| Size | Price | Stock | Qty |
|---|---|---|---|
| 1mg |
|
||
| 5mg |
|
||
| 10mg |
|
||
| Other Sizes |
| Targets |
The target of DT-2 is cGMP-dependent protein kinase (PKG), specifically PKG Ialpha and PKG Ibeta isoforms. PKG is a serine/threonine kinase that is activated by cGMP and plays a critical role in vasodilation, platelet inhibition, smooth muscle relaxation, and cardiac contractility. DT-2 binds to the substrate-binding site of PKG, acting as a competitive inhibitor. It is composed of two parts: the PKG tight-binding sequence (W45, LRKKKKKH) and a membrane-translocating sequence (DT-6, YGRKKRRQRRRPP). DT-2 inhibits both cGMP-stimulated PKG activity and basal (cGMP-independent) PKG activity. It is ~1000-fold selective for PKG over PKA.
|
|---|---|
| ln Vitro |
In vitro, DT-2 (10-1000 nM) potently inhibits PKG-catalyzed phosphorylation with a Ki of 12.5 nM. It inhibits both cGMP-stimulated and basal PKG activity. DT-2 (1-100 uM) inhibits PKG-mediated VASP (vasodilator-stimulated phosphoprotein) phosphorylation at Ser239 in cell lysates. It is 1000-fold selective for PKG over PKA. However, in intact cells, DT-2 loses specificity for PKG and may inhibit other kinases (e.g., ERK, p38, PKB, PKC) at higher concentrations (≥10 uM). It is not cytotoxic at concentrations up to 20 uM.
|
| Enzyme Assay |
The inhibitory activity of DT-2 is measured using an in vitro kinase assay. Purified PKG Ialpha or Ibeta (0.1-1 ug) is incubated in kinase buffer (20 mM Tris-HCl pH 7.5, 10 mM MgCl2, 1 mM DTT, 0.1% BSA) with 10 uM cGMP, 50 uM ATP (including 0.5-1 uCi [gamma-32P]-ATP), and a PKG-specific peptide substrate (e.g., VASP-derived peptide, 50-100 uM). DT-2 (0.1-1000 nM) is added, and the reaction is incubated at 30degC for 10-30 min. Phosphorylated substrate is spotted onto P81 phosphocellulose paper, washed with 0.5% H3PO4, and counted by liquid scintillation. The IC₅0 is calculated. For selectivity, PKA is used in a parallel assay. The Ki is calculated from the IC₅0 using the Cheng-Prusoff equation.
|
| Cell Assay |
For cell-based studies, cells (e.g., rat smooth muscle cells, human platelets, NRVM cardiomyocytes) are seeded in 6-well plates (3×10⁵ cells/well). DT-2 (1-50 uM) is added for 30-60 min. Cells are then stimulated with a PKG activator (e.g., ANP, 10-100 nM; SNP, 1-10 uM; 8-Br-cGMP, 100-500 uM). VASP phosphorylation at Ser239 (a PKG-specific site) is measured by Western blotting using phospho-specific antibodies. PKA-specific substrate phosphorylation (e.g., at Ser157 of VASP) is assessed to monitor PKA activity. For cell homogenates, cells are permeabilized with saponin (0.05-0.1%) and incubated with DT-2 (1-50 uM) in the presence of cGMP or cAMP. PKG and PKA activities are measured as described above. Cytotoxicity is assessed by LDH release or MTT assay.
|
| Animal Protocol |
In vivo, DT-2 can be administered to rodents to study PKG function. For example, in a rat model of vasoregulation, DT-2 (0.1-1 mg/kg) is administered intravenously (IV), and blood pressure and heart rate are monitored. DT-2 inhibits PKG-mediated vasodilation, leading to increased blood pressure. For platelet aggregation studies, DT-2 is added to whole blood ex vivo, or DT-2 is administered IV to mice (1-5 mg/kg), and platelet aggregation is assessed by aggregometry. For neuroregeneration studies, DT-2 is administered intrathecally. However, caution is required because DT-2 may lose specificity in intact cells and inhibit other kinases. No specific protocol is provided as DT-2 is a research tool.
|
| ADME/Pharmacokinetics |
DT-2 is a cell-permeable peptide. Following IV administration, it has a short plasma half-life (< 1 h). The membrane-translocating sequence (DT-6) facilitates cellular uptake. For research use, DT-2 is stored as a lyophilized powder at -20degC (powder: -20degC for 3 years, 4degC for 2 years; in solvent: -80degC for 1 year, -20degC for 6 months). It is soluble in DMSO (10-50 mM) and water. No detailed PK data is available.
|
| Toxicity/Toxicokinetics |
No specific toxicology data for DT-2 is available. In cell culture studies, DT-2 at 20 uM is not toxic (no LDH release). In animal studies at effective doses (1-5 mg/kg, IV), no overt toxicity has been reported. However, due to its ability to inhibit other kinases in intact cells, off-target effects may occur. Standard safety precautions for handling peptides apply: use PPE (gloves, lab coat, safety goggles), work in a fume hood, avoid inhalation and skin contact. Not for human consumption.
|
| References | |
| Additional Infomation |
DT-2 acetate (YGRKKRRQRRRPPLRKKKKKH; CAS# not assigned; MW 3462.17 free base) is a research-grade, cell-permeable peptide inhibitor of cGMP-dependent protein kinase (PKG). It is not an FDA-approved drug. It is a potent (Ki = 12.5 nM) and selective (>1000-fold) PKG inhibitor in vitro but loses specificity in intact cells. It is used as a tool to study PKG function in vasoregulation, platelet function, and cardiac physiology. For research use only, not for diagnostic or therapeutic applications. Storage: powder at -20degC for 3 years, 4degC for 2 years; in solvent at -80degC for 1 year, -20degC for 6 months.
|
| Molecular Formula |
C154H287N53O55
|
|---|---|
| Molecular Weight |
3761.25
|
| Related CAS # |
DT-2;1220985-78-3
|
| Appearance |
Solid powder
|
| HS Tariff Code |
2934.99.9001
|
| 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)
|
| 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
|
|---|---|
| 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 | 0.2659 mL | 1.3293 mL | 2.6587 mL | |
| 5 mM | 0.0532 mL | 0.2659 mL | 0.5317 mL | |
| 10 mM | 0.0266 mL | 0.1329 mL | 0.2659 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.
Products are for research use only; We do not sell to patients
Copyright 2020 InvivoChem LLC | All Rights Reserved
