| Size | Price | Stock | Qty |
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| 5mg |
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| 10mg |
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| Other Sizes |
| Targets |
ROCK1 5 nM (IC50) ROCK2 50 nM (IC50)
ROCK1 (Rho-associated coiled-coil containing protein kinase 1), ROCK2, MRCKalpha (myotonic dystrophy-related Cdc42-binding kinase alpha), and MRCKbeta. DJ4 is an ATP-competitive inhibitor of these kinases. |
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| ln Vitro |
DJ4 is an ATP-competitive inhibitor of ROCK1 and ROCK2 with IC₅0 values of 5 nM and 50 nM, respectively. It also inhibits MRCKalpha and MRCKbeta with IC₅0 values of 10 nM and 100 nM, respectively. In a cell-free kinase assay, DJ4 inhibits the phosphorylation of a peptide substrate by these kinases. The compound blocks stress fiber formation in cells and induces cell apoptosis.
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| ln Vivo |
In vitro, DJ4 induces cell apoptosis and blocks stress fiber formation. It effectively blocks cancer cell migration and invasion, making it a potential therapeutic agent for cancer metastasis. DJ4 can be used for the study of various cancers, including lung cancer, breast cancer, and pancreatic (PANC-1) cancer.
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| Enzyme Assay |
General cell-free protocol for kinase inhibition assays: A time-resolved fluorescence resonance energy transfer (TR-FRET) or radiometric kinase assay is used. Recombinant active ROCK1 (5 ng/well) is incubated with a biotinylated peptide substrate (e.g., S6K peptide or a specific ROCK substrate peptide) in kinase buffer (25 mM HEPES, pH 7.5, 10 mM MgCl2, 1 mM DTT, 0.01% Tween-20) containing 10 uM ATP. Varying concentrations of DJ4 (0.01 nM to 10 uM) are added. The reaction is initiated by adding the ATP and the peptide substrate. After 60 minutes at 30degC, the reaction is stopped by adding EDTA. An anti-phospho-substrate antibody labeled with a donor fluorophore (e.g., Europium) and streptavidin labeled with an acceptor fluorophore (e.g., XL665) are added. The TR-FRET signal is measured after 1 hour. The IC₅0 value is calculated from the inhibition curve. Similar assays are performed for ROCK2, MRCKalpha, and MRCKbeta. Kinase selectivity profiling is conducted using a panel of 50-100 kinases at a single concentration of DJ4 (e.g., 1 uM).
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| Cell Assay |
A general cellular protocol for assessing ROCK/MRCK inhibition and apoptosis induction: Cancer cells (e.g., PANC-1 pancreatic cancer cells, MDA-MB-231 breast cancer cells, or A549 lung cancer cells) are seeded in 6-well plates at 2×10⁵ cells/well in DMEM containing 10% FBS. After 24 hours, the cells are treated with various concentrations of DJ4 (0.1, 0.5, 1, 2.5, 5, 10 uM) for 24-48 hours. For stress fiber analysis, the cells are fixed with 4% paraformaldehyde, permeabilized with 0.1% Triton X-100, and stained with phalloidin-TRITC (to visualize F-actin) and DAPI (to visualize nuclei). Images are captured using a fluorescence microscope. The percentage of cells with stress fibers is quantitated. For apoptosis analysis, treated cells are harvested, stained with Annexin V-FITC and propidium iodide (PI), and analyzed by flow cytometry. For Western blot analysis, cells are lysed, and the lysates are probed with antibodies against cleaved PARP, cleaved caspase-3, phospho-MLC2 (myosin light chain 2, a substrate of ROCK/MRCK), total MLC2, and GAPDH. Cell viability is assessed using the MTT assay after 72 hours of treatment.
