糖原磷酸化酶抑制剂高通量筛选模型的建立

High throughput screening for glycogen phosphorylase inhibitor

目的：建立糖原磷酸化酶（GPa）抑制剂的体外高通量筛选模型。方法：用梯度离心方法提取大鼠肝脏GPa，以葡萄糖-1-磷酸作为底物，通过测定反应中磷酸根的释放量，间接反映肝脏GPa的活性。通过对反应体系的优化，调整反应条件，建立96孔微孔板的高通量筛选模型，用咖啡因对此模型进行验证，并评价高通量技术参数[Z＇-因子（Z＇-factor），信号本底比（signal to background，S／B），信噪比（signal to noise，S／N）]。用此模型对5000个样品（包括合成化合物、天然提取物）组成的随机库进行体外筛选，考察这些样品对GPa的抑制作用。结果：确定反应体系条件是：反应温度25℃，反应时间30min，底物浓度0．5mmol·L^-1，大鼠肝脏GPa的用量为250ng。用此条件测定咖啡因对GPa的抑制曲线，计算其半数抑制浓度（IC50）为（285．3±39．6）μmol·L^-1，与文献报道（IC50=240μmol·L^-1）基本一致；本模型技术参数Z＇-因子=0．79，S／B=0．45，S／N=11．32，表明此模型可以用于高通量筛选。应用此模型筛选得到有活性的化合物9个，其IC50在3．56～45．75μmol·L^-1。结论：建立的体外GPa抑制剂高通量筛选模型具有快速、微量、准确的特点，可以作为研究降糖药的工具。

Objective： To develop a high-throughput screening assay for identification of glycogen phosphorylase inhibitors. Methods： The glycogen phosphorylase （GPa） from rat liver tissues was extracted by a gradient centrifugation. GPa activity was assayed by quantifying a release rate of phosphate after an incubation of GPa with glucose-1-phosphate. A 96-microwell high throughput screening system was set up by optimizing the incubation conditions. The screening system was validated and assessed with regard to high throughput index, such as Z＇-factor, signal to background （S/B） and signal to noise （S/N）, using caffeine as the substrate. Inhibitions of GPa activities by 5 000 samples including synthetic compounds or botanic extracts were assessed using the high throughout assay. Results： The optimal incubation conditions were as followed： temperature at 25 ℃, incubation period for 30 min, substrate concentration at 0.5 mmol·L^-1 and quantity of GPa of 250 ng. The calibrated inhibition curve of caffeine on GPa activity showed an IC50 value of （285.3±39.6）μmol·L^-1, which was close to the one reported in the reference （IC50 = 240μmol·L^-1）. Other indexes included Z＇-factor of 0.79, S/B of 0.45 and S. N of 11.32. Nine out of 5 000 compounds were found to have high inhibitory activities using the assay, manifesting IC50 range of 3.56～45.75μmol·L^-1. Conclusion： This reproducible, micro-detective and accurate high throughput screening assay may be used as a tool in the investigation of GPa inhibitors.