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| 5mg |
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| Targets |
The primary target of TFLLR-NH2 is the Protease-Activated Receptor 1 (PAR1), which is also known as the thrombin receptor. PAR1 is a member of the PAR family of GPCRs and is activated by the coagulation protease thrombin. Thrombin cleaves the receptor's N-terminal extracellular domain, exposing a new N-terminus that acts as a tethered ligand, binding to the receptor's second extracellular loop to activate it. TFLLR-NH2 is a synthetic peptide that replicates this tethered ligand sequence and acts as a direct agonist at PAR1, with an EC50 of 1.9 μM. This peptide is highly selective for PAR1 over other PAR subtypes. Activation of PAR1 by TFLLR-NH2 leads to the dissociation of G proteins, typically Gαq, Gαi, and Gα12/13, which in turn activate various downstream signaling pathways, including phospholipase C (PLC), the mitogen-activated protein kinase (MAPK) pathway, and the Rho pathway. This leads to a wide range of cellular responses depending on the cell type, such as platelet aggregation, endothelial cell activation, and smooth muscle cell proliferation.
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| ln Vitro |
PAR1 agonists cause concentration-dependent increases in [Ca2+]i and neuronal ratios. In response to 10 μ m TF-NH2, 50–80% of the identified neurons showed the greatest increase in [Ca2+]i above basal values (peak 196.5±20.4 nM, n=25) [1]. Expression of E-cadherin was upregulated and vimentin was downregulated in SW620 cells cultured in TFLLR-NH2 activated platelet supernatants. A dose-dependent rise in TGF-β1 secreted by TFLLR-NH2 was found in the supernatant of an in vitro platelet culture system [2].
The primary in vitro activity of TFLLR-NH2(TFA) is its potent agonism at the PAR1 receptor. Its activity is quantitatively defined by its EC50 value of 1.9 μM. In functional assays, this peptide can stimulate PAR1-mediated signaling pathways. For example, in cells expressing PAR1, TFLLR-NH2 induces intracellular calcium mobilization, a classic readout for Gαq-coupled receptor activation. It can also stimulate the phosphorylation of downstream kinases like ERK1/2. The peptide's potency is often confirmed in concentration-response curves, where increasing concentrations of the agonist lead to a sigmoidal increase in the biological response. Its selectivity for PAR1 over PAR2, PAR3, and PAR4 is a critical characteristic that makes it a valuable research tool for specifically studying the physiological roles of PAR1 without off-target effects. In addition to its agonism, its high purity (≥99%) ensures reliable and reproducible results in experimental settings. |
| ln Vivo |
Rat paws were injected with TF-NH2, which caused a notable and long-lasting edema. Capsaicin and NK1R antagonist-induced sensory nerve ablation reduced edema by 44% in 1 hour and by 100% in 5 hours. TF-NH2 increased Evans blue extravasation 2- to 8-fold in the bladder, esophagus, stomach, intestine, and pancreas in wild-type mice, but not in PAR1−/− animals. Extravasation in the stomach, esophagus, and bladder can be completely eliminated by NK1R antagonists [1]. TFp-NH2 induced considerable contraction at 3-50 μM and significant relaxation at 0.3-50 μM in the absence of apamin. The concentration-response curve of TFp-NH2-induced contraction markedly moved to the left when 0.1 μM apamin inhibited TFp-NH2-induced relaxation [3].
In vivo studies with TFLLR-NH2 are less common than in vitro experiments, but the peptide can be used to investigate the physiological and pathophysiological roles of PAR1. When administered systemically or locally, TFLLR-NH2 can activate PAR1 in various tissues. This can lead to effects such as increased vascular permeability, modulation of inflammatory responses, and pain sensitization. The peptide can be used in animal models of thrombosis, inflammation, and cancer to study the contribution of PAR1 signaling to these disease processes. For example, it might be injected into the paw of a rodent to induce inflammation and pain, or administered intravenously to study its effects on platelet aggregation and thrombosis. The short half-life of the peptide in vivo due to proteolytic degradation requires careful consideration in experimental design. Its effects are typically rapid and transient, reflecting the nature of GPCR signaling. |
| Enzyme Assay |
In vitro enzyme or receptor binding assays for TFLLR-NH2 are designed to quantify its interaction with the PAR1 receptor. Radioligand binding assays can be performed using membrane preparations from cells that overexpress PAR1. In these assays, a fixed concentration of a radiolabeled PAR1 antagonist (e.g., [³H]-haTRAP) is incubated with increasing concentrations of unlabeled TFLLR-NH2. The amount of bound radioligand is measured after separating bound from free ligand. The concentration of TFLLR-NH2 that displaces 50% of the specific binding (IC50) can be calculated, which provides a measure of its affinity for the receptor. However, because TFLLR-NH2 is an agonist, its affinity determined in binding assays can be influenced by the receptor's conformational state. Functional assays are typically preferred to characterize its activity.