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| Animal Protocol |
General animal protocol for evaluating anti-metastatic efficacy in a xenograft model: Female NOD/SCID mice are injected with PANC-1 luciferase-labeled pancreatic cancer cells (1×10⁶ cells in 0.1 mL PBS) via the tail vein to establish a lung metastasis model. After 7 days, the mice are randomized into treatment groups (n=10/group). DJ4 is formulated in a suitable vehicle (e.g., 10% DMSO, 40% PEG300, 5% Tween-80, 45% saline) and administered via intraperitoneal (IP) injection at doses of 5, 10, and 20 mg/kg once daily for 21 days. Tumor metastasis is monitored weekly by bioluminescence imaging (BLI) after intraperitoneal injection of D-luciferin (150 mg/kg). At the end of the study, the mice are euthanized, and the lungs are excised, fixed in Bouin‘s solution, and the number of surface metastatic nodules is counted under a dissecting microscope. The lungs are also processed for histopathological examination (H&E staining) to confirm micrometastases. Tumor tissues are collected for immunohistochemistry (Ki-67, cleaved caspase-3, p-MLC2) and Western blot analysis (ROCK/MRCK signaling pathway).
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| ADME/Pharmacokinetics |
General pharmacokinetic protocol for DJ4: Male Sprague-Dawley rats are administered DJ4 via intraperitoneal (IP, 10 mg/kg) and intravenous (IV, 2 mg/kg) injection. The compound is formulated in a vehicle such as 10% DMSO, 40% PEG300, 5% Tween-80, 45% saline. Blood samples are collected at 0.083, 0.25, 0.5, 1, 2, 4, 6, 8, 12, and 24 hours post-dose. Plasma concentrations of DJ4 are quantified by LC-MS/MS. PK parameters (Cmax, Tmax, AUC, t½, clearance, Vd, and bioavailability) are calculated using non-compartmental analysis. DJ4 has a molecular weight of 348.42 and a molecular formula of C1₉H1₆N4OS. The solubility of DJ4 in DMSO is 10 mg/mL (28.70 mM).
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| Toxicity/Toxicokinetics |
General toxicity protocol for DJ4: A 14-day repeated-dose toxicity study is performed in ICR mice. DJ4 is administered via intraperitoneal (IP) injection at doses of 5, 15, and 40 mg/kg/day for 14 consecutive days. A vehicle control group is included. Clinical signs, body weight, and food consumption are monitored daily. At the end of the study, blood samples are collected for hematology (complete blood count with differential) and serum chemistry (ALT, AST, ALP, BUN, creatinine, total protein, albumin, glucose, CPK). Gross necropsy is performed, and the weights of major organs (liver, kidney, spleen, heart, lung, brain, testes) are recorded. Histopathological examination of these organs is conducted. Bone marrow smears are prepared to assess myelotoxicity. Special attention is given to the heart due to the known role of ROCK in cardiac function.
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| References | |
| Additional Infomation |
DJ4 has a molecular formula of C1₉H1₆N4OS and a molecular weight of 348.42. The compound is an ATP-competitive inhibitor of ROCK1/2 and MRCKalpha/beta. DJ4 has been reported to be effective in studies of lung cancer, breast cancer, and pancreatic (PANC-1) cancer. The compound blocks stress fiber formation and induces apoptosis by inhibiting the ROCK/MRCK signaling pathways. The purity of DJ4 is 98.48%. For storage, the powder should be kept at -20degC for up to 3 years, and in solution at -80degC for up to 6 months or -20degC for up to 1 month. DJ4 is a valuable research tool for studying the mechanisms of cancer cell migration, invasion, and metastasis.
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| Molecular Formula |
C19H16N4OS
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| Molecular Weight |
348.42
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| CAS # |
1681020-24-5
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| Appearance |
Solid powder
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| Density |
1.36±0.1 g/cm3(Temp: 20 °C; Press: 760 Torr)(predicted)
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| LogP |
0
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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 |
| 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) |
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
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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 | 2.8701 mL | 14.3505 mL | 28.7010 mL | |
| 5 mM | 0.5740 mL | 2.8701 mL | 5.7402 mL | |
| 10 mM | 0.2870 mL | 1.4350 mL | 2.8701 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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