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In vitro transcription of capped mRNA with modified nucleotides and Poly(A) tail
TSA (Tyramide Signal Amplification), used for signal amplification of ISH, IHC and IC etc.
Separation of phosphorylated and non-phosphorylated proteins without phospho-specific antibody
A convenient and sensitive way for cell proliferation assay and cytotoxicity assay
Protect the integrity of proteins from multiple proteases and phosphatases for different applications.
SLx-2119(KD-025) is a selective inhibitor of ROCK2 with IC50 of 105 nM [1].Rho-associated protein kinase (ROCK) is a serine-threonine kinase and is involved in regulating cytoskeletal dynamics. It is associated with many intracellular processes, which are relevant to stroke. ROCK2 is the predominant isoform mainly expressed in vasculature and neurons [2].In smooth muscle cells isolated from human intestine with radiation-induced fibrosis (RE-SMC), SLx-2119 reduced mRNA level of CTGF. Over-expression of which is associated with fibrotic diseases. While, in SMC isolated from normal human intestine (N-SMC), SLx-2119 didn’t change CTGF mRNA level [1].In focal cerebral ischemia mice, after transient middle cerebral artery occlusion, KD025 reduced infarct volume in a dose-dependant way. And the efficacy maintained for at least 4 weeks. In aged male and female mice, as well as in type 2 diabetes mice, KD025 reduced infarct volume by 34%, 42% and 32% in aged male mice, female mice and diabetic mice respectively compared to vehicle in mice [2].References:[1]. Boerma M, Fu Q, Wang J, et al. Comparative gene expression profiling in three primary human cell lines after treatment with a novel inhibitor of Rho kinase or atorvastatin. Blood Coagul Fibrinolysis, 2008, 19(7): 709-718.[2]. Lee JH, Zheng Y, von Bornstadt D, et al. Selective ROCK2 Inhibition In Focal Cerebral Ischemia. Ann Clin Transl Neurol, 2014, 1(1): 2-14.
Inhibitory activities
Cell-free enzyme assays were performed to determine the selective inhibition of ROCK1 and ROCK2 by SLx-2119. Reactions were performed on non-binding surface microplates. Four mU of human ROCK1 and ROCK2 were used to phosphorylate 30 μM of the synthetic ROCK peptide substrate S6 Long (sequence: KEAKEKRQEQIAKRRRLSSLRASTSKSGGSQK), prepared at American Peptide (Sunnyvale, CA) with the addition of 10 μM ATP, containing 33P-ATP in the presence of 10 mM Mg2+, 50 mM Tris, pH 7.5, 0.1 mM EGTA and 1 mM DTT at room temperature. One unit is the amount of kinase needed to catalyze the transfer of 1 nmol phosphate/min to the peptide. The reactions were allowed to proceed for 45 minutes and then stopped with 3% phosphoric acid to a final concentration of 1%. The reactions were captured on phospho cellulose filtration microplates and washed with 75 mM phosphoric acid and methanol using a vacuum manifold. Phosphorylation was measured on a Perkin-Elmer MicroBeta 1450.
Cell lines
Human microvascular endothelial cells (HMVEC; CC-2527, Cambrex).
Preparation method
Dissolved in DMSO to obtain a stock solution of 20 mM [1]. General tips for obtaining a higher concentration: Please warm the tube at 37℃ for 10 minutes and/or shake it in the ultrasonic bath for a while. Stock solution can be stored below -20℃ for several months.
Reaction Conditions
3 ml culture media containing 10 μM or 40 μM SLx-2119; 24 h.
Applications
SLx-2119 at 40 μM significantly reduces the mRNA levels of Tsp-1 and CTGF.
Animal models
C57BL/6 mice.
Dosage form
100, 200 or 300 mg/kg; administered every 12 h for 2 days via orogastric gavage.
KD025 (formerly SLx-2119) reduces infarct volume by 30% and 40% at 100 and 200 mg/kg dose levels. KD025 (200 mg/kg 90 min before distal middle cerebral artery occlusion (dMCAO)) significantly reduces the area of perfusion defect, suggesting that ROCK2 inhibition improves cortical perfusion during acute cerebral arterial occlusion.
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]. Boerma M, Fu Q, Wang J, et al. Comparative gene expression profiling in three primary human cell lines after treatment with a novel inhibitor of Rho kinase or atorvastatin. Blood Coagul Fibrinolysis, 2008, 19(7): 709-718.
[2]. Lee JH, Zheng Y, von Bornstadt D, et al. Selective ROCK2 Inhibition In Focal Cerebral Ischemia. Ann Clin Transl Neurol, 2014, 1(1): 2-14.