B1293-1ml | Y-27632 [146986-50-7] Clinisciences

Background

Y-27632 is a specific inhibitor of Rho-associated kinases (ROCK) family with Ki values of 0.22 μM and 0.30 μM for ROCK1 and ROCK2, respectively.

Y-27632 has shown selectivity of inhibition by comparing their Ki values for other Rho effector kinases, citron kinase and PKN, as well as PKCα. The Ki values of Y-27632 for citron kinase and PKN are least 20-fold higher, and Ki values for PKCα are about 200-fold higher than those for ROCK kinases. In addition, Y-27632 has been reported to inhibit ROCK1 and ROCK2 by competing with ATP for binding to the kinase in HeLa cells. Besides, Y-27632 has also shown the inhibition of stress fibers in Swiss 3T3 cells when the concentration of Y-27632 is 10 μM [1].

References:
[1] Ishizaki T1, Uehata M, Tamechika I, Keel J, Nonomura K, Maekawa M, Narumiya S. Pharmacological properties of Y-27632, a specific inhibitor of rho-associated kinases. Mol Pharmacol. 2000 May;57(5):976-83.

Product Citation

1. Longping V Tse, Rita M Meganck, et al. "Genomewide CRISPR knockout screen identified PLAC8 as an essential factor for SADS-CoVs infection." Proc Natl Acad Sci U S A. 2022 May 3;119(18):e2118126119. PMID: 35476513
2. Ciaran A Shaughnessy, Pamela L Zeitlin, et al. "Net benefit of ivacaftor during prolonged tezacaftor/elexacaftor exposure in vitro." J Cyst Fibros. 2022 Mar 2;S1569-1993(22)00043-1. PMID: 35248469
3. Heidi J Nick, Pamela L Zeitlin, et al. "Measurements of spontaneous CFTR-mediated ion transport without acute channel activation in airway epithelial cultures after modulator exposure." J Cyst Fibros. 2022 Mar 2;S1569-1993(22)00043-1. PMID: 34799640
4. Nina Compera, Scott Atwell, et al. "Upscaling of pneumatic membrane valves for the integration of 3D cell cultures on chip." Lab Chip. 2021 Aug 7;21(15):2986-2996. PMID: 34143169
5. Mirza, Amar Naeem. "The Regulation of GLI1 in Basal Cell Carcinoma." Stanford University. 2021.
6. Saeed Pazhoohan, Ehsan Aref, et al. "Inhibition of Rho-kinase improves response to deep inspiration in ovalbumin-sensitized guinea pigs." Iran J Basic Med Sci. 2020 Dec;23(12):1584-1589. PMID: 33489033
7. Preston E Bratcher, Sangya Yadav, et al. "Effect of apical chloride concentration on the measurement of responses to CFTR modulation in airway epithelia cultured from nasal brushings." Physiol Rep. 2020 Oct;8(19):e14603. PMID: 33038073
8. Haiyang Yu, Shan Lu, et al. "TDP-43 and HSP70 phase separate into anisotropic, intranuclear liquid spherical annuli." bioRxiv. March 29, 2020.
9. Dakhama A, Al Mubarak R, et al. "Tollip Inhibits ST2 Signaling in Airway Epithelial Cells Exposed to Type 2 Cytokines and Rhinovirus." J Innate Immun. 2019 Mar 29:1-13. PMID: 30928973
10. Zhou N, Lee JJ, et al. "Rho Kinase Regulates Aortic Vascular Smooth Muscle Cell Stiffness Via Actin/SRF/Myocardin in Hypertension." Cell Physiol Biochem. 2017 Nov 23;44(2):701-715. PMID: 29169155
11. Pazhoohan S, Raoufy MR, et al. "Effect of Rho-kinase inhibition on complexity of breathing pattern in a guinea pig model of asthma." PLoS One. 2017 Oct 31;12(10):e0187249. PMID: 29088265
12. GUANGQI ZHOU, XINMEI LIU, et al. "Homeobox B4 inhibits breast cancer cell migration by directly binding to StAR-related lipid transfer domain protein 13." ONCOLOGY LETTERS,August 3, 2017.
13. Wang E, Wang D, et al. "Growth of hollow cell spheroids in microbead templated chambers." Biomaterials. 2017 Oct;143:57-64. PMID: 28763630
14. Jones BC, Kelley LC, et al. "Dual Targeting of Mesenchymal and Amoeboid Motility Hinders Metastatic Behavior." Mol Cancer Res. 2017 Jun;15(6):670-682. PMID: 28235899
15. Lamborn IT, Jing H, et al. "Recurrent rhinovirus infections in a child with inherited MDA5 deficiency." J Exp Med. 2017 Jul 3;214(7):1949-1972. PMID: 28606988
16. Wang Y, Gunasekara DB, et al. "A microengineered collagen scaffold for generating a polarized crypt-villus architecture of human small intestinal epithelium." Biomaterials. 2017 Jun;128:44-55. PMID: 28288348
17. Jones BC, Kelley LC, et al. "Dual Targeting of Mesenchymal and Amoeboid Motility Hinders Metastatic Behavior." Mol Cancer Res. 2017 Feb 24. PMID: 28235899
18. Chan W, Akhbanbetova A, et al. "Topical delivery of a Rho-kinase inhibitor to the cornea via mucoadhesive film." Eur J Pharm Sci. 2016 Aug 25;91:256-64. PMID: 27196964