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| Cell Assay |
In vitro cell-based assays are the primary method for characterizing the activity of TFLLR-NH2. A common assay is the measurement of intracellular calcium mobilization in cells expressing PAR1. Cells are loaded with a fluorescent calcium indicator (e.g., Fluo-4 AM) and then stimulated with varying concentrations of TFLLR-NH2. The increase in fluorescence is measured using a fluorescence plate reader. The resulting concentration-response curve yields an EC50 value, which is a standard measure of the compound's potency (e.g., 1.9 μM). Another functional assay measures the activation of the MAPK pathway, for instance by quantifying the phosphorylation of ERK1/2 using a cell-based ELISA or western blotting. In a typical experiment, cells are serum-starved and then treated with TFLLR-NH2 for a short period (e.g., 5-15 minutes), and the lysates are analyzed for phospho-ERK. These assays are robust, reproducible, and provide a direct measure of the compound's biological activity, making them essential for studying PAR1 pharmacology.
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| Animal Protocol |
In vivo animal studies with TFLLR-NH2 are often conducted to study its role in pain and inflammation. For instance, the peptide can be injected subcutaneously into the hind paw of a mouse to induce mechanical and thermal hyperalgesia (increased sensitivity to pain). The animal's response to a painful stimulus, such as a von Frey filament or a hot plate, is then measured to quantify the pain threshold. This model is used to study the role of PAR1 in pain signaling. In another model, the peptide may be injected intravenously or intraperitoneally to study its effects on platelet aggregation and thrombosis. The time to occlusion of a blood vessel in a thrombosis model can be measured. These experiments typically involve a control group receiving a vehicle and a treatment group receiving TFLLR-NH2. The peptide is typically administered at a specific dose (e.g., 1-10 mg/kg) and the physiological or behavioral endpoints are measured.
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| ADME/Pharmacokinetics |
The pharmacokinetic properties of TFLLR-NH2 are characteristic of a peptide. As a peptide, it is susceptible to rapid proteolytic degradation by peptidases in the blood and tissues, which results in a very short half-life. It is not expected to be orally bioavailable and is typically administered via injection. Its volume of distribution is likely limited to the extracellular space. Clearance occurs primarily through metabolism. In research settings, these properties are important for designing experiments, as the compound's effects are typically acute and require direct administration. The TFA salt form is used to enhance the peptide's solubility and stability, which is critical for preparing stock solutions for in vitro and in vivo studies. The peptide should be stored as a lyophilized powder at -20°C to maintain its integrity.
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| Toxicity/Toxicokinetics |
The toxicological profile of TFLLR-NH2 is not extensively documented, as it is primarily a research tool rather than a therapeutic agent. In cell-based assays, its effects are mediated through its pharmacological target, PAR1, and at high concentrations, it can lead to receptor desensitization and potential cellular stress. In vivo, the peptide's toxicity is expected to be related to its pharmacological activity, with potential side effects including inflammation, pain, and thrombosis due to excessive PAR1 activation. For laboratory safety, standard precautions for handling peptides should be observed. This includes wearing gloves and eye protection to avoid skin contact and inhalation of the powder. The compound is intended for research use only and is not for human therapeutic or diagnostic use.
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| References |
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| Additional Infomation |
TFLLR-NH2 is a widely used research tool for studying PAR1 signaling. Its selectivity and potency make it a key compound for dissecting the role of PAR1 in various biological systems. The peptide's sequence, Thr-Phe-Leu-Leu-Arg-NH2, is derived from the natural tethered ligand of PAR1. It is often used in combination with PAR1 antagonists to validate the specificity of observed effects. The TFA salt is the most common form, providing enhanced solubility in aqueous buffers. The compound is available from major chemical suppliers and is typically provided with a purity of 98% or higher. It is an essential tool for researchers studying hemostasis, inflammation, and cancer. It is important to note that the peptide is strictly for research use.
