A ROCK1 and ROCK2 inhibitor
GSK269962A is a potent ROCK inhibitor with IC50s of 1.6 and 4 nM for recombinant human ROCK1 and ROCK2 respectively.
GSK269962A IC50 values of 1.6 nM toward recombinant human ROCK1. GSK269962A also exhibits more than 30-fold selectivity against a panel of serine/threonine kinases. GSK269962A induces vasorelaxation in preconstricted rat aorta with an IC50 of 35 nM. Both are highly potent toward human ROCK1 with IC50 of 1.6 nM for GSK269962A. On the other hand, GSK269962A has a significantly improved kinase selectivity profile with at least >30-fold selectivity against the panel of protein kinase tested[1].
Oral administration of GSK269965A (0.3, 1, and 3 mg/kg) induces a dose-dependent reduction in blood pressure in spontaneously hypertensive rat (SHR). The reduction of blood pressure is acute and substantial. The maximal effect on blood pressure is observed approximately 2 h after oral gavages for both compounds. Under a similar setting, oral administration of Y-27632 (10 and 30 mg/kg) also induced a dose-dependent decrease of blood pressure. For all three Rho kinase inhibitors, the reduction of blood pressure is accompanied by an acute, dose-dependent increase in heart rate, presumably due to the activation of baroreflex mechanism[1].
参考文献:
[1]. Doe C, et al. Novel Rho kinase inhibitors with anti-inflammatory and vasodilatory activities. J Pharmacol Exp Ther. 2007 Jan;320(1):89-98.
Kinase experiment: | The enzyme activity and kinetics of the purified ROCK1(3-543) are determined using scintillation proximity assay. In this assay, purified ROCK1 is incubated with peptide substrate (Biotin-Ahx-AKRRLSSLRA-CONH2), and 33ATP and the subsequent incorporation of 33P into the peptide is quantified by streptavidin bead capture. For IC50 determination, test compounds are dissolved at 10 mM in 100% DMSO, with subsequent serial dilution in 100% DMSO. Compounds are typically assayed over an 11-point dilution range with a concentration in the assay of 10 μM to 0.2 nM in 3-fold dilutions. For dose-response curves, data are normalized and expressed as percentage inhibition using the formula 100×[(U-C1)/(C2-C1)], where U is the unknown value, C1 is the average of the high signal (0%) control wells, and C2 is the average of the low signal (100%) control wells. Curve fitting is performed The results for each compound are recorded as pIC50 values[1]. |
Animal experiment: | Rats[1] Male Sprague-Dawley rats (350-400g) are anesthetized with 5% isoflurane in O2 and killed by exsanguination. Aortic rings, approximately 2 to 3 mm in length, are suspended by two 0.1-mm diameter tungsten wire hooks in 10 mL tissue baths containing Krebs of the following composition: 112 mM NaCl, 4.7 mM KCl, 2.5 mM CaCl2, 1.2 mM KH2PO2, 1.2 mM MgSO4, 25 mM NaHCO3, 11.0 mM dextrose, 0.01 mM indomethacin, and 0.01 mM L-NAME. Krebs is maintained at 37°C and aerated with 95% O2, 5% CO2, pH 7.4,. Changes in isometric force are measured under optimal resting tension (1 g) using FT03 force-displacement transducers coupled to model 7D polygraphs. After a 60-min equilibration period, the vessels are treated with standard concentrations of KCl (60 mM) and phenylephrine (1 μM). Cumulative concentration-response curves to phenylephrine are obtained for each tissue by dosing at 0.5-log unit intervals (1 nM to 10 μM). After several washes, each vessel is contracted to equilibrium with an EC80 concentration of phenylephrine, and tone is reversed by adding cumulative amounts of either GSK269962A or SB-772077-B at 0.5-log unit intervals (0.1 nM to 1 μM). Responses are expressed as percentage reversal of the tone established with phenylephrine. |
参考文献: [1]. Doe C, et al. Novel Rho kinase inhibitors with anti-inflammatory and vasodilatory activities. J Pharmacol Exp Ther. 2007 Jan;320(1):89-98. | |
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