GSK429286A (RHO-15) is a specific inhibitor of ROCK1/2 (IC50: 14/63 nM).

ROCK1:14 nM, ROCK2:63 nM

GSK429286A (3-30 mg/kg, p.o.) significantly reduces the mean arterial pressure in spontaneous hypertensive rats in a dose-dependent manner, with a maximum reduction of 50 mmHg approximately 2 hours after administering a dose of 30 mg/kg. In male Sprague-Dawley rats, the oral bioavailability of GSK429286A is 61%.

GSK429286A at a concentration of 1 μM significantly reduces the activity of ROCK2 by 20-fold, whereas it only reduces MSK1 activity by approximately 5-fold at the same concentration. This compound exhibits higher selectivity as an ROCK2 inhibitor compared to the commonly used inhibitor Y-27632, showing negligible inhibition against LRRK2 even at 30 μM, with an IC50 that is 500 times higher for ROCK2. Similar to GSK269962A, GSK429286A at 10 μM can eliminate phosphorylation at Thr850 of MYPT induced either by the baseline or by the G14V-Rho mutant in HEK-293 cells, demonstrating effects similar to those of H-1152 and Y-27632 and consistent with ROCK-mediated phosphorylation. Regardless of the presence of G14V-Rho, GSK429286A does not affect the phosphorylation of ERM proteins. Additionally, GSK429286A mildly inhibits RSK (IC50: 0.78 μM) and p70S6K (IC50: 1.94 μM). It also significantly inhibits the dilation of rat aortic rings (IC50: 190 nM).

Binding affinity assays: TR-FRET-binding affinity assays are performed for BCL-2, BCL-XL, and MCL-1 in 4.52?mM monobasic potassium phosphate, 15.48?mM dibasic potassium phosphate, 1?mM sodium EDTA, 0.05% Pluronic F-68 detergent, 50?mM sodium chloride, and 1?mM DTT (pH 7.5). For MCL-1 assays, GST-tagged MCL-1 (1?nM) is mixed with 100?nM f-Bak, 1?nM Tb-labeled anti-GST antibody, and compound at room temperature (RT) for 60?min. Fluorescence is measured on an Envision plate reader using a 340/35?nm excitation filter and 520/525 (f-Bak) and 495/510?nm (Tb-labeled anti-GST antibody) emission filters.