Rho激酶抑制剂的高通量筛选模型建立与应用

Development of High Throughput Screening Method for Rho Kinase Inhibitors

目的建立重组人Rho激酶(Rho-associated coiled-coil forming protein kinase,ROCK)的表达、纯化方法,并建立基于荧光素酶发光法的ROCK抑制剂的高通量筛选(high throughput screening,HTS)模型,通过大规模筛选发现其选择性抑制剂,为心脑血管、神经系统等疾病的预防和治疗提供新的治疗策略。方法用分子生物学的技术,构建ROCK-Ⅰ/pET32a表达质粒,转化入大肠杆菌BL21中,筛选阳性克隆,诱导目的蛋白表达。纯化可溶性蛋白后,用Kinase-Glo Luminescent Kinase Assay进行活性鉴定,用Z’因子评价HTS方法的稳定性,并对3万个化合物和中药提取物进行了HTS。结果重组人ROCK-Ⅰ蛋白显示较好的激酶活性,建立的HTS模型Z’因子为0.75,基于此模型筛选出4个活性化合物和5个活性提取物。结论本实验建立了一个简单而快速的人重组ROCK-Ⅰ表达系统,该系统具有低成本、稳定、表达量大等特点。ROCK-Ⅰ的化学发光检测方法适用于HTS技术,具有较高的稳定性、灵敏性和可重复性。

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.