Rho kinase activity assay

The inhibitory effect of DL0805-2 on ROCK1 was detected by ELISA according to the instruction of the Rho kinase assay kit (CY-1160, Cyclex). In brief, DL0805-2 was pre-incubated in a system wherein ROCK1 (0.02 ng/μL) phosphorylates the kinase substrate myosin-binding subunit (MBS) pre-absorbed onto the microplate in the presence of Mg²⁺ and ATP. The phosphorylated form of threonine-696 on MBS can be recognized specifically by the detector antibody called AF20, which has been bound with horseradish peroxidase. Horseradish peroxidase catalyzes the conversion of the chromogenic substrate tetra-methylbenzidine (TMB) from a colorless solution to a blue solution. The absorbance at a wavelength of 450 nm is measured after adding H₂SO₄ to stop the reaction. The color reflects the relative level of ROCK1 activity in the sample. The values of OD_{450 nm} were used to calculate the percent inhibition and nonlinear regression was used to obtain the IC₅₀ value.