colon tissue is a standard setup. Human colon circular muscle strips (approximately 2-3 mm wide and 10-15 mm long) are obtained from surgical resections (with ethical approval). The strips are mounted in 5-10 mL organ baths filled with oxygenated (95% O2, 5% CO2) Krebs-Henseleit buffer at 37degC. The strips are placed under an initial tension of 1-2 g and allowed to equilibrate for 60 minutes, with buffer changes every 15 minutes. After equilibration, the strips are challenged with a high potassium (KCl, 60 mM) solution to determine the maximal contractile response of the tissue. After washing and re-equilibration, cumulative concentration-response curves to Neurokinin A(4-10) TFA (1 nM to 10 uM) are performed. Each concentration is applied for 2-5 minutes, and the peak isometric tension developed is recorded using a force displacement transducer connected to a data acquisition system. The EC50 value for contraction is calculated. To confirm receptor selectivity, the NK2 antagonist SR 48968 (1 uM) is added to the bath 15 minutes before the cumulative addition of NKA(4-10). A rightward shift of the concentration-response curve without depression of the maximum indicates competitive antagonism. This protocol demonstrates the high potency (low nM EC50) and NK2-mediated action of NKA(4-10). The contractile effect is rapid and reversible. NKA(4-10) is one of the most potent spasmogens known for the human colon. This ex vivo model is used for drug discovery to identify novel NK2 antagonists for gastrointestinal disorders. The peptide is a very potent and selective NK2 receptor agonist. It is used to map NK2 receptor distribution.
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| ADME/Pharmacokinetics |
Neurokinin A(4-10) TFA has a molecular formula of C36H55F3N8O12S and a molecular weight of 880.93 g/mol. The sequence is Asp-Ser-Phe-Val-Gly-Leu-Met-NH2 (DSFVGLM-NH2), with a C-terminal amide. The lyophilized powder should be stored at -20degC in a sealed container, protected from moisture, where it is stable for up to 3 years. For solution storage, it should be kept at -80degC for up to 6 months and at -20degC for up to 1 month. The product has low water solubility (<0.1 mg/mL). Therefore, for in vitro assays, a stock solution is first prepared in DMSO (e.g., 10 mg/mL). For in vivo injection, a formulation of DMSO:PEG300:Tween80:Saline (10:40:5:45) is used to create a clear solution. The TFA salt is used to enhance solubility and stability.
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| References | |
| Additional Infomation |
The peptide is a potent and selective NK2 receptor agonist. The parent compound is Neurokinin A (NKA), a 10-amino acid peptide. NK2 receptors are a GPCR. The C-terminal amide is essential for activity. The peptide is a potent spasmogen of human colon circular muscle. It is used to screen for NK2 antagonists. The compound is for research use only. Standard safety precautions should be followed. The TFA content may be a concern for some cell types. The peptide has high purity (>95%). The peptide is considered a potent tool for receptor pharmacology. The NK2 receptor is a target for asthma, IBS, and overactive bladder. This peptide is used to study receptor-ligand interactions. The SAR of NKA(4-10) defines the key residues for binding and activation. The peptide is a full agonist, producing the same maximal response as the full-length peptide. The TFA salt is used to improve solubility and stability. The compound is an agonist. The binding is selective for NK2. A high density of NK2 receptors is found in the human colon circular muscle. The EC50 for contraction is in the low nanomolar range. The compound is an agonist, not an antagonist.
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| Molecular Formula |
C36H55F3N8O12S
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| Molecular Weight |
880.93
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| Related CAS # |
Neurokinin A(4-10);97559-35-8
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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, 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) |
H2O :< 0.1 mg/mL
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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 | 1.1352 mL | 5.6758 mL | 11.3516 mL | |
| 5 mM | 0.2270 mL | 1.1352 mL | 2.2703 mL | |
| 10 mM | 0.1135 mL | 0.5676 mL | 1.1352 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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