以FK506结合蛋白52为靶点的高通量药物筛选模型的建立及应用

Development and application of a high-throughput screening assay for inhibitors of human FK506 binding protein 52

目的发现新的FKBP52抑制剂类药物,建立以人FKBP52为靶点的高通量药物筛选模型。方法通过大肠埃希菌表达系统重组表达FKBP52蛋白,并进行纯化。基于FKBP52的肽基-脯氨酰顺反异构酶(peptidyl-prolyl cis-trans isomerase,PPIase)的活性特性,建立FKBP52高通量药物筛选体系。对本公司化合物库中的31558个放线菌及真菌次级代谢产物粗提物进行筛选。结果成功构建重组表达菌株BL21(rosetta)/pET-30a/FKBP52。纯化后的重组表达人FKBP52蛋白纯度大于90%,并具有很好的PPIase活性。阳性药FK506能剂量依赖地抑制FKBP52活性,IC50为12.035ng/m L。筛选得到121个抑制率大于80%的样品,其中11株放线菌的次级代谢产物中含FK506,6株放线菌的次级代谢产物中含FK520,7株放线菌的次级代谢产物中含雷帕霉素。对随机抽取的100块96孔板筛选数据进行统计,模型的信号/本底比平均值为2.35±0.26,Z'因子平均值为0.78±0.1。结论本研究成功建立了人FKBP52抑制剂高通量药物筛选模型,该方法具有快捷、灵敏度高、稳定性好的特点,不仅可以快速进行新FKBP52抑制剂的筛选,还可以实现对FKBP52已知抑制剂如FK506、FK520和雷帕霉素等产生菌的定向筛选。

Objective To screen novel FKBP52 inhibitors, and develop a high-throughput screening （HTS） assay. Methods The human FKBP52 gene was cloned by RT-PCR from human peripheral blood lymphocyte and recombinantly expressed in E. coli. Recombinant human FKBP52 （rhFKBP52） was purified by Ni2＋-chelating affinity chromatography column. The screening assay was established using Suc-Ala-Leu-Pro-Phe-pNA as colorimetric substrate. 31,558 microbial metabolite extracts were screened. Results Recombinant expression strain BL21 （rosetta）/pET-30a/FKBP52 was successfully constructed. The purity of rhFKBP52 was over 90% by SDS-PAGE analysis. And the concentration was 0.862mg/mL. FKBP52 inhibitor FK506 showed an IC50 value of 12.035ng/mL. One hundrund and twenty-one samples were demonstrated to have high inhibitory activities against FKBP52. Further analysis by HPLC-MS, among these positive samples, eleven actinomycete extracts were proved to contain FK506, six actinomycete extracts contain FK520, and another seven actinomycete extracts contain rapamycin. Based on 100 96-plate screening data, the assay produced the signal/background ratio of 2.35±0.26, the average of Z＇ factors was 0.78±0.1. Conclusion This HTS assay for rhFKBP52 inhibitor is robust and sensitive, and this screening assay is not only adaptable for novel rhFKBP52 inhibitors but also for the microbial producing strains of known FKBP52 inhibitors, such as FK506, FK520 and rapamycin.