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Chemical Properties

Storage	Store at -20°C
M.Wt	247.34
Cas No.	146986-50-7
Formula	C14H21N3O
Solubility	≥24.7 mg/mL in DMSO; insoluble in Chloroform
Chemical Name	4-[(1R)-1-aminoethyl]-N-pyridin-4-ylcyclohexane-1-carboxamide
SDF	Download SDF
Canonical SMILES	CC(C1CCC(CC1)C(=O)NC2=CC=NC=C2)N
Shipping Condition	Small Molecules with Blue Ice, Modified Nucleotides with Dry Ice.
General tips	We do not recommend long-term storage for the solution, please use it up soon.

Protocol

Kinase experiment [1]:
Binding assays	Recombinant ROCK-I, ROCK-II, PKN, or citron kinase was expressed in HeLa cells as Myc-tagged proteins by transfection using Lipofectamine, and was precipitated from the cell lysates by the use of 9E10 monoclonal anti-Myc antibody coupled to G protein-Sepharose. Recovered immunocomplexes were incubated with various concentrations of [32P] ATP and 10 mg of histone type 2 as substrates in the absence or presence of various concentrations of either Y-27632 at 30°C for 30 min in a total volume of 30 mL of the kinase buffer (50 mM HEPES-NaOH, pH 7.4, 10 mM MgCl₂, 5 mM MnCl₂, 0.02% Briji 35, and 2 mM dithiothreitol). PKCa was incubated with 5 mM [32P] ATP and 200 mg/mL histone type 2 as substrates in the absence or presence of various concentrations of either Y-27632at 30°C for 10 min in a kinase buffer (50 mM Tris-HCl, pH 7.5, 0.5 mM CaCl₂, 5 mM magnesium acetate, 25 mg/mL phosphatidyl serine, 50 ng/mL 12-O-tetradecanoylphorbol-13-acetate and 0.001% leupeptin) in a total volume of 30 mL. Incubation was terminated by 43 Laemmli sample buffer (10 mL).
Cell experiment [1]:
Cell lines	HeLa cells, Swiss 3T3 cells
Preparation method	The solubility of this compound in DMSO is >10 mM. General tips for obtaining a higher concentration: Please warm the tube at 37°C for 10 minutes and/or shake it in the ultrasonic bath for a while. Stock solution can be stored below -20°C for several months.
Reacting condition	0.3 and 1 μM, 30 min or 24 h
Applications	Y-27632 inhibited the kinase activity of both ROCK-I and ROCK-II in vitro, and this inhibition was reversed by ATP in a competitive manner. Y-27632 abolished stress fibers in Swiss 3T3 cells at 10 μM, but the G1-S phase transition of the cell cycle and cytokinesis were little affected at this concentration. In Swiss 3T3 cells, prior treatment of cells with Y-27632 for 30 min prevented cell shape and actin-stress fibers changes in a concentration-dependent manner; almost complete inhibition was observed at 10 μM. 10 and 100 μM Y-27632 prolonged the lag time and delayed the appearance of BrdU-labeled cells in a concentration-dependent manner, delays of about 1 and 4 h. In HeLa cells, 30 μM Y-27632 inhibited cytokinesis.
Other notes	Please test the solubility of all compounds indoor, and the actual solubility may slightly differ with the theoretical value. This is caused by an experimental system error and it is normal.
References: [1]. Ishizaki T, Uehata M, Tamechika I, et al. Pharmacological properties of Y-27632, a specific inhibitor of rho-associated kinases[J]. Molecular pharmacology, 2000, 57(5): 976-983.