H-1152
目录号 : GC36207
A ROCK inhibitor
Cas No.:451462-58-1
Sample solution is provided at 25 µL, 10mM.
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Quality Control & SDS
- View current batch:
- Purity: >98.00%
- COA (Certificate Of Analysis)
- SDS (Safety Data Sheet)
- Datasheet
Kinase experiment: | Inhibitors (including H-1152) are 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. The reactions are started by the addition of [γ-32P]ATP and carried out at 30°C for 5 min. The Michaelis-Menten equation is used to calculate the Michaelis constant (Km) and maximal velocity (Vmax) of Rho-kinase. Data are further analyzed with secondary plot to calculate the inhibitory constant (Ki)[2]. |
Cell experiment: | Briefly, cells are routinely plated on poly-d-lysine/laminin coated 96 well plates or in 16 well glass culture slides. Control medium contained Dulbecco's modified Eagles medium/Hams F12(1:1) (DMEM/F12), 2 mM l-glutamine, N2 mix (1:100 dilution), 0.63 mL of 45% glucose for each 100 mL of DMEM/F12, neurotrophin 3 (NT3; final concentration, 8 ng/mL), BDNF (final concentration 8 ng/mL), and 10% fetal bovine serum heat inactivated before use. LIF cultures contain control medium+LIF (50 ng/mL). BMP4 cultures contain control medium+bone morphogenetic protein 4 (BMP4; 25 ng/mL). Total volume of culture is 110 μL. ROCK inhibitor H-1152 is diluted in water and added in an additional 10 μL to cultures 24 h after plating. Water is added to controls. Eighteen hours after the addition of inhibitor, cultures are 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 are 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]. |
References: [1]. Tamura M, et al. Development of specific Rho-kinase inhibitors and their clinical application. Biochim Biophys Acta. 2005 Dec 30;1754(1-2):245-52. Epub 2005 Sep 12. | |
Rho-associated kinase (ROCK), activated by GTP-
1.Sasaki, Y., Suzuki, M., and Hidaka, H.The novel and specific Rho-kinase inhibitor (S)-(+)-2-methyl-1-[(4-methyl-5-isoquinoline)sulfonyl]-homopiperazine as a probing molecule for Rho-kinase-involved pathwayPharmacol. Ther.93225-232(2002) 2.Ikenoya, M., Hidaka, H., Hosoya, T., et al.Inhibition of Rho-kinase-induced myristoylated alanine-rich C kinase substrate (MARCKS) phosphorylation in human neuronal cells by H-1152, a novel and specific Rho-kinase inhibitorJ. Neurochem.819-16(2002) 3.Davies, S.L., Gibbons, C.E., Vizard, T., et al.Ca2+-sensing receptor induces Rho kinase-mediated actin stress fiber assembly and altered cell morphology, but not in response to aromatic amino acidsAm. J. Physiol. Cell Physiol.290C1543-C1551(2006) 4.Johnson, R.P., El-Yazbi, A.F., Takeya, K., et al.Ca2+ sensitization via phosphorylation of myosin phosphatase targeting subunit at threonine-855 by Rho kinase contributes to the arterial myogenic responseJ. Physiol.587(11)2537-2553(2009) 5.Rattan, S., and Patel, C.A.Selectivity of Rho kinase (ROCK) inhibitors in the spontaneously tonic smooth muscleAm. J. Physiol. Gastrointest. Liver Physiol.294(3)G687-G693(2008) 6.Fuentes, E.O., Leemhuis, J., Stark, G.B., et al.Rho kinase inhibitors Y27632 and H1152 augment neurite extension in the presence of cultured Schwann cellsJ. Brachial Plex. Peripher. Nerve Inj.3(19)(2008)
| Cas No. | 451462-58-1 | SDF | |
| Canonical SMILES | CC1=CN=CC2=C1C(S(=O)(N3[C@@H](C)CNCCC3)=O)=CC=C2 | ||
| 分子式 | C16H21N3O2S | 分子量 | 319.42 |
| 溶解度 | DMF: 15 mg/ml,DMSO: 12.5 mg/ml,Ethanol: 20 mg/ml,PBS (pH 7.2): 10 mg/ml | 储存条件 | Store at -20°C |
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1 mg | 5 mg | 10 mg |
| 1 mM | 3.1307 mL | 15.6534 mL | 31.3067 mL |
| 5 mM | 0.6261 mL | 3.1307 mL | 6.2613 mL |
| 10 mM | 0.3131 mL | 1.5653 mL | 3.1307 mL |
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动物平均体重 | g | |
每只动物给药体积 | ul | |
动物数量 | 只 |
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| % DMSO % % Tween 80 % saline | ||||||||||
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H-1152 effects on intraocular pressure and trabecular meshwork morphology of rat eyes
J Ocul Pharmacol Ther 2008 Aug;24(4):373-9.PMID:18665808DOI:10.1089/jop.2008.0029.
