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目的:建立以蛋白酶Neprilysin(NEP)为靶点的高通量药物筛选模型,应用该模型筛选抑制剂。方法:利用毕赤酵母表达系统。构建重组质粒p PICZα-A-NEP,表达载体通过与酵母菌X-33基因组染色体发生同源重组,将外源基因整合于染色体后实现目的蛋白的表达。应用荧光共振能量转移法(FRET)检测蛋白酶活性,优化反应条件,建立药物筛选体系,筛选抑制剂。结果:成功构建表达载体p PICZα-A-NEP;建立了以NEP为靶标的药物筛选模型,获得模型反应动力学参数Vmax=3.6μM/s,Kcat/Km=4.5×105M-1s-1,测定模型Z-因子为0.89,说明体系稳定可用于以NEP为靶标的药物的高通量筛选;并用该模型对天然产物组分库进行筛选,在0.5mg/ml的药物浓度下,得到抑制率较高的药物为4种,并测得半数抑制浓度IC50值,其中MDCNCL01000242的IC50值最低,为(8.31±0.03)μg/ml。结论:建立的药物筛选模型较为理想,适用于NEP抑制剂的筛选,可促进药物的研发。
OBJECTIVE: To establish a high-throughput drug screening model targeting protease Neprilysin (NEP), and to screen for inhibitors using this model. Methods: Pichia pastoris expression system was used. Construction of recombinant plasmid p PICZα-A-NEP, expression vector by yeast X-33 genome homologous recombination, the integration of foreign genes in the chromosome to achieve the expression of the target protein. The activity of protease was measured by fluorescence resonance energy transfer (FRET) method, the reaction conditions were optimized, drug screening system was established, and inhibitors were screened. Results: The expression vector p PICZα-A-NEP was constructed successfully. A drug screening model targeting NEP was established. The kinetic parameters of the model were Vmax = 3.6μM / s and Kcat / Km = 4.5 × 105M-1s-1. The Z-factor of the model was 0.89, indicating that the system was stable and could be used for high-throughput screening of drugs targeting NEP. The library of natural product components was screened by this model. The inhibition rate of the model was 0.5mg / ml The high drug was 4 kinds, and the half-value IC50 value was measured. The IC50 value of MDCNCL01000242 was the lowest (8.31 ± 0.03) μg / ml. Conclusion: The established drug screening model is ideal for the screening of NEP inhibitors, which can promote the development of drugs.