OBJECTIVE To screen new Rho kinase (Rho-associated coiled-coil forming protein kinase， ROCK) inhibitors for the preventation and treatment of the cardiovascular diseases and nervous system diseases and to establish a luminescent based high throughput screening (HTS) model. METHODS The cDNA encoding the amino acids 3-543 of human ROCK-Ⅰ was amplified and subcloned into pET32a expression vector to express the protein in Escherichia coli (E. coli) BL21 (DE3) as soluble protein. The kinase activity of the purified ROCK-Ⅰ was determined with Kinase-Glo Luminescent Kinase Assay kit. Z’ was used to evaluate the assay quality of HTS. RESULTS Recombinant human ROCK-Ⅰ showed well kinase activity. The parameter Z’ was 0.75， which showed a high feasibility and stability of the assay. Total compounds about 30 000 were screened， and 4 compounds and 5 extracts showed the inhibition for Rho kinase activity. CONCLUSION An easy and rapid procedure was established to harvest a large quantity of active recombinant human ROCK-Ⅰ from E. coli. The luminescent assay is stable， sensitive， reproducible and well suited for HTS of Rho kinase inhibitors.
GONG Li-li;FNG Lin-hu;PENG Jin-ho;HE Guo-rong;DU Gun-hu.
Development of High Throughput Screening Method for Rho Kinase Inhibitors[J]. Chinese Pharmaceutical Journal, 2010, 45(8): 580-584
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