Purpose: The aim of this study was to elucidate the effects of the Rho-kinase inhibitor, H-1152, on cultured human trabecular meshwork (HTM) cells, TM morphology, and intraocular pressure (IOP) in rats. Methods: Cultured HTM cells were treated with H-1152. Changes in cell morphology and the organization of the actin cytoskeleton and focal adhesions were evaluated by microscopy and immunofluorescence. H-1152 was administered topically to the eyes of conscious rats, and IOP was measured with a commercially available tonometer before and after treatment. The eyes were enucleated 1 h after treatment, fixed, and processed for morphologic analysis by light and electron microscopy. Results: Exposure of the cultured HTM cells to 20 microM of H-1152 induced elongation and separation of cells, deterioration, and loss of actin stress fibers and focal adhesions within 2 h. Topical administration of H-1152 resulted in a significant decrease in IOP from 0.5 to 6 h, with the maximum IOP reduction of 28.1% at 1 h post-treatment (P < 0.001; n = 10). H-1152 caused an expansion of the intercellular spaces and loss of extracellular material in the juxtacanalicular region of the TM in rat eyes. Conclusions: The IOP-lowering effect of H-1152 in rat eyes is likely due to changes in TM-cell morphology, the actin cytoskeleton, and cellular adhesions in the conventional outflow pathway. H-1152 has potential as a new antiglaucoma medication.
Accelerated neurite growth from spiral ganglion neurons exposed to the Rho kinase inhibitor H-1152
Neuroscience 2010 Aug 25;169(2):855-62.PMID:20478368DOI:10.1016/j.neuroscience.2010.05.020.
Upon the death of their hair cell synaptic partners, bipolar cochlear spiral ganglion neurons either die or retract their peripheral nerve fibers. Efforts to induce the regrowth of the peripheral neurites have had to rely on limited knowledge of the mechanisms underlying spiral ganglion neurite regeneration and have been restricted by the impracticality of undertaking large numbers of manual analyses of neurite growth responses. Here we have used dissociated cultures of postnatal mouse spiral ganglia to assess the effects of the Rho kinase inhibitor H-1152 on neurite growth and to determine the utility of automated high content analysis for evaluating neurite length from spiral ganglion neurons in vitro. In cultures of postnatal mouse spiral ganglion, greater than 95% of the neurons develop bipolar, monopolar or neurite-free morphologies in ratios dependent on whether the initial medium composition contains leukemia inhibitory factor or bone morphogenetic protein 4. Cultures under both conditions were maintained for 24 h, then exposed for 18 h to H-1152. None of the cultures exposed to H-1152 showed decreased neuronal survival or alterations in the ratios of different neuronal morphologies. However, as measured manually, the population of neurite lengths was increased in the presence of H-1152 in both types of cultures. High content analysis using the Arrayscan VTi imager and Cellomics software confirmed the rank order differences in neurite lengths among culture conditions. These data suggest the presence of an inhibitory regulatory mechanism(s) in the signaling pathway of Rho kinase that slows the growth of spiral ganglion neurites. The automated analysis demonstrates the feasibility of using primary cultures of dissociated mouse spiral ganglion for large scale screens of chemicals, genes or other factors that regulate neurite growth.
Proerectile effects of the Rho-kinase inhibitor (S)-(+)-2-methyl-1-[(4-methyl-5-isoquinolinyl)sulfonyl]homopiperazine (H-1152) in the rat penis
J Pharmacol Exp Ther 2005 Oct;315(1):155-62.PMID:15976017DOI:10.1124/jpet.105.086041.
