| Size | Price | Stock | Qty |
|---|---|---|---|
| 5mg |
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| 10mg |
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| Other Sizes |
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| Targets |
ROCKII:12 nM (IC50); CaMKII:0.18 μM (IC50); PKG:0.36 μM (IC50); AuroraA:0.745 μM (IC50); MLCK:28.3 μM (IC50); EGFR:50 μM (IC50); MKK4:16.9 μM (IC50); GSK3α:60.7 μM (IC50); PKA:3.03 μM (IC50); Src:3.06 μM (IC50); PKC:5.68 μM (IC50); Abl:7.77 μM (IC50); AMPK:100 μM (IC50) P38α:100 μM (IC50)
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|---|---|
| ln Vitro |
Rho reducing agent H-1152 dihydrochloride has an IC50 of 12 nM for ROCK2. Lower inhibition of CaMKII, PKG, AuroraA, PKA, Src, PKC, MLCK, Abl, EGFR, MKK4, GSK3α, AMPK, and P38α Activity is also demonstrated by H-1152 dihydrochloride (H-1152 dihydrochloride P); the IC50 values are 0.180, 0.360, 0.745, 3.03, 3.06, 5.68, 28.3, 7.77, 50.0, 16.9, 60.7, 100, and 100 μM [1]. With a Ki of 1.6 nM, 1152 dihydrochloride potently inhibits Rho, but its Ki of 0.63, 9.27, and 10.1 μM very mildly inhibits PKA, PKC, and MLCK. H-1152 dihydrochloride (0.1-10 μM) exhibits a strong inhibitory effect on MARCKS phosphorylation in LPA-treated cells, with an IC50 value of 2.5 μM; however, no discernible effect is observed in PDBu-treated cells [2]. Neuronal persistence rates do not recover in response to H-1152 dihydrochloride (0.5–10 μM). Additionally, H-1152 dihydrochloride (1, 5, or 10 μM) did not change the percentage of various neuronal morphologies. Furthermore, in BMP4 and LIF cultures, 10 μM of H-1152 dihydrochloride lengthens neurites [3].
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| Enzyme Assay |
Protein kinase assay[2]
To determine the effect of inhibitors on Rho-kinase activity in vitro, inhibitors was added at the indicated concentrations to 50 µL of the assay mixture 50 mm Tris-HCl (pH 7.5), 5 mm MgCl2, 1 mm EDTA, 1 mm EGTA, 1 mm dithiothreitol, 40 µm S6-peptide, various concentrations of [γ-32P]ATP and purified Rho-kinase, which was purified as described previously (Nagumo et al. 2000). The reactions were started by the addition of [γ-32P]ATP and carried out at 30°C for 5 min. The Michaelis–Menten equation was used to calculate the Michaelis constant (Km) and maximal velocity (Vmax) of Rho-kinase. Data were further analyzed with secondary plot to calculate the inhibitory constant (Ki). The effects of Rho-kinase inhibitors on protein kinase A (PKA) and C (PKC) were assayed according to our method with minor modifications (Tokumitsu et al. 1991). The reaction for PKA was carried out at 37°C for 5 min in a 20-µL mixture containing 25 mm Tris-HCl (pH 7.0), 10 mm MgCl2, 2 mm EGTA, 50 µm peptide of cyclic AMP response element binding protein and 10 µm[γ-32P]ATP (150 cpm/pmol). The reaction for PKC was carried out for 5 min in 30 µL of a mixture that contained 50 mm Tris-HCl (pH 7.0), 10 mm MgCl2, 0.5 mm CaCl2, 0.2 mg/mL histone type III-S, 50 µg/mL phosphatidyl serine and 10 µm[γ-32P]ATP (150 cpm/pmol). The effect of Rho-kinase inhibitors on myosin light chain kinase (MLCK) was assayed under the conditions previously described elsewhere (Asano et al. 1989), in 30 µL of a reaction mixture that contained 40 mm HEPES (pH 7.0), 5 mm magnesium acetate, 0.5 mm CaCl2, 0.1% (v/v) Tween-80, 5.3 nm calmodulin, 51 µm myosin light chain peptide, 1.95 nm MLCK and [γ-32P]ATP (150 cpm/pmol)[2]. |
| Cell Assay |
ROCK inhibitor H-1152 was diluted in water and added in an additional 10 μl to cultures 24 h after plating. Water was added to controls. Eighteen hours after the addition of inhibitor, cultures were fixed in 4% paraformaldehyde (1 h at room temperature for peroxidase-linked labeling and 20 min at room temperature for fluorescence labeling). For ArrayScan/Cellomics automated analysis: Cells were plated in a total volume of 50 μl on 384 well plastic plates previously coated with poly-d-lysine/laminin, and cultured in the same medium[3].
