Development of High Throughput Screening Method for Rho Kinase Inhibitors
Guanhua Du · Zhōnghuá yàoxué zázhì · 2010
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.