KD025(SLx-2119)

SLx-2119 is a selective inhibitor of ROCK2 with an IC50 of 105 nM.

Signaling Pathways >> Cell Cycle/DNA Damage >> ROCK

Signaling Pathways >> Stem Cell/Wnt >> ROCK

Signaling Pathways >> TGF-beta/Smad >> ROCK

Research Areas >> Cancer

ROCK2:105 nM (IC50)

ROCK1:24 μM (IC50)

SLx-2119 (40 µM) induces significant down-regulations of Tsp-1 and CTGF mRNA levels in PASMC. The microarray hybridized with aRNA from HMVEC treated with SLx-2119, shows a 5-times higher background than the other arrays[1].

KD025 (100, 200 or 300 mg/kg, i.p.) dose-dependently reduces infarct volume after transient middle cerebral artery occlusion. KD025 is at least as efficacious in aged, diabetic or female mice, as in normal adult males[2].

Cell-free enzyme assays are performed to determine the selective inhibition of ROCK1 and ROCK2 by SLx-2119. Reactions are performed on non-binding surface microplates. Four mU of human ROCK1 and ROCK2 are used to phosphorylate 30 µM of the synthetic ROCK peptide substrate S6 Long, prepared at American Peptide 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 are allowed to proceed for 45 minutes and then stopped with 3% phosphoric acid to a final concentration of 1%. The reactions are captured on phospho cellulose filtration microplates and washed with 75 mM phosphoric acid and methanol using a vacuum manifold. Phosphorylation is measured on a Perkin-Elmer MicroBeta 1450.

Western blots are used to determine whether HMVEC, NHDF and PASMC express ROCK1 and ROCK2. The cells are incubated for 24 hours in 3 mL culture media containing SLx-2119. All cells are collected at passage 3 and lysed on ice in 25 mM Tris-HCl pH 7.5, 150 mM NaCl, 0.5% tritonX-100, 10% glycerol, 10 mM NaF and a protease inhibitor cocktail. Protein concentration is determined using a BCA protein assay reagent. Cell lysates (35 µg) are separated on 7.5% or 12.5% SDS-PAGE polyacrylamide gels and transferred to PVDF membrane filters. Membranes are blocked in 5% non-fat milk in TBS containing 0.1% Tween 20. Blots are probed with antibodies to ROCK1, ROCK2 or actin and washed well before incubation with HRP-conjugated secondary antibodies and visualization with an enhanced chemiluminescence (ECL) kit.

Young adult (C57BL/6, 2-3 months old, male 22-30 g, female 16-23 g), aged (C57BL/6, 12 months old, 33-52 g) are used in all experiments. Vehicle (0.4% methylcellulose) or KD025 (100, 200 or 300 mg/kg) is administered every 12 h via orogastric gavage. The dosing paradigm is chosen based on the pharmacokinetic profile after oral administration in mice. Atorvastatin (4 mg/mL) is dissolved in phosphate-buffered saline (pH 7.4) containing 45% 3-hydroxypropyl-B-cyclodextrin and 10% ethanol, and administered at a dose of 20 mg/kg per day as a single daily intraperitoneal injection for 2 weeks..

[1]. Boerma, M., 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): p. 709-18.

[2]. Lee, J.H., et al. Selective ROCK2 Inhibition In Focal Cerebral Ischemia. Ann Clin Transl Neurol, 2014. 1(1): p. 2-14.

[3]. Yang W, et al. Critical role of ROCK2 activity in facilitating mucosal CD4⁺ T cell activation in inflammatory bowel disease. J Autoimmun. 2018 May;89:125-138.

[4]. Chen W, et al. Screening RhoA/ROCK inhibitors for the ability to prevent chronic rejection of mouse cardiac allografts.Transpl Immunol. 2018 Jun 6. pii: S0966-3274(18)30029-7.

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