To investigate the effects of the protein kinase inhibitors (e.g. H-1152 ), the cells were preincubated with the inhibitors for 15 min and then stimulated with LPA for 1 min or with PDBu for 5 min. At various determination points after the stimulation, 5% (v/v) trichloroacetic acid was added to the cultures and cells were scraped off with a rubber scraper. After sedimentation, the precipitate was washed with acetone containing 6 mm dithiothreitol to remove trichloro-acetic acid and then dried. The dried cell powder was placed in sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS–PAGE) sample buffer containing 125 mm Tris (pH 7.4), 8 m urea, 10% (w/v) sucrose, 0.02% (w/v) bromophenol blue, 4% (w/v) SDS and 2-mercaptoethanol, and the preparation was then extracted by heating at 95°C for 5 min. The samples were passed through a 0.22-µm centrifugal filter (Millipore, Bedford, MA, USA) to remove indissoluble materials. The samples (2.5 × 105 cells) were subjected to SDS–PAGE (7.5%)/western blotting analysis. Proteins in the gel were electrotransferred to a polyvinylidene difluoride membrane. After incubation with 5% (w/v) skimmed milk, the membrane was blotted with pS159-Mar-Ab, followed by anti-mouse IgG antibody. Immunoreactivity was detected by enhanced chemiluminescence-western blotting detection reagents, and its extent was measured by scanning densitometry of the X-ray film using KODAK 1D Image Analysis Software, and expressed as a percentage versus control under each condition[2]. |
| References |
|
| Additional Infomation |
(S)-2-methyl-1-(4-methylisoquinoline-5-sulfonyl)-1,4-diazepane dihydrochloride is a hydrochloride salt resulting from the reaction of (S)-2-methyl-1-(4-methylisoquinoline-5-sulfonyl)-1,4-diazepane with 2 mol eq. of hydrogen chloride. An ATP-competitive inhibitor of Rho kinase (ROCK). It has a role as an EC 2.7.11.1 (non-specific serine/threonine protein kinase) inhibitor. It contains a (S)-2-methyl-1-(4-methylisoquinoline-5-sulfonyl)-1,4-diazepane(2+).
A group of intracellular-signaling serine threonine kinases that bind to RHO GTP-BINDING PROTEINS. They were originally found to mediate the effects of rhoA GTP-BINDING PROTEIN on the formation of STRESS FIBERS and FOCAL ADHESIONS. Rho-associated kinases have specificity for a variety of substrates including MYOSIN-LIGHT-CHAIN PHOSPHATASE and LIM KINASES. |
| Molecular Formula |
C16H23CL2N3O2S
|
|---|---|
| Molecular Weight |
392.3437
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| Exact Mass |
391.088
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| Elemental Analysis |
C, 48.98; H, 5.91; Cl, 18.07; N, 10.71; O, 8.16; S, 8.17
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| CAS # |
871543-07-6
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| Related CAS # |
H-1152;451462-58-1;Glycyl H-1152 hydrochloride;913844-45-8
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| PubChem CID |
11560225
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| Appearance |
Typically exists as White to yellow solids at room temperature
|
| LogP |
4.867
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| Hydrogen Bond Donor Count |
3
|
| Hydrogen Bond Acceptor Count |
5
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| Rotatable Bond Count |
2
|
| Heavy Atom Count |
24
|
| Complexity |
476
|
| Defined Atom Stereocenter Count |
1
|
| SMILES |
S(N1CCCNC[C@@H]1C)(C1C=CC=C2C=NC=C(C=12)C)(=O)=O.Cl
|
| InChi Key |
BFOPDSJOLUQULZ-GXKRWWSZSA-N
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| InChi Code |
InChI=1S/C16H21N3O2S.2ClH/c1-12-9-18-11-14-5-3-6-15(16(12)14)22(20,21)19-8-4-7-17-10-13(19)2;;/h3,5-6,9,11,13,17H,4,7-8,10H2,1-2H3;2*1H/t13-;;/m0../s1
|
| Chemical Name |
4-methyl-5-[[(2S)-2-methyl-1,4-diazepan-1-yl]sulfonyl]isoquinoline;dihydrochloride
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| Synonyms |
H-1152 Dihydrochloride; 871543-07-6; H-1152 (dihydrochloride); H1152; 4-methyl-5-[[(2S)-2-methyl-1,4-diazepan-1-yl]sulfonyl]isoquinoline;dihydrochloride; brd7446 dihydrochloride; Rho Kinase; H 1152 dichloride;
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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 : ~35.71 mg/mL (~91.02 mM)
DMSO : ~10 mg/mL (~25.49 mM) |
|---|---|
| Solubility (In Vivo) |
Solubility in Formulation 1: ≥ 2.08 mg/mL (5.30 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 20.8 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: ≥ 2.08 mg/mL (5.30 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 20.8 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: ≥ 2.08 mg/mL (5.30 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: 50 mg/mL (127.44 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 | 2.5488 mL | 12.7440 mL | 25.4881 mL | |
| 5 mM | 0.5098 mL | 2.5488 mL | 5.0976 mL | |
| 10 mM | 0.2549 mL | 1.2744 mL | 2.5488 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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