The Rho-kinase pathway mediates Ca2+ sensitization in the penile circulation, which maintains the penis in the flaccid state. We aimed to investigate the functional effect of a novel Rho-kinase inhibitor, H-1152 [(S)-(+)-2-methyl-1-[(4-methyl-5-isoquinolinyl)sulfonyl]homopiperazine], both in vitro and in vivo as well as to demonstrate the expression of Rho guanine nucleotide exchange factors (RhoGEFs) in the rat corpus cavernosum (CC), by using a semiquantitative reverse transcription-polymerase chain reaction assay to measure their mRNA expression. Cumulative addition of H-1152 (0.001-3 microM) or Y-27632 [0.01-30 microM; (R)-(+)-trans-N-(4-pyridyl)-4-(1-aminoethyl)-cyclohexanecarboxamide] caused sustained relaxations of precontracted CC strips, which were not affected by inhibition of the nitric oxide signaling pathway. Addition of H-1152 (0.1 microM), Y-27632 (1 microM), or sodium nitroprusside (SNP; 0.1 microM) caused rightward shifts in the curves to phenylephrine (PE), but it had little effect on the contractions mediated by electrical field stimulation (EFS). It is noteworthy that when H-1152 or Y-27632 was combined with SNP, a marked synergistic inhibition was noted both on PE- and EFS-induced contractions. Intraperitoneal administration of H-1152 (100 nmol/kg) had a discrete effect on mean arterial pressure and significantly enhanced erectile responses evoked by stimulation of the cavernous nerve. The mRNA expression for PDZ-RhoGEF, p115RhoGEF, and leukemia-associated RhoGEF in cavernosal segments was visualized by electrophoresis on agarose gel. The results indicate that H-1152 is a powerful Rho-kinase inhibitor, giving rise to its therapeutic potential in the treatment of erectile dysfunction. The regulator of G-protein signaling-containing RhoGEFs may represent key components of the molecular mechanisms associated with the abnormal function of the cavernosal smooth muscle.
Inhibition of rho-kinase-induced myristoylated alanine-rich C kinase substrate (MARCKS) phosphorylation in human neuronal cells by H-1152, a novel and specific Rho-kinase inhibitor
J Neurochem 2002 Apr;81(1):9-16.PMID:12067241DOI:10.1046/j.1471-4159.2002.00801.x.
The functions of small G protein Rho-associated kinase (Rho-kinase) have been determined in muscle and non-muscle cells, but, particularly in neuronal cells, its effector(s) has not been well known. Recently, we preliminarily reported that Rho-kinase phosphorylates the Ser159 residue in myristoylated alanine-rich C kinase substrate (MARCKS) in vitro, but it remains obscure in vivo. To further clarify this point, we developed an isoquinolinesulfonamide derivative, H-1152, that is a more specific, stronger and membrane-permeable inhibitor of Rho-kinase with a Ki value of 1.6 nM, but poor inhibitor of other serine/threonine kinases. H-1152 dose-dependently inhibited the phosphorylation of MARCKS in human neuroteratoma (NT-2) cells stimulated by Rho-activator lysophosphatidic acid (LPA), which was determined by phosphorylation site-specific antibody against phospho-Ser159 in MARCKS, whereas it hardly inhibited the phosphorylation stimulated by phorbol-12,13-dibutyrate (PDBu). In contrast, two other Rho-kinase inhibitors, HA-1077 at 30 microM and Y-27632 at 10-30 microM, inhibited the phosphorylation of MARCKS in the cells stimulated by LPA and PDBu. A PKC inhibitor Ro-31-8220 selectively inhibited PDBu-induced phosphorylation of MARCKS. Taken together with our previous results, the present findings strongly suggest that Rho/Rho-kinase phosphorylates MARCKS at Ser159 residue in neuronal cells in response to LPA stimulation and that H-1152 is a useful tool to confirm Rho-kinase function(s) in cells and tissues.
Mini-Review; Deriving Avian Stem Cells by Small Molecules
Curr Stem Cell Res Ther 2021;16(3):238-242.PMID:32867659DOI:10.2174/1574888X15999200831155607.
Avian embryos and related cell lines have found wide applications in basic and applied sciences. The embryonated egg is a great host for monoclonal antibodies and recombinant proteins. Avian cell lines derived from embryonated eggs have been used for the production of transgenic birds and virus inoculation in vaccine preparation. Hitherto, many efforts have been invested to develop efficient avian stem cell culture. Under the conventional conditions, there are various challenges, such as the type of feeder layers, conditioned medium, serum, and growth factors. Researchers have investigated different conditions to solve these problems. Recent studies have shown that targeted strategies using small molecule inhibitors could be used as alternatives to multi-growth factor delivery approaches. Since small molecule inhibitors were used for mammalian pluripotent stem cells (PSC), several kinds of research have examined the effect of the small molecule on self- -renewal and maintenance of avian PSC. Avian PSC can be derived from early blastodermal cells (stage X), circular primoridial germ cells (PGC; stage HH17), gonadal PGC (stage HH28), and embryonic germ cells (EGC; HH28). Previous studies have shown that the use of small molecule drugs such as PD0325901, SB431542, SC1, IDE1, Z-VAD, Blebbistatin, H-1152, and IDE1 could be an efficient method for the derivation of avian stem cells. This mini-review covers the recent development of avian stem cell culture by small molecules.