GSK429286A

ROCK1

432, 37

Male spontaneously hypertensive rats (SHRs)

Formulated in 6% Hydroxypropyl-beta-Cyclodextrin (pH4.0) and 5% DMSO solution

GSK429286A slightly inhibits RSK and p70S6K with IC50 of 0.78 uM and 1.94 uM, respectively. GSK429286A significantly inhibits rat aortic ring dilation with IC50 of 190 nM. [1] GSK429286A at 1 uM reduces ROCK2 activity over 20-fold, under conditions in which the only other kinase tested that is significantly inhibited is MSK1 whose activity is reduced ca.5-fold. GSK429286A is a more selective ROCK2 inhibitor than the widely utilized ROCK inhibitor Y-27632 as assessed on kinase-specificity panel, and does not significantly inhibit LRRK2 even at doses as high as 30 uM (500-fold higher than IC50 of inhibition of ROCK2). Like GSK269962A but not sunitinib, GSK429286A treatment at 10 uM ablates basal or G14V-Rho mutant induced phosphorylation of MYPT at Thr850 in HEK-293 cells to a similar extent as H-1152 and Y-27632, consistent with ROCK mediating this phosphorylation, whereas GSK429286A does not inhibit ERM protein phosphorylation either in the presence or absence of G14V-Rho. [2]

ROCK1 kinase assays, GSK429286A is dissolved at 10 mM in 100% DMSO, with subsequent serial dilution in 100% DMSO. ROCK1 inhibitor activity is determined using human recombinant His-tag ROCK1 kinase domain (amino acid 2-543) expressed in Sf9 cells. Assays of ROCK1 activity are performed in opaque, white walled, 384 well plates, in a total assay volume of 20 uL. The assays contain: 1 nM hROCK1, 1 uM biotinylated peptide (biotin-Ahx-AKRRRLSSLRA-CONH2), 1 uM ATP, 1.85 kBq per well ATP(gamma-33P), 25 mM Hepes pH 7.4, 15 mM MgCl2, 0.015% BSA, increasing concentrations of GSK429286A ranging from 50 uM to 0.8 nM. The reactions are incubated at 22C for 120 minutes, and then terminated by the addition of a 50 uL solution containing 60 mM EDTA and streptavidin PVT SPA beads. The SPA beads are added to a concentration of 0.14 mg per well. The plates are allowed to incubate at 22C for 10 minutes before centrifugation at 1500 rpm for 1 minute. P33 incorporation is quantified by scintillation counting in a Packard TopCount. IC50 value is calculated by bespoke curve fitting software.

GSK429286A (RHO-15) is a selective inhibitor of ROCK1 and ROCK2 with IC50 of 14 nM and 63 nM, respectively.

More selective than Y-27632.