In Vitro Biochemical Assays of ROCK1 and ROCK2 Activities

The effects of compounds on the activity of the human ROCK1 and ROCK2 were quantified by measuring the phosphorylation of the substrate Ulight-RRRSLLE (PLK) using a human recombinant enzyme and the LANCE detection method. Both assays were performed at the contract research organization company Eurofins Pharma Discovery Services.⁴²

The test compound, reference compound, or water (control) were mixed with the enzyme (8.2 ng of ROCK1; 4.52 ng of ROCK2) in a buffer containing 40 mM 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid/Tris (pH 7.4), 0.8 mM ethylene glycol-bis(β-aminoethyl ether)-N,N,N′,N′-tetraacetic acid/Tris, 8 mM MgCl₂, 1.6 mM dithiothreitol, and 0.008% Tween 20. Thereafter, the reaction was initiated by adding the substrate Ulight-RRRSLLE (50 nM for ROCK1; 100 nM for ROCK2) and ATP (1 μM for ROCK1; 10 μM for ROCK2), and the mixture was incubated (20 min for ROCK1; 15 min for ROCK2) at room temperature. For control basal measurements, the enzyme was omitted from the reaction mixture. Following incubation, the reaction was stopped by adding 13 mM ethylenediaminetetraacetic acid. After 5 min, the anti-phospho-PLK antibody labeled with europium chelate was added. After 60 more min, the fluorescence transfer was measured at λ_ex = 337 nm, λ_em = 620 nm, and λ_em = 665 nm using a microplate reader (Envision, PerkinElmer). The enzyme activity was determined by dividing the signal measured at 665 nm by that measured at 620 nm (ratio). The results were expressed as a percent inhibition of the control enzyme activity. The standard inhibitory reference compound used was staurosporine, which was tested in each experiment at several concentrations to obtain an inhibition curve from which its IC₅₀ value was calculated.