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| Molecular Formula |
C33H54F3N9O8
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|---|---|
| Molecular Weight |
761.83257818222
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| Exact Mass |
761.404
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| CAS # |
1313730-19-6
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| Related CAS # |
TFLLR-NH2;197794-83-5
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| PubChem CID |
71311619
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| Appearance |
White to off-white solid powder
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| Hydrogen Bond Donor Count |
10
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| Hydrogen Bond Acceptor Count |
13
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| Rotatable Bond Count |
20
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| Heavy Atom Count |
53
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| Complexity |
1110
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| Defined Atom Stereocenter Count |
6
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| SMILES |
C(F)(F)(F)C(=O)O.[C@@H](NC(=O)[C@@H](N)[C@H](O)C)(C(=O)N[C@@H](CC(C)C)C(=O)N[C@@H](CC(C)C)C(=O)N[C@H](C(=O)N)CCCNC(N)=N)CC1C=CC=CC=1
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| InChi Key |
QVNWOGSDGQDGHP-MKVNCOEFSA-N
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| InChi Code |
InChI=1S/C31H53N9O6.C2HF3O2/c1-17(2)14-22(27(43)37-21(26(33)42)12-9-13-36-31(34)35)38-28(44)23(15-18(3)4)39-29(45)24(16-20-10-7-6-8-11-20)40-30(46)25(32)19(5)41;3-2(4,5)1(6)7/h6-8,10-11,17-19,21-25,41H,9,12-16,32H2,1-5H3,(H2,33,42)(H,37,43)(H,38,44)(H,39,45)(H,40,46)(H4,34,35,36);(H,6,7)/t19-,21+,22+,23+,24+,25+;/m1./s1
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| Chemical Name |
(2S)-N-[(2S)-1-[[(2S)-1-amino-5-(diaminomethylideneamino)-1-oxopentan-2-yl]amino]-4-methyl-1-oxopentan-2-yl]-2-[[(2S)-2-[[(2S,3R)-2-amino-3-hydroxybutanoyl]amino]-3-phenylpropanoyl]amino]-4-methylpentanamide;2,2,2-trifluoroacetic acid
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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 : ~100 mg/mL (~131.26 mM)
DMSO : ~100 mg/mL (~131.26 mM) |
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| Solubility (In Vivo) |
Solubility in Formulation 1: ≥ 2.5 mg/mL (3.28 mM) (saturation unknown) in 10% DMSO + 40% PEG300 + 5% Tween80 + 45% Saline (add these co-solvents sequentially from left to right, and one by one), clear solution.
For example, if 1 mL of working solution is to be prepared, you can add 100 μL of 25.0 mg/mL clear DMSO stock solution to 400 μL PEG300 and mix evenly; then add 50 μL Tween-80 to the above solution and mix evenly; then add 450 μL normal saline to adjust the volume to 1 mL. Preparation of saline: Dissolve 0.9 g of sodium chloride in 100 mL ddH₂ O to obtain a clear solution. Solubility in Formulation 2: ≥ 2.5 mg/mL (3.28 mM) (saturation unknown) in 10% DMSO + 90% (20% SBE-β-CD in Saline) (add these co-solvents sequentially from left to right, and one by one), clear solution. For example, if 1 mL of working solution is to be prepared, you can add 100 μL of 25.0 mg/mL clear DMSO stock solution to 900 μL of 20% SBE-β-CD physiological saline solution and mix evenly. Preparation of 20% SBE-β-CD in Saline (4°C，1 week): Dissolve 2 g SBE-β-CD in 10 mL saline to obtain a clear solution. View More
Solubility in Formulation 3: ≥ 2.5 mg/mL (3.28 mM) (saturation unknown) in 10% DMSO + 90% Corn Oil (add these co-solvents sequentially from left to right, and one by one), clear solution. Solubility in Formulation 4: 33.33 mg/mL (43.75 mM) in PBS (add these co-solvents sequentially from left to right, and one by one), clear solution; with ultrasonication. |
| Preparing Stock Solutions | 1 mg | 5 mg | 10 mg | |
| 1 mM | 1.3126 mL | 6.5631 mL | 13.1263 mL | |
| 5 mM | 0.2625 mL | 1.3126 mL | 2.6253 mL | |
| 10 mM | 0.1313 mL | 0.6563 mL | 1.3126 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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