Molecule

Description

Cancer

Description

GSK429286A is a selective inhibitor of ROCK1 and ROCK2 with IC50 of 14 nM and 63 nM, respectively.

Targets

IC₅₀

In Vitro

GSK429286A slightly inhibits RSK and p70S6K with IC50 of 0.78 μM and 1.94 μM, respectively. GSK429286A significantly inhibits rat aortic ring dilation with IC50 of 190 nM. ^[1] GSK429286A at 1 μM 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 ~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 μM (500-fold higher than IC50 of inhibition of ROCK2). Like GSK269962A but not sunitinib, GSK429286A treatment at 10 μM 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]

In Vivo

GSK429286A has 61% oral bioavailability in male Sprague-Dawley rats. Oral administration of GSK429286A at single doses of 3-30 mg/kg dramatically reduces mean arterial pressure in the spontaneously hypertensive rats (SHRs) in a dose-dependent manner, with a maximum decrease of 50 mmHg after approximately 2 hours treatment at dose of 30 mg/kg. ^[1]

Clinical Trials

Features

More selective than Y-27632.

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 μL. The assays contain: 1 nM hROCK1; 1 μM biotinylated peptide (biotin-Ahx-AKRRRLSSLRA-CONH2); 1 μM ATP; 1.85 kBq per well ATP(γ-³³P); 25 mM Hepes pH 7.4; 15 mM MgCl₂; 0.015% BSA; increasing concentrations of GSK429286A ranging from 50 μM to 0.8 nM. The reactions are incubated at 22°C for 120 minutes, and then terminated by the addition of a 50 μL 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 22°C 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.

ROCK2 kinase assays

ROCK2 kinase assays are set up in a total volume of 40 μL with recombinant His6–ROCK2-(2-543) (80 ng, corresponding to 30 nM ROCK2 in the final kinase assay) in 50 mM Tris/HCl (pH 7.5), 0.1 mM EGTA, 10 mM MgCl₂, 0.1 mM [γ-³²P]ATP (~500-1000 c.p.m./pmol), indicated concentration of peptide substrate, and increasing concentrations of GSK429286A dissolved in DMSO at 0.1% of the reaction volume. After incubation for 15 minutes at 30 °C, reactions are terminated by applying 35 μL of the reaction mixture on to P81 phosphocellulose paper and immersion in 50 mM phosphoric acid. After extensive washing, reaction products are quantified by Cerenkov counting. IC50 value is calculated using non-linear regression analysis using GraphPad Prism.

Animal Models

Male spontaneously hypertensive rats (SHRs)

Formulation

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

Doses

~30 mg/kg

Administration

Oral gavage